Home

ASTRA V User's Guide

Contents

1. BY ok HK Cancel 4 Click the Run experiment icon on the icon toolbar at the top of the win dow The template completes its data processing 5 Expand the Results node and double click the Report line The report will display the new aRI calibration constant and new aRI offset Save the ASTRA V file You may also want to print the report M1000 Rev H 8 29 Chapter 8 Editing Procedures 6 Goto the System tab on the Optilab rEX tab to the Constants button and press Enter Enter the new values into the Optilab rEX in the Constants dialog Tab to the Apply button and press Enter Tab to the Close button and press Enter The Optilab rEX aRI is now recalli brated 7 Measure and record the aRI value for pure ethanol which should be near 1 35 after the correct constants are in place and after purging the rEX with ethanol as described in the section Introduce Solvents on page 8 28 Setting Absolute RI Calibration Properties This procedure has the following properties Table 8 8 Absolute RI Calibration Properties Field Description New Abs RI Calibration The resulting absolute RI calibration constant These are measured in refrac tive index units RIU per pixel on the photodiode New Abs RI Offset The resulting absolute RloffsetinRIU Old Abs RI Calibration The previously used absolute RI calibration constant from the Optilab rEX Old Abs RI Offset The previously u
2. Experiment KE Configuration Procedures Results Save As Export as Text or CSV Save as Template Delete Available only in Experiment Builder Mode The sections in this chapter listed below correspond to the actions shown in the previous diagram Some commands behave differently depending on whether you are using ASTRA V Basic or a version that uses an experi ment database ASTRA V with Research Database or ASTRA V with Security Pack Table 6 1 Actions to Perform on Experiments Action Description See Create From Default Make a new experiment based on a template that was saved as page 6 4 the default template Create From Template Make a new experiment based on a configuration procedure page 6 5 and results template Create Blank Make a new experiment with no default configuration procedure page 6 6 or results Open Database Open an experiment from the experiment database page 6 7 Basic Open an experiment from a file This may include experi and ments saved with previous versions of Wyatt software page 6 8 Import Database Open an experiment saved in a file This may include page 6 9 experiments saved with previous versions of Wyatt software M1000 Rev H 6 3 Chapter 6 Creating amp Running Experiments Table 6 1 Actions to Perform on Experiments Action Description See Export Save an experi
3. Samples or Collection in the sample set tree in the Sample Sets tab 2 3 4 You can set properties by typing selecting from a list or checking a box Use the or next to a property to expand or hide lists of related properties You can move to other tabs in the dialog to view or set properties Click Apply or OK to make the changes The remaining sections of this chapter contain details about the properties you can set in the various tabs The Sample Set property dialog has tabs for the following items Configuration Sets global properties for the sample set configuration Samples Sets properties for samples in each well of the sample tray Also specifies the experiment template to be used for each sample Collection Shows collection data as samples are being run Configuration Tab The Configuration tab of the Sample Set property dialog looks like this WW Sample Sets Sample Set 1 Description Sample set 12 09 2004 efault Experiment Template My Templates ai tests rex ai D Humber of Samples 12 Configuration Samples Collection BY OK HK Cancel You can set the following properties for a sample set configuration Table 9 1 Experiment Configuration Properties Field Description Description Description of the sample set configuration Default Experiment Template The experiment template to assign initially to all the samples The template can be overri
4. Conventional Calibration Expanding this row shows the A coefficients of the following linear regres Function sion equation where M is the polymer molar mass and V is the elution volume log M Ag A V Ac AsV Note that a column profile can contain results both for a conventional and universal calibration Universal Calibration Expanding this row shows the A coefficients of the following linear regres Function sion equation where M is the polymer molar mass 77 is the intrinsic vis cosity and V is the elution volume 3 log M n Ap A V AV AgV Note that a column profile can contain results both for a conventional and universal calibration M1000 Rev H 7 27 Chapter 7 Configuring Experiments Connection Profiles A connection is an interface between two instruments There are three types of connections e A fluid connection represents a piece of physical tubing that routes the solution from one instrument to the next e An AUX connection represents a physical wire from the AUX output of one instrument to the AUX input of another instrument e An auto inject connection represents a physical connection between the auto inject output of an injector and the auto inject input of an instrument A connection profile stores information about a connection between two specific instruments Connection profiles are referenced by configurations Connections must be specified in an experime
5. UV Extinction Coefficient The extinction coefficient in mL g cm The extinction coefficient is used when the concentration of the sample is to be determined using a UV absorption instrument The value entered here is used as a default value when peaks are set for the data Concentration 7 30 The concentration of the sample in g mL An injector configuration always has a sample configuration associated with it in a profile See Injector Profiles on page 7 25 An autoinjector configuration always has a configuration for a sample associated with it in a profile The properties tab for samples has a table with a row of the properties in Table 7 19 for each sample well See Auto injector Connection Profiles on page 7 29 M1000 Rev H Sample Profiles Molecular Standard Profiles A molecular standard profile describes a commonly used sample such as BSA monomer that has well known properties Such profiles are used as reference standards for processes such as normalization with a light scat tering instrument Molecular standard profiles are associated with a peak in the data The values set for the molecular standard profile will be used in the peak You can set the following properties for a molecular standard Table 7 20 Molecular Standard Properties Field Description Name Name of the standard If you have already created a system profile for this sample click and select
6. gt Max Fit Delay Time gt Suppress Peaks Below Data with a higher delay time than the value you type is not used in the fit to the correlation function The default is 1 Type a size in nanometers below which peaks should be omitted from the analysis gt Show Residuals Check this box if you want the correlation function graph to show residu als The default it to omit residuals gt Use Disabled Slices ASTRA normally discards the entire slice if the avalanche photo diode APD is triggered So any measurement where the APD was triggered is excluded from analysis unless you specifically check the Use Disabled Slices check box See Rh from QELS Data on page 8 77 for more about the avalanche photo diode APD gt Prefilter If the Prefilter box is checked data points in the correlation function view that fall outside the minimum or maximum delay times the Min Fit Delay Time and Max Fit Delay Time fields are discarded from the fit You can see the fit line stop short of these points and the points themselves change color to red The rest of the points are still included in the analysis If this box is unchecked data points outside the range are used in the fit In the cumulants plot the average hydrodynamic radius and the distribu tion values at one standard deviation are presented This creates an error bar appearance for the graph but here the error bars indica
7. Dt cumulative intensity plot This graph shows the cumulative intensity as a function of translational diffusion Again this graph shows that the bulk of the intensity is contributed by fast moving smaller particles 8 93 Chapter 8 Editing Procedures Mass from Column Calibration This procedure calculates the mass of a sample based on the elution volume of a peak through a column You must have determined the response of the column to a series of known standards prior to using this procedure See Calibrate Column on page 8 37 for details Two main types of column calibration are available Conventional cali bration and universal calibration For conventional calibration the analyzed polymer must be the same as the polymer used for calibration For universal calibration the polymers may be different For example experiments that perform this procedure choose File gt Open gt Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the universal calibration or conventional calibration experiment in the Sample Data gt Analyzed Experiments folder For an experiment template choose File gt New gt Experiment From Template and open the universal calibration or determine column calibration template in the System Templates gt Vis cometry folder For more go to http www wyatt com solutions software ASTRA cfm and follow the links to
8. Experiment Builder Experiment Builder mode allows you to modify the configuration and pro cedure sequences in a template The icon to the left identifies portions of this manual that apply only if you turn on Experiment Builder mode by choosing System Preferences Experiment Builder Mode This mode allows you to open multiple procedure windows at once However you should be careful with this feature since changing and applying prop erties in one window does not generally result in changes to other open procedure windows To see such changes reflected in other procedure windows you should close and reopen them User Account Levels Security As part of the 21 CFR Part 11 compliance of ASTRA V with Security Pack all users must to log in with a unique user id and password The adminis trator sets up accounts with one of the following user account levels es ASTRA Administrator Can change database settings and can create modify and delete experiment files Also has privileges of Researchers Technicians and Guests e ASTRA Researcher Can create and modify experiment files Can connect to networked computers and instruments Also has privileges of Technicians and Guests e ASTRA Technician Can run a given experiment procedure sequence and save the resulting data Also has privileges of Guests e ASTRA Guest Has read only access to experiments and results Where necessary the user level required to perform an action i
9. Note No pre defined instrument profiles are provided with ASTRA In order to have instruments available you need to first save instrument profiles in your system database as described in Creating Profiles on page 12 3 Replacing an Entire Configuration You can replace the entire configuration with an experiment configuration that has been saved as a system profile For example you might have a standard experiment configuration that you want to use in many different experiments You can import that configuration into an existing experiment To import a completely new configuration follow these steps 1 If you have more than one experiment open make sure the one you wish to modify is selected in the experiment tree of the workspace 2 Choose Experiment Configuration Replace Configuration You see the Select Profile dialog In the Of Type field select Experiment Configurations Browse the system database for a profile to import A number of con figurations are provided with ASTRA V in the Example Configurations folder When you find a profile select it and click Open If you select the profile of an instrument or connection the item is added to your experiment If you select the profile of an entire configu ration that configuration replaces the existing one For more information about replacing a configuration or part of a configu ration with a system profile see Replacing an Experiment Configuration o
10. mean square radius mean square radius fitted molar mass molar mass fitted C rms radius Be aware that all the data items selected must be graphable against the same x axis index values For example both molar mass and concentration can be graphed against volume if that was the abscissa unit selected in the experiment configuration However the fit of RMS radius vs molar mass can only be graphed against an x axis of the molar mass 8 When you select an item in the Tag Properties checkmark list the Tag Proper Value Name molar mass ties fields change to describe me Nare le SSCS aspects of that data Some fields Freer ioe Ss are modifiable for some items Some fields vary depending on From Daa je the item selected Index Start fo 9 Modify values as needed for the Index Space jo data items you selected All the Index Steps 0 changes you make for various I Show Uncertainties items will be saved when you later click OK The properties in the list are as follows Table 11 3 Data Set Definition Properties Field Description Value Name The name of the selected item The y axis values Index Name The index against which the item can be plotted The x axis values Column Name Some data set items have column names that describe the type of data From Data If you have selected a function tag this box specifies whether the function is cal culated from the x axis data va
11. 0 P O 1 and we have Equation 20 R 0 K c M K n M since the concentration of mass in the it slice is ci n M If the elements of the particle whose molar mass is M are of uniform density and occupy a volume V then the number of particles per mL in the ith slice nj is pro portional to the extrapolated zero angle Rayleigh ratio divided by the square of the particle s volume 1 e Equation 21 n x R O V Therefore we can write the number fraction of particles within slice i as n D where Equation 22 D vin l j j 1 P J Wyatt New Insights into GPC Combined with MALS Waters Corporation GPC Sympo sium Proceedings San Diego 1996 M1000 Rev H E 7 Appendix E Particles Theory E 8 is the summation taken over all slices in the selected region or peak of the eluting fractions Note that although M is the molar mass of the parti cles that value is proportional to the mass of the particles Both are proportional to the volume of the particles if the volume is of uniform density Although the analysis of each slice results in a corresponding value of rz there may be other slices with similar sizes due to experimental fluctua tions in the derived values The expected monotonic variation of rg with elution volume may be obtained by fitting the calculated values to a selected functional form using a least squares procedure Alternatively the slice data may be sorted into a set of size
12. Cancel Type the domain you want to use to authenticate users Click Validate to confirm that the domain name you typed is accessi ble on the network 4 If you want users to be able to use ASTRA when the domain is not available check the Local Authentication box 5 Click OK 28 M1000 Rev H Running ASTRA Running ASTRA To run ASTRA do one of the following e Double click the ASTRA V icon on your desktop e Choose Programs Wyatt Technology ASTRA V from the Windows Start menu It may take a minute or so for ASTRA to open Avoid closing the initial startup window while waiting e e If you are using ASTRA V with Security Pack you will be prompted to log ecurity in Use a User Name Password combination set up as described in Setting Up User Accounts on page 2 6 If ASTRA privilege groups were set up on your local computer type the name of your local computer for the domain Otherwise if the ASTRA privilege groups were added for your networked account type the domain of your networked account User Name jastrauser Password Cancel Domain Loo Database By default ASTRA V with Security Pack and ASTRA V with Research Database store experiments in a Microsoft Access database called ASTRA Experiment A user with ASTRA Administrator privileges can change to another database by following the steps in Connecting to a Database on page 4 3 If you plan to change the database it is best t
13. Chapter 8 Editing Procedures Rh from VS Data This procedure calculates the hydrodynamic radius Rh using the viscos ity data You can use this procedure if your experiment configuration includes a viscometer You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines Rh If you place multiple methods that deter mine Rh in a procedure only the first one will be valid There are no properties to set for this procedure It runs without prompt ing for any values Experiment Builder This procedure is hidden in Run mode It is performed automatically as 8 80 part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode For more about viscometry data collection and analysis go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Viscometry Branching This procedure calculates the branching ratio between a linear and branched polymer sample This analysis is based on the fact that at a constant molar mass the molecular size decreases with increasing degree of branching Branching can be characterized by either the radius based branching ratio g which is defined as the ratio of the mean square radius of a branched and linear molecule or the intrinsic viscosity based branchi
14. Correlation function plot This graph gives an idea of how well the data fits the average correlation function over the peak Peaks 1 and 2 match pretty well while peak 3 shows some variation Generally the more species of different sizes you have the worse the correlation function matches the data Rh diff intensity plot This graph gives an idea of the number of each type of species by showing the intensity fraction as a function of hydrodynamic radius For peak 1 there seems to be a lot of particles with a radius of 4 nm and a much smaller number with a 300 nm size Peak 2 shows again a high number of 4 nm particles and a smaller number of 50 nm particles The larger particles are on the right side of the graph Rh diff weight plot This graph shows the distribution of Rh by weight fraction In peaks 1 and 2 the larger particles don t even register on this view Dt diff intensity plot This graph shows the intensity fraction as a function of translational diffusion That is the particles that move the most are toward the right side of the graph while the slower particles are toward the left side Rh cumulative intensity plot This graph gives an idea of the overall intensity produced by constituents below a specific size For peak 1 most of the intensity seems to be delivered by particles in the range of 4 nm through 300 nm Rh cumulative weight plot This graph shows the contribution to weight fraction for different sizes
15. Determine Interdetector delay Iw L jw d v DP v F o a v 2 S T 17 0 18 0 19 0 20 0 time min Delay min DAWH EOS to ViscoStar DAWU EOS to Optilab rEX Determine Delays Using Peaks in Selection BOK Z Cancel EH Apply The relative heights of the peak for the traces are auto scaled to match each other 4 Click the Determine Delays button The delays between instruments are automatically calculated This procedure stores the interdetector delays in the properties of the fluid connections in the experiment con figuration 5 Alternately you can type delay values directly in the property field The graph shows the peaks corrected for the delay values currently entered This procedure has the following properties Table 8 4 Interdetector Delay Properties Field Description Instrument relation A separate row is shown for each fluid connection between the light scattering instrument and other instruments Delay The time or fluid volume between the two instruments The units are determined by the Abscissa Units property of the experiment configuration Determine Delays Click this button to automatically calculate the delays between instruments Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Bui
16. H 8 78 For an example experiment that determines Rh from QELS data choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the band broadening example BSA vaf experiment in the Sample Data gt Analyzed Experi ments folder For an experiment template choose File gt New Experiment From Template to open a template in the System Templates gt Light Scattering gt With QELS folder The procedure has the following dialog WW gels batch regularization bsa insulin Procedures Rh from QELS data SEE results graph control graph Iw count rate at corr func Fe 0 010 3 1 20 S 50410 0 005 2 amp 2 lt 5 A 40x10 115 Si Z 3 0x10 0 000 g gt 1 40 SIS 2 0x10 4 05 0 0057 8 1 0x10 0 0 1 00 0 010 1 0x100x100x10 0 0010 01 Di 2 0 40 6 0 6 0 10 0 tau Sech time min Peak Number QELS Slice Index Abscissa Position 3 466 min Rh 4 2 0 1 nm Dt 5 79 0 19 e 7 cm fsec Processing Conditions M Temperature C 25 00 Viscosity gcm zech 8 9451e 003 APD State on Min Rh Threshold nm 1 000 Max Rh Threshold nm 300 000 Processing Parameters Min Fit Delay Time sec 0 000e 000 Max Fit Delay Time sec 1 000e 000 Use Disabled Slices Show Residuals E e The graph is used to show the fit to the QELS correlation function Use the graph on the right to select a peak and slice The left graph shows the cor relation funct
17. Plate Count The column manufacturer provides the initial plate count as documenta tion but this value changes over time You may enter the current plate count here as documentation when you perform an experiment The plate count is sometimes called Efficiency It quantifies the separat ing efficiency of the column in terms of the number of theoretical plates N The specific calculation varies by column manufacturer but generally measures how well the column is packed and its kinetic performance In general higher plate counts indicate more efficient columns More efficient columns yield narrower peaks than less efficient ones Asymmetry Factor The column manufacturer provides the initial asymmetry factor as docu mentation but this value changes over time You may enter the current asymmetry factor here as documentation when you perform an experi ment The asymmetry factor describes the shape of peaks generated by the col umn The distance between the elution volume at the peak apex Va and the front of the chromatogram at 10 of the peak apex Vj is divided by the distance between V and the backside of the chromatogram at 10 of the peak apex Vp A value greater than one indicates a tailing peak in which the bulk of material elutes after the apex Likewise a value less than one indicates a leading peak in which the bulk of the material elutes prior to the apex Resolution The column manufacturer provide
18. on page 2 10 Dilution Factor Capillary Volume Specific Viscosity Mode If you are using a UV detector plumbed before the ViscoStar in the flow sequence use the default dilution factor of 1 00 If you are using an RI detector or any other instrument plumbed after the ViscoStar see Measuring the Dilution Factor on page 7 22 to determine the value to enter here The internal capillary volume of the ViscoStar instrument This value is used to correct for certain types of mixing effects This does not include the volume of the adjustable reservoir You can compute the internal capillary volume using the provided experiment template If you are not using a concentration detector with the ViscoStar check the Specific Viscosity Mode box Intrinsic viscosity can only be calculated using both specific viscosity and concentration data Batch Mode Band Broadening gt Enable Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broad ening parameters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band
19. 5 Click Auto Baseline to automatically set baselines at the same collec tion times for all detectors 6 Check the automatic baseline settings by examining the baseline for each light scattering detector in turn If necessary you can modify the baseline for an individual detector 7 Ifyou are using other detectors RI UV or viscometer you should set baselines for them independently Other detectors are affected quite differently by chromatographic details such as injection peak pump fluctuations and baseline stability 8 Click OK to continue running the experiment You can see the details of the baselines selected for each detector by expanding the Details list You can clear all baseline settings by deleting the baseline for the source detector and then clicking the Auto Baseline button When you position a baseline the properties set for each detector are as follows Table 8 14 Baseline Properties Field Description Auto Baseline Click this button to automatically set baselines based on the selected detector Visible This box is checked if the data from this detector is shown in the graph Style None indicated no baseline is set Snap Y indicates the Y value of the end point is calculated based on the Y value of the surrounding data points Manual indicates that the X and Y endpoints are manually specified and are not taken from the Y value of the endpoint data X1 Sh
20. Abscissa Position 4 845 mL Concentration 1 633 0 806 e 7 g mL Conjugate dnide 0 247 0 121 mL q Conjugate UV ext 5523 8 4106 0 mL fg cm BY OK St Cancel The properties for this procedure are as follows Table 8 32 Copolymer Analysis Properties Field Description Molar Mass Shows the total molar mass for the currently selected peak Protein Fraction Shows the protein fraction for the currently selected peak Protein Molar Mass Shows the molar mass of the protein indicated by the selected peak Modifier Molar Mass Shows the molar mass of the protein modifier indicated by the selected peak Peak Number Click on the peak for which you want to view values Slice Index Shows the current slice index which is indicated by a vertical blue line in the graph Fit Degree Shows the fit degree selected for this peak in the Peaks procedure Abscissa Position The value on the x axis for the selected peak and slice Concentration The concentration at the selected slice Conjugate dn dc The dn dc at the selected slice Conjugate UV ext The UV extinction at the selected slice M1000 Rev H 8 87 Chapter 8 Editing Procedures 8 88 Protein Conjugate Analysis This procedure analyzes protein conjugates which are an important class of copolymers This analysis requires the use of a light scattering detector in conjunction with both a UV and RI detector The Protein Conjugate
21. E online a2 200k ps Procedures Basic collection strip chart online a2 200k ps Ga 4710101111 50 0 100 0 150 0 200 0 time min Calculated Duration min Trigger on Auto Inject Set properties Duratig 240 00 1 000 min olfection Interval sec OELS Interval sec E Details Collection Recycle Injection Delay min Laser Saver Mode COMET Run Duration min BY OK Post Collection Recycle HK Cancel 2 000 waste 0 00 0 00 recycle The properties you can set are as follows Table 8 2 Basic Collection Properties Field Collection Operator Description The user at the time data collection is started See Operator Names in Reports on page 10 2 for details Calculated Duration Shows the total duration of the collection based on the Duration Injection Delay and COMET Run Duration Trigger on Auto Inject Check this box if an auto inject signal will signal the start of data collection Duration The time or fluid volume for which data is to be collected The units are deter mined by the Abscissa Units property of the experiment configuration Collection Interval sec How often the light scattering or other instrument should collect data The default is every 0 25 seconds This interval may be set to a multiple of 0 125 seconds This interval is used to set the collection interval for Wyatt LS instruments Vis coStar
22. No pre defined instrument profiles are provided with ASTRA In order to have instruments available you need to first save instrument profiles in your system database M1000 Rev H Modifying Profiles Modifying Profiles The system profiles are contained in the system database To work witha system profile you have already created or one of the profiles provided with ASTRA V you first open the profile in the System Profiles tab of the workspace Then you can view edit or rename the profile Opening a Profile Shortcuts M1000 Rev H If you just created a system profile it is open and listed in the System Profiles tab of the workspace If the profile is not listed you need to open it before you can modify it To open a system profile follow these steps 1 Select File gt Open System Profile Press Ctrl Alt O Click the down arrow next to the D icon Right click on any profile item in the workspace and choose Open 2 In the Open dialog locate the profile you want to open C My Profiles C3 Profile Sample Configurations C System Solvents mw Of Type LA Types D Cancel Profiles may be stored in any of the following folders e My Profiles A handy place to save profiles you create Example Configurations A set of complete configurations System Solvents A collection of solvent profiles 3 You can select the type of profile you want to find in the Of Type list As in standard file select
23. Picture Native Data Series Include all a Point Index Iw Point Labels om Iw Header Text Point Colors C XML C HTML Table Delimiter Excel Tab Text Quotes 4 Select the output format you want text XML HTML table or Microsoft Excel If you select Text you can select a Delimiter to sepa rate the fields Delimiters are commonly used if you will be importing the data into a spreadsheet or database You can also specify a quote character usually blank or to use around text in the output 5 Select any other information you want to include in the data file such as headers 6 Once you have set the format and other options click one of the follow ing buttons Copy Store the data on the clipboard in this format for pasting into another application Save Send the data to a file of this type You are prompted for the file name and location The default file extension matches the for mat you selected in the Edit Graph window Send Send the data to an application such as Microsoft Outlook This is typically used to email the data If you do not find the organization of data output useful try the output described in Exporting an Experiment on page 6 17 11 18 M1000 Rev H Working with System Profiles This chapter explains how to create and use system profiles in ASTRA V CONTENTS PAGE About Profiles is 62 20 caccsststd besasdeaededsotict aaria aaiae SR 12 2 Cr
24. Select this check box to use a linear model rather than imported data to per form the analysis Imported Linear Data Select the data to use for the linear sample Choose Experiment Copy From in the menus to copy the linear data into the experiment Then click to select the copied data for the branching procedure The data you select is used if the Use Linear Model box is unchecked Plot Select the type of plot to view Model Select either trifunctional tetrafunctional star or comb branching Slice Type Select monodisperse or polydisperse Note that the star and comb branching Repeat Unit Molar Mass models require a monodisperse slice type Specify the repeat unit molar mass for long chain branching in g mol Method Linear Mark Houwink Sakurada K Parame ter Select radius mass or viscosity as the method If you select mass you must also specify the Linear Mark Houwink Sakurada a and Drainage parameters If the current experiment does not have a light scattering detector the radius option is not available Likewise if a viscometer is not present the viscosity option is not available Specify this parameter if you chose the viscosity method and checked the Use Linear Model box Linear Mark Houwink Sakurada a Parameter Specify this parameter if you chose the mass or viscosity method and checked the Use Linear Model box Drainage Parameter e
25. Table 8 9 UV Calibration Properties Field Description Percentage to Keep The percent of the marked peak data to use for calibration If the plateau is flat not drifting in the peak range using the default value is recommended Enabled Peaks This list shows the enabled peaks Normalization If an experiment has already been run you can open the Normalization procedure view by choosing Experiment Configuration Normalize Normalization is the process by which the various detectors signals are related to the 90 detector signal and the instrument calibration constant By definition the 90 detector always has a normalization coefficient of 1 This is detector 5 on a DAWN 8 and detector 11 on all other DAWNs Good normalization is an important component in achieving good results from a DAWN When to Normalize The first time you use ASTRA after installing your DAWN or miniDAWN you need to normalize the detectors Thereafter you will need to normalize only under certain conditions e Whenever you collect data from a sample whose solvent is different from that used for the previous normalization e For aqueous solvents whenever the solvent offsets you just collected are better than those used for computing normalization coefficients earlier Better means lower voltages e Whenever you reinstall the DAWN flow cell e Whenever you alter the laser alignment Laser alignment is necessary only for DAWN
26. The value is shown in the Report in ml g cm RESULTS Calculated UV extinction assuming 100 mass recovery Peak 1 UV ext mL g cm 5 645e 1 M1000 Rev H B 15 Appendix B System Templates Viscometry B 16 The following templates and folders are provided in the Viscometry folder Calibration The following template is provided in the Viscometry gt Calibration folder Determine Universal Column Profile CT Analysis of a sample using universal or conventional column calibration takes place in two distinct phases First the response of a column to a set of standards with known molecular weights must be measured Once this determine universal column profile phase is complete the unknown sample can be analyzed This template allows you to set up the column profile for Universal Calibration With Light Scattering The following templates are provided in the Viscometry gt With Light Scat tering folder Branching VS LS AT This template lets you determine the branching ratio using either viscos ity or light scattering data It does not require conventional or universal calibration as it measures the mass using the light scattering signal Online CT This is the standard online template for use when you have a light scatter ing detector and a viscometer Diagnostics The following template is provided in the Viscometry gt Diagnostics folder VS Noise CT This template measures the noi
27. definition of D 4 LS 8 19 RI 8 21 UV 8 31 CFR Part 11 icons 1 4 Chart tab Editing dialog 11 14 chromatography systems 9 5 Close command File menu 6 15 Coated sphere fit model 8 55 col file extension 8 13 collection procedures list of 8 10 raw data produced by 11 7 Collection tab sample sets 9 8 column calibration 7 26 columns Index 2 definition of 7 26 profiles for 7 26 Command log tab Diagnostic Manager 5 9 commands log of viewing 5 9 parameters for 5 8 sending to instruments 5 8 Commandes tab Diagnostic Manager 5 8 computers adding to ISI list 2 11 removing from ISI list 2 12 viewing in instrument list 2 10 Concentration Source 7 10 concentrations converting refractive indexes to 8 56 converting UV data to 8 56 configuration adding connections to 6 24 adding instruments to 6 24 definition of 1 6 3 6 6 2 7 2 editing 3 7 example of 7 4 exporting to system profiles 12 4 Configuration folder Experiments tab 6 2 6 24 see also configuration configuration procedures list of 8 14 see also experiment configuration Configuration tab sample sets 9 6 Connect to Database Database Adminis tration menu command 4 5 connections adding to configuration 6 24 adding to experiment configuration 7 6 definition of 7 28 profiles for 7 28 types of 7 28 contact information 1 9 Contents command Help menu 3 15 conventional column calibration 7 26 conventions us
28. gt Cascade Arrange open views in cascading fashion All Window Tile Horizontal Arrange open views in column wide views All Window Tile Vertical Arrange open views in row tall views All Window Windows Open a list of windows to select from All Help Menu The Help menu contains the following commands Command Keyboard Description Shortcut Help Contents F1 Open help table of contents See page 3 15 All Help Search Open search tab of online help All Help Index Open index tab of online help All Help Wyatt Online Open Wyatt Support Center website All Help Check for Look for newer versions of ASTRA available All Updates for download See page 2 3 Help About ASTRA Open version and copyright information All about ASTRA V A 8 M1000 Rev H System Templates This appendix provides an overview of the ASTRA V System Templates CONTENTS PAGE OVEVIEW ee eieiei eege esoe ee de eE eg B 2 GSM TEEN B 3 Light SCAtte rig sek seis antecceecscceaaethbdaesteeshs Feansheees aa i a B 4 RI Meas rement mesinin aa van ENEE Ee B 11 UV M as remet ssis a E E B 15 ViSCOME UY ossiani nnan a aa Aa ebana Aaa aaa EEE B 16 M1000 Rev H B 1 Appendix B System Templates Overview Hint B 2 This appendix provides an overview of the ASTRA V system templates These templates are stored in five different folders General RI Measure ment Viscometer Light Scattering UV Measurement This appendix is organized according to t
29. list of commands in A 8 wmf file extension 11 17 en ex Index 11 Index workspace definition of 1 8 hiding 3 14 repositioning 3 14 Workspace command View menu 3 14 wtc_field tags 10 3 Wyatt Technology Corporation contacting 1 9 WyattQELs ISI installation for 5 2 profiles for 7 15 X x axis units see Abscissa Units property experiment configuration XML files 10 3 Z Zimm fit method 8 62 D 13 Zimm fit model 8 55 zooming graphs 11 14 procedure graphs 8 5 Index 12 M1000 Rev H
30. tions should be collected during the same analysis run strip chart New Online A2 from online a2 5 col 0 0010 aP aa U A aim ve e 0 0005 detector voltage V Y Ba 2d A Ab See H H H DH ii 0 0000 90 0 400 0 unn 120 0 130 0 time min 4 Set baselines as you would normally X pU SANORYas Eu LS3 LS4 LS5 LS6 LS7 LS8 LS9 LS 10 LS 11 LS 12 LS 13 LS 14 LS 15 LS 16 LS 17 LS 18 FM dRI Select peak ranges for each of the peaks It is important to make the peaks widths and thus the analyzed volumes as similar as possible Since the higher concentration sample peaks may be wider you may wish to use this as the width to set all peaks Typically you should start the leading edge of the peak right before the signal begins to rise from the baseline Likewise you should choose the trailing edge of the peak to corre spond to either the baseline or the inflection point if the signal rises towards a secondary peak M1000 Rev H 8 67 Chapter 8 Editing Procedures For example the following peak range was selected for Peak 5 starting just before the peak rises from the baseline and ending at the point where the concentration signal passes through zero slope to start the second peak 125 0 126 0 127 0 128 0 time min Note that this corresponds to a peak
31. CSCC inst e D 6 C I Carr Jr and B H Zimm Absolute Intensity of Light Scattering from Pure Liquids and Solutions J Chem Phys vol 18 p 1616 1950 J J Hermans and S Levinson Some Geometric Factors in Light Scattering Apparatus J Opt Soc Am vol 41 p 460 1951 M1000 Rev H Note M1000 Rev H Measured Quantities and Calibration When you perform a calibration ASTRA calculates a configuration specific constant from Eq 3 but you never see this number It is immedi ately converted to the instrument constant Aar via Eq 7 Eq 8 or Eq 9 depending on the cell type The A value is reported as the Calibration Constant and is the value entered in the DAWN or miniDAWN profile If at some later time you change Aar in the instrument profile ASTRA will recalculate Acgcc ASTRA also recalculates Acgcc if the solvent or cell type is changed This process may sound complicated but it enables you to calibrate with one solvent and make measurements with another and the software efficiently handles all the details Since changing the sample cell may require a realignment of the laser we recommend you always recalibrate afterwards The calibration measurements should be made with great care as the accuracy of all other measurements depends upon them As long as the system is left undisturbed it is not necessary to recalibrate but we advise making occasional checks using a standard polymer as
32. Forward Monitor and none Laser Monitor The light scattering signals are divided by the laser monitor which corrects for fluctuations in the laser intensity due to power fluctuations Forward Monitor The light scattering signals are divided by the for ward laser monitor which corrects for both laser intensity fluctuations and absorbance by the sample none No correction is performed for laser intensity fluctuations or sample absorbance Use this option only if signal levels are so low that digital noise from the laser monitor signal can contaminate data No additional wiring is required for the DAWN HELEOS or DAWN HELEOS 8 to collect forward laser monitor data Disable Collection Check this box to disable data collection for this instrument For example if the light scattering instrument has the QELS option it is possible to disable the DAWN collection and collect QELS data alone Polarization Analyzer Check this box if the polarization option is currently installed on the instru ment See the DAWN hardware manual for details QELS gt Option Check this box if the DAWN instrument has a detector replaced with a QELS fiber QELS gt Replaced Detector If QELS is enabled type the number of the detector replaced for the QELS fiber Band Broadening gt Enabled Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms
33. Sakurada 8 83 sample set logs viewing 4 8 9 10 Sample Set menu list of commands in A 6 see also specific menu commands Sample Set menu see specific menu com mands Sample Set Import menu command 9 4 sample sets Collection tab 9 8 Configuration tab 9 6 creating 9 3 exporting 9 11 file type 9 4 importing 9 4 opening 9 4 running 9 9 Samples tab 9 7 stopping 9 10 samples definition of 7 30 profiles for 7 30 Samples tab sample sets 9 7 Save As command File menu 6 15 6 16 6 18 9 10 12 6 Save As dialog 6 15 6 16 9 10 Save command File menu 6 15 6 16 9 10 also Mark Houwink Index 10 Save Configuration As Experiment Con figuration menu command 7 8 12 4 Script Collection procedure 8 12 scrolling graphs 11 14 Search command Help menu 3 15 second virial coefficient 8 65 security 2 8 Security command System menu 2 8 Security Pack operating tier 1 4 Security Pack version 1 3 Select an item to delete dialog 4 8 6 19 9 11 12 7 Sign off dialog 6 12 Sign Off procedure 8 100 sign offs 4 7 6 12 8 100 signal to noise ratio increasing 8 44 peaks and 8 52 signal to noise ratio and concentration 8 33 Smoothing procedure 8 43 solvents definition of 7 32 for normalization 8 33 profiles for 7 32 specific viscosity converting to intrinsic 8 57 Sphere fit model 8 55 spikes noise removing from collected da ta 8 42 SQL Server database 4 4 ss file
34. Setting Normalization Properties You can set properties for this procedure before or during the experiment Double click on the Normalization procedure to open its property dialog Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode This procedure has the following properties Table 8 10 Normalization Properties Field Description Peak Number Type the number of the peak that corresponds to your normalization standard Radius The radius of the normalization standard in nm Action Click the Normalization button in this row when you are ready Coefficients for The Old column shows previous normalization coefficients for each detector The Detectors New column shows the computed normalization coefficients Normalization Type The type of normalization to use Options are standard and area e Standard normalization uses the Rayleigh Ratio peak apex as the basis for normalizing In effect it divides the peak apex for each detector angle by the peak apex value for the 90 degree detector e Area integrates Rayleigh Ratios over the entire peak The Rayleigh Ratio peak areas for each detector angle are divided by the area for the 90 degree detector to yield the normalization coefficient This method provides better perform
35. This list has a checkmark next to detectors whose data is used in the calculation You can disable individual detectors by removing the checkmark Enabled Peaks FOM gt 1 ton You can omit peaks from the plot by removing the checkmark next to the peak number The FOM value shown for each peak is a Figure of Merit which is a unitless value that reflects the ability to measure A2 accurately If the FOM for a peak is less than 1 or slightly greater than 1 then the peak will help measure A2 accurately The Online A2 model is based on a series expansion of the Zimm model in concentration It is best suited for small molecules for which the Berry model is not applicable You cannot specify the light scattering fit model for this analysis 8 66 M1000 Rev H Determining A2 Using the Online A2 Procedure Analysis Procedures The Online A2 procedure extends the analysis to include the third virial coefficient Az and removes the need for a band broadening correction To analyzing a sample for Ap follow these steps 1 Create a new experiment using the System Templates gt Light Scatter ing folder gt online A2 template 2 Adjust the configuration to reflect your light scattering and concentra tion detectors the default is a HELEOS and an Optilab rEX 3 Run a data collection consisting of a series of injections of varying con centrations The following example shows five samples These injec
36. e ASTRA Guest Has read only access to experiments and results Where necessary the user level required to perform an action is identified in this manual Lines above and below the Security icon in the left margin as shown here highlight such information Security information is specific to ASTRA V with Security Pack There are no access restrictions when ASTRA V is used in other operating tiers Security How This Manual is Organized The first three chapters of this manual provide an overview of ASTRA explain how to install ASTRA and prepare it for use and how to get started using ASTRA 1 4 M1000 Rev H Using This Manual The remaining chapters in this manual correspond to items in the ASTRA environment as shown in Figure 1 2 ASTRA Fie Edit View Experiment System Window Help Chapters 4 amp 5 BEA Pa AEE dp SE a ez Chapter 6 7 Experiments E SampleSets S System Profiles ies Experiments Ceper 7 Ti E a Configuration EOS batch O 4 DAWN EOS DAWN Ge AA Solvent water Chapter 8 Sa Sample untitled SY Procedures F2 Basic collection Despiking Procedure Define baselines Define peaks 2 Determine mass and radius from LS data Chapter 10 CH Results gt E Report summary Chapters D E Report detailed E Graph untitled Si leie For Help press F1 Figure 1 2 Workspace Items and the Chapters that Discuss Them Manual Conventions
37. ie g r B B expl 2I t T E 3 Here the decay time is now the average for the distribution while the higher moments correspond to the variance or width of the distribution Ko the skewness of the distribution K and so on 1 B Chu Laser Light Scattering Basic Principles and Practice Academic Boston 1991 2 D E Koppel Analysis of macromolecular polydispersity in intensity correlation spectroscopy The method of cumulants J Chem Phys 57 4814 4820 1972 M1000 Rev H F 3 Appendix F QELS Theory In practice it usually only possible to determine the first two moments of the expansion in Eq 6 that is the average and variance These are often referred to as the first and second cumulant In this simplest form the method of cumulants then boils down to fitting the correlation function to a Gaussian distribution of decay times only the average and width of the distribution are obtained Application of the Method of Cumulants In the ASTRA software a variant of Eq 6 is used to obtain the first and second cumulants in a nonlinear least squares fit of the correlation func tion This variation was derived by Frisken and is given by Equation 7 3 e r B Bexp af a T Se 3 Here the moments Z correspond to the K terms in Eq 6 and are the physical moments about the mean J Eq 7 is inherently more stable than Eq 6 when fitting at large delay times 7 thus leading to a more robust
38. m5 Experiments E System Profiles Experiments CH Experiment1 9 Configuration Profile LS Online SY Procedures E Basic collection Despiking Procedure Define baselines Define peaks Convert to physical units Convert RI to concentration Determine mass and radius from LS data Fit mass or radius procedure 2 Determine distributions and moments QJ Data Set Definitions Results Lit ii iii fii fii i EI gt v A procedures status is indicated by its icon as follows Collection proce dures have a special two page icon for all states E Procedure has not been run since the procedure was last modified Procedure has been run successfully z F Procedure is currently running P E necessary data to run Procedure is in an invalid sequence location or does not have the M1000 Rev H About Procedures About Data Processing in ASTRA When ASTRA runs a procedure the data is modified in the sequence spec ified by the set of procedures in the experiment The following figure shows a typical sequence Config Profile b Collection Procedure Collection Script DN original Data Additional Transforms Baselines Procedure Analysis Procedure Initially a collection procedure is responsible for gathering data through the ISI from instruments specified in the configuration The original data
39. specify the viscosity of the solvent in P Poise at the reference temperature Viscosity Model gt Model Parameter 2 If the Viscosity Model is Linear set this parameters using the following model where n T is the viscosity as a function of temperature nT F P T P e P is the viscosity in P at the reference temperature e Po is linear temperature dependence of the viscosity P C e Pg is the reference temperature for the model in C e T is the temperature as determined by the appropriate device s tempera ture probe in C If the Viscosity Model is Exponential set this parameters using the following model where n T is the viscosity as a function of temperature n T P exp P T P e P is the viscosity in P at the reference temperature e Pp is the exponential temperature dependence of the viscosity in 1 C e Pg is the reference temperature for the model in C e Tis the temperature as determined by the appropriate device s tempera ture probe in C Viscosity Model gt Reference Temp If the Refractive Index model is Linear or Exponential set the Reference temperature P3 in the previous equations C for which the reference vis cosity is valid M1000 Rev H 7 33 Chapter 7 Configuring Experiments 7 34 M1000 Rev H Editing Procedures This chapter explains how to set up your experiment in ASTRA V to collect and analyze data This is d
40. theta 2 Figure 8 3 Debye plot showing accurate normalization coefficients for all angles results graph 36x10 34x107 32x10 R theta 30x10 28x10 26x10 0 0 0 2 0 4 0 6 08 sin theta 2 Figure 8 4 Debye plot showing incorrect normalization coefficient for detector 9 8 36 M1000 Rev H Configuration Procedures Calibrate Column M1000 Rev H ASTRA provides this procedure for developing a column profile Such profiles can be used to compare the absolute molar masses derived from light scattering results to the relative molar masses derived from conven tional size exclusion chromatography Such comparisons can illustrate possible errors generated by relative molar mass measurements and may be useful for characterization of branching This procedure determines the calibration constants stored in the generic column profile see page 7 26 Two main types of column calibration are available Conventional cali bration and universal calibration For conventional calibration the analyzed polymer must be the same as the polymer used for calibration For universal calibration the polymers may be different Technique Conventional ti eil Conventional Universal without Viscometer Data Universal with Viscometer Data The calculation for universal calibration requires either intrinsic viscosity data or known Mark Houwink Sakurada K and a coefficients for the polymers used for calibrat
41. 0 1850 0 1850 A2 mol mL g 0 0000e 000 0 0000e 000 0 0000e 000 x Cancel b Use your mouse to click on one end of a peak range Then drag to the other end of that peak range 3 10 M1000 Rev H Performing a Simple Light Scattering Experiment c Continue adding peak ranges for the rest of the collected data A number is shown for each peak that corresponds to the column for that peak below the graph The selected peak is shaded For exam ple these are peaks selected for a batch experiment Note If you want to zoom in on the graph hold down the Ctrl key and use your mouse drag an outline around the area you want to see To zoom back out hold down the Ctrl key and click your right mouse button d Click OK to continue running the experiment 5 The experiment runs to completion and all the 3 procedure icons in the experiment show that they have been run A procedure s state is always indicated by its icon as follows Collec tion procedures have a special two page icon for all states Procedure has not been run since the procedure was last mod ified E Procedure has been run successfully g Procedure is currently running E Procedure is in an invalid sequence location For more about running experiments see Chapter 6 Creating amp Running Experiments M1000 Rev H 3 11 Chapter 3 Getting Started Viewing Reports To view a report simply double click on it in the
42. 10 The Instrument Server Interface ISI allows you to access instruments connected locally or across your internal Local Area Network LAN That means the instruments you use with ASTRA need not be directly con nected to the computer you are using Next generation instruments from Wyatt Technology Corporation such as the DAWN HELEOS Optilab rEX and ViscoStar can be controlled directly over the network or a local USB connection They have an inte grated ISI that can be accessed directly through the network You do not need to install an ISI on your local computer to access these instruments The following instruments can also be accessed locally or through the network in much the same way that a printer can be shared on a LAN For these instruments you must install the ISI on the computer to which the instrument is connected See Installing the ISI on Other Computers on page 5 4 e DAWN EOS DSP and DSP F miniDAWN e WyattQELs Other instruments including the following may be connected through the AUX input of another instrument As a result the instruments will be shown as part of an experiment configuration e Optilab DSP e Optilab 903 M1000 Rev H M1000 Rev H Instrument Server Interface Overview e Generic RI Instrument e Generic UV Detector e Generic Viscometer Figure 5 2 summarizes these types of connections local PC nne to AUX input of rEX or ViscoStar onnection or on local
43. 11 Working With Graphs s eeeeeeeeeeeeeees 11 1 About ASTRA Graphs eg eege ee neg 11 2 Using TEE EE 11 3 Using Custom Plots and Data Set Definitions 11 7 Data Collection and Storage 11 7 Creating Data Set Definitions ececeeeeeceeeeeeeeeeeeeeaeeeeaeeeeeeeeeeaaeeseeeeeessaeeseenees 11 8 Creating Custoni Te 11 10 Creating Surface Plots cccecccceceseeceeeeeeceeeeeeeeeeceaaeeeeaaeeceaeeeeeaaaeseeeeeeseaeeeeeneeesaas 11 12 Viewing and Modifying Graphs AEN 11 13 Customizing Line Colors and Widths eccccececeeeeeeeeeeeeeeeeeeaeeseeeeeesaeeeseneeeeeas 11 13 Zooming In and Out Graphs 10 eeeeeeeececeeeeeeeeeeeeeeeeeaeeseeeeeesaaaeeeeeeeesseeeseeeesas 11 14 Repositioning KETTEN 11 14 AXIS SOUS EE 11 14 PHI REI 11 15 Copying REENEN 11 16 Exporting Cra S EE 11 16 Exporting Pictures veiene aisha e a a ae ate ARA 11 16 Giele RRE 11 17 Chapter 12 Working with System Profiles c00 12 1 About Profiles eege eege Eed 12 2 Creating e 12 3 Creating a New Profile A 12 3 Exporting a System Profile ccccccsecceeeeeeeeeeeeeeeeeeeeeeeeeaaeeeeeeeeeeeaeeeseeeeseceeeseaeeeee 12 4 6 M1000 Rev H Contents Modifying Se UE 12 5 Opening a Gite 12 5 Editinig A re 12 6 leie E Nd UE 12 6 Duplicating a Profile with Save AS cccccccceeseeeeeeeeeeeeeeeeeeeeaeeeeeaeseeeeeeeteaeeneeeeeee 12 6 EI E Nd UE 12 7 Deleting a Ne UE 12 7 Re Re 12 8 Adding an Item to a C
44. 33 Protein Conjugate Analysis Properties Field Description Slice Index Shows the current slice index which is indicated by a vertical blue line in the graph Model Shows the model selected for this peak in the Peaks procedure Fit Degree Shows the fit degree selected for this peak in the Peaks procedure Abscissa Positio Concentration n The value on the x axis for the selected peak and slice The concentration as the selected slice Conjugate dn dc The dn dc at the selected slice Conjugate UV ext The UV extinction at the selected slice Enabled Detectors The detectors to enable for this analysis Regularization 8 90 This procedure regularizes the results of a QELS batch experiment using the DYNALS regularization algorithm from ALANGO See Regulariza tion on page F 6 The Regularization procedure supports the reporting the results that were previously available in the separate QELSBatch program This procedure now reports the mean peak and standard deviation of the reported values hydrodynamic radius and translational diffusion The regularization procedure provides a way to analyze a batch sample a vial of some mixture of substances Rather than setting up a chromatog raphy system to separate the components using a column or membrane system you can use the QELS data to identify the various Rh values of the mixture in the vial For an example experiment that performs regulari
45. 75 F F1 key 3 15 F5 key 8 5 FAX support 1 9 Feature Activation command System menu 2 5 Feature Activation dialog 2 5 file extensions for experiment files 6 8 6 16 9 10 for exporting experiments 6 17 for graphics files 11 17 for importing experiments 6 9 for sample sets 9 4 for scripts 8 13 File menu see also specific menu commands File menu list of commands in A 2 firewall issues 5 4 Fit Mass or Radius procedure 8 57 fit methods 8 62 D 12 D 13 D 14 fit models 8 55 flow mode see online mode fluid connections definition of 7 28 profiles for 7 28 Forward Monitor 7 10 baseline peak 7 10 8 52 selecting signal 7 12 7 13 7 15 fractionated mode see online mode fractionation 1 6 From Default Experiment menu com mand 6 4 From Template Experiment menu com Index 5 Index mand 3 5 6 5 From Template Sample Set menu com mand 9 3 G gel permeation chromatography see GPC GIF files exporting graphs to 11 17 GPC gel permeation chromatography D 2 graphics files exporting graphs to 11 16 graphs adding to experiment 6 28 11 10 adding to report 10 5 axis settings for 11 14 coordinates 11 13 of data from multiple experiments 11 10 exporting to data file 11 17 exporting to graphics file 11 16 modifying 8 6 11 6 11 13 11 14 printing 11 15 of procedures 8 5 saving format 11 13 scrolling around 11 14 viewing real time 5 5 zooming in and out o
46. 8 2 icons in 3 11 8 2 8 8 Processing Operator property experiment configuration 7 9 Profile New menu command 12 3 Profile Open menu command 12 5 profiles definition of 1 7 see also configuration system profiles properties of instruments 5 10 Properties Database Administration menu command 4 5 Property tab Diagnostic Manager 5 10 Protein conjugate analysis procedure 8 88 pump HPLC pump with injector 8 23 syringe pump 8 23 pumps definition of 7 25 profiles for 7 25 Q QELS Quasi Elastic Light Scattering 1 7 QELS data determining Dt from 8 77 determining Rh from 8 77 Quasi Elastic Light Scattering see QELS R radius see A2 radius hydrodynamic radi us rms radius Radius from LS Data procedure 8 71 Random Coil fit method D 14 Random Coil fit model 8 55 M1000 Rev H Index raw data 8 3 11 7 raw data after transform 11 7 Rayleigh ratio converting voltages to 8 56 equations for D 3 measurement and calibration of D 4 red circle blinking in Diagnostic Manager 5 7 red X icon in Procedures folder 8 8 refractive index see RI entries Regularization procedure 8 90 Remove Connections property experiment configuration 7 10 Remove Instruments property experiment configuration 7 10 Replace Configuration Experiment Con figuration menu command 7 7 report including graphs 10 5 reports adding to experiment 6 28 10 4 copying to clipboard 10 6 customizing 10 3
47. ASTRA V crash recovery file See page 6 14 rat File saved by ASTRA version 4 70 or higher fora DAWN EOS DAWN DSP or DAWN DSP F See Importing ASTRA 4 Files on page 6 9 for information about fixing problems with these files mdf File saved by ASTRA 4 for a miniDAWN nwf File saved by DNDC 5 rw File saved by RICAL 5 Imported experiments have a complete set of the configuration items pro cedures data set definitions and results needed to view the experiment Importing ASTRA 4 Files M1000 Rev H ASTRA V can automatically import most ASTRA 4 experiments However some issues may occur when importing certain files For this reason an Import Wizard allows you to attempt to fix such problems when importing the file Typical issues that may cause problems are that in some ASTRA 4 files the smoothed data is stored but the original data is not available Also 6 9 Chapter 6 Creating amp Running Experiments 6 10 some files may not contain information about how AUX channels were used to receive RI or UV data for concentration calculations Click the Details button for a description of the results fitting changes needed by your experiment When you open or import an ASTRA 4 ASTRA 4 Import Wizard file adf or mdf you see this dialog The default is to Import Method Results Fitting C Automatic V Change necessary e Specify AUX Detectors F perform an auto one D
48. Add To Experiment menu option 5 Open the Transform category and add the Blank Baseline Sub traction procedure to the experiment 6 Drag this added procedure to position it right after the Despiking pro cedure SY Procedures Sp Basic collection EI Despiking 8 Blank Baseline Subtraction EI Baselines E Interdetector delay 7 Open the Blank Baseline Subtraction procedure Value Blank Baseline Data Source C Program Files WTC Astra 5 3 Sample DatalAnalyzed E Blank Baseline Subtraction Preview LS 11 EN Instruments to Subtract 8 Click the Import button on the right end of the first row to open the Copy From dialog 8 48 M1000 Rev H Transformation Procedures 9 In the Source list select the blank experiment and click Import E Copy From m Destination PS Close C AProgram Files WT C Astra 5 355 ample Data 4nalyzed Experiments gels Import Source blank collection H gels batch regular C Program Files WTC Astra 5 355 ample DatasAnaly gi 10 In the Blank Baseline Subtraction procedure click the Browse button for the Blank Baseline Data Source Choose one of the Available data sets and move it to the Included list Typically a good choice is raw data despiked Then click OK Data Set Definition Blank Baseline Data Ta __ Cancel R Cancel Available Included raw data copied from
49. Analysis dialog allows you to see the total mass and protein fraction on a slice by slice basis ASTRA calculates the size of the complex mass of the complex and masses of the constituents and the uncertainties for these values For an example experiment that performs a protein conjugate analysis choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the protein conjugate experiment in the Sample Data gt Analyzed Experiments folder For an experiment template choose File New Experiment From Template to open the protein conjugate template in the System Tem plates gt Light Scattering folder or the protein conjugate template in the System Templates gt Light Scattering gt With QELS folder For more about protein conjugate analysis go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Protein conjugates and copolymers This procedure should be placed in the sequence with the analysis proce dures but before the fit mass and radius procedure if it occurs M1000 Rev H The procedure has the following dialog Analysis Procedures WW protein conjugate membrane protein Procedures Protei DBR results graph control graph 420x107 118x10 cR theta 116x10 lt K 144x107 00 02 04 06 08 sin theta 2 Molar Mass rms radius Protein Fraction Protein Molar Mass Modifier Mo
50. C Program Files WTC As 11 In the Instruments to Subtract list choose which instruments should have their baseline subtracted M1000 Rev H 8 49 Chapter 8 Editing Procedures 12 You can then use the Blank Baseline Subtraction Preview to see how the blank subtraction affects the signal Blank Baseline Subtraction Procedure o dR o BlankdRl o Difference 8 0x10 6 6 0x10 6 a S H 2 ZA meurt z G TUIL fo S ZS te oY S 2 D 3 6 EI 2 0x10 a 4 Ei oorp un on on mn oun 500 time min 13 You can fine tune the baseline subtraction by using the standard Baselines procedure to specify the zero point for the signal A comparison of the blank subtracted and raw data files shows that in this case the unsubtracted sample underestimates the molar mass 8 50 M1000 Rev H Transformation Procedures Peaks M1000 Rev H After collecting data on your sample and setting baselines you need to define the peak regions This is done by marking the beginning and end point of every peak you want to include in processing When this procedure runs you see a message that says peaks need to be specified Set peaks by following these steps 1 Click OK to open the dialog for setting peaks E Zimm plot 200k PS vaf ProceduresWefine peaks Define peaks v T o a v 2 S o 14 0 16 0 time min Start min 9 7583 11 9333 13 8916 15 5333 Stop min 10 6333 12 4833 14 4
51. Chromatogram 2 If you want to plot a third set of data choose an item here and check the Show Chromatogram 2 property A dashed line is used for this data set The options are the same as for Chromatogram 1 Chromatogram 3 If you want to plot a fourth set of data choose an item here and check the Show Chromatogram 3 property The options are the same as for Chromatogram 1 Scaling Log Scale Choose whether to use normalized or relative scaling Normalized means all data of a particular type e g RI data is scaled against the largest value of that type across all experiments Relative means values in each experiment are scaled from 0 to 1 allowing all traces to have the same magnitude regardless of the actual values compared between experiments Check this box if you want a log scale used for the y axis of the graph The axis that log scaling applies to depends on the type of plot Abscissa Units Choose the x axis scale to use for a Distribution Type of time or volume The options are min minutes mL milliliters h hours sec seconds or msec milliseconds To further customize the appearance of an EASI graph right click on the graph and choose Edit from the pop up menu This opens the Edit Graph dialog which looks like this M1000 Rev H Edit Graph Graph Marker Size Very small Draw Mark Every f4 Cancel Line Point Size 1 Copy Iw Show chromatogram 1
52. DSP and DSP F instruments See Normalization on page D 8 for details on how normalization coeffi cients are used in calculations Selecting a Normalization Standard The normalization standard you use should have a low molar mass and a low polydispersity It should be highly concentrated and use the same solvent you plan to use for your experiments The issues related to these requirements are discussed in the following list e Isotropic Scattering The important concept to understand about normalization is that a very small molecule scatters light isotropically meaning with equal intensity in all directions So if we inject a very small molecule into the DAWN flow cell we might expect to measure equal voltages at all detectors This is not the case for several reasons Different detectors are collimated differently to improve perfor mance and thus do not see equal lengths of the flow cell bore e Refractive index effects come into play and change the light inten sity and scattering angles 8 32 M1000 Rev H M1000 Rev H Configuration Procedures Individual detectors vary somewhat in sensitivity We overcome these conditions by injecting a very small molecule and computing factors to force the light intensity to be equal for all detec tors As long as we inject a molecule whose size is too small to be mea sured accurately by the DAWN smaller than 10 nm the exact size does not matter A good isotrop
53. Data Analysis gt Conventional and Universal Column Calibration You can place this procedure with other analysis procedures and after all transformation procedures A procedure sequence can contain only one procedure that determines the mass from column calibration If you place multiple methods that determine mass in an experiment only the first one will be valid Your experiment may or may not contain the procedure Cal ibrate Column on page 8 37 If your experiment does not contain the Calibrate Column procedure you should import the results of a column calibration using Column Profiles on page 7 26 This procedure has the following dialog WW universal calibration PS in THF Procedures Wass from col Belt Value Mark Houwink Sakurada K mL g pe ee Mark Houwink Sakurada a 6 903e 001 Flow Marker mL none E Flow Marker Peak Peak 1 BY OK St Cancel 8 94 M1000 Rev H Analysis Procedures The properties for this procedure are as follows Table 8 35 Mass from Column Calibration Properties Field Description Mark Houwink Sakurada K Mark Houwink Sakurada a Type the known Mark Houwink Sakurada K parameter of the polymer used for calibration This is only used if the Universal without Viscometer Data calibration technique was used Type the known Mark Houwink Sakurada a parameter of the polymer used for calibration This is only used if the Universal without Viscometer Data calib
54. Description The sequence number in the sample set This field is non editable Well The number of the injection well in the sample tray This is for informational purposes only You need not use this field Enable If this box is checked the sample is enabled for the sample set run Name Name of the file to be generated by the sample set The sample set name is appended to this file name If multiple injections are requested for a sam ple the injection number is also appended to the name If no name is spec ified ASTRA generates a unique name for the generated file Description Description of the sample which typically contains more information than the Name Inj The number of injections for the sample Template The experiment template to use as the source for instrument configuration procedure and result formatting information for this sample The default experiment template is set in the Configuration tab but you can override it on a sample by sample basis here If the experiment template itself is modified after you choose it here but before the sample set is run the modified version of the experiment tem plate is used For information on choosing experiment templates see Creating Experi ments from Templates on page 6 5 For information on creating experi ment templates see Creating a Template on page 6 18 M1000 Rev H 9 7 Chapter 9 Using Sample Sets
55. Detectors GG a OK SL cancel E Apply You can place this procedure with other analysis procedures and after all the transformation procedures M1000 Rev H 8 71 Chapter 8 Editing Procedures The graph display is a standard Debye plot The properties for this proce dure are as follows Table 8 23 Radius from LS Data Properties Field Description Radius geometric or RMS Shows the calculated radius The type of radius RMS or geometric displayed depends upon the LS fit model specified for the peak This field is display only Peak Number Displays the number of the peak for the Debye plot Abscissa Position Slice Index Displays the index for the slice displayed in the Debye plot Alternately you can type a slice index here Model This field shows the light scattering model selected for this peak in the Peaks dia log This field is display only Fit Degree This field shows the fit degree selected for this peak in the Peaks dialog It is valid only if the Zimm Debye or Berry model is selected This field is display only This field shows the position on the x axis for the peak and slice selected This field is display only Enabled Detectors gt 1 18 This list has a checkmark next to detectors whose data is used in the calculation You can disable individual detectors by removing the checkmark Distributions and Moments This procedure calculates the
56. EOS II EOS IT A Solvent water Sample untitled SY Connections Za Auto inject connection profile17 SQ Sample Trays Generic sample tray profile3 amp Samples M1000 Rev H Creating Profiles Creating Profiles There are several ways to create system profiles These profiles are stored in the system database rather than in separate files Experiments use copies of system profiles but modifying the portion of an experiment con figuration that came from a profile does not affect the system profile itself Creating a New Profile Shortcuts M1000 Rev H To create a profile with settings used in your experiments using the default profiles as a starting point follow these steps 1 Choose File gt New System Profile You will see the Profiles dialog Press Ctrl Alt N Click the down arrow next to the D icon Right click on any profile item in the workspace and choose New E Profiles CC Connections f Auto inject connection f Aux channel connection f Fluid connection 5 Samples As Molecular standard Sample S Experiment configuration amp Solvent ok Cancel In the Profiles dialog select the type of profile you want to create Click OK In the Save As dialog type a name for the profile You can also select a folder in the system database to contain this profile 5 Double click the item you created in the System Profiles tab of the workspace to open its proper
57. Equation 42 I d 7 i SI 4B al T Tar For each of these relations the left hand side gu is known already see the previous sections The appropriate equation is solved for B for each slice which produced a reasonable value of gu Note that if 8y falls outside the range 0 to 1 no value of B will be calculated for that slice 1 B H Zimm and W H Stockmayer ibid M1000 Rev H D 21 Appendix D Light Scattering Theory When plotted in ASTRA the legend is labeled with the specified function ality 3 for Trifunctional branching or 4 for Tetrafunctional branching n for Monodisperse slices or w for Polydisperse For each branched file to be plotted select the branching functionality and whether the slices are monodisperse or polydisperse in the Branching property view The next figure a plot of Eqs 41 44 shows how the B s are related to amp y for the various branching options Note that these relations assume randomly branched polymers Also note that different assumptions about functionality and dispersity yield quite different values of B for the same value of gj Thus some knowledge of the type of branching is necessary for a plot of branches per molecule to have any meaning 1 0 P 0 8 O S 06 Ey F 0 4 he D g2 0 0 0 01 1 10 10 Branching per Molecule B Figure D 2 Branching ratio gu as a function of B for various branching options Long Chain Branching The long chain br
58. Experiment Builder Mode Extinction from UV Data This procedure calculates the extinction value of the sample and the lin earity of the result using data from a UV detector For more about determining UV extinction go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt dn de and UV extinction determination You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the UV extinction If you place multiple methods that determine UV extinction in a procedure only the first one will be valid M1000 Rev H 8 75 Chapter 8 Editing Procedures The procedure has the following dialog Determine extinction from UY data Iw data IV fit KI KI ki Ki Ed ki 0 0005 0 0010 concentration g mL Extinction 5 8633 0 1211 e 2 mL g cm Linearity 4 2675 4 4717 e 1 mL g cm 92 7218 Fit Degree Percentage To Keep 100 Linearity View Enabled Peaks BH OK S Cancel Si Apply The graph shows a fit to the UV absorbance and concentration data The properties for this procedure are as follows Table 8 26 Extinction from UV Data Properties Field Description Extinction Shows the calculated extinction This field is display only Linearity Shows the linearity of the extinction result This field is display only Fit Degree Typ
59. Export Iw Show chromatogram 2 Iw Show chromatogram 3 Experiment Experiment3 v Color E Marker Type E Square gt x Axis Shift 0 0000 Advanced 11 5 Chapter 11 Working With Graphs This dialog has the following fields Table 11 2 Edit Graph Fields Field Description Marker Size Draw Marker Every Choose the marker size you want to use The options are very small small medium and large Use Marker Size for lines made up of individual data points use Line Point Size for fitted curves Choose how often you want markers to appear on the line The options are every 1 2 3 5 10 20 30 or 50 data points Line Point Size Set the width of the line when the line is a fitted curve Use Marker Size if the line is made up of individual data points Show Chromatogram 1 Check this box to show a line for the first Chromatogram data set The default is on Show Chromatogram 2 Check this box to show a line for the second Chromatogram data set The default is off Show Chromatogram 3 Check this box to show a line for the third Chromatogram data set The default is off Experiment Color Choose the experiment for which you want to graph data Select the line or marker color you want to use in the graph Changing this prop erty changes the line color for all chromatogram traces Marker Type X Axis Shift Select the marker type you want to use in th
60. File Menu The File menu contains the following commands Command Keyboard Description Modes Shortcut File gt New Experiment from Create an experiment from the default tem T R A Default plate See page 6 4 Experiment from Ctrl Alt T Create an experiment from a template See T R A Template page 6 5 Blank Experiment Ctrl N Create an empty skeleton of an experiment Builder See page 6 6 R A Sample Set from Create a sample set from a template See T R A Template page 9 3 Blank Sample Set Ctrl Shift N Create an empty skeleton of a sample set R A See page 9 3 System Profile Ctrl Alt N Create a system profile See page 12 3 R A File gt Open Experiment Ctrl O Open an existing experiment See page 6 7 All and page 6 8 Sample Set Ctrl Shift O Open an existing sample set See page 9 4 All System Profile Ctrl Alt O Open an existing system profile See All page 12 5 A 2 M1000 Rev H File Menu Command Keyboard Description Modes Shortcut File gt Close Close the active item See page 6 15 and All page 12 7 File Close All Close all items in the current tab See All page 6 15 File Save Ctrl S Save the selection See page 6 15 and TRA page 6 16 File gt Save As Save the selection with a new name See T R A page 6 15 and page 6 16 File Save As Save the experiment as a template See T R A Template page 6 18 File Been Injection Ctrl Shift V Create an e
61. For example you can start a collection in your lab then monitor the progress in your office over the network Alarms and other state infor mation are reported directly to the Diagnostic Manager and you can use the Diagnostic Manager to send commands and configuration information to any instrument available within ASTRA This manager is not intended for viewing and interpreting data Instead it can be used to determine if your instruments are connected and func tioning correctly Viewing Graphical Data with the Diagnostic Manager The Data graphical tab of the Diagnostic Manager allows you to view a real time graph of data received from an instrument The type of data col lected is different for each type of instrument The following figure shows data collected by a DAWN HELEOS instrument Diagnostic Manager Data graphical Data numeric Alarm Commands Command log Property Instrument wpatt sft h DAWN HELEOS D Refresh aa en Moritaing w a ba A CH gt ke CO e o 01 e a 5 3 25 19p 3 26 19p 3 27 19p 3 28 19p 3 29 19p 3 30 1 time M1000 Rev H 5 5 Chapter 5 Interfaces to Instruments To monitor data follow these steps 1 Select an instrument to monitor from the Instrument drop down list If you add computers using ASTRA s Instrument list or you connect additional instruments after opening the Diagnostic Manager click Refresh to update the drop down list 2 Select
62. IP psi If you chose differential pressure alone specify the fixed inlet pressure for the viscometer in psi Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 7 22 M1000 Rev H Viscometry Instrument Profiles Table 7 11 Generic Viscometer Profile Fields Field Description Band Broadening gt Enable Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broad ening parameters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt See Band Broadening on page 8 14 for an explanation of the instrumen Instrumental Term tal term The units are in microliters Band Broadening gt See Band Broadening on page 8 14 for an explanation of the mixing Mixing Term term The units are in microliters M1000 Rev H 7 23 Chapter 7 Configuring Experiments UV Absorption Instrument Profiles A UV absorption instrument measures the absorbance of a sample in the ultra violet region of the spectrum The absorbance can be converted to a concentr
63. M1000 Rev H If you are having problems with your experiment database you can choose System Database Administration Automatic Maintenance to attempt to correct the problem We do not recommend that you use this command unless you are having problems with the experiment database 4 9 Chapter 4 ASTRA Administration 4 10 M1000 Rev H Interfaces to Instruments This chapter explains the Instrument Server Interface ISI the Diagnos tic Manager and the WCS Client These can all be accessed through ASTRA The ISI allows you to connect to administrate and acquire data from instruments connected locally or over your network CONTENTS PAGE Instrument Server Interface Overview 5 2 Installing the ISI on Other Computers sssesseeseeesseeeseeeeieeseesriesrrresnns 5 4 Using the Diagnostic Manager 5 5 WCS Client Application 0 ceecccccceceeseeeeeeeeeeeeeeeeeeeeseeaeseeneeeetaeteaeeene 5 12 Chapter 5 Interfaces to Instruments Instrument Server Interface Overview ASTRA can collect data from various instrument types More importantly it can combine this data with analysis procedures Connections to instru ments can be made either locally or through the network e e Ken DAWN and miniD AWN cy SS Optilab rEX ES tO ge Ki Figure 5 1 ASTRA Instrument Connections ViscoStar WyattQELs For instructions for connecting to instruments see Accessing and Viewing Hardware on page 2
64. Physical Units on page 8 56 e Convert to Concentration on page 8 56 e Convert Specific to Intrinsic Viscosity on page 8 57 e Fit Mass or Radius on page 8 57 Despiking The Despiking procedure removes spurious noise spikes from the collected data Such spikes are likely caused by dust in the solvent This procedure may be placed at any point in the experiment sequence before the analysis procedures that determine the final results This pro cedure runs automatically without prompting for a value You can set the property for this procedure before running the experiment or you can modify it after running the experiment and re run the experi ment to see the effects of changing the setting If your light scattering data is noisy you may want to run one of the experiment templates provided with ASTRA for diagnostic purposes For an experiment template choose File New Experiment From Template to open a template in the System Templates gt Light Scattering gt Diagnostics folder The LS noise template characterizes baseline detector noise 8 42 M1000 Rev H Transformation Procedures Double click on the procedure to open its property dialog E conventional calibration PS in THF ProceduresWesp E If Despiking Procedure M detector 1 Iw detector 2 detector 3 Iw raw refractive index data op nHononoe D e gt S 5 2 S 15 0 20 0 volume mL Value D
65. That manager will perform the fol lowing actions which are described in this chapter Connecting to a Database on page 4 3 Managing User Accounts on page 4 6 Using the System and Experiment Logs on page 4 7 Deleting Experiments on page 4 8 atabase ASTRA features related to the use of an experiment database to store experiments and sample sets are available in both ASTRA V with Security Pack and ASTRA V with Research Database The icon to the left identifies information that applies to both of these software versions Database For more about 21 CFR Part 11 compliance in ASTRA V including a detailed white paper go to http www wyatt com solutions soft ware ASTRA cfm and follow the link to Compliance Connecting to a Database ASTRA V uses two databases M1000 Rev H System database This database stores experiment templates sample set templates system profiles system solvents and molecular standards A system database is used by all ASTRA V operating tiers Each installation of ASTRA V uses its own local system database You cannot specify a different database to use as the system database See Migrating the System Database on page 2 3 for information about updating the system database Experiment database This database stores information about experiments database actions warnings and more This database logs list all activities performed with ASTRA that must be logged for 21 CFR Part
66. You SCH have at least Researcher access to create an experiment from scratch You can modify templates and save experiments as templates so it is unlikely that you will want to work starting from an empty experiment However if you want to create an empty experiment follow this step e Choose File gt New Blank Experiment Shortcuts Press Ctrl N Click the down arrow next to the D icon Right click Experiments in the workspace and choose New 6 6 M1000 Rev H Opening an Experiment from the Database Opening an Experiment from the Database Database You can open and work with any experiment you have saved If you are using ASTRA V with Research Database or ASTRA V with Security Pack experiments are stored in the experiment database and you open experiments from that database To open experiments stored in separate files such as exported experiments or experiments saved with ASTRA 4 see Importing an Experiment from a File on page 6 9 Security There are no access level restrictions on opening an experiment To open an experiment follow these steps 1 Choose File gt Open Experiment Shortcuts Press Ctrl O Click the down arrow next to the DG icon Right click Experiments in the workspace and choose Open Open a recently used experiment from the list in the File menu 2 Inthe Open dialog select the experiment you want to import Unless you have created a folder in the database all the e
67. a Sample Set Log ecccceeceeeceeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeeeseaaeeeeaaeesecaeeesaeeseneees 4 8 Workmo Kane e CC 4 8 Deleting Experiments gegen ek ccechsvauehiapais EENS aeestnens aetivaeineae tv werdenegeeds 4 8 Performing Database Maintenance cccceseseccceeeeeeeeeeeeceeeeeeeeeeessseeeeeeeeeese 4 9 Chapter 5 Interfaces to InstruUMeNtS cccceeesseeeeeeeeeeeees 5 1 Instrument Server Interface Overview secceeceeesseeceeeeereeeeeeseeeeseneeeseenentenes 5 2 Installing the ISI on Other Gomputers1 23 ice inte eae endian ht 5 4 Using the Diagnostic Manager cccccesssseeeecceeeeeeseseeeeeeeeeeeesesseeeeeseeeeneeees 5 5 Viewing Graphical Data with the Diagnostic Manager ccccsseeeeeeeeeseeeeeeeees 5 5 Viewing Numeric Data with the Diagnostic Manager ccscceeeesteeeeeeenteeeeeeeeee 5 6 Viewing Alarms with the Diagnostic Manager ec cscceeeeeseeeeeeeennaeeeeeeenaeeeeeeeaaaes 5 7 Sending Commands with the Diagnostic Manager cccccceeeteeeeneeeeeteeeeeneeeees 5 8 Viewing the Command Log with the Diagnostic Manager s 5 9 Viewing and Setting Properties with the Diagnostic Manager ssssseseseeseeseeese 5 10 WCS Client Application WEE 5 12 Chapter 6 Creating amp Running Experiments c ccee 6 1 Abo t Eu EE 6 2 2 M1000 Rev H Contents Creating New Experiments AAA 6 4 Creating Default Experiments 0 e
68. a profile to use Description Description of the standard which typically contains more information than the Name Reference Wavelength dn dc The wavelength at which the dn dc or UV extinction value is accurate nm dn dc value associated with the sample mt o A2 Molar Mass Second viral coefficient A2 value associated with the sample mol mL g Molar mass value associated with the sample g mol Intrinsic Viscosity Radius gt Type Radius gt Value A measure of the capability of a polymer in solution to enhance the viscos ity of the solution Derived using specific viscosity and concentration data Type of radius specified May be RMS Hydrodynamic or Geometric Radius value associated with the sample nm UV Extinction Coefficient M1000 Rev H The extinction coefficient in mL g cm The extinction coefficient is used when the concentration of the sample is to be determined using a UV absorption instrument 7 31 Chapter 7 Configuring Experiments Solvent Profiles A solvent is a substance in which another substance is dissolved forming a solution The solution is placed in or flows through a sample cell A solvent profile stores information about solvents used in experiments such as toluene Profiles for common solvents water toluene THF etc are supplied with ASTRA You cannot change the values in existing solvent profiles You can build and modify custom prof
69. also a vector based format Bitmap GIF JPEG PNG and PCX are all pixel based image formats with different types of compression PDF is the Adobe Acrobat format PostScript is an output format used by many printers Since encapsulated PostScript is created some applica tions can import graphics in this format 6 The Options and Size tabs offer different settings depending on the format you select Once you have set the format and options click one of these buttons Copy Store the graphic on the clipboard in this format for pasting into another application Save Send the graphic to a file of this type You are prompted for the file name and location Send Send the graphic to an application such as Microsoft Out look This is typically used to email the graphic Exporting Data M1000 Rev H To quickly export graph data double click on a graph to open the Edit Graph dialog Click the Export button and choose a data output file type See Working with Procedure Graphs on page 8 5 for details To export a graph to a data file that you can use with a spreadsheet with more control over the output follow these steps 1 Double click on a graph to open the Edit Graph dialog Then click Advanced 2 Choose the Export tab from the top row of tabs 11 17 Chapter 11 Working With Graphs 3 Choose the Data tab from the second row of tabs Edit Graph Chart Series Tools Export Print OpenGL Themes
70. analysis of the correlation function than has traditionally been obtained from the method of cumulants The results obtained from the fit in the QELSBatch cumulant analysis are the first two moments and Ug in Eq 7 as well as the baseline B and amplitude J The baseline and amplitude values are used in the data fil tering algorithm to reject QELS correlation functions after the initial cumulants analysis However the first two cumulants are the quantities of interest for assessing the polydispersity of the sample The first two moments define a Gaussian distribution in decay times where the first cumulant gives the mean of the distribution and the square root of the second cumulant gives the standard deviation In terms of a distribution for sizes the decay time distribution can be converted to hydrodynamic radius via equations 3 through 5 Since the radius is inversely proportional to the decay time the distribution in radius is no longer a symmetric Gaussian This can be seen in Figure F 2 decay time distnbution gaussian Rh distribution skewed average Figure F 2 Cumulants distributions in decay time and hydrodynamic radius 1 B J Frisken Revisiting the method of cumulants for the analysis of dynamic light scattering data Applied Optics 40 4087 4091 2001 F 4 M1000 Rev H Cumulants In the cumulants analysis results the fitted first and second moments that is the decay time distribution av
71. analyzed via the equation Equation 2 A iq g tr B Pexpl 2I r where B is the baseline of the correlation function at infinite delay Jis the correlation function amplitude at zero delay and J is the decay time A nonlinear least squares fitting algorithm can be applied to Eq 2 to retrieve the correlation function decay time J This is exactly what is done in the ASTRA QELS analysis From this point J can be converted to the diffusion constant D for the particle via the relation Equation 3 E Here q is the magnitude of the scattering vector and is given by Equation 4 47 q m sin 8 2 Wi where n is the solvent index of refraction 2 is the vacuum wavelength of the incident light and is the scattering angle Finally the diffusion constant can be interpreted as the hydrodynamic radius r for a diffusing sphere via the Stokes Einstein equation Equation 5 kT oe 32ND where k is Boltzmann s constant and 77is the solvent viscosity The previous equations provide the tools for analyzing a correlation function from a monodisperse sample but do not address the effects of polydispersity on the correlation function One of the first attempts to analyze such data was the method of cumulants First proposed by Koppel the method of cumulants involves expanding Eq 2 into the various moments of a distribution In its simplest expression this expan sion turns Eq 2 into the following Equation 6
72. and follow the links to Data Analysis gt Distributions Copolymer Analysis Like the Protein Conjugate Analysis on page 8 88 this analysis tech nique allows you to differentiate between two polymers with the same molecular size This analysis procedure requires use of a viscometer in conjunction with a UV and RI detector The Copolymer Analysis dialog shows the total mass and protein fraction on a slice by slice basis ASTRA calculates the size of the complex mass of the complex and masses of the constituents and the uncertainties for these values This procedure should be placed in the sequence with the analysis proce dures but before the fit mass and radius procedure if it occurs For an experiment template that performs a Copolymer Analysis choose File gt New Experiment from Template and open the Copolymer Analysis experiment in the System Templates gt Viscometry folder 8 86 M1000 Rev H Analysis Procedures This procedure has the following dialog E PSAN_PMMAY_CA Procedures Copolymer Analysis EBR Copolymer Analysis UV absorbance data Iw intrinsic viscosity peak number 1 intrinsic viscosity peak number 2 jw differential refractive index data relative scale 20 0 30 0 volume mL Value Molar Mass Protein Fraction 1 617 1 202 Protein Molar Mass 5 517 4 101 e 7 g mol Modifier Molar Ma 2 105 1 565 e 7 g mol Peak Number Slice Index Fit Degree
73. and graph are displayed Determine dnde from RI NW a wa Nn J 25x10 20x10 15x10 1 Gei differential sali active index 50x10f 0 0000 0 0005 0 0010 00015 cencentratiom q ml nek 01436 0 0001 nly Unes tty 0 1447 0 0004 nijo 40 ies TR Degree 1 Percentage To Ko 10 Unes ity View Endod Peaks B 13 Appendix B System Templates Copolymer Analysis AT This template is for experiments without a light scattering instrument To use this template a conventional column calibration profile is required The molar mass is determined from the elution time and not from light scattering data For example you might use this template to determine the quantity of monomer A and monomer B RI Peak Areas AT When this template is applied to an experiment only the refractometer peak area for each selected peak is shown in the report RESULTS Peak 1 Peak 2 Peak 3 RI Instrument RIU min Peak Area 2 943e 5 3 170e 6 4 204e 7 B 14 M1000 Rev H UV Measurement UV Measurement The following folders is provided in the UV Measurement folder 100 Mass Recovery Methods The following template is provided in the UV Measurement gt 100 Mass Recovery Methods folder UV extinction from peak AT The UV extinction coefficient is calculated under the hypothesis that 100 of the injected mass is recovered To perform the calculation ASTRA needs to know the flow rate and the injected mass
74. and its contents nm Calibration Constant Type the Instrument Specific Calibration Constant ISCC value 1 V cm Light scattering instruments use the ISCC in the computation of the Config uration Specific Calibration Constant CSCC See LS Calibration on page 8 19 for a way to determine this value Normalization Coefficients 1 3 Type the normalization coefficients for the detectors or use the normaliza tion procedure to set these values Detector 2 always has a normalization coefficient of 1 Normalization is the process by which each detector signal is related to the 90 detector signal and the Instrument Specific Calibration Constant Click the Import button to import normalization coefficients from an open experiment Comet Cell Cleaner Check this box if a COMET cell cleaner is to be used with the minIDAWN instrument Please see the COMET hardware manual for more information about the COMET cell cleaner Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 7 14 M1000 Rev H Light Scattering Instrument Profiles Table 7 4 minIDAWN and miniDAWN TREOS Instrument Properties Field Description Divide by Laser Monitor Fwd Mon
75. are entered for the band broad ening parameters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the instrumen tal term The units are in microliters See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters DAWN EOS DAWN DSP DAWN DSP F and DAWN 8 Profiles The properties that may be defined for a DAWN EOS DAWN DSP DAWN DSP F and DAWN 8 are identical You can set the following properties for a DAWN instrument Table 7 3 DAWN Instrument Properties Field Description Name Name of the instrument If you have already created a system profile for this instrument click on the far right and select a profile to use Description Description of the instrument which typically contains more information than the Name Physical Instrument Choose an instrument from the drop down list If your instrument is not listed choose Browse to open the Instruments dialog See Accessing and Viewing Hardware on page 2 10 Sample Cell Wavelength Type of sample cell used during data collection The options are K5 F2 Scintillation Vial MicroCuvette and Magic glass The
76. are molar mass RMS radius hydrodynamic radius and translational diffusion Start The starting point on the x axis of the range Stop The ending point on the x axis of the range Use Limits Check this box if you want to specify specific percentages in the cumulative num ber fraction trace for the beginning and end of the range Low If you check the Use Limits box specify the cumulative number fraction at which you would like to begin the range If you do not check the box this field shows the cumulative number fraction at the start of the range you created with your mouse High If you check the Use Limits box specify the cumulative number fraction at which you would like to end the range If you do not check the box this field shows the cumulative number fraction at the start of the range you created with your mouse Cumulative Moments gt Number Averaged This property shows the difference between the High and Low which is the percent of the sample that falls within this range This field reports the number averaged value of the moment for the selected range Moments gt This field reports the weight averaged value of the moment for the selected Weight Averaged range Moments gt This field reports the Z averaged value of the moment for the selected range Z Averaged For more about distribution analysis go to http www wyatt com solu tions software ASTRA cfm
77. broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the instrumen tal term The units are in microliters See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters The purge valves on ViscoStar instruments are automatically closed at the start of data collection M1000 Rev H 7 21 Chapter 7 Configuring Experiments Measuring the Dilution Factor If an RI detector or other instrument is plumbed after the ViscoStar in the flow sequence the sample exiting the ViscoStar is diluted by approxi mately a factor of 2 Therefore the RI detector does not measure the same concentrations that flowed through the LS and ViscoStar instruments To correct for this you should measure the dilution factor experimentally To measure the dilution factor use a sample that is known to elute 100 The detailed report shows the resulting Dilution Factor which you can enter in the ViscoStar or Generic Viscometer profile You should check the dilution factor occasionally since it will change over time as samples that coat the tubing slowly build up To learn more see the Measuring the System Dilution Factor section in the ViscoStar User s Guide Generic Viscometer Profiles You can create a Generic Viscometer profile f
78. click on the graph and choose Edit from the pop up menu Then click the Copy button in the Edit Graph dialog Exporting Graphs You can export graphs as pictures or data for use in other applications You do this with the dialog you see when you double click on a graph The Experiment Graph Export command exports experiment data rather than graph data Exporting Pictures To quickly export a graph image double click on a graph to open the Edit Graph dialog Click the Export button and choose the JPEG output file type See Working with Procedure Graphs on page 8 5 for details Edit Graph Graph OK Marker Size Line Point Size Moo Cancel Series e sl Export Color Red Marker Type E quare Advanced To export a graph to a graphics file with more control over the output follow these steps 1 Double click on a graph In the Edit Graph dialog click Advanced 2 Choose the Export tab from the top row of tabs 11 16 M1000 Rev H 3 Choose the Picture tab from the second row of tabs Edit Graph Chart Series Tools Export Print OpenGL Themes Picture Native Data Format Options Size as Metafile as SVG as PostScript as PDF as POX as GIF as PNG as JPEG Colors Default M Monochrome Exporting Graphs 4 Select a file format to export Metafile is a vector based Windows Metafile wmf used by applications such as Microsoft Word SVG is
79. computer network Optilab DSP Generic RI or UV detector WCS EOS miniDAWN to AUX input of DAWN or miniD AWN Figure 5 2 Connections Between ASTRA the ISI and Instruments Figure 5 3 shows the ASTRA V with Research Database and ASTRA V with Security Pack architecture in more detail The circles represent inter faces presented by the ASTRA core the ISI and ODBC for use by other parts of the architecture WCS EOS miniDAWN QELS Server _ WyattQELs Se Optilab rEX a gt f ViscoStar export import n Astra 4 and V Eclipse files Figure 5 3 ASTRA System Architecture console scripting The core of ASTRA is used by the ASTRA user environment Console and scripting applications also access the ASTRA core The ISI presents an interface that is used by both the ASTRA core and the Diagnostic Manager The ISI in turn connects to various instruments If you use ASTRA V Basic the ODBC database shown in Figure 5 3 would be replaced by file access to separate experiment files 5 Chapter 5 Interfaces to Instruments Installing the ISI on Other Computers When you install the ASTRA software on a computer the ISI software is automatically installed along with ASTRA The ASTRA installation CD allows you to install only the diagnostic manager and the appropriate instrument controllers If you choose to do this the ISI is installed without the graphical inter
80. computers on your network Browse for Computer Select a remote computer with an ASTRA installation Network Neighborhood A Entire Network E Microsoft Terminal Services P Microsoft Windows Network gy XYZNETWORK mi COMPUTER 1 mi COMPUTER_2 E Web Client Network Selection ox Cancel Instruments and computers that you add to the Instrument dialog are still connected to ASTRA in subsequent ASTRA sessions unless you delete them Any supported instruments connected to those computers will be available within ASTRA See Chapter 5 Interfaces to Instruments for more about using the ISI Instruments and Diagnostic Manager dialogs Once instruments are visible in the Instruments dialog ASTRA is ready to use for collecting data Please note that ASTRA can still be used for the analysis of already collected data files without any connection to an instrument Removing an Instrument or Computer from the Instrument List To remove an instrument or computer from the Instrument list opened with System Instruments select the name of that resource in the instrument list and click Remove 2 12 M1000 Rev H Getting Started This chapter shows you how to create and run a simple experiment It assumes that ASTRA has been set up as described in Chapter 2 Install ing and Setting Up ASTRA CONTENTS PAGE Eilen 3 2 ASTRA Tutorials on the Support Center 3 4 Performing a Simple Light Scattering Experim
81. constants should make the line essentially flat when looking at the scattering from a solvent You can use this feature as a diagnostic tool when trying to set the orientation of a scintillation vial on the DAWN Rotate the vial view the live data and try to put the vial in a position such that the live data line is flat For the remaining fields change values for any properties you want to modify The properties shown are different for each instrument type Refer to the hardware documentation for details Click Apply 5 11 Chapter 5 Interfaces to Instruments WCS Client Application 5 12 For additional diagnostics you may choose to run the WCS Client which reports on the activities of the Wyatt Communications Server This server manages communication between ASTRA and instruments You can start the WCS Client from the Windows Start menu by choosing Programs Wyatt Technologies gt ASTRA gt WCS Client Typically you would use this in cooperation with Wyatt Technical Support M1000 Rev H Creating amp Running Experiments This chapter tells how to work with ASTRA experiments The details of items contained in an experiment are covered in other chapters This chapter describes actions you perform with the entire experiment such as creating a new one saving it running it or exporting it CONTENTS PAGE About Experiment S sseni 6 2 Creating New Experiment ccccecceeeeeeeeeeteeeeeeeeeeeaeseseeeeeese
82. created by the collection procedure is then forever kept with the experi ment in an unmodified state The same procedure then performs preprocessing on this original data based on the configuration to create what is called the raw data For example the original data contains the AUX channel traces from an instrument but the raw data uses the exper iment configuration to route that data to the appropriate instrument specified by the AUX connection After a transformation procedure runs such as setting baselines the data used by subsequent procedures has the transform applied A number of transformations can be applied in sequence to the data After an analysis procedure runs the experiment also contains analyzed data that can be displayed in reports M1000 Rev H 8 3 Chapter 8 Editing Procedures Working With Procedures Security Most users will not need to add remove or resequence procedure items The templates provided with ASTRA V contain procedures for most common experiments You must have at least Researcher access to add procedures and at least Technician access to modify existing procedures If you are a Guest you have read only access to procedures Editing Procedure Settings To set properties of a procedure follow these steps 1 Double click on a procedure in the experiment This opens the dialog for that procedure The dialog shows different types of information depending on the type of procedure S
83. dialog behaves the same as the graph in the Basic collection procedure dialog 8 12 M1000 Rev H Collection Procedures To select a script for the procedure click the button for the File Name property In the Open dialog select the script you want to use and click OK Collection scripts typically have a file extension of col You can type a script directly in the Script property row but using a separate text editor is recommended WW Experiment2 Procedures Script Collection SEE detector voltage VI File Hame SY OK strip chart Experiment2 LS2 LS3 0 000E 00 0 0000 time min C COMET col Script to run the COMET duration 300000 lsInst LSInstrument Create II lsInst LaserOn false Turn the COMET on lsInst SendCommand SetSwitch COMET lsInst StartCollection Collection Start Collection SetDuration duration Turn the COMET off lsInst SendCommand SetSwitch COMET F Collection Stop x Cancel Si Apply M1000 Rev H 8 13 Chapter 8 Editing Procedures Configuration Procedures 8 14 You may need to calibrate your instruments or measure various aspects of their behavior These procedures may be used in separate calibration experiments or integrated into other experiments The following configuration procedure types are available e Band Broadening on page 8 14 e Interdetector Delay Alignment on page 8 17 e L
84. dn dc value necessary for determining molar mass in light scattering measurements physical units Units of measurement that have scientific meaning For example the DAWN instrument produces voltage signals that must be converted to the physical units of Rayleigh ratio before they can be analyzed to determine mass and radius procedure ASTRA s representation of a process in the collection and analysis of the data A procedure can be either for collection data transformation data analysis display instrument configuration or administrative purposes profile A description of a physical entity in an ASTRA experiment For example an instrument solvent or sample QELS Quasi Elastic Light Scattering This is also known as dynamic light scattering or photon correlation spectroscopy The Wyat tQELS instrument measures rapid fluctuations in scattered light intensity to determine the translational diffusion coefficient and hydrodynamic radius for macromolecules in solution RI Refractive index Used to describe differential refractometer instruments or data from the Optilab rEX DSP or 908 system database The database in which ASTRA stores templates and profiles This is separate from the experiment database ViscoStar On line differential viscometer that measures the intrinsic viscosity and Mark Houwink Sakurada MHS parameters of polymers 1 7 Chapter 1 About ASTRA V 1 8 system profile An ASTRA profile saved in
85. experiment tree You can scroll down to read the results of the data analysis For more about setting up and viewing results see Chapter 10 Working With Reports Summary In a few minutes you ve created and run an experiment You have modified the properties of a configurations and set properties such as baselines for a procedure These are the main types of tasks you will perform when setting up and running your own experiments As you become a more advanced user you may want to learn to perform tasks that are available in Experiment Builder mode For details see User Modes on page 3 13 3 12 M1000 Rev H More About the ASTRA Environment More About the ASTRA Environment This section explains some general tasks you may perform within ASTRA that were not covered in the sample experiment in the previous section such as customizing the ASTRA environment and getting help For an overview of ASTRA features go to http www wyatt com solu tions software ASTRA cfm and follow the link to Features User Modes You can use ASTRA in Run mode or Experiment Builder mode Run mode makes it easier to learn to use ASTRA and may be the mode you prefer even after you are an experienced user In Run mode you create experiments using the configuration and procedure templates provided with ASTRA You can modify properties of the configuration and proce dures but cannot add or delete instruments or procedures
86. explanation of the mixing term The units are in microliters Optilab 903 Profiles 7 18 You do not select a Physical Instrument for the Optilab 903 because ASTRA V does not support a direct data connection to this instrument When using the Optilab 903 it is necessary to add an AUX connection to the experiment configuration to indicate which AUX channel and instru ment are to be used to read the Optilab 903 signal See AUX Connection Profiles on page 7 29 for details M1000 Rev H Refractive Index Instrument Profiles You can set the following properties for a Optilab 903 instrument Table 7 8 Optilab 903 Properties Field Description Name Name of the instrument If you have already created a system profile for this instru ment click and select a profile to use Description Description of the instrument which typically contains more information than the Name Wavelength The wavelength of the light used in the instrument nm Cell Type of sample cell used during data collection The options are P2 P2L P10 P100 P10L P20 P12 and ENGRCELL Scale The scale corresponds to the scale selected on the Optilab 903 instrument Possi ble values are 2 5 10 20 50 and 100 Offset The offset voltage is determined during the Optilab 903 setup procedure Please see the Optilab 903 hardware manual for instructions on determining the offset Batch Mode Check this box if the instrument i
87. extension 9 4 Standard Toolbar command View menu 3 14 Status Bar command View menu 3 14 status bar hiding 3 14 Stop command Experiment menu 6 13 Stop command Sample Set menu 9 10 support technical 1 9 surface plot 11 12 syringe pump 8 23 system database 2 3 definition of 1 7 4 3 deleting 2 3 saving templates to 6 18 system log copying event text to clipboard 4 8 definition of 4 7 navigating 4 8 viewing 4 7 System Log command System menu 4 7 System menu list of commands in A 7 M1000 Rev H see also specific menu commands system profiles 12 2 creating 12 3 creating by exporting from configura tion 12 4 creating by exporting from experiment configuration 7 8 definition of 1 8 deleting 12 7 duplicating 12 6 editing 12 5 importing into experiment configura tion 7 7 12 8 12 9 renaming 12 6 system requirements 2 2 system templates 6 5 T technical support 1 9 Technician group 1 4 2 6 3 3 3 13 4 6 Technician user level A 2 telephone support 1 9 templates creating 6 18 creating experiments from 3 5 6 5 default 6 18 6 19 deleting 6 19 for graphs 6 29 11 11 for reports 6 29 10 3 10 4 import templates 6 5 my templates 6 5 9 3 system 6 5 third virial coefficient 8 65 tiers operating 1 3 toolbars hiding 3 14 repositioning 3 14 transformation procedures effect on data 8 3 list of 8 42 raw data produced by 11 7 translational diffusion Dt calculat
88. files If a results fitting method has been selected ASTRA uses the fitted data from MM vs Volume and RMS Radius vs Volume directly in the branching calculations For a number of points 300 points per decade of molar mass Eq 36 is applied In order to obtain useful branching information the two files linear and branched should overlap as much as possible in molar mass The branch ing ratio gy can only be calculated in this region of overlap since only in this region can radii be found at the same molar mass To use this method select the Radius method in the Branching properties view See Branching on page 8 80 Branching Ratio Mass Method If the molecular radii are too small to be calculated accurately then we must use another method Assuming the Flory Fox equation is valid it can be shown that Equation 87 an lin M br y Buzz where Jl and Mp are the molar masses of a linear and branched polymer respectively a is the Mark Houwink Sakurada parameter for the linear polymer and e is the drainage parameter ranging from 0 5 fora non draining polymer to 1 0 for a free draining polymer to 1 5 for a Flory 1 B H Zimm and W H Stockmayer J Chem Phys 17 1801 1949 2 L P Yu and J E Rollings J Appl Polym Sci 33 1909 1921 1987 M1000 Rev H D 19 Appendix D Light Scattering Theory Fox polymer A value of 0 5 1 0 seems most used in the literature The effect of the choice
89. function The resolution value can range between 0 and 1 where 0 corresponds to the noisiest data and 1 corresponds to the least noisy data In ASTRA V the optimal value of the resolution is taken from the DYNALS algorithm and reported in the data window for the regularization analysis window The results of the regularization are an intensity distribution in hydrody namic radius However in light scattering the intensity distribution does not give an accurate representation of the number distribution Therefore intensity information can be converted to relative number by choosing a mass model for the particles and applying a correction factor for the intensity The mass models in ASTRA V are sphere and random coil Interpreting Regularization Results Regularization analysis results are more physical than results for the cumulants method However some care must be taken in interpreting these results First low size peaks lt 1 nm often appear in the regulariza tion results These are sometimes attributed to solvent scattering but are most likely due to avalanche photodiode afterpulsing picked up by the cor relator To exclude this from the correlation function try setting a longer minimum delay time for the correlation function in ASTRA Large size peaks are also common in the final distribution These are usually real and correspond to dust Another issue of concern in interpreting regularization results is deter mining whethe
90. graph 20 40 60 time min 8 0 10 0 The left graph shows various plots depending on the Plot property The right graph shows the defined peaks for the batch experiment 8 91 Chapter 8 Editing Procedures The properties for this procedure are as follows Table 8 34 Regularization Properties Field Description Peak Number Click on a peak in the graph or type a number here to select a peak to regularize Plot Select the type of plot you want to view in the left graph The options are corre lation function Rh diff intensity Rh diff weight Dt diff intensity Rh cumula tive intensity Rh cumulative weight and Dt cumulative intensity Mass Model Select the mass model you want to use in the computation Options are sphere and random coil Subpeak Summary of individual components identified by ASTRA in each analytical peak You cannot modify these values gt Rh Mean The mean hydrodynamic radius of the subpeak gt Rh Peak The hydrodynamic radius at the peak of the subpeak gt Rh Std Deviation The standard deviation of the peak hydrodynamic radius from the mean gt Peak Area The area under the subpeak as a percentage of the entire peak Processing Conditions Conditions per peak during the regularization analysis You cannot modify these values gt Temperature gt Viscosity gt Refractive Index Shows the temper
91. is performed automatically as 8 56 part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode Convert to Concentration This procedure converts the refractive indexes measured by an RI instru ment or UV absorbance data to concentrations It is only visible in Experiment Builder mode The Experiment Configuration see page 7 9 contains a Concentration Source field that allows you to choose between RI and UV data if both are available You may place this procedure in a location after the collection procedure and before the analysis procedures M1000 Rev H Transformation Procedures If the dn dc value is specified for a peak region any procedures that follow this one display RI data as concentrations for each peak region If the UV extinction coefficient is specified for the peak region any proce dures that follow this one will display UV data as concentrations for each peak There are no properties to set for this procedure It runs without prompt ing for any values Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode Convert Specific to Intrinsic Viscosity This procedure converts the specific viscosity measured by a viscometer such a
92. it and click Open The experiment con figuration you selected replaces the existing one To replace an individual item in the configuration with another item of the same type follow these steps 1 Right click on a node in the configuration and choose the Replace command for that item from the pop up menu 2 Browse the database for an item to import You can only select from items of the corresponding type That is you can replace a sample with a sample a solvent with a solvent and so on for connections and instruments If you are replacing an instrument you can select any type of instrument No pre defined instrument profiles are provided with ASTRA In order to have instruments available you need to first save instrument profiles in your system database as described in Creating Profiles on page 12 3 7 8 3 When you find a profile select it and click Open The item you selected replaces the existing item If you later edit properties of items you imported there is no effect on the system profile from which it was imported Likewise modifying a system profile does not affect experiments that imported that system profile Exporting a System Profile One way to create a system profile is to export items from an experiment To do this follow these steps 1 If you have more than one experiment open make sure the one you want to export from is selected in the experiment tree of the work space 2 Select the it
93. item to delete dialog select a profile to delete Profiles are usually in the My Profiles folder W Select an item to delete Close C Example Configurations C Import Templates E My Profiles Sy My Templates Ba collection only SS empty sample set E System Solvents CI System Templates Click Delete You are asked if you are sure you want to delete the selected profile Click Yes if you are sure 5 Click Close when you are finished deleting profiles M1000 Rev H 12 7 Chapter 12 Working with System Profiles Using Profiles Note 12 8 The benefit of creating system profiles is that you can use them to save time when configuring your experiments If you later edit properties of items you import or copy from a system profile there is no effect on the system profile from which it was obtained For more about using profiles see Using Configurations on page 7 5 Adding an Item to a Configuration You might want to add a component to an experiment For example you may have added a UV instrument to an experiment To add an item to an experiment configuration using a system profile follow these steps 1 If you have more than one experiment open make sure the one you wish to modify is selected in the experiment tree of the workspace 2 Choose Experiment Configuration Replace Configuration You see the Select Profile dialog 3 In the Of Type field select the type of item you are loo
94. less than about 10 nm molecules this small scatter nearly isotropically For polystyrene in toluene or THF this corresponds to a molar mass of less than about 50 000 g mol M1000 Rev H Normalization In practice we need not measure the various detector dark offsets Mag dark in Eq 10 This is because the instrument is typically used to study samples in solution not solvents by themselves Thus we are interested in the excess Rayleigh ratio of the eluting sample compared with the baseline of solvent alone We therefore use an alternative form of Eq 10 Equation 11 q Ho R _ N A V Vo baseline 0 VeAcscc Viser eg dark where Rais taken to be the excess Rayleigh ratio and Vgpaseline is the detector voltage far away from the sample peak The quantity Vo baseline 18 due to the scattering from pure solvent cf Eq 2 and the diode dark offset Eq 11 is the one used in ASTRA Implementation M1000 Rev H ASTRA provides two normalization techniques Standard normalization which uses the normalization calculation used since ASTRA 4 and Area normalization which uses a new method based on integration over the peak Standard Normalization In practice Ma Vo baseline iS not determined from a single data slice but from the result of following the steps below 1 Select a sample peak to use for normalization 2 Using the data points for the center half of the peak that is the half of the peak centered
95. mass of the sample injected in grams If you do not enter a value in this field and you have provided all the necessary parameters Concentration and sample volume ASTRA computes the Injected Mass Alternately you can specify the value here Procedures that use this injected mass value account for viscometer dilution factor effects if the concentration detector is downstream from a viscometer Set this parameter if you are performing any of the following analysis proce dures Dn dc from Peak UV Extinction from Peak Eta Analysis gt Model Specify the model to use for intrinsic viscosity calculations for this peak The model may be Huggins Kraemer or Solomon Gatesman The default is Hug gins ASTRA has historically used the Huggins relation with a Huggins Con stant of zero For more about ETA analysis go to http Awww wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Intrinsic Viscosity Models Eta Analysis gt Huggins Constant k If you select the Huggins model you can specify a Huggins constant here to be used in the calculation The default is zero Eta Analysis gt Kraemer Constant k If you select the Kraemer model you can specify a Kraemer constant here to be used in the calculation The default is zero Molecular Standard If you wish to specify a molecular standard for the peak select the system pro file for that standard from the drop down list The values of
96. medium and large Use Marker Size for lines made up of individual data points use Line Point Size for fitted curves Line Point Size Set the width of the line when the line is a fitted curve Use Marker Size if the line is made up of individual data points Series Choose the data series for which you want to change the color or marker type M1000 Rev H 8 5 Chapter 8 Editing Procedures Table 8 1 Edit Graph Fields Field Description Color Select the line or marker color you want to use in the graph Changing this prop erty changes the line color for all chromatogram traces Marker Type Select the marker type you want to use in the graph The default is square When 8 6 You see the effects of your changes as you make them without closing this dialog Click OK to save your changes Graph customizations such as line weight color marker style and title changes are not saved when you close procedure dialogs that contain graphs If you click Copy the graph is in your clipboard and you can paste the graph into another program If you click Export you can choose to save the graph in one of the follow ing formats e data saved as Microsoft Excel file xls e data saved as comma delimited text file csv e data saved as tab delimited text file txt e data saved as tagged XML file xml e image saved as JPEG file jpg If you click Advanced you have much more co
97. number of experiment configurations are provided with ASTRA You can also save your own experiment config urations as described in Creating Profiles on page 12 3 For example you may have a standard experiment configuration you want to use in many different experiments You can also replace a single instrument or other item with a saved system profile No pre defined instrument profiles are provided with ASTRA In order to have instruments available you need to save instrument profiles in your system database as described in Creating Profiles on page 12 3 See Chapter 12 Working with System Profiles for more about system profiles To import a complete configuration follow these steps 1 Choose Experiment Configuration Replace Configuration Or right click on the Configuration node in the experiment and choose Replace Configuration You see the Select Profile dialog Experiments Dn conventional calibration ps in thf S D A arch Dawnc Edit Configuration d ven LEET a Procedures Save Configuration As J Data Set De Results 2 Browse the system database for a configuration to import In addition to any experiment configurations you have saved ASTRA provides a number of configurations in the Example Configurations folder These are organized by the experiment type and instruments involved 7 7 Chapter 7 Configuring Experiments Note 3 When you find a profile select
98. of any size To derive the fit method for the various modeled form factors sphere coated sphere rod we fit the Lorenz Mie equation to Rg vs sin 2 Note that the Mie fit yields a geometric radius Light Scattering by Small Particles Wiley New York 1957 M1000 Rev H Radius Moments Radius Moments ASTRA calculates the following radius moments for each peak The specific type of radius RMS radius geometric radius or hydrodynamic radius depends on the type of analysis being performed All summations are taken over one peak RMS Radius M1000 Rev H Number Average Equation 8 e R r e ve Weight Average Equation 9 R r Z Average Equation 10 S ai AC z 3 AN The quantities R lt r gt and V in these equations are respectively the Rayleigh Ratio mean square radius and volume of the it slice The RMS radii are simply the square roots of the appropriate mean square radii Uncertainty Weighted Average Equation 11 e DIa r 1 Zou O gt Where lt r2 gt is as defined previously and r is the uncertainty in the mean square radius measurement The error in this calculation is defined as follows Equation 12 Appendix E Particles Theory Geometric and Hydrodynamic Radius Number Average Equation 18 R Van L n Ry Vi Weight Average Equation 14 rk P o i V R yR V Z Average Equation 15 R gt Roti Z XR The quantities R
99. of 100 mass recovery and the aim is to find the calibration constant needed to have the calculated mass equal to the injected mass Zero dRI CT This template sets the dRI calibration constant value to zero Orbit On CT This template places the Orbit device in Recycle mode for the Optilab rEX Purge On CT We recommend that you purge the Optilab rEX when not running samples the Purge On template is a convenient way to automate this as part of a sample set Purge Off CT You can use this template in combination with the Purge On template in a sample set to close the purge valves on an Optilab rEX instrument The purge valves on Optilab rEX instruments are automatically closed at the start of data collection The exception to this is when absolute RI analysis is conducted where the Optilab rEX purge valve must be left open Diagnostics The following template is provided in the RI Measurement gt Diagnostics folder Grimace AT The Grimace template shows an overlay to display it use an EASI graph Molar masses are plotted The template allows you to determine if there is secondary band broaden ing If there is secondary band broadening for a monodisperse sample the flat line will become a curve that is a grimace shape Conventional Calibration CT Use this template for configurations that contain only a refractometer Inject several monodisperse samples with well known molar masses T
100. peaks 11 Click Export in the Calibrate Column dialog to open the Save Cali brated Column Profile dialog You can click Export only after you have clicked Apply 12 Select a folder for your column profile and type a name for the column Click Save to store the profile In future experiments you can import this profile in the column profile see page 7 26 Setting Column Calibration Properties Experiment Builder This procedure is hidden in the Experiment tree in Run mode However you can still open it in Run mode by choosing Experiment Configura tion Calibrate Column M1000 Rev H 8 39 Chapter 8 Editing Procedures You can set properties for this procedure after you run the experiment col lection Double click on the Calibrate column procedure to open its property dialog W conventional calibration PS in THF vaf Procedures Calibrate e EI x log molar mass g mol Calibrate colurnn Iw m data V fit 10 0 11 0 12 0 13 0 volume mL Value Equation log molar mass 12 6132 0 86901 v 0 0175873 v Standard Error 0 0770042 R42 0 995631 R 0 997813 Technique Conventional Fit Order 2 Concentration Source DI Flow Marker mL From positive peak Lele Lele Le Flow Marker Peak 4 34 MD Mark Houwink K mL g 0 000e 000 Mark Houwink a 0 000e 000 Column Operations Import Peak Data Ex Source Experiment Enabled BY OK SH ca
101. photodiode sensi tivity may change with age The calibration should be performed with HPLC grade toluene filtered through the smallest available filter 0 02 um immediately before making the measurement using the ASTRA program The cleanliness of the cell is vital for this purpose Be sure to leave the DAWN or miniDAWN instrument and the laser switched on for one hour before making any measurements D 7 Appendix D Light Scattering Theory Normalization D 8 At this point we have calibrated the 90 detector in an absolute sense the calibration is totally independent of any sample we might wish to study In other words we can measure Rgaccurately for any solvent or sample assuming it gives a large enough signal Furthermore the calibration can be traced directly to the scattering from pure well understood solvents So far we have ignored all angles except 90 Each detector has its own geometrical factors and angular sensitivity to measured light intensity Furthermore these effects vary from solvent and sample to solvent We would like to quantify this effect so that we can correct for it If not we will mistake solvent and geometric readhead effects for characteristics of our sample resulting in poor data Therefore we use a set of normalization coefficients Ng to relate each detector to the 90 detector These coefficients must be determined using the same flow rate same pressure and the same solvent that are used f
102. please contact Wyatt Technology Corpo ration for assistance M1000 Rev H Running an Experiment Running an Experiment Once you have set up an experiment in ASTRA and the corresponding instruments connections solvents and samples are ready you can run the experiment Security You must have at least Technician access to run experiments Validating an Experiment Shortcuts You can validate an experiment s procedure sequence and configuration by choosing Experiment Validate Validation checks the procedure sequence for conflicts If the experiment collects data validation also checks that the necessary instruments are connected and available It checks to make sure the experiment configura tion contains a solvent and a sample In addition validation checks the collection script If you use the basic collection procedure the collection script is built automatically and validation never finds any problems with the script Press Ctrl Shift v Right click any folder in the tree and choose Manage gt Validate If any procedure in the sequence has a red X on its icon it is in an invalid location in the experiment sequence or the configuration is missing instru ments that produce data needed by the procedure Modify the sequence as described in Sequencing Procedures on page 6 27 or revise the experi ment configuration to include the appropriate instruments A procedure s state is always indicated by i
103. procedure calculates the dn dc of the sample and the linearity of the result using data from an RI detector The procedure has the following dialog E rEXrange recovered vaf ProceduresWetermine dn dc from RI data SEE Determine dude from RI data V data v fit x a Ki E 5 K 3 e 3 S Ki 0 010 0 015 concentration g mL dnide 0 1677 0 0002 mio Linearity 0 1728 0 0002 mL g 3 0191 Fit Degree 1 Percentage To Keep 100 Linearity View Enabled Peaks BH ok RK cancel E A The graph shows a fit to the differential refractive index and concentration data M1000 Rev H 8 73 Chapter 8 Editing Procedures For an example experiment that determines dn dc from RI data choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the dndc measure ment vaf experiment in the Sample Data gt Analyzed Experiments folder You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the dn dc or RI calibration If you place multiple methods that determine dn dc or RI calibration in a procedure only the first one will be valid The properties for this procedure are as follows Table 8 25 dn dc from RI Data Properties Field Description dn dc Shows the calculated dn dc This field
104. r and V in these equations are respectively the Rayleigh Ratio radius either geometric or hydrodynamic and the volume of the i slice Uncertainty Weighted Average Equation 16 2 Dun r SE i avg 2 gt CO 7 where r is as defined previously and O is the uncertainty in the radius measurement The error in this calculation is defined as follows Equation 17 1 E 6 M1000 Rev H Theory Theory This section discusses how the distribution plots are calculated and why a model is needed The mean square radius is given by Eq 18 where the distances r are measured from the particle s center of mass to the mass element m E ion 18 Equation 18 pss K fram gt iM M Eq 18 refers to a single particle whereas the quantity actually measured from an ensemble of particles may be shown to be a so called LS average mean square radius Were the particles random coils in a theta solvent then this would be the so called z average mean square radius We assume that the particle size distribution within each slice of an eluting sample following separation is essentially monodisperse Therefore the particles in slice i each of mass JM are assumed to have the same mean square radius We define the root mean square radius as the square root of the mean square radius or simply r lt r gt The Rayleigh Gans Debye approximation RGD Equation 19 Ke 1 Kg VM P can be re written in the limit as
105. ratio em H Eq 13 is the basis of the calculations in ASTRA as well as of the Zimm plot technique which is often implemented in a batch sample mode Eq 13 assumes vertically polarized incident light and is valid to order ch The task is now to determine for each slice the molar mass and mean square radius It is possible to solve Eq 13 in a variety of ways leading to a number of different fit methods We shall consider the Debye Zimm Berry and Random Coil methods M1000 Rev H D 11 Appendix D Light Scattering Theory Debye Fit Method First construct a Debye plot that is a plot of Rg Kc vs sin 2 Second fit a polynomial in sin 2 to the data and thereby obtain the intercept at zero angle Ry K C as well as the slope at zero angle m d K c dain gi Dhar Note that as approaches zero the form factor P approaches unity Therefore Eq 13 becomes Equation 14 Roo Ry M 2A cM Kc Ke If Ay 0 then Equation 15 M a Ke Otherwise solving Eq 14 for M yields Equation 16 R 2 i aal Ke Care must be exercised in solving Eq 14 If Ag or c is too large there will be no real solution which means that a higher order formulation of Eq 13 is required In addition only one of the two solutions of Eq 14 is physi cally reasonable Also the standard solution to the quadratic equation is susceptible to round off error as A gt 0 Therefore we use an alter nate form of E
106. screen e What you were doing when the problem occurred e How you tried to solve the problem Contact Information Website http www wyatt com E Mail Support astra support wyatt com FAX Support 805 681 0123 Telephone Support 805 681 9009 Corporate Headquarters Wyatt Technology Corporation 6300 Hollister Ave Santa Barbara CA 93117 USA International Support Outside the USA you may use one of the contact methods listed here or you may contact the Wyatt Technology Distributor in the country where you bought your product M1000 Rev H 1 9 Chapter 1 About ASTRA V Where to Go from Here Continue to Chapter 2 Installing and Setting Up ASTRA to prepare ASTRA for use Be sure to read your hardware manual s before attempting to collect data using the software They contain important safety and operational information 1 10 M1000 Rev H Installing and Setting Up ASTRA This chapter provides instructions for installing ASTRA on your computer and instructions for preparing it for use The ASTRA administrator in your organization should follow the steps in all sections of this chapter to make ASTRA ready for use as described in the remaining chapters CONTENTS PAGE System Requirements cceceececeseeenseeeeseeaeeeeceeeaaeeeeseesaeeeeessetateeeeseaaes 2 2 Installing the ASTRA Software ccccescceceeceeeeceeeseeeeeeeeaeeeeeeeeeseaeeeeeenees 2 2 Setting Up User Accou
107. ssnin seed AEAEERR SOS ege 7 26 Connection Profiles echte 7 28 Fluid Connection Profiles AA 7 28 AUX Connection Profiles A 7 29 Autoinjector Connection Profiles c cccecccceeeeeeeeeeeeeceeeeeeeeeeseaaeeseaeeeseeeeesaeessnees 7 29 Sample P ofileS pin a e a Ea E Ee 7 30 Sample Profile Siei a a a E Ee EEOAE 7 30 Molecular Standard Profiles cccccccceseeeeeeeeeceeaeeeeeeeeeceaeeeeeeeeeceaeeeseeeeseeeeeeaaeeees 7 31 Solvent PROUMES eege dee ee ee 7 32 Chapter 8 Editing Procedures ssseeseeeeseeeeeeeeeeees 8 1 ADout Ee ee EE 8 2 About Data Processing in AST TRA esssesseessssiressrrsrrrssrrrsrsrsrrissrrnssrisstnnssrnsssrnsnnt 8 3 Working With Stee IT 8 4 Editing Procedur Se ttingS is stcieiecessadeveiies Geavebltetiieennhidlveesndaesten shed Ee Zu 8 4 Working with Procedure Graphs cccccececceeeceeeeeeeeececaeeeeeeeeeseaaeeeeaaeesesaeeesnaaeteneees 8 5 When to Modify Procedures seeiis aa ier EE AAN EAn EA AENEAN ARRERA 8 6 Advanced Procedure EIDEN ECK Fees bie rene eek 8 7 Adding ProCedUres EE 8 7 Rue Bee TE 8 8 Sequencing Procedures A 8 8 Validating a Procedure Sequence cceceececeeeeeeeeeeeceaeeeeaaeeseaeeesaaeeseeeeeesiaeeeteneees 8 9 Coll6Ction Procedures EE 8 10 Basic Collecti n oriana aaa aer aE aa a aa a Aaa E A a AOSA EEEa 8 10 aile Ee del WEE 8 12 Configuration Procedures EE 8 14 Band Broadening EE 8 14 Interdetector Delay Alignment sssessssreesssrres
108. the criteria for Rayleigh Debye Gans scattering As a result this is the most general method for analyzing spheres of any size See the Determination of Molar Mass and Sizes on page D 11 for a dis cussion of the fit models Broaden Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures It applies the terms calculated by the Band Broadening procedure If you want to see this procedure enable Experi ment Builder mode by choosing System gt Preferences Experiment Builder Mode For an example experiment that corrects for band broadening choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the band broadening example BSA vaf experiment in the Sample Data gt Analyzed Experi ments folder Convert to Physical Units This procedure is only visible in Experiment Builder mode It converts instrument signals to physical units if necessary For example light scat tering values in volts are converted to Rayleigh ratios You may place this procedure in a location after the collection procedure and before the analysis procedures Any procedures that follow this one will display detector data in physical units rather than voltages There are no properties to set for this procedure It runs without prompt ing for any values Experiment Builder This procedure is hidden in Run mode It
109. the file 4 In the File Name field type a name for the sample set The Save As Type field shows that the file will be saved with an exten sion of vsf if you are using ASTRA V Basic If you are using ASTRA V with Research Database or ASTRA V with Security Pack the sample set is saved in the experiment database 5 Click Save To save a sample set with a different name or location choose File gt Save As and follow steps 3 through 6 above M1000 Rev H Deleting a Sample Set Saving Sample Sets as Templates A sample set can be saved as a template by selecting Templates in the Of Type field in the Save As dialog Once you have configured a sample set save it as a template before running it You can then create new sample sets from the template without building a new one from scratch each time Exporting Sample Sets You can export a sample set from the experiment database to a file with an extension of vsf This file is a binary file that can only be imported by ASTRA V To export a sample set follow these steps 1 Select the sample set you want to export in your Sample Set tab 2 Choose File gt Export Sample Set 3 Select the directory where you want to save the file and type a file name for the sample set 4 Click Save to create the file Deleting a Sample Set Basic Database M1000 Rev H If you are using ASTRA V Basic you may delete a sample set by deleting the vsf file that contai
110. the laser monitor signal and any auxiliary input data e Raw data This is the data gathered by the data collection procedure For a light scattering experiment this is the detector voltages The raw data is kept with the experiment so that data can be reprocessed if you modify the procedure and can be viewed in reports if desired e Raw data after each transform This data has the results of despik ing smoothing baselines peaks and other transformations A separate data set for each transformation is stored in the experiment e Data after conversion This data has the results of conversions such as from detector voltages to Rayleigh ratios e Analyzed data This data has been processed to arrive at results such as molar mass RMS radius or other values M1000 Rev H 11 7 Chapter 11 Working With Graphs Creating Data Set Definitions Experiment Builder Data set definitions are visible in the experiment tree of the workspace Shortcuts 11 8 and can be created only if you enable Experiment Builder mode by choosing System Preferences gt Experiment Builder Mode You can create a data set definition before or after you run an experiment To create a new data set definition follow these steps 1 Choose Experiment Add to Experiment This opens the Add to Experiment dialog which allows you to add items to the Procedures Data Set Definitions and Results nodes of the experiment Press Ctrl Shift P Right clic
111. the molecular stan dard parameters are then used for the peak See Chapter 12 Working with System Profiles for more about creating molecular standard system profiles Extended Parameters gt Modifier dn dc If you are using the Protein Conjugate Analysis procedure specify the dn dc value in mL g fro the modifier Extended Parameters gt Modifier UV Extinction If you are using the Protein Conjugate Analysis procedure specify the extinc tion coefficient in mL g cm for the modifier Extended Parameters gt Coating Thickness If you are using the coated sphere LS model specify the coating thickness in nm Extended Parameters gt Coating Real RI If you are using the coated sphere LS model specify the real RI of the coating Extended Parameters gt Coating Imag RI Extended Parameters gt Rod Radius 8 54 If you are using the coated sphere LS model specify an RI value for the coat ing This RI value should be corrected for absorption For Rod LS model calculations ASTRA assumes that the thickness of a rod shaped particle is insignificant 0 0 nm compared to its length If the thickness is significant enter its thickness or approximate thickness in nm M1000 Rev H Transformation Procedures Table 8 15 Peak Properties Field Description Extended Parameters gt If you are calibrating a column or using molecular standards with known molar Molar Mass g mol mas
112. the system database a backup is created with a filename of ASTRA_System2 bak or ASTRA_System2_ bak where is a sequence number if you do multiple migrations You can click Show backup to open a Windows Explorer view of the Database folder of the ASTRA installation which contains the system database and backup files You can rollback a migration by deleting the new ASTRA_System2 mdb file and renaming the backup file to ASTRA_System2 mdb M1000 Rev H Activating Optional ASTRA Features Activating Optional ASTRA Features To activate optional ASTRA features such as QELS you use the Feature Activation dialog In this dialog you enter keys provided to you by Wyatt Technology Corporation based on your licensing agreement M1000 Rev H Feature Activation Feature Key ASTRA V wiSecurity Pack Uy sen seet BAL Base LS Module tttt 5 N L a Extended LS Modules Online A2 eee FEEEG KPR Particle Analysis FHEKOKKEKS SDIJ Protein Conjugate Analysis FREE EKERY YNF _ QELS Module eee eRAN OZD RI Module Mee 2 Figure 2 1 Software Activation Dialog To activate a feature follow these steps 1 2 3 Si 2 Gs 5 9 Double click the ASTRA V icon on your desktop Choose System gt Feature Activation to open the dialog above You can choose the operating tier for ASTRA by selecting either ASTRA V ASTRA V w Research Database or ASTRA V w Secu rity Pack from the drop down list Cli
113. the type of Data to monitor The choices differ depending on the type of instrument For more about viewing live data for light scat tering instruments see Viewing and Setting Properties with the Diagnostic Manager on page 5 10 3 Click Start Monitoring 4 To stop the graph click Stop Monitoring You can modify the appearance of the graph just as you would in ASTRA For details see Working with Procedure Graphs on page 8 5 Viewing Numeric Data with the Diagnostic Manager The Data numeric tab of the Diagnostic Manager allows you to view real time numeric data received from an instrument The type of data col lected is different for each type of instrument The following figure shows data collected by a DAWN HELEOS instrument Diagnostic Manager Data graphical Data numeric Alarm Commands Command log Property Instrument Channel Yoltage detector 1 1 523721 e 2 detector 4 1 545349e 2 detector 7 1 587684e 2 detector 10 1 539368e 2 detector13 1 529076e 2 detector 16 1 580804e 2 Aux channel 8 621892e 3 Aux channel 4 1 032190e 2 forward laser 11 067591 monitor wyatt sft h DAWN HELEOS Channel Voltage detector 2 1 555533e 2 detector 5 1 554793e 2 detector 8 1 560707e 2 detector 11 1 543257e 2 detector 14 1 479513e 2 detector 17 1 587328e 2 Aux channel 2 5 503916e 3 laser monitor 3 248930e 3 read head 314 000000 temperature Channel Yoltage detector 3 1 53
114. through the alignment procedure The band broadening procedure recalculates the interde tector volume when determining the band broadening parameters The resulting interdetector volume is generally different from that obtained from the alignment procedure A term that defines the degree of broadening due to instrumental effects that is not due to mixing in the band broadening calculation 8 16 M1000 Rev H Configuration Procedures Table 8 3 Band Broadening Properties Field Description Instrument Details gt A term that defines the degree of broadening due to mixing in the band broaden Mixing Term ing calculation Instrument Details gt Click Reset to change the instrumental and mixing terms back to zero and Reset update the graph Experiment Builder This procedure is hidden in the Experiment tree in Run mode It is per formed automatically as part of certain procedures If you want to see this procedure in the tree choose System Preferences Experiment Builder Mode Interdetector Delay Alignment M1000 Rev H If an online experiment has already been run you can open the Interdetec tor Delay procedure view by choosing Experiment Configuration gt Alignment ASTRA uses the volume delay between different instruments to correlate their measurements Determine the delay volumes by collecting data on a monodisperse sample then aligning the resulting peak for each instru ment
115. to measure the actual concentration and mass for each peak The RI or UV signal is used to determine the concentration and the light scattering for the intensity With this template the concentration doesn t need to be entered manually The results graph shows the mean square radius vs volume Online Zimm Plot CT This template is similar to the Online template except that the data is dis played in a Zimm plot using physical units Online A2 CT This template is similar to the Online template In addition to mass and radius the A2 value is computed and a Zimm plot is shown This is similar to the Zimm batch analysis except that it is performed in an online mode B 8 M1000 Rev H Light Scattering Protein Conjugate AT This template allows you to determine the molar mass size and relative polymer fractions of a copolymer using light scattering All that is required are two additional detectors that have differing sensitivities to the constituent polymers Traditionally light scattering has been used in conjunction with an RI and UV detector for this purpose An important class of copolymers are protein conjugates For example researchers often need to determine the fraction of protein in glycosylated and pegylated proteins as well as membrane protein detergent com plexes ASTRA has native support for protein conjugate and copolymer analysis using a light scattering detector in conjunction with a UV and RI detec
116. up for a simple batch collection You do not need to connect pumps or other instru ments at this time If you have already set up a flow experiment you may choose the appropriate online template for your setup If you don t have a DAWN or miniDAWN light scattering instrument you can still follow the steps in this example by selecting an experiment template appropriate to your instrument and setting properties that corre spond to the ones described here You must use an account with ASTRA Researcher or ASTRA Administra tor access to follow the steps in this section Checking the Instrument Connection Your ASTRA administrator has probably already set up connections to computers with instruments you will access with ASTRA You can confirm this by choosing System Instruments to open the Instruments dialog This dialog lists instruments connected to computers that can currently be accessed by ASTRA If the instrument you want to use for this experiment is not listed follow the steps in Accessing and Viewing Hardware on page 2 10 Creating an Experiment M1000 Rev H ASTRA provides a large number of experiment templates Most users will be able to find a template that defines a configuration identical or close to their own experimental setup To create an experiment follow these steps 1 Choose File New Experiment From Template Ctrl Alt T 2 In the New from Existing dialog open the System Templates folde
117. wavelength of the laser that produces scattered light from the sample cell and its contents nm 7 12 M1000 Rev H Light Scattering Instrument Profiles Table 7 3 DAWN Instrument Properties Field Description Calibration Constant Type the Instrument Specific Calibration Constant ISCC value 1 V cm Light scattering instruments use the ISCC in the computation of the Config uration Specific Calibration Constant CSCC See LS Calibration on page 8 19 for a way to determine this value Normalization Coefficients gt 1 18 1 8 for DAWN 8 Type the normalization coefficients for the detectors or use the normaliza tion procedure see page 8 32 to set these values Detector 11 always has a normalization coefficient of 1 This is Detector 5 on a DAWN 8 Normalization is the process by which each detector signal is related to the 90 detector signal and the Instrument Specific Calibration Constant Click the Import button to import normalization coefficients from an open experiment Comet Cell Cleaner Check this box if a COMET cell cleaner is to be used with the DAWN instrument Please see the COMET hardware manual for more information about the COMET cell cleaner Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batc
118. will not need to modify them You can double click on the LS calibration procedure to open its property dialog WW Experiment ProceduresWetermine LS calibration procedure TBR Calibration Peak Index 0 This procedure has the following properties Table 8 5 LS Calibration Properties Field Description Baseline Peak Index Number of the peak marker in the Peaks dialog that marks data collected with the laser off The calibration template contains a pre set baseline peak Calibration Peak Number of the peak marker in the Peaks dialog that marks data collected with Index the laser on The calibration template contains a pre set calibration peak Differential RI Calibration M1000 Rev H Most materials when dissolved in a solvent change the refractive index of the solution by an amount proportional to their concentration This pro portionality factor is called dn dc a factor that is usually independent of molar mass especially for molar masses greater than roughly 10 000 g mol The dRI detector s output is proportional to the change in refractive index which in turn is equal to the product of the concentration and dn dc For analog dRI instruments that send signals to the AUX input of another instrument the proportionality factor relating detector output voltage to n is called the calibration constant This constant is inversely proportional to dRI detector sensitivity It is the number requir
119. wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Particles and number density You can place this procedure with other analysis procedures and after all the transformation procedures 8 69 Chapter 8 Editing Procedures The procedure has the following dialog WW number density 100 nm Duke standard ProceduresWetermine number SEE results graph control graph Iw detector 90 19x10 18x10 17x10 R theta 16x10 15x10 0 0 0 2 0 4 0 6 0 8 10 0 20 0 sin theta 2 volume mL Value geometric radius 37 0 0 3nm Number Density 1 4716 0 0719 e 8 particles mL Peak Number 1 Slice Index 1276 Mie Fit Degree 5 Abscissa Position 15 950 mL E Enabled Detectors lt a ok Z Cancel The graph display is a standard Debye plot The properties for this procedure are as follows Table 8 22 Number from LS Data Properties Field Description Radius geometric or Shows the calculated radius The type of radius RMS or geometric displayed RMS depends upon the LS fit model specified for the peak This field is display only Number Density Shows the number density in particles per mL This field is display only Peak Number Displays the number of the peak for the Debye plot Slice Index Displays the index for the slice displayed in the Debye plot Alternately you can type a slice index here Model This field shows the light scatterin
120. 11 compliance An experiment database is used only in ASTRA V with Security Pack and ASTRA V with Research Database You can specify which experiment database to use The rest of this section talks about ASTRA s experiment database which is sometimes called simply the database 4 3 Chapter 4 ASTRA Administration ASTRA uses Open DataBase Connectivity ODBC to connect to data bases ASTRA has been tested with Microsoft Access and Microsoft SQL Server version 7 or higher databases ASTRA core Experiment 1 Database By default the experiment database is a Microsoft Access database called 4 4 ASTRA Experiment If you have Microsoft SQL Server Wyatt recommends that you use this for your experiment database Microsoft Access is a light weight database that cannot handle the large amounts of data generated by ASTRA the database operations will become slow as the amount of stored data increases Ideally the experiment database will be a networked database that is backed up regularly by the IT department Note that adequate database storage is required If the database is networked the database server must be accessible to the PC running ASTRA via the network If you have no networked Microsoft SQL Server installation available you can install SQL Server locally There is a free version of SQL Server called SQL Server 2005 Express Edition that is available from Microsoft For more information go to the Wyat
121. 2 7 templates 6 19 demo period for optional modules 2 5 Description property experiment configu ration 7 9 Description property sample set configu ration 9 6 Despiking procedure 8 42 detectors interdetector delay determining 8 17 normalizing 8 32 Determining Rh from QELS Data proce dure 8 77 Diagnostic Manager accessing 5 5 alarms in 5 7 definition of 5 5 monitoring graphical data with 5 5 monitoring numeric data with 5 6 sending commands to instruments 5 8 viewing and setting properties with 5 10 viewing command log with 5 9 diagnostics baseline drift 8 46 noise 8 42 differential distribution calculations D 18 Distribution analysis procedure 8 85 Distributions and Moments procedure 8 72 dn de definition of 1 6 8 21 dn dc from peak procedure 8 74 dn de from RI Data procedure 8 73 DNDC importing files from 6 8 6 9 domain setting up users 2 7 specifying when logging in 2 9 3 2 validation 2 8 drift diagnostics 8 46 Dt translational diffusion calculating 8 77 Dt cumulative intensity plot 8 93 Dt diff intensity plot 8 93 E Edit menu list of commands in A 4 Edit Experiment Configuration menu command 3 7 6 24 7 5 7 6 7 7 Edit Sample Set menu command 9 5 Editing dialog Index 4 Chart tab 11 14 Export tab 11 16 11 17 opening 8 6 11 6 11 13 11 14 Print tab 11 15 tabs in 11 14 e mail support 1 9 Empower importing sample sets 9 5 Empower Sample Set I
122. 417 16 8917 LS Analysis dn de mt ot 0 1100 0 1100 0 1100 lt j BY oK 2 Cancel Si Apply 2 You can check additional detector boxes to view multiple sets of data The colors for data shown use the following default colors light scat tering data is red refractive index data is green UV data is blue vis cosity data is gray and QELS data is magenta Multiple detector angles use colors assigned by the graphing system Click on the graph to add a peak range to the collected data Use your mouse to drag the ends of the range to appropriate locations for the leftmost peak you want to analyze 8 51 Chapter 8 Editing Procedures Note For online experiments we recommend setting peak endpoints so that the signal to noise ratio for both the light scattering and concentration detec tors is greater than or equal to 2 This may necessitate excluding an aggregate peak for which there may be a strong light scattering signal but no RI signal or a low molar mass tail of a broad distribution sample for which the light scattering signal will be small even though the RI signal is strong 5 Continue adding peak ranges for the rest of the collected data A number is shown for each peak that corresponds to the column for that peak below the graph Peaks are numbered in the order you cre ate them not necessarily from left to right The selected peak is shaded Note If you want to zoom in on the graph hold down the Ctrl ke
123. 6836e 2 detector 6 1 567 757e 2 detector 9 1 581965e 2 detector 12 1 526380e 2 detector 15 1 587573e 2 detector 18 1 533147e 2 Aux channel 3 4 384834e 3 laser curent 4 174114e 3 heated line 0 000000 Stop Monitoring temperature 5 6 M1000 Rev H Using the Diagnostic Manager To monitor data follow these steps 1 Select an instrument to monitor from the Instrument drop down list If you add computers using ASTRA s Instrument list or you connect additional instruments after opening the Diagnostic Manager click Refresh to update the drop down list Click Start Monitoring To stop the data updates click Stop Monitoring Viewing Alarms with the Diagnostic Manager When an alarm occurs for any instrument available to ASTRA via ISI a warning signal flashes in all tabs of the Diagnostic Manager This warning is a blinking circle on the bottom of the Diagnostic Manager The Alarms tab of the Diagnostic Manager shows alarms sent by all instruments available to ASTRA via ISI Only alarms sent during the current Diagnostic Manager session are shown Diagnostic Manager Data graphical Data numeric Alarm Commands Command log Property Alarm Name Severity Instru Description __ Time _ Status VIS EXTERNAL ALARM RETRANSMI ViscoStar Extemal alarm retransmit VIS PARAMETER UPDATED Minor ViscoStar Parameter updated M1000 Rev H This tab shows the following information about an alar
124. 8 amp 0 time min Trigger on Auto Inject Injection Delay min Duration min Collection Interval sec QELS Interval sec Laser Saver Mode divider COMET Run Duration min Ok 2 Cancel SR Apply 3 Type a new duration for the collection For example since you are sim ply learning to use ASTRA you might collect data for only one minute 4 Click Apply If you ever want to add procedures to the provided templates you can use Experiment Builder mode M1000 Rev H Performing a Simple Light Scattering Experiment Running the Experiment Experiment procedures prompt you for any information they need in order to run successfully To run the experiment follow these steps 1 Click the k Run icon in the ASTRA toolbar Ctrl Shift R Note If you are learning to use ASTRA without access to a light scattering instrument you can open an experiment with pre collected data by choosing File gt Open Experiment Ctrl O and opening the Sample Data folder then Practice Experiments then batch processing exam ple vaf Then skip to step 4 2 Watch the data as it appears in the Basic collection graph You can enable and disable detector displays in real time Note While you are collecting data you can work on setting up other experi ments You cannot modify the experiment that is running 3 Inject samples and or start pumps as needed to run the experiment After data is collected you s
125. 9 3 2 sign offs for 4 7 6 12 8 100 user accounts for 1 4 2 6 3 3 3 13 website for 4 2 3D surface plot 11 12 A A2 calculation 8 65 A2 mass definition of 1 6 A2 Mass and Radius from LS Data proce dure 8 63 A2 radius 1 6 A3 calculation 8 65 Abscissa Units property experiment con figuration 7 9 access levels 1 4 2 6 3 13 acknowledging an alarm 5 7 activation keys for optional modules 2 5 Add Connections property experiment configuration 7 10 Add Instruments property experiment configuration 7 10 Add to Experiment command Experiment menu adding data set definitions 6 28 11 8 adding graphs 6 28 11 11 adding procedures 6 26 8 7 adding reports 6 28 10 4 adf file extension 6 8 6 9 6 17 administration procedures 8 100 Administrator group 1 4 2 6 3 3 3 13 4 6 Administrator user level A 2 Alarms tab Diagnostic Manager 5 7 M1000 Rev H alignment procedure 8 17 Alt F4 keyboard shortcut 3 15 analysis procedures data produced by 11 7 effect on data 8 3 list of 8 59 analyzed data 8 3 definition of 11 7 Apply Template command Experiment menu 6 22 10 5 Approval category for sign offs 6 13 area under peak calculating 8 83 ASTRA activating optional modules for 2 5 customizing environment for 3 14 definition of 1 6 exiting 3 15 features of 1 2 installing 2 2 running 2 9 3 2 system requirements for 2 2 upgrading version 2 3 version 4 70 compatibility 1 2 ve
126. A calculates the following molar mass and RMS root mean square radius moments for each peak All summations are taken over one peak Number average molar mass Equation 28 K Weight average molar mass Equation 29 3 le AM z average molar mass Equation 80 Fle m Number average mean square radius Equation 81 1 J Appl Phys 22 1242 1246 1951 M1000 Rev H D 15 Appendix D Light Scattering Theory D 16 Weight average mean square radius Equation 32 Sear z average mean square radius Equation 33 gt c m moe aer The quantities c M and lt r gt in these equations are respectively the mass concentration molar mass g mol and mean square radius of the Oh slice The often quoted RMS radii are simply the square roots of the appro priate mean square radii ASTRA also calculates two polydispersity values M M and M M The uncertainty weighted average molar mass M y is calculated as follows Equation 84 A Midia Mvg Zeit Mi M is the uncertainty in the molar mass measurement The error in this calculation is defined as follows Equation 35 l OM Ze OM M1000 Rev H Uncertainties in Calculated Quantities Uncertainties in Calculated Quantities ASTRA calculates uncertainties for all reported quantities By analyzing the baseline data at the beginning and end of the chromatogram ASTRA determines the statistical f
127. ASTRA V User s Guide Version 5 3 4 M1000 Rev H CE Wyatt fechnology gt BCORPORATION Ww Copyright 2008 Wyatt Technology Corporation All rights reserved All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form by any means electronic mechanical photocopying recording or otherwise without the prior written permission of Wyatt Technology Corpo ration WYATT TECHNOLOGY Corporation makes no warranties either express or implied regarding this instrument computer software package its merchantability or its fitness for any particular purpose The software is provided as is without warranty of any kind Furthermore Wyatt Technology does not warrant guarantee or make any repre sentations regarding the use or the results of the use of the software or written materi als in terms of correctness accuracy reliability currentness or otherwise The exclusion of implied warranties is not permitted by some states so the above exclusion may not apply to you DAWN miniDAWN Optilab Wyatt Technology and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation EOS HELEOS TREOS and WyattQELS are trademarks of Wyatt Technology Corporation A variety of U S and foreign patents have been issued and or are pending on various aspects of the apparatus and methodology implemented by this instrumentati
128. ASTRA version 4 70 or higher for a DAWN EOS DAWN DSP or DAWN DSP F See Importing ASTRA 4 Files on page 6 9 for information about fixing problems with these files mdf File saved by ASTRA 4 for a miniDAWN nwf File saved by DNDC 5 rw File saved by RICAL 5 Imported experiments have a complete set of configuration items proce dures and results needed to view the experiment M1000 Rev H Importing an Experiment from a File Importing an Experiment from a File Basic This item is disabled in ASTRA V Basic since it is identical to File gt Open Experiment atabase You can import experiments stored in files This includes experiments Database saved with ASTRA 4 and ASTRA V Basic It also includes experiments exported by ASTRA V You must have at least Researcher access to import an experiment Security Shortcuts To import an experiment follow these steps 1 Choose File gt Import Experiment Press Ctrl I Right click Experiments in the workspace and choose Import Drag and drop an experiment file from Windows Explorer or the desktop to the ASTRA window 2 In the Import Experiment dialog navigate to the folder that contains the experiment you want to import 3 Select a file and click Open You can open any of the following types of files File Extension Description vat File saved or exported by ASTRA V vst ASTRA V sample set file vet
129. Advanced Procedure Editing To correct the problem drag the procedure to a location in the sequence where its icon and the icons that follow it have no red X See the section about the specific procedure in this chapter for information about its valid positions in an experiment Validating a Procedure Sequence Shortcuts M1000 Rev H To check the procedure and configuration choose Experiment Validate Right click an experiment tree folder and choose Manage gt Validate In addition to checking for a correct procedure sequence validation also tests to make sure instruments in the configuration are connected and available when you are getting ready to collect data It also validates col lection scripts for experiment builders If you use the basic collection procedure the collection script is built automatically and validation never finds any problems with the script If any procedure in the sequence has a red X on its icon it is in an invalid location in the experiment sequence or it requires data from an instru ment that is not in the configuration Modify the sequence as described in Sequencing Procedures on page 8 8 or revise the experiment configura tion to include the appropriate instruments 8 9 Chapter 8 Editing Procedures Collection Procedures 8 10 Your experiment will typically contain a collection procedure as the first procedure in the experiment sequence The following types of collection pro
130. Builder mode Run mode makes it easier to learn to use ASTRA It may be the mode you prefer even after you are an experienced user In Run mode you create experiments using the configuration and procedure templates provided with ASTRA You can modify configuration and procedure properties but cannot add or delete instruments or procedures Experiment Builder Experiment Builder mode allows you to modify the configuration and pro cedure sequences in a template The icon to the left identifies portions of this manual that apply only if you turn on Experiment Builder mode by choosing System Preferences Experiment Builder Mode In addition Experiment Builder mode allows you to open multiple proce dure windows at once However you should be careful with this feature since changing and applying properties in one window does not generally result in changes to other open procedure windows To see such changes reflected in other procedure windows you should close and reopen them Operating Tiers M1000 Rev H ASTRA V can be purchased with any of the following operating tiers e ASTRA V Basic Saves experiments to files In this manual the Basic icon applies to this mode e ASTRA V with Research Database Saves experiments to an exper iment database Does not provide 21 CFR Part 11 compliance In this manual the Database icon applies to this mode e ASTRA V with Security Pack Provides 21 CFR Part 11 compliance Th
131. Calibration CT To use this template you must have a saved column profile In the Generic Column of the experiment configuration select the corre sponding column profile by clicking the button M1000 Rev H B 17 Appendix B System Templates B 18 M1000 Rev H Data Collection with Scripts This appendix describes the scripting language you can use for script based data collection CONTENTS PAGE WALFOCDUCHI OM EE C 2 Elliot ugesi atees e deeg egen ee gege Eege EE geegegg C 3 Interacting with Instruments ceceeeeeeeeeceeeeeeeceaeeeeeeaeeseeeeeeeaeeteeees C 3 EXAMPIOS s 2isses diveced cee dadensenvadisieten evened taveese dient aea adaa dna END C 5 M1000 Rev H C 1 Appendix C Data Collection with Scripts Introduction Note C 2 ASTRA V embeds a powerful general purpose programming language called Lua in its data collection system Scripts can be written using the normal syntax and features present in the core Lua language version 5 1 3 For details about Lua see http www lua org You use scripts with the procedure described in Script Collection on page 8 12 When you run a script any syntax errors are reported in a message The following sections provide an overview of the features available for creating custom collection scripts Writing scripts is an advanced feature almost never needed for typical data collection tasks For users who want to construct novel collectio
132. EC LS Characterization This tutorial uses sample experiments that have already been run to show how to use data analysis procedures You ll need to download and unzip the data files provided The steps of the tutorial show how to set baselines normal ize the detectors set peaks set delay volumes alignment set band broadening parameters assessing whether the flow cell was clean and viewing molar mass results e ASTRA V Skill Building Exercise This tutorial contains a number of brief exercises that ask you to answer questions about the results of various experiments that have already been run It also contains ques tions that help you learn to assess the quality of data and troubleshoot detector problems using data It uses the same experiment and data files as the previous tutorial Additional information about various ASTRA features is provided at http www wyatt com solutions software ASTRA cfm 3 4 M1000 Rev H Performing a Simple Light Scattering Experiment Performing a Simple Light Scattering Experiment Note Security In this section you will use ASTRA to perform a simple batch light scat tering experiment For this experiment you need to have one of the following instruments connected to either your local computer or one that can be accessed over the network e DAWN HELEOS e DAWN EOS e DAWN DSP e DAWN DSP F miniDAWN miniDAWN TREOS For this introductory experiment it is best to set the instrument
133. Extinction from RI This procedure calculates the ultra violet extinction using the RI data You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the UV extinction If you place multiple methods that determine UV extinction in a procedure only the first one will be valid There are no properties to set for this procedure It runs without prompt ing for any values For more about determining UV extinction go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt dn de and UV extinction determination Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode Rh from QELS Data M1000 Rev H This procedure calculates the translational diffusion Dt and hydrody namic radius Rh using QELS data You can use this procedure if your experiment configuration uses the Wyatt QELS option in online mode If you use QELS in batch mode see Regularization on page 8 90 For more about analyzing online QELS data go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Online QELS 8 77 Chapter 8 Editing Procedures Correlation Function H
134. H Configuration Procedures UV Calibration The UV response factor supplied by the instrument manufacturer is gen erally accurate and the calibration constant for the UV AUX connection can be set to 1 If however you suspect that the specified response factor is incorrect you can determine the UV calibration directly The procedure for determining the UV calibration constant is very similar to that for determining the RI calibration constant The main difference is that the Known Extinction in mL g cm of the sample is used instead of the known dn dc of the sample The UV calibration constant varies with the extinction coefficient of the particular sample See Wyatt Technical Note 15 for more instructions on using a UV detector Double click on the UV calibration procedure to open its property dialog E RI cal ProceduresWetermine UV calibration procedure SEE Determine UW calibration Iw data IV fit ES s _ 3 e gt Ki EJ S T d 3 0 0005 0 0010 concentration g mL Calibration 1 0237 12 6867 Known Extinction mL g emt L tze Percentage To Keep 100 Enabled Peaks BY OK x Cancel Si Apply This procedure has the following properties Table 8 9 UV Calibration Properties EHS Calibration The resulting UV calibration constant Known Extinction The known extinction value for the calibration standard used M1000 Rev H 8 31 Chapter 8 Editing Procedures
135. In particles mode it is possible to measure the size radius and number density of a sample using just a light scattering detector without any con centration detector Note that you won t be able to measure the molar mass using this template In addition this template computes Rh using QELS data Protein Conjugate CT This template permits measurement of Rh from QELS data as well as RMS radius and molar mass using Protein Conjugate analysis Batch Debye plot CT Use this template to do batch injection of a sample of known concentration only one concentration The Debye plot is displayed together with the molar mass and radius Batch Zimm plot CT Light scattering can be used to measure the second virial coefficient A2 of a macromolecule ASTRA supports the analysis of rapid injections of small volumes of a sample This proprietary online analysis can retrieve A2 using a fraction of the sample needed for traditional measurements ASTRA uses a proprietary global fitting method to upgrade the Zimm plot to a more robust modern analysis The global fitting method removes all extrapolations from the Zimm plot helps identify inconsistent data sets and provides more precise and robust results for A2 As with all analysis procedures in ASTRA the global fit view offers immediate visual confir mation of fit quality using a visual representation of the Zimm plot You can change parameters in the grid view and view fit results imm
136. OS Comet 10min e TREOS Comet 2hour Laser off templates can only be used with a sample set In the United States the recommendation is not to turn the laser off if it s for less then 3 weeks Before using a Laser off template you need to customize it This means to perform the physical instrument connection and save the resulting experiment as a template before running a sample set s Turn EOS laser off s Turn HELEOS laser off s Turn miniDawn laser off s Turn TREOS laser off The Orbit on template places the Orbit device in Recycle mode for the TREOS and HELEOS Diagnostics M1000 Rev H The following templates are provided in the Light Scattering gt Diagnostics folder Detector Overlay AT This template allows you to visualize an overlay of the light scattering detectors It is a very useful tool for trouble shooting e You can check the photodiodes normalization e You can check for laser misalignment All the detector signals should look the same for a monodisperse sample e You can check the for a dirty cell If the cell is dirty the peaks are not the same shape for all detectors View 2 Overlay Test CT This is similar to the Detector Overlay template Appendix B System Templates B 6 Noise AT es HELEOS Noise e TREOS Noise s EOS Noise e miniDawn Noise The noise templates analyze the noise level of the light scattering detec tors To do so you must measure a stable base
137. Profiles on page 7 29 for details You can set the following properties for a generic RI instrument Table 7 9 Generic RI Instrument Properties Field Description Name Name of the instrument If you have already created a system profile for this instrument click and select a profile to use Description Description of the instrument which typically contains more information than the Name Wavelength The wavelength of the light used in the instrument nm Temperature Control gt Enable M1000 Rev H Check this box if the instrument is set to maintain a specified temperature heated or cooled 7 19 Chapter 7 Configuring Experiments Table 7 9 Generic RI Instrument Properties Field Description Temperature Control gt If this instrument is temperature controlled specify the temperature to Temperature which it is set Use C Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 Band Broadening gt Enable Band Broadening gt Instrumental Term Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broad ening parameters These parameters are usu
138. S Calibration on page 8 19 e Differential RI Calibration on page 8 21 e Absolute RI Calibration on page 8 27 e UV Calibration on page 8 31 e Normalization on page 8 32 e Calibrate Column on page 8 37 Band Broadening This procedure allows you to correct for the effects of interdetector band broadening between instruments in an online experiment Such broaden ing can distort the peak shape between instruments resulting in incorrect results for analysis methods that require comparing the signal of two dif ferent instruments The band broadening procedure allows you to correct for the broadening To do so collect data from a narrow monodisperse sample for use in deter mining the band broadening parameters For an example experiment that determines band broadening choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the band broadening example BSA vaf experiment in the Sample Data gt Analyzed Experi ments folder When to Determine Band Broadening This procedure only needs to be performed once when you connect the instruments or change the tubing between the instruments The band broadening remains the same until you change the length of tubing between the instruments or change the instrument sequence For more about how and why you need to correct for band broadening go to http www wyatt com solutions software ASTRA
139. Specify this parameter if you chose the mass method Linear Radius Model k Parameter Specify this parameter if you chose the radius or mass method and checked the Use Linear Model box Linear Radius Model b Parameter Specify this parameter if you chose the radius or mass method and checked the Use Linear Model box For more about branching analysis see Branching Calculations on page D 19 and go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Branching calculations 8 82 M1000 Rev H Analysis Procedures Parametric Plot The parametric plot procedure generates a new data set for two different types of x y data that share the same x axis For example you can use this procedure to create a plot of RMS radius vs molar mass The properties for this procedure are as follows Table 8 29 Parametric Plot Properties Field Description X Data Choose the data that will be used for the x axis of the parametric plot Y Data Choose the first data set that will be used for the y axis of the parametric plot Y Data2 Optionally choose a second data set to be used for the y axis of the parametric plot See Creating Data Set Definitions on page 11 8 for information about the dialog that appears when you click the button for either the X Data or the Y Data property Peak Areas This procedure calculates the area under the
140. Support Overvlew 4 2 Connecting to a Databaee nsen nnenn nnt 4 3 Managing User Accounts 4 6 Using the System and Experiment Loge 4 7 Deleting Experiment AA 4 8 Performing Database Maintenance 4 9 Chapter 4 ASTRA Administration 21 CFR Part 11 Support Overview Security 4 2 21 CFR Part 11 contains regulations by the U S Food and Drug Adminis tration FDA concerning electronic records and electronic signatures FDA regulated companies in pharmaceutical biotechnology and other industries are under increased scrutiny to comply with 21 CFR Part 11 Background and Reasons for Compliance Title 21 of the Code of Federal Regulations includes regulations for food and drugs regulated by the Food and Drug Administration Part 11 of this title establishes the criteria under which electronic records and signatures can be considered equivalent to paper records and handwritten signatures in processes regulated by the FDA FDA regulated industries must document that proper processes have been followed to insure that products are consistent Signed documents about various points in the manufacturing processes must be reviewed securely stored and available for review by the FDA Reviewing these records was time consuming and required manual searches 21 CFR Part 11 makes record handling more accurate and efficient for all parties because all of the records stored are digital The benefits of becoming 21 CFR Part 11 compliant include the f
141. Table 9 2 Sample Properties Field Description Duration The time or fluid volume for which data is to be collected The units are determined by the Abscissa Units property of the experiment configuration Delay The delay in time or fluid volume between injection and the start of data collection The units are determined by the Abscissa Units property of the experiment configuration The default is zero dn dc dn dc value associated with the sample in mL g The dn dc value is used when the sample concentration is to be determined using a refractive index instrument The value entered for the profile is used as a default value when peaks are set for the data A2 Second viral coefficient A value associated with the sample measured in mol mL g The value set here is used as a default value for peaks set in the experiment UV Ext The UV extinction coefficient in mL g cm The extinction coefficient is used when the concentration of the sample is to be determined using a UV absorption instrument The value entered here is used as a default value when peaks are set for the data Conc The concentration of the sample in g mL Vol The injection volume of the sample in mL In order to avoid cycling the laser frequently which will shorten its life span you should make sure the Laser Saver Mode box is not checked in the Basic Collection configuration of the templates you use for th
142. The instrument is added to your experiment configuration and you can edit its properties by double clicking it in the configuration tree To add a connection follow the same steps but click the Browse but ton in the row to add connections Importing an experiment configuration with Experiment Experiment Configuration Replace Configuration replaces the entire experi ment configuration all instruments and connections with a different experiment configuration Adding an instrument or connection adds only that item without replacing or removing other items 6 25 Chapter 6 Creating amp Running Experiments Adding Procedures You can add procedure items only if you enable Experiment Builder mode by choosing System Preferences Experiment Builder Mode The Procedures folder in the experiment tree shows actions ASTRA performs in sequence when you run the experiment For details on all types of procedures see Chapter 8 Editing Procedures x EA Experiments E System Profiles Experiments Sy Experiment1 Configuration Profile LS Online Sy Procedures Basic collection gt Despiking Procedure Define baselines 2 Define peaks Convert to physical units Convert RI to concentration Determine mass and radius from LS data Fit mass or radius procedure Determine distributions and moments LC Data Set Definitions 0 Results v i ig 9 To add a procedure to an experiment fo
143. To apply a template to multiple experiments see Applying a Template to Multiple Experiments on page 6 23 For example after using the LS batch Debye plot template when col lecting data you might want to apply the LS batch Zimm plot template to the same data so that you can view the results differently Applying a template creates a separate experiment so you do not lose any of the information in the original experiment The new experiment has a name that reflects both the original experiment and the template To apply a template follow these steps 1 Open the experiment that contains the raw data you want to use 2 Choose Experiment Apply Template The New From Existing dia log appears This is the same dialog you use to create an experiment from a template before data collection 3 Choose a template to apply to the data The procedure and result for mats in the template will be used Typically you would choose a tem plate from the System Templates or My Templates folder 4 Click Create A new experiment is created and is run automatically The name of the new experiment combines the names of the template used to create the experiment and the original experiment 5 After the applied procedure runs you can view the new results M1000 Rev H Applying a Template Applying a Template to Multiple Experiments If you want to apply the same experiment template to multiple experi ments follow thes
144. To make it easier to use this manual we have used the following conven tions to distinguish different kinds of information e Menu commands This manual indicates menus command to use as follows File gt Open This example indicates that you should open the File menu and select the Open command You will see this style wherever menu commands are described es Buttons In the text you will see instructions to click on screen buttons and to press keys on the keyboard s Key combinations A plus sign between key names means to press and hold down the first key while you press the second key For example Press ALT ESC means to press and hold down the ALT key and press the ESC key then release both keys e DAWN instrument Except where there are details for a particular instrument when the name will be given we will refer to the DAWN EOS and miniDAWN instruments simply as the DAWN M1000 Rev H 1 5 Chapter 1 About ASTRA V Molar mass versus Molecular weight The IUPAC Definition Com mittee specifies the term molar mass for the sum of the atomic weights of all atoms in a mole of molecules The term molecular weight has the same meaning You will see molar mass used in this manual Tip See Appendix A Menu Quick Reference for a complete list of keystroke alternatives to the mouse pointer for selecting menu options Glossary 1 6 The following terms are used in this manual A2 mass and radius Resul
145. Workspace to hide the workspace when it is not needed so that you will have more screen space for other dialogs When the workspace is hidden you can move your cursor to the edge of the window where the workspace is docked to display the workspace View Customize The Toolbars tab provides another way to hide various ASTRA toolbars The Experiment toolbar is the one that contains the Validate Run and Stop icons You may want to add keystrokes for various menu commands you use fre quently To add a command follow these steps 1 SY ey ge ibs 6 Choose View Customize and go to the Keyboard tab Select a menu in the Category pull down list Select a command in the Commands list Click in the Press New Shortcut Key field Use your keyboard to press a key combination The key names are shown in the field and any command to which they are already assigned is shown below To assign the key combination to the selected command click Assign Command Reference See Appendix A Menu Quick Reference lists of all menu commands tool bar buttons and key sequences provided in ASTRA 3 14 M1000 Rev H More About the ASTRA Environment Printing You can print Configuration property dialogs procedure dialogs reports and graphs from ASTRA To print choose File gt Print Ctrl P and use the Print dialog as you would in other Windows applications In addition you can choose File gt Print Setup to c
146. ach slice and hence uncertainties in the calculated molar mass and size averages Remember these uncertainties are statistical only and do not include any of the many possible systematic errors that may be present Examples are errors in dn dc the DAWN calibration constant the AUX calibration constants and the normalization coefficients Use the reported uncertainties as a measure of the statistical consistency of the data never as an absolute limit on the error in your results M1000 Rev H D 17 Appendix D Light Scattering Theory Out of Range Values Occasionally electrical noise or a very low concentration or light scatter ing signal may cause the calculated molecular weight at a particular slice to be a negative number For low molecular weights often the mean square radius at a particular slice will be negative due to random noise in the Debye plot for that slice Also noise may cause both the calculated molecular weight and the mean square radius to have uncertainties larger than the values themselves In these cases special considerations are called for When calculating molecular weight averages ASTRA first checks the cal culated molecular weight values of all slices to be included in the calculations to find out if any of them are negative ASTRA then removes slices that have negative values before calculating the averages When calculating mean square radius averages ASTRA includes values from all slices in the su
147. aeeeeeeeeees 6 4 Opening an Experiment from the Database eese eesse esere eene 6 7 Opening an Experiment from a File 6 8 Importing an Experiment from a File 6 9 Running an Experiment aerssnersarennanrno a 6 11 Closing an Experiment 6 15 Saving an Experiment to the Database nens 6 15 Saving an Experiment to ale 6 16 Exporting an Experiment 6 17 Creating a Template cccecccceceeeeeeeeeeeeeeeeeeaeeeeeeeeeeaeeeeeaeeeetiaeeseeeeeeed 6 18 Deleting an Evpermemt reinaan NaN aA 6 20 Copying RE E 6 21 Applying a TOmplale senis 6 22 Adding Elements to an Experiment 6 24 M1000 Rev H 6 1 Chapter 6 Creating amp Running Experiments About Experiments The ASTRA user environment is centered around a structure we call an experiment which contains all the information needed to run an experiment and produce results After you run an experiment the experiment structure contains the results The Experiments tab in ASTRA shows the parts of the experiment LI ixj Experiments E SampleSets S System Profiles ies Experiments amp E Configuration EOS batch E Ei DAWN EOS DAWN EOS amp Solvent water Sa Sample untitled SJ Procedures Basic collection Despiking Procedure Define baselines Define peaks Determine mass and radius from LS data SQ Results E Report summary E Report detailed For Help press F1 You can expand or collapse the folders in an
148. aks to search for the peak maximum or minimum If you select the same flow marker for each experiment it is used when combining data to yield a more accu rate curve e You can view individual peak entries by pressing the sign next to the Peak label Save the completed experiment Repeat the previous steps for any additional mixtures of known molec ular standards as many times as necessary to cover the full column range Use the flow marker you selected in each mixture Open all column calibration experiments you saved for this column M1000 Rev H Configuration Procedures 9 In the Calibrate Column dialog click the Import Peak Data button You will see a dialog that lists other open experiments that contain col umn calibration data Check the boxes next to any peaks you want to import The grid shows the peak number elution volume flow marker molar mass and intrinsic viscosity for each peak Then click OK Import Peak Data Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Source Experiment M universal calib Peak Volume 1 2 3 4 5 10 4763 11 0697 12 3702 14 6807 17 067 Flow Mark 22 5719 22 5719 22 5719 22 5719 22 5719 Molar Mass 2e 006 585000 136100 14050 1800 Intrinsic iscosits OK 444 053 179 49 60 495 12 5646 4 21115 10 Confirm that the peaks have been added to the list and adjust the curve fit order Then click Apply to store the imported
149. alibration Iw data IV fit 0 0005 0 0010 concentration g mL Calibration 1 8749 0 0043 e 4 Known dn de mt at 0 1100 Percentage To Keep 100 Enabled Peaks By OK Si Cancel This procedure has the following properties Table 8 6 RI Calibration Properties Field Description Calibration The resulting calibration constant Known dn dc The known dn dc value for the calibration standard used M1000 Rev H 8 25 Chapter 8 Editing Procedures Table 8 6 RI Calibration Properties Field Description Percentage to Keep The percent of the marked peak data to use for calibration If the plateau is flat not drifting in the peak range using the default value is recommended Enabled Peaks This list shows the peaks used in the fit to determine the calibration constant Checking or unchecking a peak adds or removes it from the fit to determine the calibration constant Note 8 26 6 Enter the known dn dc into the row titled Known dn dc mL g and click Apply The value in the Calibration row should update once this known value is entered 7 Expand the Results node of the experiment and double click the Report Summary line The report displays the new Calibration con stant Optilab rEX dRI Calibration Results For the Optilab rEX the reported Calibration Constant is actually a cor rection factor A value of 1 0000 would indicate th
150. ally determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box See Band Broadening on page 8 14 for an explanation of the instrumen tal term The units are in microliters Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters 7 20 M1000 Rev H Viscometry Instrument Profiles Viscometry Instrument Profiles A viscometer measures the specific viscometry of a solution Wyatt s ViscoStar measures specific viscosity When combined with con centration data from an RI or UV concentration detector specific viscosity can be used to calculate intrinsic viscosity Intrinsic viscosity in turn combined with data from light scattering measurements can be used to derive the hydrodynamic radius r and molecular shape information ViscoStar Profiles You can set the following properties for a ViscoStar instrument Table 7 10 ViscoStar Profile Fields Field Description Name Name of the instrument If you have already created a system profile for this instrument click and select a profile to use Description Description of the instrument which typically contains more information than the Name Physical Click and select from the Instruments dialog See Accessing and Instrument Viewing Hardware
151. ally or determined via the alignment procedure Typically the volume only needs to be set for fluid connections between instruments that collect data mL Temperature Control gt Enable Temperature Control gt Temperature Check this box if the instrument is set to maintain a specified temperature heated or cooled If this instrument is temperature controlled specify the temperature to which it is set Use C 7 28 M1000 Rev H Connection Profiles AUX Connection Profiles An AUX connection profile describes a connection from the analog output of the source instrument to the analog input of the destination instrument If there is no AUX connection and the instrument is capable of collecting data and communicating data to the PC the instrument to PC connection is implied and does not require a profile You can set the following properties for an AUX connection Table 7 17 AUX Connection Properties Field Description Name Name of the connection If you have already created a system profile for this connection click and select a profile to use Description Description of the connection which typically contains more information than the Name Source Instrument Select the type of instrument that sends analog data over this connection The drop down list shows the instrument profile types that are available for a connection Destination Instrument Select the type o
152. anager described in Using the Diagnostic Manager on page 5 5 M1000 Rev H Accessing and Viewing Hardware Adding an Instrument or Computer to the Instrument List If you are collecting from a DAWN or WyattQELS instrument connected to your local computer the first computer you should add to the instru ment list is your own local computer You should also add other computers that have or will have instruments connected to them To add an instrument to the instrument list which you open with Sys tem Instruments follow these steps 1 In the Instruments dialog click Add to open the Add Instruments dia log A search for instruments is performed automatically E Add Instruments Ze Automatic Searching 36 Ei Differential Refractometer 2 E MultiAngle Light Scattering Detector 7 2 Quasi Elastic Light Scattering Detector 3 Viscometer 1 mO Manual is d lt aT Cancel 2 Ifthe instrument you want to add was found select that instrument and click Add 3 Ifyour instrument was not found you can add a computer or instru ment by selecting the Manual option Then either type the network name of the computer or instrument or click the Browse button or select the IP Address option and type the numeric IP address Then click Add M1000 Rev H Chapter 2 Installing and Setting Up ASTRA If you click the Browse button expand the network listing so that you can see the instruments and
153. ance than standard normalization Radius Type The type of radius specified Options are RMS geometric and hydrodynamic If you are using a Mie or sphere model for the sample specify that the radius is a geometric radius Model Displays the fitting model being used Percentage to Keep If this is a batch experiment specifies the fraction of data to keep for performing the normalization If the plateau is flat not drifting in the peak range using the default value is recommended M1000 Rev H Checking the Normalization Coefficients After normalizing the detectors you need to make sure that the coeffi cients you obtained are accurate You can use the Debye plot About Debye Plots on page 8 61 to do this If you use the Debye plot inject a sample with a radius around 20 nm a linear polymer with a molar mass about 200 000 g mol is suitable Set baselines and mark the peak then use the Mass and Radius from LS Data 8 35 Chapter 8 Editing Procedures procedure page 8 59 to display the Debye plot using the Debye model and a Fit Degree of 1 It is a good idea to step through several data slices at the top of the peak use the keys to get a feeling for the random noise in the data If one detector is consistently off the fitted line above or below its normalization coefficient needs to be redetermined results graph 3 6x10 34x107 32x10 Ritheta 30x10 28x107 0 0 0 2 0 4 0 6 08 sin
154. anching per 1000 repeat units is defined for each slice as Equation 43 R de A 10008 7 where B is the branching per molecule for the slice as calculated above R is the repeat unit molar mass and M is the branched molar mass for the slice You must enter the repeat unit molar mass in the Unit MW box in the Branching property view for each branched file to be plotted D 22 M1000 Rev H M1000 Rev H Particles Theory Particles support is an add on option for ASTRA This option provides a procedure to calculate number density see Number from LS Data on page 8 69 Particles include lattices liposomes and vesicles Particle measurements are especially suited for use when a light scattering instru ment is coupled to a fractionation technique such as Field Flow Fraction ation FFF or Capillary Hydrodynamic Fractionation CHDF but concentration is not measured CONTENTS PAGE Determination Of Sizes ou ccc cece cee eececeseeseeeeceeesseauaeeeeeeeauaaeeeeeeeees E 2 Radius Moments wiiiiccnccteeediteccttedsecccaznestidvcreie lavevieesessdeebtuencesesavadeieeenieess E 5 TEO enno eae tena cenrueavantectiibes dicta iaceatio eaten aaadetanieeneeu tes E 7 E 1 Appendix E Particles Theory Determination of Sizes 1 E 2 As discussed in Appendix D Light Scattering Theory data collected by a DAWN system can be used to derive molecular parameters In Particles mode concentration is not measured instead the a
155. and graph formats along with procedures from a template to an experiment you have already run to collect data For example after using the LS batch Debye plot template when collecting data you might want to apply the LS batch Zimm plot template to the same data so that you can view the results differently M1000 Rev H Applying a template creates a separate experiment so you do not lose any of the information in the original experiment To apply a template follow these steps 1 2 Open an experiment containing raw source data you want to use Choose Experiment Apply Template The New From Existing dia log appears This is the same dialog you use to create an experiment from a template before data collection Choose a template to apply to the data Typically you would choose a template from the System Templates or My Templates folder The procedures and result formats reports and graphs in the tem plate are used in place of those in your source experiment The source data and the source experiment configuration are not changed Click Create A new experiment is created Select the new experiment and click the Run icon in the toolbar After the applied procedure runs you can view the new results 10 5 Chapter 10 Working With Reports Printing a Report Tip 10 6 To print a report do one of the following e Choose File gt Print e Select the report window right click and sel
156. angle detectors to improve the fit of the extrapolation since the curvature can be very large Berry model Uses the sqrt K c R 8 formalism It can be useful in combination with deleting high angle data when analyzing molecules with RMS radii greater than 50 nm Random coil model Uses the formula for a theoretical random coil molecule rather than a polynomial to fit the angular light scattering data Sphere model Uses the analytical formula for a sphere rather than a polynomial to fit the angular light scattering data Use this model only with known spherical samples such as lattices Note that if the spheres are aggregated this model may not fit since the aggregated particles may be of any shape Rod model Uses the analytical formula for a rod rather than a poly nomial to fit the angular light scattering data It is necessary to specify the rod radius when using this model Coated sphere model Uses the analytical formula for a coated sphere rather than a polynomial to fit the angular light scattering data It is necessary to specify the coating thickness and core and coating refractive indices when using this model 8 55 Chapter 8 Editing Procedures e Mie model Uses the Mie analysis for a sphere rather than a polyno mial to fit the angular light scattering data It is necessary to specify index of refraction when using this model If you are using the Lorenz Mie theory as this model does the particle need not satisfy
157. ard sample and verifying that the injected and calculated masses are equal This option is often much more efficient than the off line approach described below particularly with organic mobile phases Contact Wyatt Technical Support and review the section RI Calibration from Peak on page 8 75 for more information Off line dRI calibration may be performed using any substance with a well characterized dn dc value In all cases the recommended standard is anhydrous sodium chloride dissolved in pure de ionized water This solution has a dn dc of 0 172 mL g at a wavelength of 690 nm 0 181 mL g at 488 nm 0 174 mL g at 633 nm or 658 nm and 0 170 ml g at 900 nm Appropriate concentrations should be prepared using pure distilled water and clean glassware The lowest and highest recommended concentrations vary by instrument e For the Optilab rEX 0 1 mg mL and 5mg mL e For all other dRI instruments 0 1mg mL to 1 2mg mL Use six or more concentrations prepared within 1 or better accuracy to increase the precision of the final determination Wyatt Technology provides validated pre mixed NaCl solutions in the ideal concentration range for calibration of any dRI instrument Wyatt Technology part number P8400 NaCl Solutions Kit NaCl concentrations are as follows 0 0 blank 0 1 0 5 1 0 1 2 2 0 3 0 4 0 and 5 0 mg mL in nanopure water dRI instruments can differentiate between solvent that has been satu rated with ambient gase
158. ardware manual for details Not available for the DAWN 8 QELS gt Option QELS gt Replaced Detector Check this box if the DAWN instrument has a detector replaced with a QELS fiber If QELS is enabled type the number of the detector replaced for the QELS fiber Band Broadening gt Enabled Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broad ening parameters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term See Band Broadening on page 8 14 for an explanation of the instrumen tal term The units are in microliters Band Broadening gt Mixing Term M1000 Rev H See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters Chapter 7 Configuring Experiments Table 7 3 DAWN Instrument Properties Field Description Temperature Control gt Enable Check this box if the instrument is set to maintain a specified temperature heated or cooled Temperature Control gt Temperature If this instrument is temperature controlled specify the temperature to which it is set Use C Temp Controlled Line gt Enable Check this box if the plumbing line in the instrument is set to
159. at the Optilab rEX s internal dRI calibration constant has remained unchanged If the value is 1 0120 the Optilab rEX dRI calibration constant has increased by a factor of 1 2 1 Multiply the old dRI calibration constant by the correction factor to determine the new dRI calibration constant The old dRI calibration constant is viewable in the Constants window on the Optilab rEX front panel The calculation is old dRI calibration constant x ASTRA V correction factor new dRI calibration constant For example 3 3828e 3 x 1 012 3 4234e 3 You must calculate the new dRI calibration constant manually using the old dRI calibration constant and the correction factor provided by ASTRA 2 Tab to the System screen on the Optilab rEX front panel Open the Constants window and enter the new dRI calibration constant Tab to the Apply button and press Enter Optilab DSP or Third Party Instrument dRI Calibration Results Enter the reported dRI calibration constant for analog instruments in the calibration constant field of the AUX connection profile for the RI detector This value is not set in the AUX connection profile automatically M1000 Rev H Configuration Procedures Absolute RI Calibration You can calibrate the Optilab rEX aRI measurement as described in this section This procedure is also summarized in the Optilab rEX User Guide section titled Instrument Calibration for aRI We recommend that y
160. ata Random coil sphere coated sphere rod and Mie models do not require a fit degree Hence the Debye plot can be used to assess the efficacy of the fit model and flag noisy detectors or a poor normalization For the Debye Zimm and Berry models the angular data is fit to a poly nomial expansion Hence it is necessary to specify a fit degree for these models Fit degree can be set form 0 to 5 When using the Debye Zimm or Berry model and determining the fit degree it is often sufficient to choose a fit degree that gives the smallest error As an example compare the result in Figure 8 5 with that of Figure 8 6 The error in MM has increased The only difference is the Fit Degree chosen for the calculation In this example using a Fit Degree of 2 increases the error in the data and gives biased low results It is also obvious in Figure 8 6 that a fit degree of 2 is inappropriate due to the sys tematic deviations of the data with respect to the fit Debye Plot SD1250K 1 00x10 8 00x10 T L Zeng R 4 00x10 2 00x10 0 0 sin theta 2 Peak Slice 1 663 Volume 2 727 mL Fit degree 2 Conc 4 062 0 001 e 4 g mL Mw 1 554 0 020 e 6 g mol Radius 122 1 2 5 nm 90 amp AUX detectors Figure 8 6 Debye plot with Fit degree of 2 Sometimes minimizing the error in the data is not a sufficient criterion In fact several polynomials may give very similar errors Thi
161. ater having an RMS radius of 3 nm High Concentration The standard you inject for normalization is at a higher concentration than normal This is to improve the signal to noise ratio of the measurement Aim for a ratio of at least 100 1 for the normalization peak Batch Mode Issues For batch measurements you do not have the advantage of molar mass separation as you do in chromatography Any aggregates in your sample will not be separated and may cause nor malization errors Therefore we recommend higher concentrations of non aggregating lower molar mass standards for normalization in batch mode A 10 15 mg mL solution of 4000 g mol polystyrene in toluene or THF or 5000 g mol dextran pullulan or PEO in water works well All of these have RMS radii of about 2 nm 8 33 Chapter 8 Editing Procedures Running a Normalization Experiment The sample and solvent you use for normalization are important Follow these steps to normalize 1 Choose a normalization standard as described earlier in this section 2 Create a new experiment from the template appropriate for your type 8 34 of experiment Run the experiment and set baselines and peaks as described for those procedures Use a narrow peak for normalization For online experiments set a peak symmetrically over a monomer peak exclude any multimer peaks For batch mode set a peak region over the plateau corresponding to your normalization standard Perform the
162. ation choose File gt Save As and follow steps 3 through 6 above Right click the experiment name in the tree and choose Save As M1000 Rev H 6 15 Chapter 6 Creating amp Running Experiments Saving an Experiment to a File Basic It is a good idea to save experiments frequently If you are using ASTRA V Basic experiments are stored in files with an extension of vaf To save an experiment follow these steps 1 Choose File Save Shortcuts Press Ctrl S Click the NM icon Right click the experiment name in the tree and choose Save 2 If this is the first time you have saved this experiment you see the Save As dialog Otherwise you are finished saving the file 3 In the Save As dialog navigate to the folder you want to contain the file 4 In the File Name field type a file name for the experiment The follow ing characters may not be used in ASTRA file names colon question mark quote R asterisk i forward slash backslash less than lt greater than gt pipe 5 The Save As Type field shows that the file will be saved with an exten sion of vaf You can choose an older version of ASTRA V if you like To save to an ASTRA 4 tab delimited text or comma separated values format see Exporting an Experiment on page 6 17 6 Click Save To save an experiment with a different name or location choose File Save As and follow steps 3 through 6 above Sho
163. ation if the cell length of the UV absorption instrument is known and if the UV extinction coefficient for the sample is known Generic UV Detector Profiles You can create a Generic UV Instrument profile for any third party UV instrument for which data is collected through the AUX input of another instrument You do not select a Physical Instrument for a Generic UV Instrument profile because ASTRA V does not support a direct data connection to such instruments Instead add an AUX connection to the experiment configu ration to indicate which AUX channel and instrument are to be used to read the signal See AUX Connection Profiles on page 7 29 for details You can set the following properties for a generic UV instrument Table 7 12 Generic UV Instrument Profile Fields Field Description Name Name of the instrument If you have already created a system profile for this instrument click and select a profile to use Description Description of the instrument which typically contains more information than the Name Wavelength The wavelength of the light used in the instrument nm Cell Length The length of the sample cell in cm UV Response Factor The conversion factor from absorbance units AU to volts for the UV aux output Please see the hardware manual for the UV detector to determine this value Temperature Control gt Enable Check this box if the instrument is set to maintain a specifie
164. ature at which the data was collected Shows the viscosity of the solvent This value comes from the solvent profile page 7 32 Refractive index of the sample for the given peak gt Resolution A value that represents the optimal smoothing of the distribution given the noise level of the correlation function It varies from 0 for very noisy data to 1 for data with very good signal to noise In general the lower the resolution value the more uncertain the actual widths and structure of the final distribution Min Rh Threshold Fitted Rh values with a lower radius than the value you type are not used in the analysis The Min and Max Rh Thresholds are applied after all other analysis to allow you to discard results that fall outside a desired range For example you could dis card results greater than 300 nm If the Prefilter box is not checked the Min Max Rh Thresholds are ignored Max Rh Threshold Processing Parame ters Fitted Rh values with a higher radius than the value you type are not used in the analysis Various values that govern the processing of the data Users do not frequently need to change these values gt Min Fit Delay Time Type the minimum number of seconds for the fit delay The default is 0 seconds gt Max Fit Delay Time Type the maximum number of seconds for the fit delay The default is 1 second gt Suppress Peaks Below Type a size in nanometers below whic
165. au Method Enabled Detectors gt 1 18 Put a checkmark in this box if this is a batch experiment or an online experiment with plateaus rather than peaks This list has a checkmark next to detectors whose data is used in the calculation You can disable individual detectors by removing the checkmark Enabled Peaks FOM gt 1 ton You can omit peaks from the plot by removing the checkmark next to the peak number The FOM value shown for each peak is a Figure of Merit which is a unitless value that reflects the ability to measure A2 accurately If the FOM for a peak is less than 1 or slightly greater than 1 then the peak will help measure A2 accurately 8 64 M1000 Rev H Analysis Procedures Online A2 M1000 Rev H This procedure calculates the second and third virial coefficients Ay and A respectively mass and RMS radius of the sample based on light scat tering data as a function of angle and concentration For more about determining the second virial coefficient go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Second virial coefficient The procedure has the following dialog Experiment2 Procedures Online A2 Zimm plot Rithetap K c na r sin theta 2 kc Mass g mol RMS Radius 0 0 0 0 nm R 4 Radius nja A2 0 000 0 000 mol mt io A3 nja Zimm Orientation Angle Fit Degree Concentration Fit Degree Percentage To Kee
166. b page editor because they use special lt wtc_field gt tags to identify data to be shown in the report Many web page editors would remove or modify these non standard tags If you are comfortable editing web pages using a text editor you can modify the format of the provided report templates as desired For example you might want to add your company logo or department information The report formats provided with ASTRA V are located in the Reports folder under the ASTRA installation folder This is usually similar to C Program Files WTC ASTRA 5 Reports 10 3 Chapter 10 Working With Reports Adding a Report Experiment Builder You can add reports only if you enable Experiment Builder mode by choosing System Preferences Experiment Builder Mode To create a new report follow these steps 1 Choose Experiment Add to Experiment This opens the Add to Experiment dialog which allows you to add items to the Procedures Data Set Definitions and Results nodes of the experiment Shortcuts Press Ctrl Shift P Right click any folder in the experiment tree and choose Manage Add To Experiment E Add to experiment GES EI Graph Cancel E Report Procedures Lx B Dataset Definition 2 Close the Procedures nodes and select Report in the Results folder Then click OK 3 Double click the Report untitled1 item that was added to the Results node in the experiment tree 4 In the property l
167. bins to obtain the differential number fraction after dividing each such fraction by the bin size The frac tions may also be distributed over the range of size bins included within the measured standard deviation associated with the particular contribut ing fraction In any event the differential number fraction n r dr of particles in the selected peak region between r and r dr now may be calculated explicitly without any a priori knowledge of the mass concentration at each slice provided we know the particle structure and that the RGD approximation is valid For example if we know that the particles are homogeneous spheres we may replace Vi by be There are many other particle shapes where the relation between r and V is known The differential mass fractions may be generated in a similar manner without reference to a second detector What about particles whose shape is not known a priori Although we may still calculate rgasa function of elution volume the lg calibration curve we cannot determine the differential number or mass fractions Indeed if we do not know the relation between the measured Tg and the particle s hydrodynamic radius we cannot generate differential distributions Were we to add a concentration detector following the LS detector we could easily generate the differential mass fraction distributions of lg A few other points must be discussed most important among them is the applicability of the RGD appr
168. bution analysis in photon correlation spectros copy website documentation at http www softscientific com science WhitePapers dynals1 dynals100 htm F 8 M1000 Rev H Viscosity Theory This appendix reviews the theory of viscosity related calculations CONTENTS PAGE Calculating Intrinsic Viscosity ceececeeceeeeeeeneeeeeeeeeeeeeeeeeeeeeetaeeeeeeeeees G 2 Intrinsic Viscosity and Molecular Parameters eseseeeseeeseeereeere G 4 Flory Fox Relations cccs scccsseesistecsceessetecedeseasceeesvaeseeaagedndeuencusuersreneneezsees G 5 M1000 Rev H G 1 Appendix G Viscosity Theory Calculating Intrinsic Viscosity ASTRA V can process a wide variety of input viscosity sources ranging from simple devices producing only a single pressure differential to more sophisticated devices that measure specific viscosity directly Once specific viscosity is measured it is useful to compute the intrinsic viscosity Intrinsic viscosity is defined as the limit of Equation 1 Ny ol Im c gt 0 Of course all real instruments measure the specific viscosity at finite con centrations The concentration dependency of the specific viscosity is typically described using one of three formalisms the Huggins equation the Kraemer equation and the Solomon Gatesman equation In all cases the concentration of the sample must be derived from a detector such as the Optilab rEX or a UV absorption detector Huggins The Huggins equat
169. by clicking the OK button e Run the experiment Introduce Solvents 1 Flow the pure solvents directly into the Optilab rEX at a constant flow rate using a syringe pump or an HPLC pump set in the flow rate range of 0 5mL min to 1 0mL min After flowing roughly 4 5 mL of solvent toggle the purge valve to PURGE OFF for 15 seconds and then toggle it back to PURGE ON for 15 seconds Repeat this cycle for 2 3 minutes Cycling the purge valve in this manner creates just enough agitation in the flow path to displace air bubbles and thoroughly remove the previous solvent On the Optilab rEX System tab go to the LED button and press Enter Adjust the Percent max power setting so that the Light intensity is close to but not above 7 8 Volts Close the LED Intensity dialog and return to either the Main or aRI tab Complete the solvent introduction process by leaving the purge valve in the PURGE ON state for roughly one minute and then turn the syringe pump off After three minutes of stopped flow the Optilab rEX is stable During this time ASTRA V will record the stable flat no flow region Note the time of this no flow period which will be referenced during data processing Repeat this solvent introduction procedure beginning with step 1 of this list for each remaining solvent ASTRA Calculations Follow these steps in ASTRA V 1 After data collection open the Procedures section of the experiment and click Define peaks On
170. c Manager shows commands you have sent to the instrument via the Diagnostic Manager and responses provided by the instrument Only commands sent during the current Diagnostic Manager session are shown The following figure shows some commandes sent to a DAWN HELEOS instrument Diagnostic Manager Data graphical Data numeric Alarm Commands Command log Property Parameters DAWN HELEOS Command Start Monitoring Apply Help You can drag the borders between column headings to resize the columns You can copy data from this log for pasting into other applications M1000 Rev H 5 9 Chapter 5 Interfaces to Instruments 5 10 Viewing and Setting Properties with the Diagnostic Manager The Property tab of the Diagnostic Manager allows you to view and set instrument properties The list of properties you can set is different for each type of instrument The following figure shows the properties avail able for the DAWN HELEOS instrument Diagnostic Manager Data graphical Data numeric Alarm Commands Command log Property Instrument EES e NR aE Maintain data in strip chart for 5 lt j Minutes 7 for all instruments Display data in bouncing line chart as normalized format Normalization coefficients Detector 1 Detector 7 hoo Detector 13 Detector2 f Detector 8 Leg Detector 14 Detector3 fi Detector 9 bh Detector 15 Detector 4 fj Detector 10 LES Detector 16 Detector 5 Detector11 Dete
171. calculation Plot Choose whether to show the correlation function or the cumulants plot in the left graph The cumulants plot shows the log differential hydrodynamic radius Mean Rh Shows the mean hydrodynamic radius Rh for the peak You can override the calculated value by typing a known value Width Shows the width in cm2 sec Processing Conditions Conditions per peak during the regularization analysis You cannot mod ify these values gt Temperature gt Viscosity gt Refractive Index Shows the temperature at which the data was collected Shows the viscosity of the solvent This value comes from the solvent pro file page 7 32 Refractive index of the sample for the given peak Min Rh Threshold Fitted Rh values with a lower radius than the value you type are not used in the analysis The default is 1 nm The Min and Max Rh Thresholds are applied after all other analysis to allow you to discard results that fall outside a desired range For example you could discard results greater than 300 nm If the Prefilter box is not checked the Min Max Rh Thresholds are ignored Max Rh Threshold Fitted Rh values with a higher radius than the value you type are not used in the analysis The default is 300 nm Processing Parameters gt Min Fit Delay Time Data with a lower delay time than the value you type is not used in the fit to the correlation function The default is 0
172. careful to select a region of the peak that is free from contamination by other eluting species Typically the range marker should be set from a position about halfway up the lead ing edge of the peak to a point just past the peak where all detector signals have returned to the baseline Click Perform Fit to calculate the terms and update the graph 8 15 Chapter 8 Editing Procedures Examine the fit between the two traces and the values for the Instru mental Term and the Mixing Term The expected values vary depend ing on whether there is a viscometer in the instrument series and whether despiking or smoothing has been performed No Viscometer and No Despiking or Smoothing The value of the instrumental term should be small 1 ul e No Viscometer and Despiking or Smoothing Performed The value of the instrumental term will generally be larger than 1 ul but smaller then the mixing term Viscometer Used The instrumental term will generally be com parable to or larger than the mixing term Despiking and smooth ing should not make much difference if there is a viscometer If the resulting instrumental term is significantly larger than expected and the match between the peaks is not good you should repeat the fit To do this click Reset then enter seed values for the instrumental and mixing terms e No Viscometer Used Use seed values of 1 ul for the instrumen tal term and 40 ul for the mixing term Viscometer Used U
173. cattering medium and r is the distance between the scattering volume and the detector Eq 1 implies that the dimensions of Rg are length The DAWN instruments are provided with vertically polarized light sources this fact is assumed in Eq 1 and all derivations to follow When studying a solution of solvent plus solute it is convenient to use Rg to represent the excess scattering of the solution compared to that of the solvent alone Equation 2 1 E Io 0 0 solvent LV R where Ig solvent 18 the scattered intensity of the solvent For a pure solvent we use Eq 1 for a solution we use Eq 2 The philosophy behind Eq 2 is simple we are merely describing the scattering after subtracting the baseline of pure solvent In this case Rg is often called the excess Rayleigh ratio of the solute As we shall see below knowledge of Rg at a number of different angles leads directly to the weight average molar mass and mean square size of the solute molecules making Rg the most important measured quantity in light scattering D 3 Appendix D Light Scattering Theory Measured Quantities and Calibration How do we measure Rg If we try to use Eq 1 or Eq 2 directly we run into trouble immediately First the quantities we can measure directly are detector voltages not light intensities Because the measured voltages are proportional to light intensities we must calibrate our detectors This calibration is not difficu
174. ccecceeeeeeeeeeeeeceeeeeeeneeeeseeneeeeeeeeeeees D 18 8 M1000 Rev H Contents lee le D 19 Branching Ratio Radius Method 00 eeeceeeecneeeeeeenneeeeeeeaaeeeeseeaaeeeeeeeaaeeeeneeaaes D 19 Branching Ratio Mass Method A D 19 Branching Ratio Viscosity Method eeccceeeeesseceeeeenneeeeeeenaaeeeeeeeaaeeeeeeenaaeeeeneeaaes D 20 Branching Per Molecule cccscccceeeeecceceeeeeecceeeeeseececeeeneeceeeeenneeceeeeenneaeeeneennaes D 21 Long Chain Branching cccccccceceeceeceeeeecaeeeeseeeseeeeeeeaaeeseeeeeeeaaeeeeeaeeessaeeeseneeeesaees D 22 Appendix E Particles Theory ccseccceeesseseeeeseeeeeeeeeeeees E 1 Determination Of SIZES senedi geed eE aaie E 2 Debye Fit Meel tt Gaede E N hy ae a en een E 2 ZIMIMUP n WEE E 3 Berry Fit Method EE E 3 Random Coil Fit Method ceeccccceeeeeeneeeeeeeeeeeeaeeeeeeeeeeeaeeeeeeeeecaeeeeeeeeeessaeeseeaeeeee E 3 Model Fit Method NENNEN ee A EEE eee ENEE E 3 Mie Fit Method s aise ieee adidas tee E 4 Radius len E 5 RMS e EE E 5 Geometric and Hydrodynamic Radius ccccccesececeteeeeeeeeeeeeeeeeeaeeseeeeeetaeeeeeeeeess E 6 TTL EE E 7 Appendix F QELS Theory ei tienen F 1 Ee TE e F 2 TG OLY EE F 2 Application of the Method of Cumulants eeceeeeeeeeeeeeeeeeeeeececaeeeeeaeeeeeeeeseaaeeenes F 4 Interpretation of Cumulant Results ccceccceeseeeeeeeeeeeneeeeeeeeeeeaaeeseeeeeesaeeneeeeeess F 5 RegularizatiOn ci
175. cecceeecceceeeeeeeeeeeseeeeeesaeeceneeeesaeeeneeeeeesiaeeeseneeess 6 4 Creating Experiments from Templates cccccccseceeeeeeeeeeeeeeeeeeeseaeeeeeaeeeseeeeeeeeeees 6 5 Creating Experiments from Scratch ccccccceceeeeeeeeeeeeeeeeeaeeeeeeeeesaeeeseeeeeeseaeeeneneeess 6 6 Opening an Experiment from the Database ccccceceeeeseeeeeeeeeeeeeeeeeeeeeeteeees 6 7 Opening an Experiment from a File EE 6 8 Importing an Experiment from a File vsrcisnccdceteesctcc etd cekdideianiekdee dy die 6 9 Importing ASTRA 4 Files 0 cccccceceeeeeeeneeeeeeeeeeeeeeeeeeeeeeaaeeeceeeeeseaeseseaeeseeaeeeeeeaeeeeaas 6 9 Running an Experiment EE 6 11 Validating an Experiment ererrarekdarrieiaiini aiia aN aN 6 11 Starting a Data Collection RUN riscan nantan naaraana anata aa a i a aaa anina 6 11 Signing Off on an Experiment 0 eeccececeeeeeeceeeeeeeeeeceeeeeeceaeeseeeeeesaaeeseeneeessaeeeseaeees 6 12 Stopping an Experiment cccceecccececeeeeeseeeceeeeeeeaeeceeeeeeaaeeeeeeeeeseaeesseneeessaeenenaeees 6 13 Re Running an Experiment for Data Processing eessssessesssserrrsssrrrrrssrrrrrssrrenss 6 13 fa Cras M OCCU S sn air geegeeegteuge A8 aa aaa a aeaaea Ar bites araa evden aa E AE Aaa ani 6 14 Gl sing an EXperimMent E 6 15 Saving an Experiment to the Database cececceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeneeeeees 6 15 Saving an Experiment to a File sscctecsccstaptecsachesaneectidietieisnaadantheeetadeateagecticee rake 6 16 Exporti
176. cedures are available e Basic Collection on page 8 10 e Script Collection on page 8 12 Basic Collection The Basic collection procedure collects data from the instruments specified in the Experiment Configuration You can set properties for this procedure before running the experiment When you run the experiment this proce dure runs automatically without prompting for parameters You can use this dialog to view data as it is being collected If multiple instruments are collecting data you can choose to view any set of signals in the checkboxes to the right of the graph You may need to scroll to see some checkboxes Signals other that light scattering LS are shown at the end of the list This may include the forward laser monitor FM and refractive index Light scattering data is shown in red RI data is blue UV data is green QELS data is magenta and viscosity data is black The x axis of the graph uses units specified by the Abscissa Units property of the experiment configuration see page 7 9 By default the units are milliliters for an online experiment and minutes for a batch experiment The y axis of the graph uses raw data units For light scattering experi ments this is in volts The data is converted to other units by a later transformation procedure M1000 Rev H Check boxes to display various data detector voltage NI Drag separator to resize Collection Operator Collection Procedures
177. ceptable or not If the fit is not good ASTRA reports results that are meaningless You should try both polynomial and exponential fitting before deciding which one to use 8 58 M1000 Rev H Analysis Procedures Analysis Procedures The analysis procedures calculate various results using the data The following analysis procedure types are available A2 Mass and Radius from LS Data on page 8 63 Branching on page 8 80 Copolymer Analysis on page 8 86 Cumulants on page 8 98 Distribution Analysis on page 8 85 Distributions and Moments on page 8 72 Dn dc from Peak on page 8 74 Dn dc from RI Data on page 8 73 Extinction from UV Data on page 8 75 Mark Houwink Sakurada on page 8 83 Mass and Radius from LS Data on page 8 59 Mass from Column Calibration on page 8 94 Mass from VS Data on page 8 69 Number from LS Data on page 8 69 Online A2 on page 8 65 Parametric Plot on page 8 83 Peak Areas on page 8 83 Protein Conjugate Analysis on page 8 88 Radius from LS Data on page 8 71 Regularization on page 8 90 Rh from QELS Data on page 8 77 Rh from VS Data on page 8 80 RI Calibration from Peak on page 8 75 UV Extinction from Peak on page 8 77 UV Extinction from RI on page 8 77 Mass and Radius from LS Data M1000 Rev H This procedure calculates the molar mass and RMS radius of th
178. cfm and follow the links to Data Analysis gt Band Broadening correction How to Determine Band Broadening To calculate appropriate band broadening terms follow these steps M1000 Rev H Configuration Procedures 1 Open the Band Broadening procedure view If an experiment has already been run you can open this view by choosing Experiment Configuration Band Broadening The procedure view looks similar to the following WW band broadening example bsa Procedures Band Broadening SE Determine Band Broadening M1000 Rev H relative scale Iw L jw UN M AR 8 5 9 0 volume mL Reference Instrument Optilab DSP Perform Fit El Instrument Details Instrument Generic UV instrument DAWN EOS Enabled lv lv Delay mL 0 1916 0 1577 Instrumental Term uL 1 0000 1 0000 Mixing Term ut 54 4513 52 0325 Reset BY OK 2 4 X Cancel EH Apply For the Reference Instrument property select the instrument that has the broadest signal Typically this is the last instrument in the flow path However if there is a viscometer in the flow path choose the viscometer as the Reference Instrument The inherent instrumen tal broadening in the viscometer is usually greater than the broaden ing resulting from flow between instruments Drag a peak range on the graph to create a range marker to be used to determine the band broadening parameters It is important that you select a monodisperse peak Be
179. ch data collection can be triggered You can set the following properties for an injector Table 7 13 Injector Profile Fields Field Description Name Name of the instrument If you have already created a system profile for this instru ment click and select a profile to use Description Description of the injector which typically contains more information than the Name Injected The volume of the solution injected in milliliters This is the same as the sample loop Volume mL volume An injector configuration always has a sample configuration associated with it in a profile See Sample Profiles on page 7 30 If the injector provides an auto inject signal you should also have an auto injector connection in your configuration as described in Autoinjector Connection Profiles on page 7 29 Pump Profiles Pumps move the mobile phase or solvent through the experimental appa ratus Therefore a pump has a solvent profile associated with it When setting up a pump profile you select an available solvent profile to associ ate with the pump ASTRA supports profiles for generic third party pumps You can set the following properties for a generic pump Table 7 14 Generic Pump Profile Fields Field Description Name Name of the pump If you have already created a system profile for this instrument click and select a profile to use Description Description of the
180. cible with the solvent it replaces anytime the Optilab rEX is flushed with a new solvent Collecting Data for Optilab rEX aRI Calibration 1 The Optilab rEX should be purged flow liquid with the Purge on with high purity water for several minutes before beginning the aRI cali bration procedure Confirm that the temperature is stable at 25 C and operate the Optilab rEX with the Purge ON for all data collection the Purge indicator will be yellow on the Main tab 2 On the Optilab rEX front panel navigate to the System tab and open the Constants dialog Record the values listed for dRI calibration con stant dRI offset aRI calibration constant and aRI offset 8 27 Chapter 8 Editing Procedures 8 28 3 Collect data using the ASTRA V software As of the time of publication of this user s guide the ASTRA collection procedure is as follows refer to the current ASTRA V User s Guide for more information a Start the ASTRA V software and select File gt New gt Experiment from Template b In the dialog that opens select the System Templates folder then the RI Measurement folder then the Optilab rEX Specific folder and finally the absolute RI calibration template c Expand the Configuration node and verify that the proper exper imental parameters have been entered d Expand the Procedures node of the template Click on the Basic collection procedure and enter appropriate values for each of the fields Close the pane
181. cie ieee ees B 9 Paneles AL EE B 9 Number Density AT 0 cesccccceeeeccceeeeeeeeececeeeeeecceeeeeaeeeceseneeaeeeeeeenneeeeeesnsneeeeeenniaaees B 9 M1000 Rev H Contents PMG aSUre Me see tesa aa dae Meshes Naaa de Eara Taa rE E LERIN ERSA tAn S EEPE SES B 11 100 Mass Recovery Methods B 11 Optilab rEX Specific enee eebe eebe Eech Eet B 11 RE Ee EEN B 12 Conventional Calibration CT B 12 Rl Galibration Le EE B 13 Determine CC Column Profile CT B 13 Batch Determine dn dc CT u ececeeeseceeeeeceeeeeeeeeeeeeeaeeeeceneeesaeeesenaeeseeeeseaaeeneneees B 13 Copolymer Analysis AT ccceeseceeeeeeeeeeeeeceaeeeseaeeeceaeeeesaaeeseaeeessaaeeseeeeessnaeeneneees B 14 RiPeak Areas Al oieee nene n E E Otek tte NE EE AE eae iets B 14 Kleeder deg clade a E B 15 100 Mass Recovery Methods B 15 ViISCOMEtrY eegene ee dE eeh B 16 Galibratio nics ct y0 y2 ait a de Sa a eee a i B 16 With Light Scatter Ng gege eege LEE edeE E SE eelere B 16 RI et Le B 16 lte nish eevee nei A hed ee eed daa eae tee ein alate Aah a B 17 Copolymer Analysis AT c ccccesscceceeeeeeeeeeeeceaeeeeeaeeeceaeeeesaaeeseaeeessaaeeeeeeeessaeenennees B 17 le ET rette dee ceeestes EA di decslurerba ELE EE B 17 Branching from VS Data AT eeccceceeeeceeeeeeeeeee cence eeceaeeeeeeeeeseaeeeseaaeeseeeeseaeeneeaees B 17 Mass from VS Data AT scsisscnnine nia aiai dene dee B 17 Universal Calibration CT sessssssssisesrssssrnssrsrserrss
182. ck in the right column next to a module you want to activate Options for which no activation key has been entered are blank Type the registration code exactly as it is printed on your certificate Click Close after you have entered all keys Close ASTRA If you selected ASTRA V w Security Pack go to Setting Up User Accounts on page 2 6 for instructions on creating user accounts Restart ASTRA When ASTRA is installed the entire program and all modules are placed on the computer You enter a key to unlock optional modules Certain modules can be used for a 30 day demo period After 30 days have passed you can purchase an activation key to use these modules Most users receive an activation key for ASTRA and additional modules when purchasing the software If you want to add other modules contact Wyatt Technology Corporation to purchase an activation key for that module 2 5 Chapter 2 Installing and Setting Up ASTRA Setting Up User Accounts Security Security As part of the 21 CFR Part 11 compliance of this software ASTRA V with Security Pack requires that all users log in with a unique user id and password User accounts in ASTRA V with Security Pack are managed as Microsoft Windows user accounts You create the following four Windows groups and then assign each user that should be able to access ASTRA to one of the following groups ASTRA Administrator Can change database settings and can create modif
183. cndvessvadawevtesdasissneeveaavenaeveavecscaiaavivereenias 8 72 Dide from RI Datta a ae A a aasad 8 73 Dri de trom GT EE 8 74 RI Calibration from Peak 8 75 Extinction from UV Data 8 75 UNV EXtinctlon fron PG AK EE EE E E E A E genase asevaapadbabiaousdaeniadvaaedaca 8 77 UV Extinction Om WEE 8 77 Ri frOM QELS Datel EE 8 77 RA from VS Date EE 8 80 e ell WEE 8 80 Parametric PlOt EEEE EE cacaba sah sateadihio4aah dguadtanceavaajaacabtiadiseesaiadiagsasuneetees 8 83 PO AK AGA ege ee KEE ee Ps aia bh sae See ed 8 83 Mark Houwipnk Gakurada ccccccccccececcececceecececaeaecaeaueuecaseseeseeeeececeeceeeeeeeeeueueananaea 8 83 DIStTIHUNOMANAN EEN 8 85 Copolymer Analysis c cccccceececeeceneeeeeeeceeeeeeeaaeeseeeeesaaeeseeeeesaaeeeseneeessaeeeseaeees 8 86 Protein Conjugate Analysis cccccceeesceeeeeeeeeeeeeeeeeeeeeaaeeeceeeeeseaeeeeeeeeeeeaeeeseaeeeee 8 88 ee UE 8 90 Mass from Column Calibration ccccccccccccssssessscsseeeeeceeseeeceeeeeeecececeeeeeeaeaesaaaeneaes 8 94 lU CEET 8 98 Administration Procedures cccccesseceecececeececesseeseceuseeeetsueeseetseneaeeseeeaaes 8 100 SIGMOTl ebe EE 8 100 Chapter 9 Using Sample Sets ccccccccsseessssesssseeeeeeeees 9 1 ADOUES le 9 2 Creating New Sample Sets AEN 9 3 Creating Blank Sample Sets 0 0 eee eeee cere eee eeeeeeeeeeeaeseeaeeeaeessaesseesnaeeeaeeesas 9 3 Creating a Sample Set from a Template cccccceeeeeeeeeeeeeeeeeeee
184. collecting data Once delay volumes between instruments have been determined ASTRA subtracts them from each instrument to correct for the time it takes fluid to propagate between instruments When to Determine the Delay This procedure only needs to be performed once when you connect the instruments or change the tubing between the instruments The volume delay will remain the same until you change the length of tubing between the instruments or change the instrument sequence How to Determine the Delay To determine the delay volumes follow these steps 1 Prepare a monodisperse sample for data collection A monodisperse sample is necessary for determining the volume delay between the DAWN and other instruments It should have a polydis persity of less than 1 05 A suitable sample for alignment may be a narrow polystyrene polyethylene oxide or a non aggregated protein Broad standards will not have peaks overlaid even when the system is properly aligned This is perfectly normal and due to the different characteristics of molar mass detectors and concentration detectors Use the Band Broadening on page 8 14 procedure to correct for this 2 Create an experiment that includes the Interdetector Delay procedure For best results set the collection interval to 0 125 second 8 17 Chapter 8 Editing Procedures 3 The dialog for this procedure looks as follows E vs Is ri online ps standards in thf Procedures nter DER
185. ctive index is very similar to that of the flow cell windows 9 Inject the solvent into the flow cell using a syringe pump The solvent must be pure and free of particulates we recommend that you use a 0 02 um syringe filter attached to the syringe 10 Wait until solvent is flowing through the cell and the front panel dis play for the 90 detector detector 11 on most DAWNs or detector 5 on a DAWN 8 is stable The variation in the signal should be 5 mV or less 11 Choose Experiment Run to begin running the calibration experi ment The calibration constant is calculated and written to the DAWN instrument configuration property dialog and the final calibration report 12 Use the calibration constant in other experiment configurations in either of the following ways Type the calibration constant in the property dialog for the DAWN instrument in other experiments Export the calibrated DAWN profile to the system profiles then import the profile whenever you create a new experiment See Chapter 12 Working with System Profiles for details The accuracy of this constant may be improved by repeating the measure ment a few times and averaging the results DAWN DSP and DAWN DSP F ASTRA prompts the user to turn the laser on and off at the appropriate times 8 20 M1000 Rev H Configuration Procedures Setting Calibration Properties The values for this procedure are set in the calibration template Typi cally you
186. ctor17 Detector6 f Detector 12 Bi Detector 18 Properties are stored in the Windows registry of the computer to which the instrument is connected The ISI running on that computer gets the infor mation from the registry To set one or more properties follow these steps 1 Select an instrument from the drop down Instruments list If you add computers using ASTRA s Instrument list or you connect additional instruments after opening the Diagnostic Manager click Refresh to update the drop down list 2 In the Maintain data in strip chart area set fields as follows e Minutes Select the number of minutes for which you want data to be displayed in the graphical data page for each strip chart graph M1000 Rev H M1000 Rev H Using the Diagnostic Manager For all instruments Put a checkmark in this box if you want the number of minutes to apply to all instruments accessible through the Diagnostic Manager This setting affects only your view of the data this setting does not affect users on other computers Display data in bouncing line chart as normalized format This checkmark is used for Wyatt light scattering instruments only If you put a checkmark in this box the normalization coeffi cients shown here are applied in the Data graphical tab see page 5 5 when live data is selected as the data type Live data displays the detector voltages as a function of detector number Applying the normalization
187. cumulative and differential molar mass and radius distributions as well as the moments mass x length and averages for various quantities for each peak This procedure is normally used with online fractionated experiments It is used by the Distribution Analysis on page 8 85 You can place this procedure with other analysis procedures and after all the transformation procedures This procedure is hidden in Run mode It is performed automatically as part of certain procedures To see this procedure enable Experiment Builder mode by choosing System Preferences Experiment Builder Mode The procedure has the following dialog E Experiment4 ProceduresWetermine distributions and moments SEE Value Data Analysis Range EERE BY OK 8 72 X Cancel Si Apply M1000 Rev H Analysis Procedures The properties for this procedure are as follows Table 8 24 Distributions and Moments Properties Field Description Data Analysis Range Fine tunes the amount of data used in the distribution and moments calculation For large amounts of data the calculations can take a long time to perform con sequently the range is set to 99 75 by default If you wish you can set it to 100 to use the full range of data in the analysis For more about distributions go to http www wyatt com solutions soft ware ASTRA cfm and follow the links to Data Analysis gt Distributions Dn dc from RI Data This
188. d temperature heated or cooled Temperature Control gt Temperature If this instrument is temperature controlled specify the temperature to which it is set Use C Batch Mode Band Broadening gt Enable Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broad ening parameters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term See Band Broadening on page 8 14 for an explanation of the instrumen tal term The units are in microliters Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters 7 24 M1000 Rev H Injector Profiles Injector Profiles An injector consists of an injection loop that injects the sample into the flowing solvent or mobile phase stream from the pump ASTRA supports both manual and autoinjectors each of which may provide an auto inject signal from whi
189. d the concentration needs to be measured via an RI or UV instrument Flow mode is also called flow or fractionated Light scattering ultra violet RI and viscometry instruments can all be run in either batch or flow mode M1000 Rev H Getting More Help Getting More Help If you have a question about ASTRA first look in this manual or consult the online help You can also find late breaking updates and technical information about your version of ASTRA in the readme file Also be sure to register for and use your Wyatt Technology Support Center account Go to www wyatt com to log in You ll find FAQs tutori als software downloads newsletters and ways to order supplies If you still cannot find an answer please contact Wyatt Technology Tech nical Support Contacting Technical Support Please be prepared to provide the following information when you contact technical support If you e mail or fax your question in to us include all of the following information e Wyatt Technology instrument serial numbers located on the back panel e ASTRA software version number The software version number is located on the original distribution CD or you can view it by selecting About from the Help menu The version of ASTRA used to collect the data is included in all reports e The type of computer hardware you are using e Microsoft Windows version number e Exact wording of any messages that appear on your computer
190. data set The sets listed have data for multiple detectors Only data sets that can be plotted in three dimensions are listed here Click OK Both a data set definition and the 3D graph are created Double click on the new graph listed in the Results folder for this experiment in your workspace It may take some time to load graphs of large amounts of data W protein conjugate membrane protein Results Graph Instrument D DBR Instrument Data Value Instrument Data Data Set Definition Instrument Data BY OK St Cancel 11 12 M1000 Rev H Viewing and Modifying Graphs Viewing and Modifying Graphs The graphs you generate can be manipulated in a number of ways You can see the coordinates for your current mouse location in a graph by holding down the Shift key The coordinates are shown in the status bar If the Y axis has more than one scale the left axis is shown as Y L and the right axis is shown as Y R x 10 585 YL 1 491 Y R 9 124e 4 Customizing Line Colors and Widths M1000 Rev H To change the line colors and widths in a graph either double click the graph or right click and choose Edit This opens the Edit Graph dialog Edit Graph Graph OK Marker Size Very small Line Point Size ooo Cancel Copy Series Export peak number 1 e Espot Color Black D Marker Type E dee Advanced The Marker Size and Line Point Size fields let yo
191. dden on a sample by sample basis in the Samples tab For infor mation on choosing experiment templates see Creating Experiments from Templates on page 6 5 For information on creating experiment templates see Creating a Template on page 6 18 Number of Samples 9 6 The number of samples in the sample set This can be changed in the Sam ples view after the initial configuration M1000 Rev H Samples Tab Editing a Sample Set The Samples tab of the Sample Set property dialog looks like this WW Sample Sets Sample Set 1 Enable Hame Description Template Duration Delay Iv Iv BSA conc 1 1 My Templates aitests re 5 00 0 00 second BSA conc 2 1 My Templates aitests re 5 00 0 00 BSA conc 3 3 My Templates ai tests re 5 00 0 00 2 Configuration Samples Collection BY OK X Cancel 3 Apply While a sample set is being run you can change the values for a sample that has not yet been run Once a particular sample has been run its fields become noneditable If you right click on a sample row the pop up menu allows you to add delete and reorder the samples before they are run These operations are all available even while the sample set is being run Note that you must select the Apply button to apply any changes you make to the sample set while it is running You can set the following properties for a sample Table 9 2 Sample Properties Field Seq
192. defined peaks If you use the peak_areas html report template your report will contain the values cal culated by this procedure These values are peak area peak area and retention time You can place this procedure after the Peaks procedure There are no properties to set for this procedure It runs without prompt ing for any values Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode Mark Houwink Sakurada M1000 Rev H This procedure calculates the Mark Houwink Sakurada K and a fit parameters using viscosity data If you use one of the Mark Houwink Sakurada report templates your report will contain the values calculated by this procedure For an example experiment that uses the Mark Houwink Sakurada proce dure choose File gt Open Experiment or File gt Import gt Experiment if you are using ASTRA V with Research Database and open the Mark Houwink Sakurada plot PS706 vaf experiment in the Sample Data gt Analyzed Experiments folder For an experiment template choose File gt New Experiment From Template to open the online template in the System Templates gt Viscometry gt With Light Scattering folder 8 83 Chapter 8 Editing Procedures You can place this procedure at the end of the procedure sequence Th
193. destination experiments The source experi ment contains the data you want to copy You will copy the data to the destination 2 Activate the destination experiment by clicking on any part of the experiment in the workspace 3 Choose Experiment Copy From The Copy From dialog appears Destination Close branching tests B_PMMA adf Import Experiments L_PMMA linear file branching tests proteins adf Experiments 4 Select the source experiment you want to use and click Import A Data Set Definition dialog for the source experiment is shown 5 Select the data you want to copy into the destination experiment See Creating Data Set Definitions on page 11 8 for information on using this dialog 6 Click OK in the Data Set Definition dialog 7 Repeat steps 4 through 6 for any additional data you want to copy 8 When finished click Close in the Copy From dialog After you have copied data you can access it using a standard data set def inition in the source experiment The data set definition allows you to display the data in graphs In addition the data is available for proce dures such as branching M1000 Rev H 6 21 Chapter 6 Creating amp Running Experiments Applying a Template 6 22 You can apply the procedures and result formats from a template to an experiment you have already run to collect data This allows you to perform multiple procedure sequences on the same set of raw data
194. dit View Experiment System Window Help ID a Swe 4B SZ Ki gt validate Y Run aI 3 Experiments E System Profiles Define peaks detector 2 Experiments w E experiment 1 W experiment 472x10 Geer S Configuration Profile O z i 372x10 2 i detector 4 DAWN DSP DAWN DSP Processing E detector 5 2k Solvent toluene i Procedures Processing tim 2 72 x10 E experiment 1 Experiment Configurat DAR Basic collection Pacific St AN Collection time Q Graph Injection 1 Pacific Ste Despiking Procedure Fit method mo EZE Che RS Name DAWN DSP k 722x10 Description Imported from ASTRA 4 EI Smoothing dnide 0 110 EI Define baselines Concentration Physical Instrument E Define peaks Angle fit degre oe Sample Cell K5 D Convert to physical units Percent to kee Wavelength nm 632 8 EI Determine AZ mass andr Detectors used a Peaks used 1 Calibration Constant 1 9080e 005 J Data Set Definitions d zati A ee Peak Be ai oK K Cancel Normalization Coefficients CH Results 1 9 E Report summary 2 1F Comet Cell Cleaner IT E Report detailed Batch Mode lv j gt Si Ok amp Cancel B5 Apply Divide by Laser Monitor Iw detector voltage V 3 administrator Figure 1 1 ASTRA Environment How is ASTRA V Different from Earlier Versions If you created files with ASTRA version 4 70 and higher this version can imp
195. dom Coil fit method D 14 Rayleigh ratio equations for D 3 M1000 Rev H Index measurement and calibration of D 4 summary of D 2 uncertainties in calculated quantities D 17 Zimm fit method D 13 linearity of result calculating 8 73 8 75 Log command Manage menu 4 7 Log command Sample Set menu 4 8 9 10 logging in 2 9 3 2 logs see experiment logs system log long chain branching D 22 LS Calibration procedure 8 19 LS data determining A2 mass and radius from 8 63 8 65 determining mass and radius from 8 59 determining number of particles and rms radius for 8 69 determining rms radius from 8 71 fit methods 8 62 fit models 8 55 see also light scattering entries Manage menu see specific menu com mands manual injectors 7 25 Mark Houwink Sakurada coefficients 7 26 calculating 8 83 column calibration 8 37 intrinsic viscosity 8 69 Mark Houwink Sakurada procedure 8 83 mass calculating from viscosity 8 69 mass see A2 mass eluted mass molar mass Mass and Radius from LS Data procedure 8 59 Mass from Column Calibration procedure 8 94 Mass from VS data procedure 8 69 mdf file extension 6 8 6 9 menu bar repositioning 3 14 menu commands assigning keyboard shortcuts to 3 14 see also specific menu commands menus instruction conventions 1 5 list of A 2 see also specific menus Index 7 Index metafiles export graphs to 11 17 Microsoft Access database 4 4 Micros
196. e samples Instead you can add one more sample to the sample set and use one of the System Templates gt Light Scattering gt Utilities gt turn laser off templates as the last experiment template in the sample set Seq Well Enable Name Description Inj Template EI i fo lv 1 My Templates collection only ose 2 0 wi 1 My Terplates collection only ser 3 0 v 1 fix Termplates collection only ah 4 UI lv 1 Div Templates collection only BE s fo E 1 fix Templates collection only el 6 0 Iw 1 tv Termplates collection only CH 7 0 v 1 Div Templates collection only X 8 0 v 1 System Templates Light Scattering UtilBes turn laser off EA The order of precedence for values such as dn dc A2 UV extinction con centration and so on is as follows 1 Peak values defined in the specified experiments are used if they exist 2 Values from the Samples tab are used next 3 The sample profile is used if no other values are set Collection Tab 9 8 The Collection tab of the Sample Set property dialog allows you to view data as it is collected during a sample set run While a sample set is running the collection data is displayed for the current sample If an autoinject signal is expected to trigger the injection this view displays a message indicating the sate of the collection M1000 Rev H Running Sample Sets Running Sample Sets Sample sets act as sets of experiments that can be run as a group Rather than run
197. e Instrument Specific Calibration Constant ISCC value 1 V cm Light scattering instruments use the ISCC in the computation of the Config uration Specific Calibration Constant CSCC See LS Calibration on page 8 19 for a way to determine this value Type the normalization coefficients for the detectors or use the normaliza tion procedure see page 8 32 to set these values Detector 11 always has a normalization coefficient of 1 This is Detector 5 on a HELEOS 8 Normalization is the process by which each detector signal is related to the 90 detector signal and the Instrument Specific Calibration Constant Click the Import button to import normalization coefficients from an open experiment Comet Cell Cleaner Check this box if a COMET cell cleaner is to be used with the DAWN instrument Please see the COMET hardware manual for more information about the COMET cell cleaner Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instru ment configuration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 M1000 Rev H 7 11 Chapter 7 Configuring Experiments Table 7 2 DAWN HELEOS Properties Field Description Divide by Laser Monitor Select the laser monitor option you want to use The options are Laser Monitor
198. e Shows the state of the avalanche photo diode APD in the WyattQELS detec tor If the QELS count rate exceeded the APD protection threshold it is auto matically turned off This field displays the state of the APD for the correlation function If the APD was off the correlation function is probably not valid and is by default excluded from the analysis This field is display only Min Rh Threshold Fitted Rh values with a lower radius than the value you type are not used in the analysis Max Rh Threshold Processing Parameters Fitted Rh values with a higher radius than the value you type are not used in the analysis gt Min Fit Delay Time gt Max Fit Delay Time Data with a lower delay time than the value you type is not used in the fit to the correlation function Data with a higher delay time than the value you type is not used in the fit to the correlation function gt Use Disabled Slices Check this box if you want to include disabled slices that is slices for which the APD was turned off in the calculation gt Show Residuals gt ACF Display Binning Check this box if you want to show residuals from the fit to the QELS correlation function Adjust the number of auto correlation function bins to display By default data is shown as displayed by the correlator board If you choose heavy a larger number of points is shown which smooths the signal M1000 Rev H 8 79
199. e a fit degree to use for fitting the data The value may be either 1 or 2 Percentage to Keep Type the percentage of the peaks to use for calculations By default 100 is used If the plateau is flat not drifting in the peak range using the default value is recommended Linearity View Put a checkmark in this box to view the linearity plot Enabled Peaks gt You can omit peaks you marked from the plot by removing the checkmark next to 1ton the peak number 8 76 M1000 Rev H Analysis Procedures UV Extinction from Peak This procedure calculates the ultra violet extinction using peak data You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the UV extinction If you place multiple methods that determine UV extinction in a procedure only the first one will be valid There are no properties to set for this procedure It runs without prompt ing for any values Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode This procedure uses the injected mass value specified for the peak The procedure adjusts for viscometer dilution factor effects if the concentration detector is downstream from a viscometer UV
200. e experiment into logical units called profiles The types of profiles that are available are shown in Figure 7 1 Each instrument has a profile that contains parameters specific to that instru ment In addition connections between instruments are represented by profiles Finally elements used in the apparatus such as the solvent and sample are represented by profiles as well E Profiles Instruments Dawn 8 Dawn DSP DAWN DSP F J DAWN E0S DAWN HELEOS DAWN HELEGS 8 Generic column _ Column Profiles on page 7 26 Generic pump Pump Profiles on page 7 25 Generic RI instrument Refractive Index Instrument Profiles on page 7 17 Generic UV instrument UV Absorption Instrument Profiles on page 7 24 Genetic viscometer Viscometry Instrument Profiles on page 7 21 Injector Injector Profiles on page 7 25 miniDAwWN SH Light Scattering Instrument Profiles on page 7 11 E miniDAwWN TREOGS g Optilab 903 Sec Refractive Index Instrument Profiles on page 7 17 Optilab DSP Optilab Ex iscoStar Viscometry Instrument Profiles on page 7 21 wyatt QELS Light Scattering Instrument Profiles on page 7 11 Connections Connection Profiles on page 7 28 qf Auto inject connection AT Aux channel connection f Fluid connection Samples Sample Profiles on page 7 30 Sg Molecular standard Sample m Experiment configuration Experime
201. e graph The default is square Type a numeric value by which the x axis values should be shifted The number will be added to the existing times or volumes You can use a negative value for example if 15 mL of solvent were run through the system initially you might use an X Axis Shift of 15 You can see the effects of your changes as you make them without closing this dialog If you click Advanced you have much more control over the graph display is provided than is described in this manual For help on settings in the Advanced dialog move to a field and press F1 11 6 M1000 Rev H Using Custom Plots and Data Set Definitions Using Custom Plots and Data Set Definitions Data set definitions are used to create graphs When you create and use data set definitions you have access to more data sets stored within an experiment and have more control over which data is plotted Data Collection and Storage ASTRA experiments store a set of data for each procedure in the experi ment Each set represents the data after the procedure ran The following figure shows some of the typical data sets Config Profile b Collection Procedure Collection Script DN original gt gt gt Data Additional Transforms Baselines Procedure Analysis Procedure The data sets include the following e Original data In addition to raw detector voltages this data set typi cally includes
202. e procedure to a location in the sequence where its icon and the icons that follow it have no red X Chapter 8 Editing Procedures has details on the correct location in the sequence for each procedure and the data required for each procedure to be run Note that the standard experiment templates already contain the neces sary procedures for collecting and analyzing the data in the correct sequence 6 27 Chapter 6 Creating amp Running Experiments Adding Data Set Definitions Experiment Builder You can create data set definitions only if you enable Experiment Builder Shortcuts mode by choosing System Preferences Experiment Builder Mode You can see the Data Set Definitions folder in the workspace only if you opened the experiment while in Experiment Builder Mode Data Set Definitions make additional sets of data available for use by reports For details about creating and using data set definitions see Chapter 11 Working With Graphs To add a data set definition to an experiment follow these steps 1 Choose Experiment Add to Experiment This opens the Add to Experiment dialog Press Ctrl Shift P Right click any folder in the experiment tree and choose Manage Add To Experiment E Add to experiment LC Results B Dataset Definition Cancel ae ri 2 Select Data Set Definition and click OK The data set definition is added to the Data Set Definitions folder of the experiment tree S
203. e sample Both light scattering and concentration data are required For an online experiment either an RI or UV detector provides the concentration data For a batch measurement the concentration can be specified for the peak ranges For more about light scattering analysis of molar mass go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Molar mass via light scattering 8 59 Chapter 8 Editing Procedures The procedure has the following dialog WW dawn adf ProceduresWetermine mass and radius from LS d SEE results graph control graph 5 50 x10 545x10 4 Iw scattering angle 90 degrees 45x sansa 4 79 x10 H 535x10 3 79 x10 5 30 x10 5 25 x10 5 20 x10 545x10 510x10 5 05 x10 0 0328 0 5328 1 02 6 5995 16 5 sin theta 2 time min 279x10 1 79x10 K c R theta 7 88 x10 Rayleigh ratio 1 cm 242 x10 Molar Mass 1 978 0 001 e 5 g mol rms radius 17 2 0 1 nm Peak Humber 1 Slice Index 182 zimm Fit Degree 1 SO S Cancel The left graph shows the results in a Debye graph See About Debye Plots on page 8 61 The right graph shows the baseline and peaks for the selected detector You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the mass If you place multiple methods that determine mas
204. e sort of mass averaged result for the hydrodynamic radius The measured correlation function for a polydisperse sample actually contains more information than this and several strategies have been developed to extract more information about the underlying size distribution from the correlation function The next level of sophistication in QELS analysis for polydisperse unfrac tionated samples is the method of cumulants In a nutshell the method of cumulants involves fitting the correlation function not to a single decay time but to a Gaussian distribution of decay times The method of cumu lants retrieves the mean and variance for this distribution Theory The result of a QELS measurement is a second order correlation function Equation 1 II r g r UO where I t is the intensity of the scattered light at time t and the brackets indicate averaging over all t The correlation function depends on the delay 7 that is the amount that a duplicate intensity trace is shifted from the original before the averaging is performed A typical correlation function for a monodisperse sample is shown in Figure F 1 2 0 correlation function in in 0 0 1e 006 1e 005 1e 004 0 001 0 01 0 1 1 10 tau sec Figure F 1 Correlation function for a multi tau correlator like that in WyattQELS F 2 M1000 Rev H Cumulants As described in various light scattering texts the correlation function for a monodisperse sample can be
205. e steps 1 Open all the experiments to which you want to apply a new experi ment template 2 Choose File gt Batch Apply Template Batch Apply Template Template System Templates Light Scattering online A2 nn vs ri online bsa C N Sample Data 4nalyzed Experiments band broadening example bsa C Sample Data4nalyzed Experiments online a2 200k ps C Sample Data4nalyzed Experiments Iw Close original after apply Apply Close Auto save 3 Click and browse for the template you want to apply The proce dure and result formats in the template will be used Typically you would choose a template from the System Templates or My Templates folder 4 Check the boxes next to the open experiments to which you want to apply the selected template 5 Leave the Close original after apply box checked if you want to automatically close the old experiments when new experiments with the template applied are created 6 Check the Auto save box if you want the new experiments that will be created to be saved automatically 7 Click Apply A new experiment is created for each old experiment and is run automatically The names of the new experiments combine the names of the template used to create the experiment and the original experiment M1000 Rev H 6 23 Chapter 6 Creating amp Running Experiments Adding Elements to an Experiment This chapter provides simple examples for adding el
206. ea units depend on the units you have chosen in the configuration for the abscissa unit RESULTS Peak 1 Peak 2 Peak 3 RI Instrument RIU min Peak Area 2 943e 5 3 170e 6 4 204e 7 UV Instrument absorbance min Peak Area 1 034e 1 1 084e 2 1 212e 3 Viscometer specific viscosity min Peak Area n a n a n a DAWN or miniDAWN Rayleigh ratio min Detector 1 2 147e 1 4 485e 2 8 013e 3 2 2 139e 1 4 375e 2 7 568e 3 3 2 137e 1 4 356e 2 7 470e 3 UV Extinction from RI Peak AT First select the peak of interest You need to know and enter the dn dc value of the molecule and the calibration constants of the UV and RI The report shows the UV extinction coefficient in ml g cm calculated from the refractometer signal If you know the dn dc of the molecule of interest it is better to determine the epsilon using this template and not the 100 mass recovery template where the 100 mass recovery is hardly verified M1000 Rev H B 3 Appendix B System Templates The advantage of using this template is that there is no effect based on the flow rate the recovery etc That is you don t have to make assumptions for these parameters RESULTS Calculated UV extinction based upon concentration from known dn dc and RI detector Peak 1 UV ext mL g cm 6 467e 2 Light Scattering B 4 The following templates and folders are provided in the Light Scattering folder Baseline Subtraction The following template is provided in the L
207. earlier versions of ASTRA txt Tab delimited text file for exporting a data set defined by the selected data set definition This format is easily imported into most spreadsheets CSV Interpolated comma delimited text file for exporting a data set defined by the selected data set definition This format is easily imported into most spreadsheets Data traces are interpolated so that all measurements are on the same time scale allowing for easy display in software such as Excel 5 If you choose a txt or csv format you can also choose a data set defini tion to export with the experiment See Creating Data Set Defini tions on page 11 8 for information about data set definitions 6 Click Save If you do not find the organization of the tab delimited or comma delim ited output useful try the output described in Exporting Data on page 11 17 6 17 Chapter 6 Creating amp Running Experiments Creating a Template After you modify an experiment you may want to save it as a template for other experiments The saved template includes the configuration proce dure and result formats Security You must have at least Researcher access to save templates To save a template follow these steps 1 Choose File gt Save As Template Look in Ey Ki Ei a Import System Templates Templates My Templates Files of type mz D Cancel Ge UI Make Default 2 Navigate to the
208. eating Proniles siimus anenun on annaa eege eege eegene es 12 3 Modifying Profiles 12 5 Using Vi te UE 12 8 12 1 Chapter 12 Working with System Profiles About Profiles 12 2 In an experiment a set of instrument and connection profiles describes how an experiment is set up This is called the configuration You can also create profiles that are stored outside of experiments and can be copied into experiments as needed these are called system profiles The experiment templates provided with ASTRA contain commonly used configurations However if your instrument setup differs from the default templates you may find that you need to modify new experiments in the same way each time you create one System profiles allow you to save time by creating reusable blocks of infor mation about your instruments and experiment setup You can copy this information into new experiments Another way to save time is to save experiments as templates as described in Creating a Template on page 6 18 This chapter focuses on system profiles See Chapter 7 Configuring Experiments for information about configurations and for reference infor mation about the properties of all the available profile items that make up configurations and system profiles You can work with system profiles in the System Profiles tab of the workspace E B Experiments BB SampleSets E System Profiles lips System Profiles Sy Instruments S DAWN
209. echnical Support if you have a specific need to create additional collection scripts C 6 M1000 Rev H M1000 Rev H Light Scattering Theory This appendix reviews the application of the basic theory of laser light scattering to the determination of molar mass and root mean square radii The text covers basic quantities the relation to measurements calibration and normalization and determination of molar masses sizes and distribu tions CONTENTS PAGE Introduction ccccecesesscceececesssseceeeeceaeseseeeceaseseeeeeauaasseceeseeaaaseeseeeeaaaesees D 2 BasiG QUanttiE Sininen A Ee D 3 Measured Quantities and Calbraton D 4 NOFIMAN ZATION EE D 8 Determination of Molar Mass and Sizes sssesnnnnnrirrrrrrrrrrrrreesreeeesesns D 11 Molar Mass and RMS Radius Moments D 15 Uncertainties in Calculated Quantities ccccccccccececeeeeeeeeeeeeeeneeeeees D 17 Out of Range Values eseeeneeeseesnneeenessnnssrnsernnnrnnnttnnttnnnsnnnssrensrennnnnn D 18 Differential Distribution Calculations cccccccccccceceeceeeeesaeeeaeeeueeeneeeeees D 18 Branching Calculations ccccccceeceseeceeeeeeeeeeeeceeeesaaeeseaeeesecaeeesaeeesaes D 19 D 1 Appendix D Light Scattering Theory Introduction D 2 Although gel permeation chromatography GPC provides good separation of molecules by their hydrodynamic radius traditionally it has been neces sary to calibrate GPC columns using standard sampl
210. ect Print To copy a report to the Windows clipboard highlight text right click and choose Copy Then move to another application and paste To change the printer setup do the following 1 Choose File gt Print Setup 2 Make any changes in the Print Setup dialog and click OK To preview the report layout do the following 1 Choose File gt Print Preview 2 You can use the Print Preview window to change the print setup or to print the report Exporting a Report To export data from a report we recommend that you use the procedure described in Exporting Data on page 11 17 to export data from a graph The Experiment Report Export command exports the experiment data rather than the report data M1000 Rev H Working With Graphs This chapter describes how to create and use EASI graphs data set defini tions DSDs and DSD based graphs CONTENTS PAGE About ASTRA Grapns siiic cceit ivi atti dante tenangentiieieete anntiends 11 2 Using EASI Grapnsiss icssesccsdesctte decease served bendaadtetsaeediangessictenszacedacitenasede 11 3 Using Custom Plots and Data Set Definitions ssssssnnenenneeneeennen nenea 11 7 Viewing and Modifying Graphs ccceeeeeeeeeeeeeeeeeee see eeseeeeseeeeeeteaeetees 11 13 Printing ETC AE 11 15 Exporting lt GrapnsS ees etuateeeetrgeege de EENG tee taeda een 11 16 Chapter 11 Working With Graphs About ASTRA Graphs You can use the following ways to create gra
211. ection has ended follow these steps 1 ASTRA V may produce a message box informing you that baselines need to be set Click OK and the software will expand the Procedures section of the experiment and open the Baselines window Otherwise click the Run button to proceed M1000 Rev H fe 5 T S E 2 S T Ki gt Configuration Procedures 2 In the Define Baselines window define the baseline using the sol vent blanks that where injected before and after the standard solu tions Click and drag from one solvent plateau to the other such that a baseline is drawn under the sample solution plateaus Click OK to close the baselines window In the Define Peaks window define a peak for each of the flat plateau regions associated with each injected standard Click and drag over the maximum flat region for each sample injection Do not set peak regions for the blank injections As peak regions are defined a table beneath the Define peaks graph shows a new column associated with each respective peak In these columns under the Refractive index node enter the known concentra tion for each standard in the row titled Concentration g mL Once you have assigned all the peaks their respective concentrations click on the OK button at the bottom to close the pane Double click the RI Calibration procedure to open its property dialog E w410 rwf ProceduresWetermine RI calibration Determine RI c
212. ection until the string message is received instrument message by instrument The instrument is an instrument name retrieved using one of the Create commands described in the next section The only message cur rently available is ISI_INSTRUMENT_AUTOINJECT which indicates that an autoinject signal has been received by the specified instrument Interacting with Instruments To issue commands to an instrument you must first obtain an instrument reference that links the specific physical instrument in the experiment configuration to the instructions in the script This is done using one of the following commands Command Effect LSInstrument Create Get a reference to the experiment configuration s static light scattering instrument QELSInstrument Create Get a reference to the experiment configuration s dynamic light scattering instrument Rilnstrument Create Get a reference to the experiment configuration s refrac tive index instrument Vilnstrument Create Get a reference to the experiment configuration s vis cometer Once you have obtained a reference to one or more instruments you can issue commands to the instrument Instruments respond to a general set of messages as well as some instrument specific commands M1000 Rev H C 3 Appendix C Data Collection with Scripts Common Instrument Commands All instruments understand the following messages Co
213. ection without any sample ASTRA collects data about changes in baseline conditions caused by the collection environment This collec tion should have the same characteristics duration solvent instru ments etc as the sample runs and should be subject to the same conditions temperature control use of Eclipse cross flow levels etc For example this blank run shows a fluctuating dRI baseline blue strip chart 20070926__213 20070926 LS3 LS4 j LS5 2 0x10 6 LS6 LS7 LS8 5i LS9 0 020 apis SH o o tsi 6 L812 1 0x10 Q S LS13 S S LS14 z 3 LS15 S 2 LS16 0 0154 3 LS17 E S 1818 2 Lon i 3 In UW S FM S jg aR g HOER L 1 0x10 6 un 100 4 200 42 300 44400 500 time min M1000 Rev H 8 47 Chapter 8 Editing Procedures 2 Run a data collection for the samples strip chart 20071002__236 20071002 es E J T LS4 0 035 3 es H 5 hi 1 1 0x10 LS6 h LS7 D Lea 0 030 Lea e Lem o Lsi LS 12 0 025 e S L813 S it s LS14 D J H 5 3 LS15 S IA d i Fi Oog nung LS16 S sei 8 i eet Le A SS gt 5 te 8 LS18 S e ZS In UW 3 A DM Se Ss 0 0154 S o dn lel 7 0 010 i R H Ji 0 005 0 0 10 0 20 0 30 0 40 0 50 0 time min 3 Keep both experiments open in ASTRA 4 Right click on the sample experiment and select the Manage gt
214. ectors equal to the result of the integration for the current detector divided by the result for the 90 detector This forces the 90 detector to equal 1 0 D 10 M1000 Rev H Determination of Molar Mass and Sizes Determination of Molar Mass and Sizes We begin by reviewing briefly the relationship between the data collected by the DAWN system and the molecular parameters derived from them We start with Eq 13 from Zimm LI Chem Phys 16 1093 1099 1948 Equation 13 Ro mp 0 24 cM P 0 K c where e cis the mass concentration of the solute molecules in the solvent g mL e Mis the weight average molar mass g mol e Apis the second virial coefficient mol mL eh e E dl ne dn dc Agt N5 is an optical constant where ng is the refractive index of the solvent at the incident radiation vacuum wavelength J is the incident radiation vacuum wavelength expressed in nanometers N4 is Avogadro s number equal to 6 022 x 1023 molt and dn dc is the differential refractive index increment of the solvent solute solution with respect to a change in solute concen tration expressed in mL g this factor must be measured independently using a dRI detector e P is the theoretically derived form factor approximately equal to 1 2p r 314 where w 47 A sin 2 and lt r gt is the mean square radius P is a function of the molecules Z average size shape and structure e Rg is the excess Rayleigh
215. ed by ASTRA to convert the voltage output of the dRI detector into changes in refractive index units when reading the dRI instrument signal through the AUX input of another instrument The Wyatt Optilab rEX is intended to communicate digitally with ASTRA in refractive index units As such the Optilab rEX dRI calibration constant is an internal constant stored on board the Optilab rEX itself When to Calibrate dRI instruments manufactured by Wyatt Technology including the Optilab rEX and the Optilab DSP come pre calibrated 8 21 Chapter 8 Editing Procedures Note 8 22 Other dRI instrument manufacturers might or might not supply an approximate dRI calibration constant ASTRA needs an accurate dRI cali bration constant since any error in the dRI calibration constant is directly proportional to the error in molar mass The calibration constant can vary 10 15 from the manufacturer s approximate value so calibration of any third party dRI instrument is strongly recommend prior to use In all cases dRI measurement performance should be checked regularly annually against a standard with a known dn dc value Changing solvents does not affect the dRI calibration constant If the calculated mass for a known sample changes over time consider calibrating the dRI as part of your troubleshooting effort For use with certain chromatography systems and the ASTRA V software dRI calibration may be performed by injecting a suitable stand
216. ed in this manual 1 5 conversion procedures Convert RI to Concentration proce dure 8 56 Convert specific to intrinsic viscosity 8 57 Convert to Physical Units procedure 8 56 Convert UV to Concentration proce M1000 Rev H dure 8 56 data produced by 11 7 Convert RI to Concentration procedure 8 56 Convert specific to intrinsic viscosity proce dure 8 57 Convert to Physical Units procedure 8 56 Convert UV to Concentration procedure 8 56 coordinates 11 13 Copolymer analysis procedure 8 86 Copy From command Experiment menu 6 21 Copy From dialog 6 21 copy icon in log dialog 4 8 Correlation function plot 8 93 crash recovery file 6 8 6 9 6 14 csv file extension 6 17 Ctrl key zoom 8 5 Ctrl Alt N keyboard shortcut 12 3 12 5 Ctrl Alt T keyboard shortcut 3 5 6 5 Ctrl I keyboard shortcut 6 9 Ctrl N keyboard shortcut 6 6 Ctrl O keyboard shortcut 6 7 6 8 Ctrl P keyboard shortcut 3 15 Ctrl S keyboard shortcut 6 15 6 16 9 10 Ctrl Shift N keyboard shortcut 9 3 Ctrl Shift O keyboard shortcut 9 4 Ctrl Shift P keyboard shortcut 6 26 6 28 6 29 8 7 10 4 11 8 11 11 Ctrl Shift R keyboard shortcut 3 9 6 12 Ctrl Shift S keyboard shortcut 6 13 Ctrl Shift V keyboard shortcut 6 11 cumulants analysis 8 98 curve fitting model 8 57 Customize command View menu 3 14 D data after conversion 11 7 analyzed 11 7 copying to another experiment 6 21 exporting to file 11 18 monitoring while receiv
217. ediately It is recommended that you inject six concentrations of the sample For this template you need to manually enter the concentration of each injected sample Branching AT The branching characteristics of a polymer can only be determined if MALLS Multi Angle Laser Light Scattering data of a linear example of the polymer exists 2 Branching ratio g g ag R root mean square radius Branching parameter KR radius of gyration in JM M1000 Rev H B 7 Appendix B System Templates The Branching procedure shows the branched conformation plot and the slope of the linear and branched polymer branched conformation plat iV branched conformation plot F branched conformation plot ft F linear conformation piot NW r conformation phot tt fies radius aim o Dei anyine et gt pe ENNS Foe Ep pen 20x10 30x10 40x10 5 0x10 6 0x10 70x10 8 0x10 molar mass Ia melt Linear Conformation Slope Branched Conformation Slope 045 0 00 Use Linear Model Imported Lincas Daa EADewelopmet ASTR A_S 3 2 2etatsrcke Dstsbrachke Experimentsiiinear s Plot conformation plot 7 Modet Uifunctional xl Sike Type monodsosse z Repeat Unit Molar Masa grrot 100 002 v The branching ratio is displayed in the report Online CT Use this template to run basic online light scattering experiments With this template the sample is injected online using a concentration detector
218. edures see Chapter 8 Editing Procedures e Results The reports and graphs to be produced after the experiment procedure has been run For details see Chapter 10 Working With Reports 3 6 M1000 Rev H Performing a Simple Light Scattering Experiment Modifying the Configuration The experiment template sets most of the properties to values you are likely to use There are just a few properties you typically need to set QELS For the example LS Batch experiment follow these steps 1 Hame Choose Experiment Configuration Edit This opens the proper ties dialog for the configuration You can also open this dialog by dou ble clicking on any part of the Configuration tree in the Experiments tab Notice that this dialog has a tab along the bottom for each item in the configuration Select the tab for the DAWN or miniDAWN instrument Use the l4 LD ri arrows to the left of the tabs to scroll to the right until you find the tab for the light scattering instrument Value HELEOS ISS Description Physical Instrument wyatt sFt rx0 Optilab rEX Sample Cell wyatt sft rx0 Optilab EN Wavelength nm Calibration Constant 1 0000e 004 Normalization Coefficients Import Comet Cell Cleaner E Batch Mode lv Divide by Laser Monitor Laser Monitor Disable Collection E Polarization Analyzer E 4 4 gt gt Experiment configuration LS batch DAWN HELEOS HELEOS Solventi BY OK Es Cance
219. ee a message that says a baseline needs to be set Set a baseline by following these steps a Click OK to open the dialog for setting baselines W gels Is batch bsa insulin Procedures Baselines Define Baselines o gt S gt e _ S 2 S K 40 6 0 time min na Auto Baseline Source LS 11 Auto Baseline Visible Style wO X Cancel SI Apply b If you like you can select a different detector s data to view M1000 Rev H 3 9 Chapter 3 Getting Started c Use your mouse to click on the baseline of the graph at one location and drag to another location on the baseline By default baseline ends snap to the voltage level for a particular time If you hold down the Shift key you can then drag the end of a baseline to any location d Scroll down to the bottom of the property list In the Auto Baseline row select the detector for which you set the baseline For example detector 11 f Click Auto Baseline to automatically set baselines at the same collection times for all detectors g Click OK to continue running the experiment 4 You next see a message that says peaks need to be specified Set peaks by following these steps a Click OK to open the dialog for setting peaks WW gels Is batch bsa insulin Procedures Peaks Define Peaks _ M QELS r relative scale insulin Start min 7 7666 Stop min 8 8000 LS Analysis dn de mt o 0 1850
220. eeeeeeeeeeeseeeeeeeeeees 9 3 Opening a Sample Set saiia CERN elek EK 9 4 Importing an ASTRA Sample Set eeeseeseeeeeesensssenssrnnenreenenninsrnnnsrnssrnnssrnnssrnssnn 9 4 Importing an Empower Sample Set 0 0 ceceseee esses eeneeeeaeeeneecsaeesaeeseeeseeseeeee 9 5 M1000 Rev H Contents Editing a Sample Dele h aaa ieee eeedae hc kira ctces esis ood iat athe hoe tec 9 5 Config ration NET WEE 9 6 ue 9 7 GolleCtiOn Tab ET 9 8 Edi elle E EE 9 9 VEHRE Mie E RETTEN 9 9 Running a Sample Set cccscccceceseeeeeeeeeeeeaeeeeeneeeceaeeseeaaeeeeeeeeseaaeseeeeeeeecaeeeeeaeeeeeas 9 9 Stopping a Sample Set EE 9 10 Viewing a Sample Set Log ccccececeeeeseeceeeeeeceaeeeeeeeeeeaaeeeeeeesaaeseeaeeeeeceeeenaeeeeaes 9 10 e ne Bun 9 10 Saving Sample Sets as Templates cc cccccceeeeeeeeececeeeeeeaeesecaeeseaeeseeeeeessaeeseenes 9 11 Exporting Sample Get niece cee ESA gue 9 11 Deleting a Sample Set gek ceeeetiecid eetetentebeesiaeentagaeiaenaieceteeeendenindeendes 9 11 Chapter 10 Working With Reports eeseeeeeeseeees 10 1 Ab u ut EE 10 2 Operator Names in Reports cc ccccceceesececeeeeeeeeaeeeceaaeeeeaaeeceaeeeseaaeeeeeeessaeeesenees 10 2 Changing a Report un E EEN 10 3 Customizing Report Formats ccccccccceeesececeeeeeeeeeeceeeeeesaeeseeeeeesaeeeseeeeeetsaeeseenees 10 3 Adding a TEE 10 4 Applying Report lu EE 10 5 Printing asREPOM EE 10 6 Exportingia REPO EE 10 6 Chapter
221. eeeeeeeeeteeeeeeesaaeeeeaes 7 7 Exporting a System Profile cccccccsecceeeeeeeeeeeeeeeeeeceeeeeeeaeeseeeeeesaeeeseeeeseeeeeseaaeeneaes 7 8 Experiment Configuration EE 7 9 Light Scattering Instrument Profiles ereeschen ees Sea niiaiiagsa 7 11 DAWN HELEOS and DAWN HELEOS 8 Profiles 0 ccececceeeeeeeeeeneeeeeeeeeeeeeeees 7 11 DAWN EOS DAWN DSP DAWN DSP F and DAWN 8 Profiles eeeee 7 12 miniDAWN and miniDAWN TREOS Profiles 0 c ccccceeeeeeeeeeeeeeeeeeeeteeeeeeetaeeeees 7 14 WyattQELs Profiles ina accel AAA the nena ne D ck et 7 15 M1000 Rev H Contents Refractive Index Instrument Profiles AAA 7 17 OptilabrEX Profiles geet ake adeescaeeedeve fe the dee inde ddsdv aeai a en a aD eaa oaaae 7 17 Optilab DSP Profiles she asain athe Aaa Shaan 7 18 Optilab 903 Profiles rria aeeaiei aaa a a aa SE eege 7 18 Generic RI Instrument Profiles seeseeeeeeeeneeeeeseenesrerssrrssernssrrnsernsetnnnennntennnernnne 7 19 Viscometry Instrument Profiles secsiois ceca ge MEdENKEEE ENEE dvbveemeetteie taeda eens 7 21 Viscootan Gre UE 7 21 Generic Viscometer Profiles c cccccesesececeeeeeeeaeeceeeeeeeaaeeeeeeeeeeaaeesseneeessaaeeesenees 7 22 UV Absorption Instrument Profiles ise sects cteecsdeechesenes vesrdckepeseeesseredea ed 7 24 Generic UV Detector Profiles cceceeeeseeeceeeeeeeeeeeceeeeeeeeaeeseeeeeesaaeeeseneeetsaeenseaeees 7 24 UG eelere UE 7 25 FOUND Ee EE 7 25 Column Profiles ss
222. eesaessaeeseeeaaes 3 8 RUNNING He E penOepk ease ue Eden due deed SEET 3 9 Viewing Roonaan ran Aaa E EAEE EE Seege Ehe eg 3 12 SUMIMANY Ste AAR eet ates te ed bats Wien tne be ee 3 12 More About the ASTRA Environment AAA 3 13 User MOUS aaee urse u itd eceied eege et pre E a ETSA Si s ie 3 13 User Account Levels eisene egge dree r ete AA ARE AAAA AREER Ei Eii ensuite A KORAAN 3 13 Customizing the Environment cccceescceceeeeeeeeeeeceeeeeeeaeeseeeeeeeaaeeeeeneeetsaeeeeeaeees 3 14 Command Reference cccccecccececeececeeeeeeeeceaeeeeeaaeeceeeeeeeaaeeseeneeeseaaeesseeeeetaeeesenees 3 14 PIDIN aoi e NA EE E ETERRA deen ga E ENA 3 15 Getting ale EE 3 15 Exiting FOM A STRA EE 3 15 Chapter 4 ASTRA Administration cccccccessssesssseeeeeeeeees 4 1 21 CFR Part 11 Support Overview 2 5 eEEEEENNEEKNEENRNEREERAEESECEENSNEEREEENEEEAAE SEN 4 2 Background and Reasons for Compliance sssssesseesseeeeesieesiresirnssrnssrnssrnnen nnee 4 2 21 CFR Partti Support In ASTRA Voastre anean ana a aaa aaae a EES 4 3 Connectingtoa Databasen EE 4 3 Viewing the Current Experiment Database A 4 5 Creating a Microsoft Access Experiment Database A 4 5 Managing User Accounts EE 4 6 Using the System and Experiment LOgS cceecccceeeseneeeeeseeeeeeeeeeeeeeeeeeneeees 4 7 Viewing the System Log ccccccceceeeeeeeeeeeeeeeeeceaaeeeeeeeeseaaeeesaaeeseaeeesaaaeeeeeeeessaeeeseneees 4 7 Viewing an Experiment Log ae 4 7 Viewing
223. ege a a a Ra e NaS 2 7 Assigning Users to Ee ie E 2 7 Note for Networked Accounts ssesesseessssrsnessennnessnnnnesenennestnnnnnnutennenennnnnnnnnnnnnennnnnne 2 7 Setting a Validation Domain for Security eceeeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeeeesaeeeeeneeess 2 8 EE EE eee reese er ener ae ese eres meen ere eae eee eee re 2 9 Accessing and Viewing Hardware ecceeeeeeeeeeeeeeenneeeeeeeeeeeeenaaaeeeeeeeeeeenaaa 2 10 Viewing the Instrument List 20 0 0 ee ceeeee cece eee eeeeeeeeeeeeteaeeeeeeteeaeeeeeeseneeeeeeeeeeeeeeees 2 10 Adding an Instrument or Computer to the Instrument List 0 cceeeeeeeeeeeeeeeees 2 11 Removing an Instrument or Computer from the Instrument List eeeee 2 12 Chapter 3 Getting Started ccccccessssseeeeseseeeeesesessesseeeneeeeens 3 1 Staring ASTRA cncpucseecereans cet engs Ka EO a Ea aeni Eia a 3 2 ASTRA Tutorials on the Support Center ccccceeessceeeeeeseeeeeeseeeeeeeeseeeeenseaes 3 4 ASTRA V with Security Pack 1 Contents Performing a Simple Light Scattering Experiment ceecceeeeeeeeeeeeeeeeneeeeees 3 5 Checking the Instrument Connection 20 0 cece cee eeee sees eeeeeeeeaeeeaeessaesnaaesnaeeeeeesas 3 5 Creating an Experiment 0 ica Ac asin ae ai ae aa 3 5 Modifying the Configuration ccceccccesseeeeeeneeeeeeeeeeeeeeeceaeeeeeeeeecaeeeseaeeeseaeeeseaeeseaas 3 7 Modifying Procedure Settings 0 0 eee eeee creer eeeeeeeeeeeeeseeeseesseae
224. eld e Administers ASTRA accounts and database in the Description field Click Create M1000 Rev H Setting Up User Accounts Create the following additional groups in the New Group dialog and click Create after each one Make sure your use the exact capitaliza tion and spacing shown here Group name Description ASTRA Researcher Creates and modifies experiments and profiles ASTRA Technician Runs experiments and saves data ASTRA Guest Read only access to experiments and results Click Close after you have created all four groups Creating Users Security You can use existing Windows user accounts or create special accounts for ASTRA access To create a new user account follow these steps D 1 In the Computer Management window right click on Users under Local Users and Groups and select New User In the New User dialog type a User name Full name Description and Password as desired Click Create Assigning Users to Groups Security To assign user accounts to an ASTRA group follow these steps 1 In the Computer Management window right click on one of the ASTRA groups you added and select Properties Click Add In the Enter the object names to select field type a user name you want to add to this group Click OK in the Select Users dialog Click Add again if you want to add other users to this group Click OK in the Properties dialog when you have fini
225. em in the configuration you want to export Any items nested at a lower level will be exported along with the item you select For example in the following figure exporting the injector creates a system profile that contains the injector and the sample If you export the configuration item the entire configuration is saved as a system profile 3 Choose Experiment Configuration Save Configuration As Or right click on an item and choose the Save As item from its right click menu 4 In the Save As dialog choose the folder where you want to save the system profile Then type a name for the profile you are creating and click OK See Chapter 12 Working with System Profiles for more about using a profile you export M1000 Rev H Experiment Configuration Experiment Configuration An experiment configuration groups together all the profile components used in a particular experiment For information about creating a new experiment and the associated configuration see Creating New Experi ments on page 6 4 WW Experiment1 Experiment Configurations Configuration L E Description Processing Operator Abscissa Units Concentration Source E Details Fwd Monitor Baseline Start Fwd Monitor Baseline End Baseline Hoise Start Baseline Noise End E Instruments Add Remove Connections Add Remove L5 RI online Browse Browse Browse Brow
226. ements to the folders in an experiment tree Other chapters are referenced to provide details about all the various configurations procedures data sets and results that can be added and how to work with these things after adding them to an experiment Security You must have at least Researcher access to add elements to experiments Adding to the Configuration Experiment Builder You can add items to the configuration only if you enable Experiment 6 24 Builder mode by choosing System Preferences Experiment Builder Mode The Configuration tree in the Experiments tab shows the hardware config ured to be used in the experiment The templates provided with ASTRA include most instruments and connections you use in a typical experi ment Chapter 7 Configuring Experiments contains details about the properties that can be set for each instrument type E Experiments E Sample Sets S System Profiles e EASI Comparison E EASI Graph Dol Experiments D Configuration LS RI online S Generic pump pump amp Solvent water Ei Injector injector Sample untitled1 E7 DAWN EOS DAWN EOS optilab rEx Optilab rEx al Fluid connection pump to injector il Fluid connection injector to LS f Fluid connection LS to RI ig Auto inject connection O Ma m 4 To add instruments and connections you can specify them in the Experi ment Configuration tab of the Experiment Configuration properties d
227. ent 3 5 More About the ASTRA Environment 3 13 M1000 Rev H 3 1 Chapter 3 Getting Started Starting ASTRA To run ASTRA do one of the following e Double click the ASTRA V icon on your desktop e Choose Programs Wyatt Technology ASTRA V from the Windows Start menu It may take a minute or so for ASTRA to open Security If you are using ASTRA V with Security Pack you will be prompted to log in Use a User Name Password combination given to you by the ASTRA administrator This may be the same as your Windows user name and password If ASTRA privilege groups were set up on your local computer type the name of your local computer for the domain Otherwise if the ASTRA privilege groups were added for your networked account type the domain of your networked account User Name astrauseri OK Password Cancel Domain x 3 2 M1000 Rev H Starting ASTRA The account you use determines the types of actions you can perform within ASTRA The user levels are as follows e ASTRA Administrator Can change database settings and can create modify and delete experiment files Also has privileges of Researchers Technicians and Guests e ASTRA Researcher Can create and modify experiment files Can connect to networked computers and instruments Also has privileges of Technicians and Guests ASTRA Technician Can run a given experiment procedure sequence and save the resulting data Also has privileges of G
228. ents Note 7 6 No pre defined instrument profiles are provided in the ASTRA system database In order to have instruments available to select in the Add Instrument dialog as described in the following procedure you need to first save instrument profiles in your system database as described in Creating Profiles on page 12 3 To add instruments and connections you can specify them in the Experi ment configuration tab of the Configuration properties dialog To add an instrument follow these steps 1 Choose Experiment Configuration Edit This opens the proper ties dialog for the configuration which has a tab for each item in the configuration tree Alternately double click the Configuration node in the Experiment tab of the workspace 2 In the Experiment configuration tab click the Browse button in the row to add instruments WW Experiment1 Experiment Configurations Configuration L SEE Value Hame LS RI online Description Processing Operator _ Abscissa Units min E Concentration Source RI Ei Details Fwd Monitor Baseline Start 0 0000 Fwd Monitor Baseline End 0 0000 Baseline Noise Start 0 0000 Click here Baseline Noise End 0 0000 E Instruments Add Browse Remove DrOWSE El Connections Add Browse Remove Browse 3 Inthe Add Instrument dialog find the instrument you want to add to the experiment This must be an ins
229. er category Responsibility Selecting this category indicates responsibility for performing the experiment according to procedures Approval Selecting this category indicates approval of the experiment Review Selecting this category indicates review of the experiment 3 Inthe Comments field type any additional information required by your standard operating procedures 4 In the User id Password and Domain fields type the values for your valid ASTRA account as described in the section on Starting ASTRA on page 3 2 Be sure to use uppercase and lowercase correctly in your password Note that it is not necessary for the person signing off on the experiment to be the same person who logged in initially to begin the ASTRA session 5 Click OK Stopping an Experiment To stop a running experiment choose Experiment Stop Shortcuts Press Ctrl Shift S Click the Stop icon BR in the experiment toolbar Right click any folder in the experiment tree and choose Manage Stop Stopping an experiment with ASTRA stops only the selected experiment from executing This includes the collection and analysis of data It does not affect the execution of other experiments in ASTRA nor does it affect any activity going on outside of ASTRA s control See your hardware documentation for information about alarms emer gency stops and setting up safety interlocks Alarms may be monitored via the Diagnostic Manager See Vi
230. erage and variance are reported as well as the uncertainties one standard deviation from the fit The square root of the variance is used to determine the standard deviation in the decay time distribution The average the average plus the standard devia tion and the average minus the standard deviation are converted to hydrodynamic radius via Eq 3 through 5 and are included in the results Interpretation of Cumulant Results M1000 Rev H The cumulants method presents a challenge in displaying the results ASTRA present the hydrodynamic radius results from the cumulants analysis since size is more intuitive than decay time for most researchers In the cumulant analysis graph the average hydrodynamic radius and the distribution values at one standard deviation are presented This creates an error bar appearance for the graph but the error bars indicate the width of the fitted distribution They are asymmetric because the hydrody namic radius is inversely proportional to the symmetric decay time distribution as shown in Figure F 2 With cumulant results presented this way it is important to remember that there is uncertainty in the first and second moments determined from the fit This uncertainty translates into an uncertainty in the average hydrodynamic radius but more importantly an uncertainty in the effec tive width of the distribution implied by the cumulants analysis graph Therefore an uncertainty in this width is
231. erence obtained by the Create method defined in Interacting with Instruments on page C 3 Command Effect LaserOn state Set the laser status to on true or off false state is either true or false RunCOMET state Sets the COMET to on true or off false state is either true or false This command has no effect on instruments without a COMET device DitherOn state Sets the laser dithering status to on true or off false state is either true or false Dynamic Light Scattering Refractometer and Viscometer Commands There are currently no Dynamic Light Scattering Instrument Refracto meter or Viscometer specific instrument commands C 4 M1000 Rev H Examples Examples The following examples show some useful collection scripts You can learn about additional features of the Lua programming language by visiting the website at http www lua org or by reading the book Programming in Lua second edition by Roberto Ierusalimschy The following script collects data for three auto injections lsInst LSInstrument Create lsInst LaserOn true lsInst SetCollectionInterval 0 5 lsInst StartCollection for i 1 3 do Collection WaitForMessage lsInst TSI INSTRUMENT AUTOINJECT if i 1 then start collection on 1st iteration Collection Start end Collection SetDuration 30000 end Collection Stop The following script collects data for a light scatte
232. es of known molar mass in order to determine the molar mass of an unknown sample Unfor tunately appropriate standards having the same composition and conformation as the unknown specimen are often not available If the value of dn dc differential refractive index increment or the total mass of eluting solute is known light scattering measurements can provide an absolute measurement of molar mass when used in series with a concentration sensitive detector such as a refractive index RI detector Light scattering can provide a continuous measurement of molar mass if sample concentrations are high enough to provide adequate signals In essence light scattering measurements automatically provide a column calibration curve for every sample obviating time consuming conforma tion dependent calibration procedures When techniques such as reverse phase chromatography are used separation is not based on molecular size and it is often difficult or impossible to calibrate with known standards Also the situation is more complicated for certain types of heterogeneous co polymers for which dn dc is a function of the molar mass M1000 Rev H Basic Quantities Basic Quantities Note M1000 Rev H The symbol used to describe the angle dependent light scattering is Rg called the Rayleigh ratio It is defined as Equation 1 Lr where Ig is the scattered intensity Ig is the intensity of the incident beam V is the volume of the s
233. es the mean and variance for the distri bution Cumulants on page F 2 provides details on cumulants analysis Cumulants analysis is used with QELS data Thus an experiment config uration must include a light scattering instrument and Wyatt QELS You can place this procedure with other analysis procedures and after all transformation procedures A sequence cannot contain both the Cumu lants procedure and the procedure for Regularization on page 8 90 This procedure has the following dialog W batch gels cumulants Procedures Cumulants ei g differential hydrodynamic radius control graph g 4 0x10 5 3 0x10 S 0 000E 00 hydrodynamic radius nm 0 0000 20 40 60 80 100 hydrodynamic radius nm time min Peak Humber 2 Plot cumulants Mean Rh 0 0 0 0 nm Width 0 00 0 00 cm2 sec E Processing Conditions Temperature C 25 00 Viscosity g cm zech 0 0000e 000 Refractive Index 1 3301 Min Rh Threshold nm 1 000 Max Rh Threshold nm 300 000 El Processing Parameters Min Fit Delay Time sec 0 000e 000 Max Fit Delay Time sec 1 000e 000 Suppress Peaks Below nm 1 00 Show Residuals Use Disabled Slices Prefilter YOK BK Cancel Zi Apply M1000 Rev H Analysis Procedures The properties for this procedure are as follows Table 8 36 Cumulants Properties Field Description Peak Number Click on a peak in the graph or type a number here to select the peak for the cumulants
234. escription Name Name of the instrument If you have already created a system profile for this instru ment click and select a profile to use Description Description of the instrument which typically contains more information than the Name Wavelength The wavelength of the light used in the instrument nm Cell Type of sample cell used during data collection The options are P2 P2L P10 P100 P10L P20 P12 and ENGRCELL Temperature If this instrument is temperature controlled specify the temperature to which it is set Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instrument configu ration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 Band Broaden ing gt Enable Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broadening parame ters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the instrumental term The units are in microliters See Band Broadening on page 8 14 for an
235. espiking Leve FEP BY ok 2 Cancel Si Apply If data has already been collected for this experiment the graph shows the data with the currently selected despiking level applied The property you can set is as follows Table 8 12 Despiking Properties Field Description Despiking Level Choose the degree of despiking The options are none normal or heavy The default is normal Smoothing M1000 Rev H The Smoothing procedure smooths noisy data Smoothing can be useful in certain circumstances but in general it is better to remove the source of the noise such as particulates in the mobile phase and pump pulsations rather than to smooth the data ASTRA smooths data using the Savitsky Golay technique of least squares smoothing Use this procedure with care the height of very sharp peaks may be reduced somewhat by the smoothing process This procedure may be placed at any point in the experiment sequence before the analysis procedures that determine the final results This pro cedure runs automatically without prompting for a value You can set the property for this procedure before running the experiment or you can modify it after running the experiment and re run the experi ment to see the effects of changing the setting 8 43 Chapter 8 Editing Procedures Double click on the procedure to open its property dialog E experiment 2 Procedures Smoothing Smoothing V bd in nN
236. estimated by calculating the spread in possible width values based on the fitted uncertainty in the second cumulant The effective width implied by the cumulants is then compared to the spread of possible widths to derive a percentage uncer tainty in the effective width The average uncertainty in width is reported in the Width property and should provide a good measure of how much to trust the widths that result from the analysis After all is said and done the question remains how the cumulant analysis results relate to the actual polydispersity of the sample Assuming that the size distribution in Figure F 2 reflects all samples is simply incorrect Therefore the cumulant results should be taken as a semi quantitative estimate of the degree of polydispersity It would probably be safe to assume that for two samples with the same average size but different widths estimated from the cumulant analysis that the sample with the greater width is more polydisperse However trying to define a rigorous polydispersity index from the cumulant analysis would probably lead to very inaccurate results when compared to a quantitative method such as fractionation followed by light scattering to determine the underlying distribution Therefore cumulant analysis results should only be used to assess the potential relative polydispersity of samples Follow up analysis such as fractionation followed by light scattering should be used to assess the reliability
237. etails matic import and RI Detector most other options E Pest S are grayed out Model Optilab DSP v AUX To import an ASTRA C AUX2 4 file l Concentration Detector 1 If your ASTRA A file had a stan UV Detector dard configura IV Present Cell Length cm 10 tion such as C AUx1 io batch particles AUX2 SE IS or RI detector Concentration Detector alone click OK in the Import Repair Wizard to try an automatic import P 2 Ifyou had a UV detector instead of an RI detector or in addition to an RI detector choose the Specify AUX Detectors item Then specify how your RI and or UV detectors were connected Note that only one detector should have the Concentration Detector box checked For an RI detector select the model For a UV detector specify the cell length and response value Then click OK 3 Double click on a procedure with a graph such as Peaks or a results graph If the graph makes sense the experiment was imported cor rectly and you need not repair the file If the graph looks like a scatter plot when it should not close the experiment without saving and continue to the next steps 4 Reimport the ASTRA 4 file Check the box in the Repair area to remove smoothed or despiked data Click OK 5 Again check to see if the resulting graphs look valid If they do the file was imported correctly 6 If you continue to encounter difficulties importing a file created with ASTRA 4 DNDC or RICAL
238. ethod used See Appendix D Light Scattering Theory UV Extinction The UV extinction for this peak Set this parameter if you are using a UV detec tor for concentration and are performing any of the following analysis proce dures Mass and Radius from LS Data Protein Conjugate Analysis If you are performing a Protein Conjugate Analysis this parameter corre sponds to the extinction value for the protein M1000 Rev H 8 53 Chapter 8 Editing Procedures Table 8 15 Peak Properties Field Description Refractive Index gt Real The real index of refraction for the peak Set this parameter if you are perform ing any of the following analysis procedures Mass and Radius from LS Data using Mie or coated sphere Radius from LS Data using Mie or coated sphere Number from LS Data If you are using the coated sphere model this parameter corresponds to the real refractive index of the core of the coated sphere Refractive Index gt Imaginary The imaginary index of refraction Set this parameter for the same analysis procedures listed for the Real Refractive Index Concentration The concentration of the sample for this peak Set this parameter if you are performing any of the following analysis procedures A2 Mass and Radius from LS Data Dn dc from RI Data Extinction from UV Data Mass and Radius from LS Data batch mode with no concentration detector Injected Mass The
239. ewing Alarms with the Diagnostic Manager on page 5 7 for details Re Running an Experiment for Data Processing If you modify one or more procedures in an experiment you can re run the experiment using the Run command This time instead of collecting data only the procedures marked with the Si not run icon are performed M1000 Rev H 6 13 Chapter 6 Creating amp Running Experiments 6 14 If a Crash Occurs While data is being collected ASTRA stores data in a crash recovery file in your My Documents ASTRA V Recovered Files folder The filename contains the current date and time and have a file extension of vrf For example 20080324_221518237Experiment vrf When the data collection is complete and data is successfully saved this file is automatically deleted When saving over the network a problem may occur for example if the network connection fails or if you do not have permission to save to a par ticular location When this happens the save fails and the crash recovery file is retained Each time ASTRA starts it checks for crash recovery files in the My Docu ments ASTRA V Recovered Files location If a vrf file is present ASTRA opens it and creates a standard ASTRA V vaf file in the same My Docu ments location M1000 Rev H Closing an Experiment Closing an Experiment Shortcuts You can work with multiple experiments open in ASTRA To close an experiment without exiting from ASTRA fo
240. experiment as desired Each experiment contains the following categories of items e Configuration The hardware devices and connections used in the experiment For online fractionated experiments this may include a pump injector solvent sample DAWN Optilab and data connec tions For details on all types of items that may be configured and their properties see Chapter 7 Configuring Experiments e Procedures The actions to be performed in sequence when the exper iment is run There are configuration collection transformation analysis and administrative procedures For details on all types of procedures see Chapter 8 Editing Procedures e Results The reports and graphs to be produced after the experiment procedure has been run For details see Chapter 10 Working With Reports 6 2 M1000 Rev H About Experiments The actions you can perform on an experiment are shown in the following diagram The arrows that point to the main experiment show ways to open create or bring information into an experiment The arrows that point away from the main experiment show ways to close save or export information from an experiment Re Create from Template Create Blank Open Import Experiment E Results data Template KE Configuration El Procedures Results format Copy From Apply Template Add to Experiment
241. exporting 10 6 preview 10 6 printing 3 15 10 6 template format for 10 3 templates for 10 4 viewing 3 12 repositioning graphs 11 14 Research Database operating tier 1 4 Research Database version 1 3 Researcher group 1 4 2 6 3 3 3 13 4 6 Researcher user level A 2 Responsibility category for sign offs 6 13 results fitting 8 57 Results folder Add to experiment dialog 6 29 10 4 11 11 Results folder Experiments tab adding graphs to 6 28 11 11 adding reports to 6 28 10 4 definition of 3 6 6 2 Review category for sign offs 6 13 Rh hydrodynamic radius calculating 8 77 8 80 Rh cumulative intensity plot 8 93 Rh cumulative weight plot 8 93 Rh diff intensity plot 8 93 Rh diff weight plot 8 93 Rh from VS procedure 8 80 RI refractive index instruments calibration of 8 21 definition of 1 7 7 17 Index 9 Index generic profiles for 7 19 profiles for 7 17 RI calibration from peak procedure 8 75 RI Calibration procedure 8 21 RI data converting to concentrations 8 56 definition of 1 7 determining dn dc and linearity from 8 73 RICAL importing files from 6 8 6 9 rms radius calculating 8 59 8 63 8 65 8 69 8 71 curve fitting model for 8 57 light scattering theory and D 15 Rod fit model 8 55 Run button ASTRA toolbar 3 9 Run command Experiment menu 6 11 Run command Sample Set menu 9 9 Run mode 1 2 1 3 3 13 command list A 2 rwf file extension 6 8 6 9 S Sakurada see
242. f 11 14 see also data set definitions groups for user accounts assigning users to 2 7 creating 2 6 list of 2 6 4 6 number of groups a user can be as signed to 4 6 Guest group 1 4 2 6 3 3 3 18 4 6 Guest user level A 2 H Help menu list of commands in A 8 see also specific menu commands help using 3 15 Houwink see also Mark Houwink Sakura da 8 83 HPLC pump with injector 8 23 hydrodynamic radius Rh calculating 8 77 8 80 l icons Basic 1 4 Database 1 4 Security 1 4 icons in Procedures folder 3 11 8 2 8 8 ID tubing 8 23 Index 6 Import command Sample Set menu 9 4 9 5 Import Experiment command File menu 6 9 Import Experiment Configuration menu command 12 8 12 9 Index command Help menu 3 15 injector 8 23 injectors definition of 7 25 profiles for 7 25 installation of ASTRA 2 2 Instrument list accessing Diagnostic Manager from 5 5 definition of 2 10 viewing 2 10 instruments adding to experiment configuration 6 24 7 6 checking connections to 3 5 connected through AUX input 5 2 definition of 7 11 ISI installation required for 5 2 monitoring graph of data in real time 5 5 numeric data from 5 6 properties of viewing and setting 5 10 sending commands to 5 8 validating for experiment 6 11 9 9 viewing in Instruments dialog 2 10 see also specific instruments Instruments command System menu 2 10 3 5 Instruments dialog 2 10 3 5 Interdetector Dela
243. f instrument that receives analog data over this connec tion The drop down list shows the instrument profile types that are avail able for a connection AUX Channel Calibration Constant The input AUX channel number on the destination instrument Constant value by which the AUX signal should be scaled This constant can be set manually or determined through one of the calibration proce dures The default value is 1 0 See Differential RI Calibration on page 8 21 and UV Calibration on page 8 31 Autoinjector Connection Profiles An Autoinjector connection profile describes a physical connection between the auto inject output of an injector and the auto inject input of an instrument You can set the following properties for an Autoinjector connection Table 7 18 Autoinjector Connection Properties Field Description Name Name of the connection If you have already created a system profile for this connection click and select a profile to use Description Description of the connection which typically contains more information than the Name Source Instrument Select the type of instrument that sends the auto inject signal over this connection The drop down list shows the instrument profile types that are available for a connection Destination Instrument Select the type of instrument that receives the auto inject signal over this connection The drop down list shows the
244. face and other compo nents of the ASTRA V software Your network administrator may want more technical information about the ISI The ISI runs as a DCOM server on each machine where it is installed To successfully connect to an ISI the computer should allow DCOM connections This is the standard configuration for Windows 2000 and Windows XP Professional prior to Service Pack 2 Windows XP Professional SP2 and newer operating systems require manual configuration of the DCOM settings to allow external data connec tions For more on DCOM and firewall issues go to the Wyatt Support Center website www wyatt com support and follow the links to FAQs Software The ReadMe Files directory of your ASTRA installation contains addi tional information about database and network issues The ISI runs a server called the Wyatt Communications Server In the Processes tab of your Windows Task Manager this process is called WCS exe If ASTRA quits unexpectedly you may need to end the WCS process through the Task Manager M1000 Rev H Using the Diagnostic Manager Using the Diagnostic Manager If you choose System Instruments select an instrument and click View you see the Diagnostic Manager for that instrument You can also start the Diagnostic Manager from the Windows Start menu by choosing Programs Wyatt Technologies gt ASTRA Diagnostic Manager You can use the Diagnostic Manager utility to monitor and control instru ments
245. fied to the following for computation Equation 11 EE 2Ing 7 1 In Appendix G Viscosity Theory Intrinsic Viscosity and Molecular Parameters The simplest model of the intrinsic viscosity is due to Einstein and Simha They considered the case of noninteracting rigid particles They found that the viscosity can be related to the volume fraction of the fluid occupied by the particles They found Equation 12 KE A OU where d is the volume fraction and y 2 5 for spheres and larger for non spherical particles If the weight concentration of the molecule is c then the number of mole cules per unit volume is N4c M where N4 is Avogadro s number and M is the molar mass as measured by light scattering Therefore Eq 12 can be written in terms of the measured intrinsic viscosity as Equation 13 N V 7 M where V is the hydrodynamic volume of the molecules Note that M V is the molecular density so in some sense the intrinsic viscosity is measur ing the molecular density The intrinsic viscosity often differs from the bulk density due to molecular shape molecular density and the effects of adsorbed or immobilized solvent on the surface of molecule This so called hydration layer moves with the molecule so it affects measurement of the molecular density In addition when the molecule has an extended shape penetration of non immobilized solvent into the interior of the molecule similarly affects thi
246. figure the interdetector delay and make any other experiment configuration changes you normally make 6 Choose Experiment Configuration Save Configuration As to save this experiment as a template for later use 7 Now use this experiment to collect data on an unknown sample or apply this template to already collected data that was collected with the column and configuration used in the template The results are calculated using the calibrated column values 8 In the Mass from column calibration procedure choose a Flow Marker type and Flow Marker Peak if any 8 96 M1000 Rev H Analysis Procedures 9 Finally you can evaluate the molar mass of the unknown sample In the following image the mass characteristics are very close to the light scattering values Iw Alkyd_3_6 LS M Alkyd_3_6 UC z E 5 o E 10 0 12 0 14 0 16 0 time min As a counter example the following plot illustrates the false polydis persity displayed by universal calibration z 3 a S E gt E 10 5 11 0 time min M1000 Rev H 8 97 Chapter 8 Editing Procedures 8 98 Cumulants Cumulants analysis is a strategy for extracting information about the underlying size distribution of a polydisperse sample from the correlation function In a nutshell the cumulants method involves fitting the correla tion function not to a single decay time but to a Gaussian distribution of decay times This method retriev
247. for 7 18 Optilab instruments 1 7 Optilab rEX ISI integrated 5 2 profiles for 7 17 optional modules activating 2 5 demo period for 2 5 order of precedence 9 8 original data 8 3 11 7 P PCX files exporting graphs to 11 17 PDF files exporting graphs to 11 17 peak 9 8 Peak areas procedure 8 83 peaks defining 3 10 8 51 Peaks procedure 3 10 8 51 phone support 1 9 physical units 1 7 plus sign between key names 1 5 PNG files exporting graphs to 11 17 polydispersity 8 97 and normalization 8 33 PostScript files exporting graphs to 11 17 precedence 9 8 Print command File menu 3 15 10 6 printing graphs 11 15 Print Preview command File menu 10 6 Print Setup command File menu 3 15 10 6 Print tab Editing dialog 11 15 printer setup 10 6 printing configuration 3 15 graphs 11 15 reports 3 15 10 6 procedures adding to experiment 6 26 8 7 M1000 Rev H administration procedures 8 100 analysis procedures 8 59 collection procedures 8 10 configuration procedures 8 14 data processing during 8 3 data sets produced by 11 7 definition of 1 7 8 2 deleting 8 8 editing 8 4 folder listing 3 6 6 2 8 2 graphs of 8 5 modifying 3 8 8 6 sequencing 6 27 8 8 sign offs for 6 12 status icons for 3 11 8 2 8 8 transformation procedures 8 42 types of 8 2 validating 6 11 8 9 Procedures folder Experiments tab 6 2 adding procedures to 6 26 definition of 3 6
248. fy them after running the experiment and re run the experiment to see the effects of changing the settings If you set a model and fit degree for the radius the fit line is shown in graphs of RMS radius including EASI graphs 8 57 Chapter 8 Editing Procedures To set fit properties open the dialog for the procedure Mass Fit Model Fit Degree Radius Fit Model Fit Degree The properties for this procedure are as follows Table 8 16 Fit Mass or Radius Properties Field Description Mass Fit gt Model The model to use to fit the mass Options are none the default polynomial and exponential Mass Fit gt Degree The fit degree for the mass The default is 1 The allowed range is 0 to 5 This field is disabled if the fit type is None which means no fit will be performed Radius Fit gt Model The model to use to fit the radius Options are none the default polynomial and exponential Radius Fit gt Degree The fit degree for the radius The default is 1 The allowed range is 0 to 5 This field is disabled if the fit type is None which means no fit will be performed Fit the mass or radius data if you with to obtain more accurate peak moments and distribution results for mass and radius ranges that have significant scatter due to lower signal to noise ratios After choosing a fit you must view the appropriate molar mass or radius vs volume graph to visually determine whether the fit is ac
249. g open the folder that contains the experiment template you want to use You can choose from the follow ing folders System Templates These templates are provided with ASTRA for your use A set of experiment templates is provided for each Wyatt instrument These templates typically provide a starting point for most experiment types you might perform My Templates These templates are ones you have saved as described in Creating a Template on page 6 18 Import Templates These templates are used when you open an experiment created with the old ASTRA 4 or DNDC 5 software Creating new experiments from these templates is not recom mended If you read about an experiment template you want to use but don t see it in the New from Existing dialog see Migrating the System Database on page 2 3 to update your system database so you have all the latest experiment templates and system profiles 3 Select a template and click Create A new experiment is created based on the template you selected Templates you use to create experiments are stored in the ASTRA system database They are not stored in separate files in the instal lation tree M1000 Rev H 6 5 Chapter 6 Creating amp Running Experiments Creating Experiments from Scratch Experiment Builder You can create and modify blank experiments only if you enable Experi ment Builder mode by choosing System Preferences Experiment Builder Mode Security
250. g Graphs Printing Graphs M1000 Rev H To print a graph do either of the following Right click on the graph and choose Print Choose File gt Print from the menus To print a graph with more control over the output follow these steps 1 Double click on a graph to open the Edit Graph dialog Then click Advanced 2 Choose the Print tab from the top row of tabs E Editing Chart Series Data Tools Export Printer GATEWAY 1 HP3150 Orientation P e Portrait mea sque radis us volume e Landscape g Detail More Normal Proportional In the Printer list choose the printer you want to send the graph to Click Setup if you want to adjust printing properties Choose an orientation of Portrait or Landscape The default is Land scape which is appropriate for most graphs Use the Detail slider to adjust the quality of the output and number of major tick marks on the axes Put a checkmark in the Proportional box if you want the graph to have a height and width proportional to the current graph display After you check this box you can drag the dotted lines on the preview to resize the graph on the page When you are ready to print click Print 11 15 Chapter 11 Working With Graphs Copying Graphs to the Clipboard To copy a graph to the clipboard for pasting into another applications press Ctrl C while in the dialog that contains the graph Alternately right
251. g a polynomial to Rg Le vs sin 2 as in the Debye method we insert into Eq 13 the theoretical form factor P for random coils which was first derived by Debye P Debye J Phys Coll Chem 51 18 1947 Equation 25 2 Equ P e 1 u u where us LI A r sin 2 Since P is a nonlinear function of its parameter lt r2 gt we use an iterative nonlinear least squares fit Unlike the other fit methods the Random Coil method assumes the polymers are approximately random coils This can be an advantage for large random coil molecules because it allows the fit to proceed with fewer parameters than would otherwise be required in a simple polynomial fit and the result can be lower estimated errors You can select which calculation type Zimm Berry Debye or random coil you wish to employ in the Peaks procedure page 8 51 If you own a miniDAWN the calculation type must be either Zimm or Random Coil The result of these calculations is that for each slice i we have the molar mass M and the mean square radius lt r gt Technically the molar mass is weight averaged and the mean square radius is z averaged Assuming good chromatographic separation these quantities can be used together with the concentration C measured with a concentration sensitive detec tor to find the molar mass and radius moments as described next Model Fit Method To derive the fit method for the various modeled form facto
252. g dialog E band broadening example BSA ProceduresWistribution Els Distribution analysis Iw cumulative molar mass cumulative weight fraction 65859 75 69876 57 Humber Averaged Weight Averaged Z Averaged EI OK x Cancel Si Apply 6 7435e 004 6 7439e 004 6 7444e 004 Iw linear differential molar mass 40x10 Eg F e mi 2 z gt H E ZS E rm 134454 63 141561 30 ZZ Det 3754e 005 The graph shows the linear and cumulative distribution of the geometric radius or other selected data Click and drag on the graph to select a range such as a peak A separate column is created in the properties area for each range you create For an example experiment that performs a distribution analysis choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the band broadening example experiment in the Sample Data gt Analyzed Experiments folder M1000 Rev H 8 85 Chapter 8 Editing Procedures The properties for this procedure are as follows Table 8 31 Distribution Analysis Properties Field Description Name Name of the range for this column The default names are Range 1 Range 2 etc You can change these as desired Distribution Data The type of data to plot on the x axis for distribution analysis Options vary depending on the data in the experiment Examples
253. g model selected for this peak in the Peaks dia log This field is display only Fit Degree This field shows the fit degree selected for this peak in the Peaks dialog It is valid only if the Zimm Debye or Berry model is selected This field is display only 8 70 M1000 Rev H Analysis Procedures Table 8 22 Number from LS Data Properties Field Description Abscissa Position This field shows the position on the x axis for the peak and slice selected This field is display only Enabled Detectors gt 1 18 This list has a checkmark next to detectors whose data is used in the calculation You can disable individual detectors by removing the checkmark Radius from LS Data This procedure calculates the radius of the sample based on the light scat tering signal alone This procedure is normally used with online fractionated experiments The procedure has the following dialog E rons adf ProceduresWetermine radius from LS data proced SEE 1 R theta results graph control graph 175x10 1 73 x10 1 71 x10 1 69 x10 1 67 x10 1 65 x10 1 63 x10 1 61 x10 1 59 x10 xI scattering angle 90 degrees wf XIU 4 37 sin H 3 37 x10 2 37 x10 1 37 x10 3 72 x10 6 28 x10 0 0336 0 5336 1 02 2 4467 12 4467 22 4 sin theta 2 time min Rayleigh ratio 1 cm rms radius Peak Number 1 Slice Index 1171 Zimm Fit Degree 1 Abscissa Position 9 758 min Enabled
254. h mode and flow mode see Batch Mode vs Online Mode on page 1 8 Divide by Laser Monitor Fwd Monitor Aux Channel Select the laser monitor option you want to use The options are Laser Monitor Forward Monitor and none Laser Monitor The light scattering signals are divided by the laser monitor which corrects for fluctuations in the laser intensity due to power fluctuations Forward Monitor The light scattering signals are divided by the for ward laser monitor which corrects for both laser intensity fluctuations and absorbance by the sample none No correction is performed for laser intensity fluctuations or sample absorbance Use this option only if signal levels are so low that digital noise from the laser monitor signal can contaminate data To use the Forward Monitor you must route the instrument s forward laser monitor signal through one of the AUX inputs on the instrument If you selected Forward Monitor in the field above specify which AUX input on the instrument receives the forward laser monitor signal Disable Collection Check this box to disable data collection for this instrument For example if the light scattering instrument has the QELS option it is possible to disable the DAWN collection and collect QELS data alone Polarization Analyzer Check this box if the polarization option is currently installed on the instru ment See the DAWN h
255. h peaks should be omitted from the regu larization gt Show Residuals Check this box if you want the correlation function graph to show residuals The default it to omit residuals 8 92 M1000 Rev H Analysis Procedures Table 8 34 Regularization Properties Field Description gt Use Disabled Slices ASTRA normally discards the entire slice if the avalanche photo diode APD is triggered So any measurement where the APD was triggered is excluded from analysis unless you specifically check the Use Disabled Slices check box See Rh from QELS Data on page 8 77 for more about the avalanche photo diode APD gt Prefilter If the Prefilter box is checked data points in the correlation function view that fall outside the minimum or maximum delay times the Min Fit Delay Time and Max Fit Delay Time fields are discarded from the fit You can see the fit line stop short of these points and the points themselves change color to red The rest of the points are still included in the analysis If this box is unchecked data points outside the range are used in the fit For an example of the results you can view with the regularization proce dure open the QELS batch regularization BSA insulin sample file and open its Regularization procedure This procedure allows you to see the characteristics of three mixtures the three peaks in the right graph M1000 Rev H
256. he folders that contain the templates WW New from Existing General RI Viscometry Measurement Cossch Light uv Scattering Measurement Files of type Experiment Templates D Cancel Some templates are only usable with a feature activation keys Some templates need to be applied to an already existing experiment and some are used to run a new experiment This information is provided along with the name of each template using these abbreviations e CT Templates used to collect new data s AT Templates that are generally applied to an already run experi ment to analyze data in an additional way If you read about an experiment template you want to use but don t see it in the New from Existing dialog see Migrating the System Database on page 2 3 to update your system database so you have all the latest experi ment templates and system profiles M1000 Rev H General General The following templates are provided in the General folder Concentration Determination AT After you select the peaks zones and enter the dn dc value for each peak the relative concentration and calculated mass is displayed in the report for each peak RESULTS Peak 1 Peak 2 Peak 3 concentration moments g mL c avg 1 06e 4 0 0 9 12e 6 0 0 2 10e 6 0 0 calculated mass g m 6 3631e 5 6 8543e 6 9 0903e 7 Peak Areas AT After you select the peaks zones the following data is displayed in the report The peak ar
257. he other fit methods the Random Coil method assumes the polymers are approxi mately random coils This can be an advantage for large random coil molecules because it allows the fit to proceed with fewer parameters than would otherwise be required in a simple polynomial fit and the result can be lower estimated errors Model Fit Method To derive the fit method for the various modeled form factors sphere coated sphere rod we fit the Zimm equation to Rg vs sin 2 To derive the specific equation for the desired model we insert into Eq 1 the theoretical form factor P for the specific model being analyzed Form factor models have been derived for spheres coated spheres and rods 1 P Debye J Phys Coll Chem 51 18 1947 M1000 Rev H E 3 Appendix E Particles Theory 1 E 4 They are covered in the text by van de Hulst Note that the sphere and coated sphere models yield geometric radius while the rod model produces a length Sphere Equation 6 3 P sinu ucosu u Rods REES sint sin u PO a ME u where u 277 A L sin 2 and L is the rod length L gt gt rod radius Mie Fit Method In the Lorenz Mie Fit method Maxwell s equations for electromagnetic radiation are solved under the assumption of spherical particles The Mie solution does not require the particle to satisfy the Raleigh Gans Debye criteria and is therefore the most general method for analyzing spheres
258. hen do a calibration curve this will allow you to compare the elution volume for your sample If two molecules have the same molar masses but different radii there is an error in the calculation M1000 Rev H RI Measurement RI Calibration CT Use this template only with batch measurements The experiment config uration contains only an Optilab rEX Inject at least three concentration of sodium chloride which has a well known dn dc The slope allows ASTRA to determine the calibration constant Determine CC Column Profile CT Analysis of a sample using a universal or conventional column calibration takes place in two distinct phases First the response of a column to a set of standards with known molecular weights must be measured Once this determine column calibration phase is complete the unknown sample can be analyzed The determine column profile template allows you to set up the column profile for Conventional Calibration Batch Determine dn dc CT M1000 Rev H The determination of dn dc for a specific sample in a solvent is done by manually injecting several concentrations of the sample Additionally the solvent used to dissolve the sample should also be injected before and after the samples When you have run the experiment define the peaks and the concentra tion of each sample These concentrations have to be very precise In the dn dc from peak procedure the dn dc value errors
259. hnician or Guest you have read only access to profiles This chapter describes the types of profiles contained in configurations and how their properties can be modified Each profile type has a property list similar to that shown in Figure 7 2 E Experiment Experiment Configurations Configuration Profile LS SE Name injector to EOS Description Source Instrument Manual injector Destination Instrument DAWN EOS Volume mL 0 5000 Temperature Control Enable Temperature C Experiment configuration L5 Online Fluid connection injector to EOS Fluid connection EO BY OK St Cancel Figure 7 2 Typical property dialog fluid connection example M1000 Rev H 7 3 Chapter 7 Configuring Experiments Configuration Example The Configuration tree in the Experiment tab of the workspace shows both the hardware configured to be used in the experiment as well as addi tional elements such as the solvent and sample The experiment templates provided with ASTRA V include most common configurations A configu ration for an online light scattering experiment apparatus is shown in Figure 7 3 The same experiment is shown in the schematic of Figure 7 4 Comparing these two figures highlights the logical structure of the config uration and its constituent profile elements in the workspace E Experiments E Sample Sets E System Profiles ies EASI Comparison B EASI Graph Bol Experiments A ES E Configura
260. hoose a printer Getting Help The online help for the ASTRA software contains all the information in this manual Descriptions of properties in the help system are linked to dialogs so that you can quickly learn more about individual properties Note The online help system does not contain hardware related information To access the online help system do one of the following e Choose Help Contents to open the help system to the beginning topic e Choose Help Search to open the full text search for the help system e Choose Help Index to open the index for the help system e Press F1 in any dialog to open help about that dialog e Click L and then click on a dialog for context sensitive help Exiting from ASTRA When you have finished working with ASTRA exit it just as you would any other Windows application If a file is open ASTRA closes it If any changes to an open file haven t been saved you are prompted to save the changes or cancel the closing of ASTRA To close ASTRA do one of the following e Choose File gt Exit e Press ALT F4 e Press Alt F X e Click the X button in the upper right corner of the ASTRA window M1000 Rev H 3 15 Chapter 3 Getting Started 3 16 M1000 Rev H ASTRA Administration This chapter tells how to administer ASTRA V experiment databases Such databases are used in ASTRA V with Research Database and in ASTRA V with Security Pack CONTENTS PAGE 21 CFR Part 11
261. ialog To add an instrument follow these steps 1 Choose Experiment Configuration Edit This opens the proper ties dialog for the experiment configuration which has a tab for each item in the configuration tree M1000 Rev H Adding Elements to an Experiment Shortcuts Double click any part of the configuration tree in the Experiments tab Right click any part of the experiment tree and choose Manage Configuration Edit M1000 Rev H 2 In the Experiment Configuration tab click the Browse button in the row to add instruments WW Experiment1 Experiment Configurations Configuration L SEE Value Hame LS RI online Description Processing Operator Abscissa Units min E Concentration Source RI Ei Details Fwd Monitor Baseline Start 0 0000 Fwd Monitor Baseline End 0 0000 Baseline Noise Start 0 0000 Click here Baseline Noise End E Instruments Add Remove Connections Add 0 0000 Browse Drowse Browse i 3 In the Add Instrument dialog find the instrument you want to add to the experiment For example you might navigate to the Profile Instrument UV Instrument folder to select a profile of a UV instrument Profiles of many instruments are provided with ASTRA and you can create additional profiles as described in Chapter 12 Working with System Profiles 4 Select the instrument profile you want to add and click Open
262. ic standard is a sample molecule with a diameter of less than about 1 20th of the wavelength of the incident light which is the case for random coil molecules with a molar mass below 50 000 and also for most proteins Perform the normalization at the flow rate and in the solvent you intend to use to run samples Low Polydispersity It is inappropriate to normalize with a broad distribution polydispersity greater than 1 2 or unknown sample Always use a relatively narrow distribution polydispersity less than 1 1 low molar mass sample for normalization It is usually appropriate to use the same sample for normalization that you used to determine the delay volumes between the DAWN and other instruments If there are absolutely no narrow standards avail able in the solvent you are using you may be able to normalize prop erly by setting the peak region to include only the central part Nevertheless use a narrow standard if one is available Same Solvent as Samples Due to the changes in scattering angle with solvent refractive index the normalization needs to be performed in the same solvent as the samples you want to analyze For chroma tography we recommend one of the following e A 30 000 g mol narrow polystyrene in toluene or THF having an RMS radius of about 5 nm e A 20 000 30 000 g mol polysaccharide such as pullulan or dextran in water or a PEO also having an RMS radius of about 5 nm A monomer protein such as BSA in w
263. ight Scattering gt Baseline Sub traction folder Online CT Use this template to run an online light scattering experiment to measure the baseline for later baseline subtraction Calibration CT To calibrate a Wyatt Light scattering instrument you need to create a new experiment File gt New gt Experiment from Template and choose the template corresponding to your light scattering detector The calibration is done with a batch injection of pure and filtered toluene Globally the method used in the template first collects data for 30 seconds with the laser on and then does a second measurement with the laser off dark voltage The intensity is measured at the 90 angle ASTRA V analyzes the difference between both signals to convert the volt signal into meaningful units The resulting calibration constant is shown in the report Results Calculated calibration constant 1 0000e 4 1 V cm Templates are available for the following instruments e Dawn 8 e Dawn DSP e Dawn DSP F e Dawn EOS M1000 Rev H Light Scattering e HELEOS e HELEOS 8 miniDAWN s TREOS Utilities CT When you run a single experiment or sample set the Comet templates allow you to program the Comet cell cleaner for a certain amount of time shown in the name of the template Note that in these templates the laser is automatically turned off e HELEOS Comet 5min e HELEOS Comet 10min e HELEOS Comet 2hour e TREOS Comet 5min e TRE
264. ilab rEX Profiles You can set the following properties for a Optilab rEX instrument Table 7 6 Optilab rEX Properties Field Description Name Name of the instrument If you have already created a system profile for this instru ment click and select a profile to use Description Description of the instrument which typically contains more information than the Name Physical Click and select from the Instruments dialog See Accessing and Viewing Instrument Hardware on page 2 10 Wavelength The wavelength of the light used in the instrument nm Batch Mode Check this box if the instrument is to be used in batch mode Checking this box associates a single sample and solvent configuration with the instrument configu ration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 Band Broaden ing gt Enable Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broadening parame ters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term See Band Broadening on page 8 14 for an explanation of the instrumental term The units are in microliters Band Broadening gt Mi
265. iles for any other solvents you use You can set the following properties for a custom solvent Table 7 21 Solvent Properties Field Description Name Name of solvent profile Typically this is the name of the chemical You can click to choose from a list of common solvents in the System Solvents folder or solvent configurations you have saved as system profiles Description Description of the solvent which may show more information than the Name Refractive Index Displays the computed refractive index of the solvent at the wavelength used in the experiment This property is shown only if this profile is part of an exper iment configuration as opposed to when you are editing a system profile Viscosity Displays the computed viscosity of the solvent at the temperature used in the experiment This property is shown only if this profile is part of an experiment configuration Rayleigh Ratio Displays the computed Rayleigh ratio of the solvent at the wavelength used in the experiment This property is shown only if this profile is part of an experi ment configuration Refractive Index Model The model used to specify the refractive index May be Fixed or Polyno mial Set the model type before setting the parameters required for that model e If Fixed specify the Reference Refractive Index e f Polynomial specify the Refractive Index Model Parameters These are used to compute the Refrac
266. ill change and hence it is necessary to correct for them if A is to be independent of solvent and cell glass The reflection correction can be considered in terms of the transmitted intensity between media 1 and 2 with indices of refraction n and na respectively Transmitted intensity is given by the Fresnel equations as Equation 5 Ann n n If g represents the flow cell glass S represents the solvent and a repre sents air then the reflection correction can be written as Equation 6 ReflectionCorrection ee T Nunc Sg ga D 5 Appendix D Light Scattering Theory where Nunc is the number of uncoated glass air interfaces the incident and scattered light have to traverse It is assumed that the reflective losses at a coated i e antireflection coated interface are negligible The Geometry Correction for a sample cell is not so straightforward to determine as the reflection correction There are examples of analytical expressions derived for simple cell geometres but there are no simple analytical expressions for more complicated geometries such as that of the K5 and F2 flow cells In addition these analytical expressions are valid for instances where the source of scattered light is either a point source or a completely illuminated volume neither of which hold for the instance of scattered light being collected from a line source like a collimated laser beam Therefore the geometry correc
267. ination Then you can use those values in this procedure to determine the mass You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the mass If you place multiple methods that determine mass in a procedure only the first one will be valid The procedure has the following dialog WW Experiment4 ProceduresWetermine mass from VS data pro Els Value Mark Houwink Sakurada K mL g pg RTR Mark Houwink Sakurada a 0 000e 000 BY OK St Cancel The properties for this procedure are as follows Table 8 21 Mass from VS Data Properties Field Description Mark Houwink Sakurada K The K fit parameter for the Mark Houwink Sakurada analysis Mark Houwink Sakurada a The a fit parameter for the Mark Houwink Sakurada analysis For more about viscometry data collection and analysis go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Viscometry Number from LS Data M1000 Rev H This procedure calculates the radius and the number of particles per mL density in the sample It is necessary to specify the refractive index of the sample in the Peaks procedure to determine the number density This pro cedure is normally used with online fractionated experiments For more about determining and using number density go to http www
268. indicate that the cell has been fully flushed with sample solution A syringe pump fitted with a large syringe may be used instead of the HPLC pump Syringe Pump Infusion Another option is to connect the Optilab rEX as shown in Figure 8 2 using a syringe pump Use 0 02 in ID tubing for both connections A recom mended flow rate is 0 1 0 2 mL minute The syringe must be rinsed and dried thoroughly or replaced between samples to avoid contamination of one concentration by the next sample When the syringe is disconnected to change samples the pressure change and injected air may cause an unstable baseline for several minutes PEEK tubing RI syringe detector syringe pump waste flask Figure 8 2 Setting up a syringe pump for calibration 8 23 Chapter 8 Editing Procedures 8 24 Collecting Data The dRI instrument s sample and reference cells should be flushed with high purity water first solvent to be analyzed for several minutes before making measurements Confirm that the temperature is stable and activate the instrument s purge feature while flowing pure water for several minutes Deactivate the purge flow with water for several more minutes then zero the instrument Collect data using ASTRA V software as follows 1 Start the ASTRA V software and select File gt New gt Experiment from Template In the dialog box that opens select the System Templates folder then the RI Measuremen
269. ine Start End configuration for your light scattering instrument See page 7 11 you should specify the start and end points for a region that corresponds to pure solvent This pure solvent region acts as a baseline for the forward laser monitor sig nal If you do not specify a region the average of the forward laser monitor signals for the first 10 of the collected data is assumed to be the default pure solvent range for calculating the average forward monitor signal Details gt Baseline Noise Specify the start and end points for a region to be used for the baseline noise Region Start End computation If you do not specify a region the first and last 10 of the run are used to assess baseline noise These fields allow you to override the default if there are artifacts in these regions Add Instruments Click Browse to select an instrument profile to add to the experiment This property and the ones that follow are available only in Experiment Builder Mode Add Connections Click Browse to select a connection profile to add to the experiment Remove Connections Click Browse to select a connection profile to remove from the experiment Experiment Builder Buttons to add and remove instruments and connections are visible only in Experiment Builder mode which you enable by choosing Sys tem Preferences Experiment Builder Mode If you have already opened the Configuration properties dialog close and reopen it after e
270. ing 8 TT TREOS profiles for 7 14 tubing between injector and Optilab rEX 8 23 for syringe pump infusion 8 23 txt file extension 6 17 U universal column calibration 7 26 Unsigned category for sign offs 6 13 updating 2 3 M1000 Rev H Index upgrading ASTRA 2 3 user accounts assigning to groups 2 7 creating 2 7 displaying current user 4 6 list of groups for 1 4 2 6 3 3 3 13 managing 4 6 number of groups assigned to 4 6 setting up 2 6 user authentication 2 8 user level definition of 1 8 UV absorption instruments definition of 7 24 profiles for 7 24 V Calibration procedure 8 31 JV data determining extinction value and lin earity from 8 75 UV extinction from peak procedure 8 77 UV extinction from RI procedure 8 77 V vaf file extension 6 8 6 9 6 16 6 17 Validate command Experiment menu 6 11 8 9 9 9 View button Instrument list 5 5 View menu see also specific menu commands list of commandes in A 4 viscosity converting specific to intrinsic 8 57 determining mass from 8 69 ViscoStar definition of 1 7 ISI integrated 5 2 profiles for 7 21 voltages converting to Rayleigh ratios 8 56 vrf file extension 6 8 6 9 6 14 VS data determining mass from 8 69 vsf file extension 6 9 9 4 9 11 WwW warning signal in Diagnostic Manager 5 7 Waters Empower importing sample sets 9 5 websites 21 CFR Part 11 compliance 4 2 Wyatt Technology Corporation 1 9 Window menu
271. ing Procedure X means the procedure is in an invalid location in the sequence or it requires data from an instrument that is not in the configuration A procedure s state is always indicated by its icon as follows Collec tion procedures have a special two page icon for all states Procedure has not been run since the procedure was last mod ified E Procedure has been run successfully E Procedure is currently running Ex Procedure is in an invalid sequence location or does not have the necessary data to run 5 Drag the procedure to a position in the sequence where its icon changes to show it is in a valid location Deleting Procedures Experiment Builder You can delete procedure items only if you enable Experiment Builder mode by choosing System Preferences Experiment Builder Mode To delete a procedure from an experiment select the procedure and press the Delete key Sequencing Procedures Experiment Builder You can resequence procedure items only if you enable Experiment Builder mode by choosing System Preferences gt Experiment Builder Mode If a procedure is in an invalid location in the experiment sequence it will have a red X on its icon SQ Procedures E Basic collection 2 Despiking Procedure E Define baselines 3 Define peaks B Convert to physical units E Determine 42 mass and radius from LS data 2 Smoothing Procedure 8 8 M1000 Rev H
272. ing from instru ments 5 5 5 6 original 11 7 precision of increasing 8 44 processing of while procedure runs 8 3 raw 11 7 raw after each transform 11 7 Data graphical tab Diagnostic Manager 5 5 M1000 Rev H Index Data numeric tab Diagnostic Manager 5 6 data file exporting graphs to 11 17 data set definitions adding to experiment 6 28 creating 11 8 definition of 11 7 exporting 11 10 see also graphs Data Set Definitions folder Experiments tab 6 28 data sets definition of 1 6 11 7 list of 11 7 database system 2 3 Database operating tier 1 4 Database Properties dialog 4 5 databases see experiment database sys tem database DAWN calibrating 8 19 definition of 1 6 see also miniDAWN DAWN DSP calibration and laser alignment 8 19 ISI installation for 5 2 profiles for 7 12 turning laser on off and calibration 8 20 DAWN DSP F ISI installation for 5 2 profiles for 7 12 DAWN EOS ISI installation for 5 2 profiles for 7 12 DAWN HELEOS profiles for 7 11 DCOM settings 5 4 Debye fit method 8 62 D 12 Debye fit model 8 55 Debye plot checking normalization coefficients us ing 8 35 definition of 8 61 error bars in D 17 procedures using 8 61 default template 6 18 6 19 delay volumes determining 8 17 Delete command File menu 12 7 Delete Items menu command 4 8 6 19 9 11 12 7 deleting Index 3 Index experiments 4 8 sample sets 9 11 system profiles 1
273. instrument profile types that are available for a connection M1000 Rev H 7 29 Chapter 7 Configuring Experiments Sample Profiles A sample is the substance being tested It is dissolved in the solvent forming a solution The solution is placed in or flows through a sample cell A sample may be injected or may be a molecular standard used as a reference standard A sample profile stores information about samples to be used in experi ments Sample profiles are used by injector profiles and by instruments configured to run in batch that is standalone mode Sample Profiles A sample profile describes a sample for which you are determining properties You can set the following properties for a sample Table 7 19 Sample Properties Field Description Name Name of the sample If you have already created a system profile for this sample click and select a profile to use Description Description of the sample which typically contains more information than the Name dn de dn dc value associated with the sample in mL g The dn dc value is used when the sample concentration is to be determined using a refractive index instrument The value entered for the profile is used as a default value when peaks are set for the data A2 Second viral coefficient A value associated with the sample in mol mL g The value set here is used as a default value for peaks set in the experiment
274. ion about saving graphs to image files M1000 Rev H Using EASI Graphs Using EASI Graphs M1000 Rev H EASI graphs are intended to be used to quickly visualize results They are not saved as part of an experiment You can only have one EASI graph To begin creating an EASI Graph do one of the following e Choose Experiment EASI Graph e Right click an experiment tree folder and choose Manage gt EASI Graph from the right click menu The EASI Graph window opens and the Experiments tab lists an EASI Graph item that you can use to reopen this window during the current session B Experiments e EASI Comparison RES Bel Experiments YS LS RI online PS standards in THF vaf Although you cannot save it you can print an EASI graph by right clicking on the graph and choosing Print from the pop up menu To customize an EASI Graph follow these steps 1 Change any properties in the EASI Graph dialog 2 Click Apply to see the effects of your changes A message is shown if the experiment does not contain the data for the selected graph type 11 3 Chapter 11 Working With Graphs The EASI Graph dialog looks similar to this E FASI Graph LU CH relative scale ao CH chromatograms Iw vs ls ri online ps standards in thf time min chromatograms Distribution Type vs time or volume Chromatogram 1 Rayleigh ratio Chromatogram 2 differential refractive index Chr
275. ion and fit results for the peak and slice you select The quality of the fit can be determined from the left graph M1000 Rev H Analysis Procedures You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines Rh If you place multiple methods that deter mine Rh in a procedure only the first one will be valid Data points excluded from the calculations by the max and min properties are shown in red QELS data collection and analysis tolerates gaps that may occur in QELS data collection due to instrument problems The properties for this procedure are as follows Table 8 27 Rh from QELS Data Properties Field Description Peak Number The peak number of the displayed correlation function QELS Slice Index Abscissa Position Rh Displays the slice number for the correlation function You may type a slice number here This field shows the position on the x axis for the peak and slice selected This field is display only Shows the calculated hydrodynamic radius Rh value This field is display only Dt Shows the calculated translational diffusion Dt value This field is display only Processing Conditions gt Temperature Shows the temperature at which this slice was collected This field is display only gt Viscosity Shows the viscosity at this slice index gt APD Stat
276. ion and the polymer to be analyzed The dn dc value is required for universal calibration as it is necessary for intrinsic viscosity calculations but not for conventional calibration You can see example experiments that perform column calibration by choosing File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and opening the conven tional calibration or universal calibration vaf file in Sample Data gt Analyzed Experiments For an experiment template choose File gt New Experiment From Template and open the universal cal ibration template in the System Templates gt Viscometry folder For more go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Conventional and Universal Column Calibration When to Calibrate This procedure only needs to be performed when a new column is used or if you think the column has aged to a point that its behavior needs to be reassessed Analysis of a sample using universal calibration takes place in two logical phases First the column profile must be determined by measuring the behavior of a set of known molecular standards when passed through the column Once this determine column calibration phase is complete the unknown sample can be analyzed in a separate experiment 8 37 Chapter 8 Editing Procedures In an experiment this procedure must come after the Baselines and Peaks procedure
277. ion dialogs you can click the icon to change the view of the list of experiments in the database In the detail view the last data and time the experiment was modified is shown 4 Click Open You see the profile listed in the System Profiles tab of the workspace 12 5 Chapter 12 Working with System Profiles Editing a Profile To modify a system profile that you have opened follow these steps 1 Double click the item you created in the System Profiles tab of the workspace to open its property dialog If you created a profile type that has multiple components there will be a tab for each component For example a light scattering instru ment in batch mode has a tab for a solvent and a sample This infor mation is stored with and imported with the instrument profile 2 Edit the properties as needed See Chapter 7 Configuring Experi ments for details about the properties of all profile types WW System Profiles Samples Molecular standard std4 DER Description Reference Wavelength nm dn de mL g A2 mol mt 07 0 0000e 000 Molar Mass g mol 0 0000e 000 Value nm UV Extinction Coefficient mlg cm 0 0000e 000 Molecular standard std4 Si ok BK cancel SP Apply 3 Click OK or Apply to save your changes Information from a system profile is copied when it is used in an experi ment After being copied there is no link between the system profile and the experiment So editing a s
278. ion is specified as Equation 2 Ny nk k InFe D SIM The coefficient k is the Huggins constant For random coil polymers in good solvents the Huggins constant typically has a value between 0 0 and 0 3 In size exclusion chromatography the concentration of the sample is usually so dilute that one can ignore the concentration terms of third power or above and use the approximation Equation 3 l c Solving Eq 2 for intrinsic viscosity yields Equation 4 24 H UaK ln at ON Eq 4 is simplified to the following for computation Equation 5 1 An bk 1 yl 47 k In G 2 M1000 Rev H Calculating Intrinsic Viscosity Kraemer The Kraemer equation is Equation 6 1 1 n n a 7 Glaf Solving Eq 6 for intrinsic viscosity yields Equation 7 n 1 2 k y7 y x 0 n3 C C The expansion shows that for small values of specific viscosity which is almost always the case for chromatography the two formalisms are related Equation 8 k 1 24k Eq 7 is simplified to the following for computation Equation 9 S ail 4k In 7_ sp ZE In Solomon Gatesman M1000 Rev H The advantage of the Solomon Gatesman equation is that it does not require empirical constants However for values of specific viscosity much less than one it reduces to the Huggins Equation with a value of k 1 8 Equation 10 1 oui DEE 0 n c 3c Eq 10 is simpli
279. ion would appear as in Figure F 4 that is only two bins would have any intensity The correlation function is then modeled by adding the correlation functions for the two separate bins It is more complicated than this since there is cross correlation between the various components but for the sake of pedagogy the concept of adding is adequate 1 S W Provencher Inverse problems in polymer characterization Direct analysis of polydis persity with photon correlation spectroscopy Makromol Chem 180 201 209 1979 F 6 M1000 Rev H Regularization fractional intensity 0 1 1 10 100 1000 10000 Rh nm Figure B 2 Mode histogram for bimodal size distribution Figure F 4 Model histogram for bimodal size distribution More complicated correlation functions from more polydisperse samples could be modeled by the histogram method Intensity would be shifted between bins until the right match was found In so doing the underlying distribution would be revealed albeit in a somewhat jagged fashion from the bins We can remedy this jaggedness by making our bin sizes smaller and smaller until we get the true distribution In reality the histogram method breaks down long before enough bins can be added to accurately represent a distribution The problem is that as more bins are added the number of possible solutions explodes There is not enough information in the correlation function to accurately distribute the inten
280. ional to these parameters RI Calibration from Peak CT This template is used to calibrate any refractometer Wyatt or generic EXCEPT for the Optilab rEX which uses a special template Note that the main difference is in the experiment configuration For this template there is a pump injector light scattering instrument and refractometer To perform the calibration you need to enter the precise injected mass and to know the dn dc of the solvent The calculation of the calibration constant is based on finding the same calculated mass Optilab rEX Specific M1000 Rev H The following templates are provided in the RI Measurement gt Optilab rEX Specific folder Absolute RI Calibration CT The template is specifically for calibrating the Optilab rEX for absolute refractive index measurements Absolute measurements means that there is no reference to a standard So for these analyses the purge valve must be ON Inject at least three different solvents with known and different refrac tive indexes e g toluene THF and water RI Calibration from Peak AT This template is only used to calibrate the Optilab rEX For this template there is a pump injector and an Optilab rEX No other instruments are necessary Appendix B System Templates B 12 To do the calibration you need to enter the precise injected mass and to know the dn dc of the molecule in the specific solvent The calculation is based on the assumption
281. is procedure can be used in the same experiment as Distribution Analysis and other analysis procedures The procedure has the following dialog WW Mark Houwink plot PS706 vaf Procedures Mark Houwink Sele Marke Houwink plot Iw Mark Houvink plot jw Mark Houwink fit d Ei Be fe E bg o e o 2 gt 2 a T molar mass g mol K mLa EEEE a 7 350 0 001 e 1 Sa OK x Cancel E Apply The diagram shows the fit for the Mark Houwink Sakurada plot The pro cedure selects K and a parameters to make the red line as close to straight as possible The values of the K and a parameters vary depending on the polymer solvent and temperature The properties for this procedure are as follows Table 8 30 Mark Houwink Sakurada Properties Field Description K The resulting K fit parameter for the Mark Houwink Sakurada analysis a The resulting a fit parameter for the Mark Houwink Sakurada analysis For more about viscometry data collection and analysis go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Viscometry 8 84 M1000 Rev H Distribution Analysis Analysis Procedures This procedure analyzes the distribution of the geometric radius or other selected data item within the sample for an online experiment You can place this procedure at the end of the procedure sequence The procedure has the followin
282. is currently installed on the instru ment See the hardware manual for details Available for the miniDAWN TREOS only Temperature Control gt Enable Check this box if the instrument is set to maintain a specified temperature heated or cooled Available for the miniDAWN only Temperature Control gt Temperature If this instrument is temperature controlled specify the temperature to which it is set Use C Available for the miniDAWN only Band Broadening gt Enabled Band Broadening gt Instrumental Term Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broad ening parameters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box See Band Broadening on page 8 14 for an explanation of the instrumen tal term The units are in microliters Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters Auxiliary Channel Gains gt 1 2 Select the auxiliary channel gains for channels 1 and 2 The options are 1 10 100 and 1000 Available for the miniDAWN only WyattQELS Profiles A WyattQELS device is a Quasi Elastic Light Scattering device You can set the following properties for a WyattQELS ins
283. is display only Linearity Shows the linearity of the dn dc result This field is display only Fit Degree Type a fit degree to use for fitting the data The value may be either 1 or 2 Percentage to Keep Type the percentage of the peaks to use for calculations By default 100 is used If the plateau is flat not drifting in the peak range using the default value is recommended Linearity View Enabled Peaks gt You can omit peaks you marked from the plot by removing the checkmark next to 1ton the peak number Put a checkmark in this box to view the linearity plot For more about determining dn dc go to http www wyatt com solu tions software ASTRA cfm and follow the links to Data Analysis gt dn de and UV extinction determination Dn dc from Peak This procedure calculates the dn dc using the peak data from a batch mode run You can use data from a batch mode RI run to calculate either the calibration constant if dn de is known or dn de if the calibration constant is known See the manual for your RI instrument for details You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the dn dc or RI calibration If you place multiple methods that determine dn dc or RI calibration in a procedure only the first one will be valid There are no properties to set for this procedure It runs without prom
284. is includes saving experiments to an experiment database user accounts with access levels and sign off procedures In this manual the Database and Security icons both apply to this mode 1 3 Chapter 1 About ASTRA V This manual identifies information that is specific to these operating tiers using the following icons Table 1 1 Operating Tier Icons Icon Description z Identifies information that applies only to the ASTRA V Basic Basic operating tier Identifies information that applies to both of the followin Database pp S operating tiers ASTRA V with Research Database ASTRA V with Security Pack e Identifies information that applies only to the ASTRA V Security with Security Pack operating tier User Account Levels Security As part of the 21 CFR Part 11 compliance of ASTRA V with Security Pack all users must log in with a unique user id and password The administra tor sets up accounts with one of the following user account levels e ASTRA Administrator Can change database settings and can create modify and delete experiment files Also has privileges of Researchers Technicians and Guests e ASTRA Researcher Can create and modify experiment files Can connect to networked computers and instruments Also has privileges of Technicians and Guests e ASTRA Technician Can run a given experiment procedure sequence and save the resulting data Also has privileges of Guests
285. ist for the report expand the Template list and type a Description to appear at the top of the report Graphs Template Description summary File ong For the Template File click the button 6 Select a report file to use as a template for the report A number of summary and detailed templates are provided as XML web page files in the Reports folder under the ASTRA installation folder To custom ize reports see Customizing Report Formats on page 10 3 10 4 M1000 Rev H Applying Report Formats If you want to add any graphs to your report expand the Graphs list and check the boxes for any graphs you want to include The report displays graphs in the sequence you check the boxes The list and for mat of the graphs comes from the graphs in the Results section of your experiment and any graphs in the procedures in your experiment See Creating Custom Plots on page 11 10 for information on adding a graph to your experiment Graphs V Mean square radius vs volume Despiking Collection Note that you cannot add EASI graphs to reports because they are ageregates of multiple experiments and cannot be part of a particular experiment s report Click OK Choose Experiment Run to run or re run the experiment After the procedure runs successfully the report and other results are gener ated Applying Report Formats You can apply report
286. ith Research Database log information about actions performed that relate to experiments There are two types of logs created by ASTRA e System log Shows global actions that include logging in logging out creating importing exporting opening saving and deleting an exper iment and signing off on an experiment e Experiment logs Shows actions that relate to a particular experi ment These include actions involving procedures data set definitions reports templates and more This log shows when the procedures that make up an experiment were run As ASTRA Administrator you should communicate to researchers about the Sign Off procedures they should add to experiments they create These are likely required by your 21 CFR Part 11 policies and procedures Sign offs can store information about the user responsible for the experiment the user who approved the experiment or the user who reviewed the experiment Viewing the System Log To view the system log choose System Log Open WW System Log Event User PP Information 1 28 2008 11 15 094M database Experiment database connection 1 28 2008 11 15 094M database Experiment database disconnection COMP 1 28 2008 11 14 10 4M database Experiment database connection COMP 1 28 2008 11 14 10 4M database Experiment database disconnection COMP 1 28 2008 11 01 34 AM system Login COMP 1 28 2008 11 01 224M database Experiment database connection pre lo 1 28 2008 11 01 22 AM database S
287. itor Aux Channel Select the laser monitor option you want to use The options are Laser Monitor Forward Monitor and none Laser Monitor The light scattering signals are divided by the laser monitor which corrects for fluctuations in the laser intensity due to power fluctuations Forward Monitor The light scattering signals are divided by the for ward laser monitor which corrects for both laser intensity fluctuations and absorbance by the sample none No correction is performed for laser intensity fluctuations or sample absorbance Use this option only if signal levels are so low that digital noise from the laser monitor signal can contaminate data To use the Forward Monitor with the miniDAWN but not the minIDAWN TREOS you must route the instrument s forward laser monitor signal through one of the AUX inputs on the instrument No additional wiring is required for the minIDAWN TREOS to collect forward laser monitor data If you selected Forward Monitor in the field above specify which AUX input on the instrument receives the forward laser monitor signal Used for the miniDAWN only Disable Collection Check this box to disable data collection for this instrument For example if the light scattering instrument has the QELS option it is possible to disable the collection and collect QELS data alone Polarization Analyzer Check this box if the polarization option
288. ively Dark offsets are obtained with the laser turned off Note that dividing by the laser monitor signal compensates for any changes in laser intensity due to power supply fluctuations tempera ture drift laser aging etc As an example suppose we calibrate with toluene Pure filtered toluene has a Rayleigh ratio of 1 406 x 10 cm at a wavelength of 632 8 nm Suppose that using our DAWN or miniDAWN sample cell we observe a 90 scattering signal of about 1 V The laser monitor signal is factory set to be near 5 V and the dark offsets are much smaller than this so Eq 3 implies 1 Acscc is not displayed in the software 2 W Kaye and J B McDaniel Applied Optics vol 13 No 8 1974 pp 1984 1937 D 4 M1000 Rev H M1000 Rev H Measured Quantities and Calibration Acscc 7 0 x 10 cm Of course this is just an example and the measured constant may be quite different depending on the instrument and conditions laser wavelength etc Toluene provides a large scattering signal in fact toluene has the highest Rayleigh ratio of any of the common solvents and is thus highly desirable for use as a calibrator Many other solvents can theoretically be used for calibration of the DAWN or miniDAWN but we do not recommend them The astute reader will point out that since we know the Rayleigh ratio for toluene and since the scattering from toluene is relatively large we ought to be able to calibrate with toluene mea
289. k any folder in the experiment tree and choose Manage Add To Experiment E Add to experiment Procedures Lo 39 EI Graph Cancel E Report B Dataset Definition 2 Close or scroll past the Procedures node select Data Set Definition and click OK 3 Double click the untitled item that is added to the Data Set Defini tions folder in the experiment tree 4 Inthe Data Set Definition dialog type a name for this data set Typi cally the name should describe what you want to graph For example molar mass vs volume 5 In the Available list set one or more data set stages you want to graph data from Each of these stages represents the data after an individual procedure runs See Data Collection and Storage on page 11 7 M1000 Rev H Note Using Custom Plots and Data Set Definitions 6 Click the gt gt button to move the selected items to the Included list Available Included instrument data in physical units des mass and radius data mass and radius data fitted concentration data despiked sr A distributions and moments a original data gt gt raw data gt gt raw data despiked raw data despiked smoothed raw data despiked smoothed b lt II gt E gt 7 Select an item you have moved to the Tice ore IBEERE a Included list In the list of data in that stage place checkmarks next to the data you want to graph concentration
290. king for Connections or Instruments 4 Browse the system database for a profile to import 5 When you find the profile select it and click Open The item is added to your experiment If you want to import an entire configuration see Replacing an Entire Configuration on page 12 9 No pre defined instrument profiles are provided with ASTRA In order to have instruments available you need to first save instrument profiles in your system database as described in Creating Profiles on page 12 3 Replacing a Single Configuration Item You can copy all the properties set for a system profile into the property dialog for an item in an experiment configuration This has the effect of replacing the item in the experiment with a system profile To copy from a system profile to an experiment follow these steps 1 Double click on an item in Configuration tree in the Experiments tab of the workspace to open its property dialog M1000 Rev H Using Profiles 2 Click the browse button to the right of the Name property Name pump to injector Description Source Instrument Generic pump Ei Destination instrument Manual injector EI 3 In the Copy System Profile dialog find the profile you want to copy from The Of Type field is automatically set to match only the type of item you are editing 4 Click Copy The values of properties in the system profile are copied to the experiment
291. l a M1000 Rev H In the Physical Instrument row select an instrument to use for data collection from the drop down list Select the tab for the solvent In the Name row click the button on the far right In the Copy System Profile dialog open the System Solvents folder and select the solvent you are using For example you may be using toluene Then click Copy The properties for the solvent you select automatically replace those of the default solvent Click Apply or OK at the bottom of the properties dialog 3 7 Chapter 3 Getting Started 3 8 Just by setting two properties you have created an experiment that can be run The templates provided with ASTRA make it as simple as possible to get to the point where you can run an experiment You can set other properties for the experiment instrument or solvent if you like Configurations are described in detail in Chapter 7 Configuring Experiments Modifying Procedure Settings At this point you could run the default experiment However to show you more about using ASTRA we ll set the duration of the data collection Follow these steps 1 In the Procedures node double click the Basic collection procedure This opens the Basic collection dialog 2 If necessary resize the dialog to see the Duration property You can also drag the divider between the graph and the property list to resize the graph zH apa punoo qdadadaadaaadaad 40 60
292. l at once 11 If you checked the box to iterate select the items to iterate 12 Click OK to save your changes Using Data From Multiple Experiments If you want to graph data from multiple experiments you can copy data from one experiment to another The copied data is shown in the Avail able list in the Data Set Definition dialog See Copying Data on page 6 21 for details Other Uses for Data Set Definitions When you export an experiment you can choose to export the data matching a data set definition as a tab delimited or comma delimited text file For details see Saving an Experiment to a File on page 6 16 or Exporting an Experiment on page 6 17 Creating Custom Plots In Run mode you can add custom graphs by choosing Experiment Graph Add Custom Plot from the menus This opens the Data for Custom Plot dialog and you can specify the data to display Both the data set definition and results graph are created You can also add graphs as follows if you enable Experiment Builder mode by choosing System Preferences Experiment Builder Mode 1 Create a data set definition for the data to be graphed as described in Creating Data Set Definitions on page 11 8 M1000 Rev H Using Custom Plots and Data Set Definitions 2 Choose Experiment Add to Experiment This opens the Add to Experiment dialog which allows you to add items to the Procedures Data Set Definitions and Results folde
293. l cleaner for a specified duration You should also check the Comet Cell Cleaner box in the configuration dialog for the light scattering instrument See the COMET hardware manual for more informa tion about the COMET cell cleaner Details gt Recycle Valve Post Collection Controls how the Recycle valve is set at the end of the collection This may be set to waste or recycle During a collection the Recycle valve is always set to waste You can change the Duration while an experiment is running However you cannot change the collection interval or auto inject signals after collec tion has started The Basic Collection procedure automatically closes all purge valves switches recycle valves to waste by default and deactivates the COMET feature prior to data collection The exception to the closing of purge valves is when absolute RI analysis is conducted the Optilab rEX purge valve is left open as required Script Collection You can customize your data collection by using a script collection To learn more about writing collection scripts and the scripting language see Appendix C Data Collection with Scripts Several simple collection scripts are included with ASTRA V and are used for example in the experiment templates for light scattering calibration and in utility tem plates for turning off the later and for using the COMET cell cleaner The graph in the Script collection procedure
294. lace Configuration Import a configuration item or entire con R A figuration from a system profile See page 7 7 and page 12 4 Save Configuration As Export a configuration item or an entire R A configuration to a system profile See page 7 8 and page 12 3 gt Alignment Opens procedure dialog for determining R A the interdetector delay See page 8 17 Band Broadening Opens procedure dialog for correcting R A effects of fluid mixing between instru ments See page 8 14 Normalize Opens procedure dialog for relating R A detector signals to the 90 degree detector signal and the instrument calibration con stant See page 8 32 Calibrate Column Perform a column calibration for a SEC R A column See page 8 37 Experiment Add To Ctrl Shift P Add a procedure report graph or data Builder Experiment set definition to an experiment See R A page 8 7 page 10 4 and page 11 10 Experiment Copy From Copy data from one experiment to the cur R A rent experiment See page 6 21 Experiment Apply Apply procedures and results to the cur T R A Template rent experiment creating a new experi ment See page 6 22 Experiment Validate Validate the experiment procedure T R A sequence and instrument availability See page 6 11 Experiment Run Ctrl Shift R Start the experiment run See page 6 11 T R A Experiment Run Run the experiment data collection until TRA Indefinitely manually stopped See page 6 12 Experiment S
295. lar Ma Peak Number Slice Index Model Fit Degree Abscissa Position Concentration Conjugate dn dc Conjugate UV ext Enabled Detectors BY OK XK Cancel Iw Iv Iw detector 90 UV absorbance data differential refractive index data o in relative scale CH CH 5 0 10 0 volume mL 15 0 8 573 0 096 e 4 g mol 10 2 4 5 nm 0 601 0 000 5 155 0 058 e 4 g mol 3 419 0 104 e 4 g mol 1 917 zimm 1 7 583 mL 6 328 0 001 e 4 g mL 0 165 0 000 mL g 1172 4 0 2 mL fg cm The left graph shows a Zimm plot of the results The right graph shows collection data and peaks The properties for this procedure are as follows Table 8 33 Protein Conjugate Analysis Properties Field Description Molar Mass Shows the total molar mass for the currently selected peak Radius Shows the calculated radius for the currently selected peak The type of radius Protein Fraction peak This field is display only Shows the protein fraction for the currently selected peak RMS or geometric displayed depends upon the LS fit model specified for the Protein Molar Mass Modifier Molar Mass The molar mass of the protein indicated by the selected peak The molar mass of the protein modifier indicated by the selected peak Peak Number M1000 Rev H Click on the peak for which you want to view values 8 89 Chapter 8 Editing Procedures Table 8
296. larm names Get controller computer name Read dark current Get list of detector angles Get the attributes of the instrument Get current state of the instrument Get the current reporting interval Get IP address and mask Get any parameter by name Get list of parameters in config file Get description of data slice contents Get interval between data slices Get list of switches Get temperature controller parameters Get time constant of the filters Set Parameters To send a command follow these steps 1 Select an instrument from the drop down Instruments list If you add computers using ASTRA s Instrument list or you connect additional instruments after opening the Diagnostic Manager click Refresh to update the drop down list Select the command you want to send to the instrument If the command you selected requires any parameters the Send Com mand button is disabled until you set those parameters If this is the case click Set Parameters to open a dialog that asks you for the appropriate parameter values M1000 Rev H Using the Diagnostic Manager For example if you set the collection interval you will be asked to pro vide the new interval in seconds Set Parameters Parameter Name Parameter Value Interval Cancel Type or select the parameter value and click OK 4 Click Send Command Viewing the Command Log with the Diagnostic Manager The Command log tab of the Diagnosti
297. lates include a summary report and a detailed report Typically these reports will need little or no modification You can simply view and print the reports produced when you run an experiment To view a report double click on its name in the Results list of the experi ment tree in the workspace Operator Names in Reports Basic If you are using ASTRA V Basic the operator names shown in reports are assigned as follows Processing Operator This is determined at the time the report is generated It is the Microsoft Windows currently logged in user So on the Aqueous 1 machine this will show up as Aqueous 1 Collection Operator This is determined at the time data collection is started It is also the currently logged in user obtained from Microsoft Windows This username is stored with the collected data so that it is retained even if processing and report generation is per formed by another user Database If you use ASTRA V with Research Database or ASTRA V with Security Pack the operator names shown in reports are assigned as follows 10 2 Processing Operator This is determined at the time the report is generated It is the username for the user currently logged into ASTRA The Microsoft Windows currently logged in user is ignored Collection Operator This is determined at the time data collection is started It is also the username for the user currently logged into ASTRA The Microsoft Wind
298. lder Mode 8 18 M1000 Rev H Configuration Procedures LS Calibration M1000 Rev H A DAWN instrument needs to be calibrated to enable ASTRA to convert its signals to Rayleigh ratios You must determine its calibration constant before using ASTRA to calculate absolute molar masses Calibration should be performed in batch mode that is before connecting the DAWN to a fractionation system This section describes the behavior of the separate procedure that performs calibration In practice this procedure is not used in isolation Instead you create an experiment using the calibration template for your instrument That experiment contains a number of procedure items that run in sequence to turn the laser on and off set peaks and more When to Calibrate Wyatt Technology calibrates each DAWN during manufacture and includes the calibration constant on the Quality Control report shipped with the instrument However you should calibrate the DAWN in your own lab and compare the value you obtain with the value on the QC report to verify that no internal damage occurred during shipment A DAWN should be recalibrated for any change that may affect the value of the scattering signal at the 90 detector Calibrate if you e Disassemble the flow cell e Change the 90 detector photo diode e Change the laser gain e Change the jumper setting on the amplifier PCB for the 90 detector If you do this make sure you change the correspo
299. ler radius so g M will lie between 0 and 1 g M The Radius and Viscosity branching ratios are related by a term called the drainage parameter e as follows geg 1 B H Zimm and R W Kilb J Polym Sci 37 19 1959 D 20 M1000 Rev H Branching Calculations ASTRA uses the raw intrinsic viscosity vs MM data for both the linear and branched files For a number of points 300 points per decade of molar mass Eq 38 is applied In order to obtain useful branching information the two files linear and branched should overlap as much as possible in molar mass The branch ing ratio g M can only be calculated in this region of overlap since only in this region can intrinsic viscosity be found at the same molar mass To use this method select the Viscosity method in the Branching proper ties view See Branching on page 8 80 Branching Per Molecule The number of branches per molecule is related to the branching ratio but some knowledge of the type of branching is necessary You can choose either trifunctional Y or T or tetrafunctional X branching and monodis perse or polydisperse slices These formulas relate 8m to B for randomly branched polymers Trifunctional e Polydisperse Equation 39 P 6 US 1 2 H 8 1 g y y i B 2 K 2 SI B e Monodisperse Equation 40 Z S L a 4B g ek Ee Tetrafunctional e Polydisperse Equation 41 In 1 B e Monodisperse
300. line Some criteria are already entered in the template in the LS Noise procedure WW Experiment2 Procedures S Noise Value Short Term Hoise Interval min 0 500 Short Term Hoise Limit V 1 000e 005 Wander Interval min 5 000 Wander Limit V 1 000e 005 Drift Limit V min 4 000e 006 Peak Number 0 whole range 1 The report indicates whether the measured noise levels are within specification With QELS Batch CT Use this template to do batch QELS measurements Inject the sample directly into the light scattering detector The Rh from QELS graph is displayed in the Rh from QELS procedure Online CT Use this template to do online QELS measurements Run the sample through the chromatography system and detectors The Rh from QELS graph is displayed in the Rh from QELS procedure Regularization AT Apply this template to perform a regularization regression which permits the calculation of the size distribution of a sample For example if you have a bad separation and several entities leave the column at the same time regularization reveals this co elution because there will be several peaks with the regularization Cumulants AT Similarly to the Regularization analysis applying this template calculates the size distribution of the sample This template fits the correlation function data to a cumulant distribution M1000 Rev H Light Scattering Particles AT
301. llow these steps 1 Choose Experiment Add to Experiment This opens the Add to Experiment dialog which allows you to add items to the Procedures Data Set Definitions and Results folders of the experiment Shortcuts Press Ctrl Shift P Right click any folder in the experiment tree and choose Manage Add To Experiment 6 26 M1000 Rev H Adding Elements to an Experiment 2 Open a folder under Procedures and select the procedure you want to add See Chapter 8 Editing Procedures for descriptions of all proce dures E Add to experiment Cancel ee SEN Collection LC Configuration B Absolute RI calibration B Calibrate column Determine band broadening Determine interdetector delay Determine LS calibration ou a a Determine Fil calibration B Determine UY calibration EB Normalization Transform Results B Dataset Definition 3 Click OK Sequencing Procedures Experiment Builder You can change the sequence of the procedure only if you enable Experi M1000 Rev H ment Builder mode by choosing System Preferences Experiment Builder Mode If a procedure is in an invalid location in the experiment sequence it will have a red X on its icon SQ Procedures Basic collection 2 Despiking Procedure 2 Define baselines Define peaks 12 Convert to physical units 2 Determine A2 mass and radius from LS data 34 Smoothing Procedure To correct the problem drag th
302. llow these steps 1 In the experiment tree select an item in the experiment you want to close 2 Choose File Close Right click the experiment name in the tree and choose Close Choose le Close All to close all experiments at once unless data col lection is in progress 3 Ifyou have made unsaved changes you are asked whether you want to save them Saving an Experiment to the Database Database If you use ASTRA V with Research Database or ASTRA V with Security Pack experiments are saved in the ASTRA database To save experiments in separate files see Exporting an Experiment on page 6 17 It is a good idea to save experiments frequently Security You must have at least Technician access to save an experiment Shortcuts To save an experiment follow these steps 1 Choose File gt Save Press Ctrl S Click the NM icon Right click the experiment name in the tree and choose Save Shortcuts 2 If this is the first time you have saved this experiment you see the Save As dialog Otherwise you are finished saving the file Type a name for the experiment If you want to store this experiment in a subfolder click the cl New Folder icon and type a name for the folder Then open the folder 5 Make sure the Of Type field shows Experiments For information about saving templates see Creating a Template on page 6 18 6 Click Save To save an experiment with a different name or loc
303. location in the system database where you want to save this template The recommended location is the My Templates folder or a subfolder you create in My Templates You can create a subfolder by clicking the cl New Folder icon and typing a folder name Then open the new folder 3 Check the Make Default box if you want this template to be the default for use with the File New Experiment from Default command 4 Click Save Note Templates are saved in the system database This database is separate from the experiment database 6 18 M1000 Rev H Creating a Template Setting a Default Template You can specify the default experiment template by choosing System Preferences Set Default Template In the Select Experiment Template dialog choose an existing template from the My Templates folder or the System Templates folder and its subfolders and click OK E Select Experiment Template Look in IC Light Scattering e Es e i Baseline Utilities L d Er Ej Subtraction plot File name fonine Files of type Experiment Templates D Cancel Current online A2 The Current line shows the currently selected default template When you choose File New Experiment from Default the template you selected will be used to create a new experiment The default template is also used when you create a blank sample set as the default template for all samples in the set You can also set the default template when saving an experime
304. lt and will be explained shortly A more serious problem is that due to the geometry of the sample cell in the DAWN or miniDAWN each detector sees a different scattering volume V and subtends a different solid angle with respect to the scatter ing volume Refractive index differences among various solvents and sample cells exacerbate this problem Calculating V for each detector is thus extremely complex and prone to inaccuracies The solution is to use the proportionality between Rgand Ig in Eq 1 to derive a calibration factor which gives the correct value of Rg for a known scatterer Fortunately some common solvents have been thoroughly studied and their Rayleigh ratios are well known allowing us to use the GPC solvent itself as the calibration standard Using pure solvent as the scattering standard makes the calibration completely independent of any polymer sample Let us first consider scattering at 90 We absorb not only the detector sen sitivity but also all the geometrical volume and solid angle factors into a single Configuration Specific Calibration Constant called Acgcc By using the proportionality between detector voltage and light intensity Eq 1 may be written as Equation 8 Voo Voo dark l V laser laser dark Ry Al where Mag and Mag dark are the 90 detector signal voltage and dark offset voltage respectively and Viggo and Mise dark are the laser monitor signal and dark offset respect
305. luctuation in each detector s output including all photodiodes and the AUX signals Each detector is weighted based on the fluctuations noise seen in the first and last 10 of the data points up to 100 data points Whichever end is least noisy is used to calculate the weighting factor For batch mode cal culations data points within each plateau are used to calculate the detector weighting factors for each concentration The error bars in the Debye plot do not represent this weighting factor directly The Debye plot involves performing an nth order polynomial fit Ry Kc to for the Debye Fit Method Ke R for the Zimm Fit Method JK ei R 0 for the Berry Fit Method or P for the Random Coil Fit Method The error bar calculation therefore involves the weight ing factor the normalized R theta value as well as a concentration uncertainty factor and the Chi squared value returned from the fit If the normalization is off for some detectors then the Chi squared value from the fit tends to increase causing all error bars to grow Hence changes to the normalization coefficients will affect the error bars shown in the Debye plot as well as the uncertainties in the overall peak results The different errors combine according to the usual rules for propagation of errors to yield a standard deviation depending on calculation method for each slice These in turn allow calculation of uncertainties in the molar mass and size for e
306. lues If not you need to specify the index start and increment in the following fields Index Use This property is not yet implemented M1000 Rev H 11 9 Chapter 11 Working With Graphs Table 11 3 Data Set Definition Properties Field Description Index Value This property is not yet implemented Column Use If you are using a matrix tag you can specify how the columns are to be used Column Value Select which column values to use The options are DSD_USE_ALL Use all column values DSD_CONTROL_VARIABLE This option is not yet implemented DSD_AT_VALUE Use only one column with the value specified below If you are using a matrix tag and are using only one column this is the value of the column to use For example 90 degrees for the right angle detector Index Start If you are using a function tag and are not calculating the function from data specify the starting index Index Space If you are using a function tag and are not calculating the function from data specify the range the x axis should span Index Steps If you are using a function tag and are not calculating the function from data Show Uncertainties specify the total number of index points Check this box if you want the graph to contain uncertainty error bars for this item 10 Put a checkmark in the Iterate Experiment Data Over Injection box if there are multiple injections in the experiment that are to be dis played al
307. m e Alarm Name This column tells what type of alarm occurred e Severity The level of alarm severity Instrument Which instrument sent the alarm e Description More information about the alarm Time The time the alarm occurred e Status Shows whether you have acknowledged an alarm Acknowl edging an alarm hides the blinking circle on the bottom of the Diagnostic Manager window To acknowledge an alarm right click on this column and select Acknowledged You can resize the dialog and its columns to see more text in a column 5 7 Chapter 5 Interfaces to Instruments Sending Commands with the Diagnostic Manager The Commands tab of the Diagnostic Manager allows you to send commands to instruments The list of commands you can send is different for each type of instrument The following figure shows the commands available for the DAWN HELEOS instrument Diagnostic Manager Data graphical Data numeric Alarm Commands Command log Property Instrument wyatt sft h DAWN HELEOS 5 8 CommitDisk ConfigHeater ConfigNetwork control Get larmDescription GetComputerN ame GetDarkCurrent GetDetectorList Getlnstrumentinfo GetlnstrumentStatus Getlnterval GetNetContig GetParameter GetParamList GetSliceDescription GetSlicelnterval GetSwitchList GetTempCtrl GetTimeConstant lt Set heater P and parameters Config IP address and mask Control uncontrol instrument Get list of a
308. maintain a specified temperature heated or cooled Temp Controlled Line gt Temperature If the line is temperature controlled specify the temperature to which it is set Use C Detector Amp Gains gt 1 18 1 8 for DAWN 8 Select the detectors gains to use for each laser detector The options are 1 21 and 101 Auxiliary Channel Gains gt 1 2 Select the auxiliary channel gains for channels 1 and 2 The options are 1 10 100 and 1000 miniDAWN and miniDAWN TREOS Profiles You can set the following properties for a miniIDAWN or miniDAWN TREOS instrument Table 7 4 minIDAWN and miniDAWN TREOS Instrument Properties Field Description Name Name of the instrument If you have already created a system profile for this instrument click and select a profile to use Description Description of the instrument which typically contains more information than the Name Physical Instrument Sample Cell Wavelength Choose an instrument from the drop down list If your instrument is not listed choose Browse to open the Instruments dialog See Accessing and Viewing Hardware on page 2 10 Type of sample cell used during data collection The options for the miniDAWN are K5 F2 and MicroCuvette The options for the minIDAWN TREOS are K5 F2 Scintillation Vial MicroCuvette and Magic glass The wavelength of the laser that produces scattered light from the sample cell
309. ment to a file or save experiment data to a tab page 6 17 delimited or comma separated values file Close Close the current experiment page 6 15 Save Database Save an experiment to the experiment database page 6 15 Basic Save an experiment to a file and page 6 16 Save As Database Save experiment to the database with different name page 6 15 Basic Save an experiment to a file with a different name and page 6 16 Export as Text or CSV Create a text or comma separated file containing experiment page 6 17 data Save As Template Save the configuration procedure and results formats so that page 6 18 they can be used as the basis for future experiments Delete Delete the experiment from the experiment database page 6 20 Run Run the experiment procedure page 6 11 Run Indefinitely Run the experiment procedure ignoring the Duration page 6 11 Copy From Copy results data from one experiment to another page 6 21 Apply Template Create a copy of the experiment Then apply the analysis proce page 6 22 dures and result presentation from the selected template to the copy of the experiment Add to Experiment Add items to the configuration procedure or reports Experi page 6 24 ment Builder mode only Creating New Experiments The recommended way to create an experiment is from a system template provided with ASTRA For more customization you may want to save experiments as templates and use those Experiment Builders may choo
310. ments Debye Fit Method First construct a Debye plot excluding the optical constant and concentra tion terms that is a plot of Rovs sin 0 2 Second fit a polynomial in sin 0 2 to the data and thereby obtain the intercept at zero angle Ro as well as the slope at zero angle my d R dl sin 6 2 Equation 2 37 Whe ER 0 A 167 R J Chem Phys 16 1093 1099 1948 M1000 Rev H Determination of Sizes Zimm Fit Method To compute mean squared radius from the Zimm fit we must fit 1 Rg vs sin 0 2 Fit the Zimm equation to these data and thereby obtain the intercept at zero angle R and the slope at zero angle mg d 1 Rg dl sin 6 2 J Equation 3 2 Am R Ce 167 Berry Fit Method To perform calculations with the Berry method we must fit to 4 1 R vs sin 2 and thereby obtain the intercept at zero angle Ro and the slope at zero angle my d 1 R df sin 0 2 Equation 4 1 3g hf Ry Ja Random Coil Fit Method To derive the Random Coil fit method we go back to Eq 1 but instead of fitting a polynomial to Rgvs sin2 2 as in the Debye method we insert into Eq 1 the theoretical form factor P for random coils which was first derived by Debye Equation 5 POs 1a u where u 47 A sin 0 2 Since P is a nonlinear function of its parameter lt r gt we use an itera tive nonlinear least squares fit to the Zimm formalism Unlike t
311. mmands are issued using the following syntax instrument command where instrument is the instrument reference obtained by the Create method defined in Interacting with Instruments on page C 3 For example the following commands turn the recycle valve of a light scattering instrument to the waste position lsInst LSInstrument Create Turn the recycle valve off lsInst SendCommand SetSwitch Recycle F The complete set of instrument commands is shown in the following table Command Effect GetInstrumentLabel Get a string version of the instrument s name Use ful for generating messages to the user Enabled Return true or false indicating whether the Dis able Collection flag in the instrument configuration is enabled SetCollectionInterval seconds Set the instrument data collection interval in to the specified number of seconds SendCommand command Tell the instrument to perform a particular com mand command is an instrument command string described elsewhere StartCollection Tell the instrument to begin transmitting data to ASTRA This provides finer control over when instruments begin transmitting collection data Static Light Scattering Instrument Commands All static light scattering instruments understand the following messages Commands are issued using the following syntax instrument command where instrument is the instrument ref
312. mmation If the sum of the mean square radii is positive ASTRA will calculate the root mean square averages If it is neg ative the resulting root mean square averages will be set to zero In addition to the above if any of the slices to be included in the averages have uncertainties larger than the values themselves ASTRA will exclude them from the averages When plotting data in the Distribution Plots ASTRA removes any slices that have negative values Differential Distribution Calculations D 18 ASTRA V uses an adaptive binning technique for determining the differ ential distributions It works both with the direct results and with data that has been fit with results fitting M1000 Rev H Branching Calculations Branching Calculations ASTRA performs a number of sophisticated branching calculations These are described below Branching Ratio Radius Method The branching ratio gy is formally defined as Equation 36 2 br Zur r Din M where lt r gt p and lt r gt are the mean square radii of branched and linear unbranched polymer samples to be compared Note that the ratio is taken at the same molar mass not at the same volume In general for a given molar mass the branched polymer will have a smaller radius so gu will lie between 0 and 1 ASTRA calculates gy this way If no results fitting method has been selected ASTRA uses the raw RMS Radius vs MM data for both the linear and branched
313. mn item 3 In the Calibration Technique field select the type of calibration you performed on this column M1000 Rev H 8 95 Chapter 8 Editing Procedures 4 Press the button next to the Column name and select a column calibration you performed earlier Z Experiments E Sample Sets 4 gt e EASI Comparison EB EASI Graph Dal Experiments Bn Experiment3 S Configuration L5 Y5 bio column Ei Generic pump pump 0 75 Description H Injector injector 200 uL Plate Count DAWN HELEOS HELEOS Asymmetry Factor i EA ViscoStar viscoStar 100 E Optilab DSP Optilab DSP Resolution 0 0000 E Calibration Technique Universal with Viscometer Data wf Fluid connection pump to Flow Marker mL 10 4763 A Fluid connection injector l Mark Houwink Sakurada K mL g 1 832e 002 EN Fluid connection LS to v5 Mark Houwink Sakurada a 6 903e 001 wf Fluid connection V5 to RI Conventional Calibration Function PT Aux channel connection F gf Auto inject connection LS Universal Calibration Function WW Experiment3 Experiment Configurations Com qf Fluid connection column t e G Procedures Mlali Solvent thf Generic column column Optilab DS C Data Set Definitions a LC Results SN OK E Cancel The column profile is updated with the coefficients from the known sample run 5 Select the particular instruments used at your location con
314. mport menu com mand 9 5 Exit command File menu 3 15 Experiment Builder Mode command list A 2 Experiment Builder mode 1 2 1 3 3 13 experiment configuration adding connections to 7 6 adding instruments to 7 6 column profiles 7 26 connection profiles 7 28 definition of 3 6 6 2 editing 7 5 exporting to system profiles 7 8 importing system profiles into 7 7 12 8 12 9 injector profiles 7 25 light scattering instrument profiles 7 11 properties in 7 9 pump profiles 7 25 refractive index instrument profiles 7 17 sample profiles 7 30 solvent profiles 7 32 UV absorption instrument profiles 7 24 see also configuration procedures experiment database changing 2 9 4 5 database types supported by 4 4 default name for 2 9 4 4 definition of 1 6 4 3 files as alternative to 5 3 opening 6 7 saving experiments to 6 15 viewing current database 4 5 experiment files definition of 5 3 exporting experiments to 6 17 extensions for 6 16 9 10 file extensions for 6 16 6 17 importing 6 9 opening 6 8 saving experiments to 6 16 M1000 Rev H types of 6 8 6 9 experiment logs copying event text to clipboard 4 8 definition of 4 7 navigating 4 8 viewing 4 7 Experiment menu list of commands in A 5 see also specific menu commands Experiment Open menu command 6 7 6 8 experiments baselines for 3 9 8 45 closing 6 15 configuring see configuration proce dures experiment configu
315. n Stop The x axis ending point for the peak The units are determined by the Abscissa Units property of the experiment configuration LS Analysis gt Model Set this parameter when you perform any of the following analysis procedures Radius from LS Data Mass and Radius from LS Data Number from LS Data Protein Conjugate Analysis The available models are Zimm the default Debye Berry random coil sphere Mie coated sphere and rod See Choosing a Fit Model on page 8 55 for details LS Analysis gt Fit Degree dn de If you selected Zimm Debye or Berry as the fit model you must specify the fit degree here The fit degree default is 1 The range is 0 to 5 for the DAWN and 0 or 1 for the miniDAWN See Choosing a Fit Model on page 8 55 for details The dn dc value for this peak Set this parameter if you are performing any of the following analysis procedures Mass and Radius from LS Data Differential RI Calibration UV Extinction from RI Protein Conjugate Analysis If you are performing a Protein Conjugate Analysis this parameter corre sponds to the dn dc value for the protein A2 The second viral coefficient for this peak Set this parameter if you are perform ing any of the following analysis procedures Mass and Radius from LS Data Protein Conjugate Analysis Caution If you enter too large a second virial coefficient the molar mass may become negative or artificially large depending on the m
316. n be monitored in the Samples tab After a sample has been run its row in the Samples tab is shown with a blue background After the full sample set has been run a message says Sample set run complete 9 9 Chapter 9 Using Sample Sets Stopping a Sample Set Shortcuts To stop a running sample set choose Sample Set Stop Click the Stop Sample Set icon in the experiment toolbar A message says Sample set run manually stopped Stopping a sample set with ASTRA stops only the collection and analysis of data It does not affect any activity going on outside of ASTRA s control See your hardware documentation for information about alarms emer gency stops and setting up safety interlocks Alarms may be monitored via the Diagnostic Manager See Viewing Alarms with the Diagnostic Manager on page 5 7 for details Viewing a Sample Set Log Database To view a log for a sample set choose Sample Set Log gt Open Double click on a line to see more details You can choose Sample Set Log gt Save As to save the log to a file Saving Sample Sets Shortcuts Basic Database 9 10 To save a Sample Set follow these steps 1 Choose File gt Save Press Ctrl S Click the NM icon 2 If this is the first time you have saved this sample set you see the Save As dialog Otherwise you are finished saving the file 3 In the Save As dialog navigate to the folder you want to contain
317. nabling Experiment Builder mode 7 10 M1000 Rev H Light Scattering Instrument Profiles Light Scattering Instrument Profiles An instrument is any hardware device used in an experiment Light scat tering instruments measure the molar mass RMS radius and second virial coefficient of a sample via Rayleigh scattering DAWN HELEOS and DAWN HELEOS 8 Profiles The DAWN HELEOS is the default light scattering instrument in most light scattering experiment templates You can set the following proper ties for a DAWN HELEOS or DAWN HELEOS 8 instrument Table 7 2 DAWN HELEOS Properties Field Description Name Name of the instrument If you have already created a system profile for this instrument click on the far right and select a profile to use Description Description of the instrument which typically contains more information than the Name Physical Instrument Choose an instrument from the drop down list If your instrument is not listed choose Browse to open the Instruments dialog See Accessing and Viewing Hardware on page 2 10 Sample Cell Select the type of sample cell used during data collection The options are K5 F2 Scintillation Vial MicroCuvette and Magic glass Wavelength The wavelength of the laser that produces scattered light from the sample Calibration Constant Normalization Coefficients gt 1 18 1 8 for HELEOS 8 cell and its contents nm Type th
318. ncel X Apply The graph shows a plot of the data and the linear regression The red fit line provides visual feedback as to the quality of the fit This procedure has the following properties Table 8 11 Column Calibration Properties Field Description Equation Shows the resulting equation from the linear regression The coefficients are the ones that will be stored in the Column profile Standard Error 8 40 Also known as the residual standard deviation this shows the standard devia tion of the observed data from the fit values If the fit degree equals the number of degrees of freedom this is zero the fit is the same as the observed values M1000 Rev H Configuration Procedures Table 8 11 Column Calibration Properties Field Description R 2 Shows the adjusted R squared value from the fit This quantity can be used to gauge the quality of a linear fit The closer this is to one the better the fit If the fit degree equals the number of degrees of freedom this is set to zero to indi cate that statistical interpretation of the results is not possible Shows the square root of the adjusted R squared value The closer this is to one the better the fit If the fit degree equals the number of degrees of free dom this is set to zero to indicate that statistical interpretation of the results is not possible Technique The type of column calibration to perform The opti
319. nces Experiment Builder Mode You must have at least Researcher access to add delete or sequence procedures Adding Procedures You can add procedure items only if you enable Experiment Builder mode Shortcuts M1000 Rev H by choosing System Preferences Experiment Builder Mode To add a procedure to an experiment follow these steps 1 Choose Experiment Add to Experiment This opens the Add to Experiment dialog which allows you to add items to the Procedures Data Set Definitions and Results nodes of the experiment Press Ctrl Shift P Right click any folder in the experiment tree and choose Manage Add To Experiment E Add to experiment SES Lox GC Analysis Cancel 9 Collection C Configuration B Absolute RI calibration B Calibrate column Determine band broadening Determine interdetector delay Determine LS calibration Determine AI calibration B Determine UY calibration EB Normalization Transform LC Results B Dataset Definition 8 7 Chapter 8 Editing Procedures 2 Open a folder under Procedures and select a procedure to add 3 Click OK The procedure is added to the end of the experiment 4 The procedure is likely to E Procedures have a red X on its icon Basic collection when it is placed at the end 2 Despiking Procedure of the experiment The red 2 Define baselines 3 Define peaks Convert to physical units 5 Determine 42 mass and radius from LS data 2 Smooth
320. nco csc a ae Sette es a kaaa AAEE EEREN Enako F 6 THOON EE F 6 Implementation of Regularization in ASTRA V ssesssesssesesiessreseessriesrrrssrrsssrnsens F 7 Interpreting Regularization RESUItS usssssesssssrrssserrreseirrrssrrirnnssriennnnnennsnstennnne F 8 Appendix G Viscosity Theory sssssssssnssnnnnnnnnnnnnnnnnnnnnnnnnnnnnn G 1 Calculating Intrinsic Viscosity EE G 2 Leift LEE ENG G 2 IKPASINOl PE T E ees TA EEEE Eege E tema ee teed eet eer G 3 Solomon Gatesman ceccecececeeceeeeeeceaeeeeaaeeceeeeeeeaaeeceeaeseeaaeeseeeeeseaaeesseneeessaeeseeneees G 3 Intrinsic Viscosity and Molecular Parameters AA G 4 FlOry FOX ReIATIOM EE G 5 M1000 Rev H Contents 10 M1000 Rev H About ASTRA V This chapter provides an overview of the ASTRA V software and this manual It also tells you how to contact Wyatt Technology for support CONTENTS PAGE KOL 1 2 Using This Mangal ged E EE EdeEegd ege eiai 1 3 Glossa ege i ei ee eed 1 6 Getting M re El i gegteeefegeh iaaii fiaweetebasarens akaid teni sanien aek 1 9 Where to Go from Here 1 10 1 1 Chapter 1 About ASTRA V What is ASTRA The ASTRA software collects and processes data from dilute macromolecu lar solutions It uses this data to calculate the molar mass radius moments and other results In addition it controls the data acquisition performed by various instruments ASTRA experiment 1 Experiment Configurations Configuration Profile DDAR E E
321. nding detector gain in the DAWN profile e Have not calibrated in the last 30 days The passage of time may affect the signal e Realign the laser beam older DAWN DSP models only See Measured Quantities and Calibration on page D 4 for a discussion of calibration theory How to Calibrate To perform a calibration experiment follow these steps 1 Set up your equipment for a batch non flow experiment 2 Create a new calibration experiment by choosing File gt New Exper iment From Template 3 In the New from Existing dialog open the System Templates folder then Light Scattering then Calibration and then the experiment for your light scattering instrument 4 Click Create Double click the light scattering instrument in the configuration 8 19 Chapter 8 Editing Procedures 6 In the properties dialog select the appropriate physical instrument sample cell and wavelength Select the Solvent tab or double click the Solvent in the configuration Click the button for the Name property and open the System Sol vents folder Then select your solvent from the list of solvent profiles and click Copy You should calibrate the instrument using a pure solvent with a well characterized Rayleigh ratio We recommend calibrating with HPLC grade toluene for the following reasons e It has a high and accurately determined Rayleigh ratio Itis generally a dust free solvent Its refra
322. ng With Procedures Working with Procedure Graphs A number of procedure dialogs contain graphs You can manipulate these graphs in the following ways Select Detectors or Data If there is a list of detectors or data sources above or to the right of the graph you can use checkmarks to indicate the data you want to view Some graphs can display multiple data sets in different colors for example collection Others display only one data set at a time for example defining baselines Zoom In Hold down the Geboter Ctrl key and your left Edit mouse button Drag a rect Copy image to clipboard angle around the data you Print want to view larger Or Autoscale press Ctrl F5 to zoom in Zoom In Ctrl F5 Press F5 to open the Scale Zoom Out Ctrl Shift F5 Graph dialog Zoom Out Hold down the Ctrl key and click your right mouse button Each click undoes one zoom in action Alternately you can press Ctrl Shift F5 to zoom out one level Edit You can modify a variety of aspects of a graph s styles by double clicking a graph and using the Edit Graph dialog Edit Graph Graph ox Marker Size z Line Point Size Moo SH Cancel Copy Series is Export Color B Fei Marker Type ws quare Advanced This dialog has the following fields Table 8 1 Edit Graph Fields Field Marker Size Description Choose the marker size you want to use The options are very small small
323. ng an e 6 17 Creating a Fenteng eeneg EE 6 18 Setting a Default Template cececcccceeceeeeeeceeeeeeeeeeeseaaeeeeaeeeseeeeesaaeeseeeeeesnaeeseenees 6 19 Deleting ERC ue EC 6 19 Deleting an Experiment 27 kgt SEENEN NENNEN 6 20 COPYING Data EE 6 21 Applying Ee 6 22 Applying a Template to Multiple Experiments 0 cc cceceeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 6 23 Adding Elements to an Experiment 2 ecccceeeeeeeeseeeeeeeeeeeeeeseeeeeeeeeeeeeeees 6 24 Adding to the Configuration cccccceceeceeeeeeeeeeeeeeeeeeeeeeaeeeceeesaaaeeseaeeeseaeeeeaeeeeaes 6 24 Adding Mal tee EE 6 26 Sequencing Procedures A 6 27 Adding Data Set Definitions 20 2 cececeeeceeeeeeeeeceeeeeeeeeeeaaeeeeaaeeseeaeeesaeeseeeeeesnaeeesines 6 28 Adding Reports and Graphs mreti saaran aean aa aeaiia aaa ia aaa haa aaaea 6 28 Chapter 7 Configuring Experiments seesseessees 7 1 About Configurations and de UE 7 2 Configuration Example smene enirar ana ia aiia ai a a ia a i aaia 7 4 Using Comigurations screams eaei n CERS 7 5 Editing a Configuration eesseeeseeeeseeesrressrrsstnrstnnstnttinttnntstinsttnsstnnssrnsssnnnstnnnnnnnnn 7 5 Adding Instruments and Connections cccceeseceeeeeeeeeaeeeceeeeceaaeeeeaaeeeeeeeessaeeeseneees 7 5 Removing Instruments and Connections cccecceeecceeeeeeeeeeeeeeeeaeeeeeeeeseeaeeessaaeeeeaes 7 7 Replacing an Experiment Configuration or Item cceeceeeeeeeeee
324. ng ratio e which is defined as the ratio of the intrinsic viscosities of a branched and linear molecule In both cases the linear and branched molecules must have the same molar mass The two types of branching ratios are related by a term called the drainage parameter e Linear Models Branching analysis requires the comparison of a branched sample to a linear reference of the same molar mass The linear reference data can be obtained by performing a collection for a sample with the requisite molar mass and then importing the data into the branching procedure However in many cases it is sufficient to use an ideal linear model for comparison A linear model is used when the Use Linear Model check box is enabled Depending on the type of analysis and instruments in the experiment you can use a model based on radius data or intrinsic viscosity The models may be based on measurements made in earlier experiments where a con firmation or Mark Houwink Sakurada plot were created or from published values M1000 Rev H Analysis Procedures e Radius Model The radius model is specified using a slope k and offset b for a conformation plot of RMS radius as a function of molar mass e Viscosity Model The viscosity model is specified using the Mark Houwink Sakurada K and a parameters For an example experiment that determines Rh from QELS data choose File gt Open Experiment or File gt Import Experiment if you are
325. ngular variation of the scattered light is measured and used to characterize particle shape As in Appendix D we start with the following equation from Zimm Equation 1 R MP 0 2A cM P 0 where e cis the mass concentration of the solute molecules in the solvent g mL e Mis the weight average molar mass g mol e Agis the second virial coefficient mol mL g2 e K dn n dn dc Ac Ny is an optical constant where ng is the refractive index of the solvent at the incident radiation vacuum wavelength Ag is the incident radiation vacuum wavelength expressed in nanometers N4 is Avogadro s number equal to 6 022 x 102 mol and dn dc is the differential refractive index increment of the solvent solute solution with respect to a change in solute concen tration expressed in mL g this factor must be measured independently using a dRI detector e P is the theoretically derived form factor approximately equal to 1 2u lt r2 gt 8 where u 47 A sin 6 2 and lt r gt is the mean square radius P is a function of the molecules z average size shape and structure e Rg is the excess Rayleigh ratio tem Ju In the absence of a concentration detector the molar mass is not deter mined However the angular dependency of the scattered intensity can be used to determine particle shape As in regular light scattering analysis different formalisms may be used to derive these measure
326. ning each separate experiment from the Experiments tab you run the collection from the Sample Sets tab Validating a Sample Set Shortcuts You can validate an entire sample set by choosing Sample Set Validate Validation checks for conflicts in the procedure sequences for all samples in the sample set If an experiment collects data validation also checks that the necessary instruments are connected and available In addition validation checks the collection scripts If your templates use the Basic Collection procedure the collection script is built automatically and vali dation never finds any problems with the script Click the Validate Sample Set icon in the experiment toolbar Procedures are not shown in the Sample Sets tab You can view them by creating new experiments from templates as described in Creating Exper iments from Templates on page 6 5 For information about modifying the procedure sequence see Sequencing Procedures on page 6 27 Running a Sample Set Shortcuts M1000 Rev H To start the sample set run follow these steps 1 Begin by turning on warming up and stabilizing your experimental apparatus When everything is ready to go continue with the following steps in ASTRA 2 Choose Sample Set Run Click the Run Sample Set icon El inthe experiment toolbar 3 During a sample set run the live data can be viewed in the Collection tab and the state of the sample set execution ca
327. normalization by choosing Experiment Configura tion Normalize You see the property view for the procedure W vs Is ri online ps standards in thf Procedures Nor TBR A Peak Name Radius nm Action Hormalize Import Coefficients for Old Hew Detector 1 1 000 1 000 Detector 2 1 976 1 921 Detector 18 1 120 1 146 El Details Hormalization Type standard Radius Type rms Model random coil Percentage to Keep nia a ok Z Cancel EH Apply In the property view specify the peak and radius for the sample If you are using a Mie or sphere model for the sample specify that the radius is a geometric radius If this is a batch experiment specify the fraction of data to keep for the normalization Click the Normalization button The newly calculated normalization coefficients are displayed next to the previous coefficients If you wish to use the new normalization coefficients click OK or Apply Otherwise click Cancel M1000 Rev H Configuration Procedures 9 You can view the calculated normalization coefficients in the Experi ment Configuration dialog for the DAWN or miniDAWN Importing Normalization Coefficients Instead of performing a normalization you can import normalization coef ficients from another experiment by clicking the Import button The source experiment must contain normalization coefficients and must be open before you click the Import button
328. ns involving significant interaction with the instrument settings valve posi tion laser power levels and so forth it can provide a powerful means of interacting with your Wyatt instruments Scripts allow you to issue commands to instruments to prepare a collec tion collect data for a specific period of time and so forth This provides a powerful mechanism for customizing data collection Internally scripts form the foundation of the Basic Collection procedure which is simply a graphical interface to the features of the script collection interpreter M1000 Rev H Collection Collection The overall collection is controlled by issuing commands in the Collection namespace These commands are used to interact with the user and to control the collection status Commands are issued using the following syntax Collection command where command is one of the following commands Command Effect PromptUser text Pop up a message to the user that must be responded to via the OK button for the collection to proceed Start Tell ASTRA to begin listening for data from instruments SetDuration milliseconds Wait for the specified number of milliseconds while col lecting data SetInjectToCollectDelay Set the number of milliseconds ASTRA will wait after milliseconds receiving an auto injection signal before proceeding Stop Tell ASTRA to stop listening for data WaitForMessage Hold the coll
329. ns the sample set If you are using ASTRA V with Research Database or ASTRA V with Security Pack delete a sample set by following these steps 1 Choose System Database Administration Delete Items This opens the Select an item to delete dialog E Select an item to delete 9 Example Configurations Import Templates CI My Profiles Sy My Templates R collection only g empty sample set CI System Solvents C System Templates 2 Highlight the sample set you wish to delete in the list 3 Click Delete 4 Click Close when you have finished deleting sample sets 9 11 Chapter 9 Using Sample Sets Security If you are using ASTRA V with Research Database or ASTRA V with Security Pack you must have Administrator access to delete a sample set You can delete sample set templates by choosing System Database Administration Delete Items and using the dialog to delete sample set templates you no longer need 9 12 M1000 Rev H M1000 Rev H Working With Reports This chapter explains how to create customize and print reports with ASTRA CONTENTS PAGE About Reports aceite seed Eege EE dE 10 2 Changing a Report Format 10 3 Adding EE EE 10 4 Applying Report Format A 10 5 PRINTING a Report seissioun a eanna Ra aa aaaea naaa 10 6 10 1 Chapter 10 Working With Reports About Reports The templates provided with ASTRA V include reports that show the results computed for the experiment Most temp
330. nt template by checking the Make Default box Deleting a Template Security You must have at least Researcher access to delete templates M1000 Rev H To delete an experiment template follow these steps 1 Choose System Database Administration Delete Items This opens the Select an item to delete dialog WW Select an item to delete CI Example Configurations E Import Templates CI My Profiles Sy My Templates collection only empty sample set EI System Solvents EI System Templates 6 19 Chapter 6 Creating amp Running Experiments 2 Find and highlight the template you wish to delete Templates are generally in the My Templates and System Templates folders Click Delete Click Close when you have finished deleting templates Deleting an Experiment Security If you are using ASTRA V with Security Pack experiments can only be deleted by an ASTRA administrator See Deleting Experiments on page 4 8 for details Basic If you are using ASTRA V Basic any user may delete an experiment by deleting the vaf file that contains the experiment 6 20 M1000 Rev H Copying Data Copying Data You can copy data generated for one experiment into another experiment You might do this to create combined plots of results from several experi ments or to use the data in a procedure such as branching To copy data into an experiment follow these steps 1 Open both the source and
331. nt Configuration on page 7 9 d Solvent Solvent Profiles on page 7 32 Light Scattering Instrument Profiles on page 7 11 Figure 7 1 Profile types available in ASTRA V 7 2 M1000 Rev H About Configurations and Profiles In an experiment the set of profiles describe how an experiment is set up This is called the configuration You can also create profiles that are stored outside of experiments and can be copied into experiments as needed these are called system profiles This chapter focuses on using profiles in configurations The experiment templates provided with ASTRA contain commonly used configurations However as you gain more experience using ASTRA you may want to use system profiles in conjunction with your experiments See Chapter 12 Working with System Profiles when you are ready to learn more about using system profiles In Run mode you use the configuration items provided in the templates While you can modify the properties of items you cannot add items to or remove items from a configuration You can add items to a configuration only if you enable Experiment Builder mode by choosing System Preferences Experiment Builder Mode If you have already opened the Configuration properties dialog you need to close and reopen it after enabling Experiment Builder mode You must have at least Researcher access to work with configurations and Security i system profiles If you are a Tec
332. nt that uses more than one type of instrument The WyattQELS instrument is an exception since it is associated with a DAWN or miniDAWN instrument See Figure 7 3 in Configuration Example on page 7 4 for a diagram that shows the typical connections in an online light scattering experiment Fluid Connection Profiles A fluid connection profile describes a plumbing tubing connection through which solvent or a solution flows between instruments Experiment Builder Fluid connections are hidden in Run mode To see them you must enable Experiment Builder mode by choosing System Preferences Experi ment Builder Mode You can set the following properties for a fluid connection Table 7 16 Fluid Connection Properties Field Description Name Name of the connection If you have already created a system profile for this connection click and select a profile to use Description Description of the connection which typically contains more information than the Name Source Instrument Select the type of source instrument The drop down list shows the instru ment profile types that are available for a connection Destination Instrument Select the type of destination instrument The drop down list shows the instrument profile types that are available for a connection Volume mL The fluid volume displacement that is a result of the plumbing tubing between instruments This can be set manu
333. ntrol over the graph display is provided than is described in this manual For help on settings in the Advanced dialog move to a field and press F1 For more about modifying graph displays see Viewing and Modifying Graphs on page 11 13 to Modify Procedures Some procedures prompt you to perform some action such as marking baselines when the experiment runs Other procedures have default values for all their properties For procedures other than data collection you can easily modify the prop erties after the initial experiment run Procedures not affected by your changes still show the run icon Procedures that need to be re run show the Si not run icon Then you can re run the experiment using the Run command This time instead of collecting data only the procedures marked with the Si not run icon are performed M1000 Rev H Advanced Procedure Editing Advanced Procedure Editing The following subsections discuss advanced ways of managing procedures in an experiment Most users will not need to add remove or resequence procedure items The templates provided with ASTRA V contain procedures for most common experiments You may contact Wyatt Technical Support if you are having difficulty creating an experiment template for your setup Experiment Builder If you want to add remove or resequence procedures as described in this Security section enable Experiment Builder mode by choosing System Prefer e
334. nts 2 6 RUMNING AS TRA geegek eebe aiaa gebeten 2 9 Activating Optional ASTRA Features ssennesesnnneneeernnneernnnnrnrnnnren nn 2 5 Accessing and Viewing Hardware 2 10 M1000 Rev H 2 1 Chapter 2 Installing and Setting Up ASTRA System Requirements In order to use the ASTRA software you must have the following Microsoft Windows 2000 Service Pack 2 or higher Microsoft Windows XP Professional 82 bit or Microsoft Windows Vista Internet Explorer 5 5 or higher At least 150 MB free hard disk space At least 512 MB of RAM 1 GB recommended Microsoft Access or Microsoft SQL Server 7 or higher for ASTRA V with Research Database or ASTRA V with Security Pack only A CD ROM drive Available communication ports as required for data collection See the manuals for your instruments for details A Windows supported printer and or plotter A Windows supported mouse Additionally adequate experiment storage space is required Each experi ment run with ASTRA will use on average of about 0 5 MB of storage Installing the ASTRA Software This section provides basic instructions for installing ASTRA You must use the ASTRA installation program to install ASTRA rather than simply copying the files to your hard disk To install ASTRA do the following 1 Log in to Windows using an account with Administrator or Power User privileges Place the ASTRA disk in your CD drive On most systems the ASTRA setup p
335. o collect data from a sequence of injections usually from an autosampler The sample set creates a new experiment from a predefined template for each injection and then runs the experiment to collect and analyze the data Therefore a sample set can create many experiments Security You must have at least Researcher access to create sample sets If you are a Technician you can run sample sets If you are a Guest you have read only access to sample sets ASTRA V has a Sample Sets tab in the workspace that allows you to create edit and run sample sets E Experiments GB Sample Sets o System Profiles e Sample Sets BD Sample Set 1 ER Configuration SS Samples Sa Graph Collection The procedures run for a particular sample are determined by referencing an experiment template for each sample The samples may reference the same or different experiment templates 9 2 M1000 Rev H Creating New Sample Sets Creating New Sample Sets You can create sample sets starting from a blank configuration or from a template you have created Blank sample sets are easy to work with If you often perform experiments with the same set of samples using a template can save time in setting up the sample set Security You must have at least Researcher access to create sample sets Creating Blank Sample Sets Create an empty sample set by following this step 1 Choose File gt New Blank Sample Set Shortcuts Press Ctrl Shif
336. o do so before you start using ASTRA for experiments M1000 Rev H 2 9 Chapter 2 Installing and Setting Up ASTRA Accessing and Viewing Hardware Note 2 10 ASTRA s instrument list provides the following capabilities e Allows you to add and delete computers or instruments that run an Instrument Server Interface ISD to the set available to ASTRA on your computer e Shows instruments connected to your network and allows you to launch the Diagnostic Manager for each instrument This section gives step by step instructions for the initial actions you need to perform to make instruments visible to ASTRA For more details about the ISI and the Diagnostic Manager see Chapter 5 Interfaces to Instruments Viewing the Instrument List To view the list of instruments available to your copy of ASTRA choose System Instruments from the ASTRA menus You will see the Instru ments dialog Instruments 8 RD02 HHCS 172 20 2 66 DAWN HELEOS GELS B WYATT 310 HHC 172 20 1 152 Optilab Ex Refresh The list shows the instruments and computers with an ISI installed that your copy of ASTRA knows about You see instruments that support a direct data connection to ASTRA or instruments that are connected via USB to computers you have added to the list If you physically connect an instrument to the network after opening this dialog click Refresh to update the list The View button opens the Diagnostic M
337. of e on the results can be seen in the figure below which shows gu for various values of e using a Mark Houwink Sakurada parameter a of 0 7 typical for a random coil Notice that the ratio in Eq 37 is taken at constant elution volume V If no results fitting method has been selected ASTRA uses the raw MM vs Volume data for both the linear and branched files If a results fitting method has been selected ASTRA uses the fitted data from MM vs Volume in the branching calculations For each slice of the branched file ASTRA obtains the linear molar mass from the slice in the linear file having the elution volume closest to that of the branched slice 1 0 a 0 7 250 5 A 0 8 IL a07 ei i EE KE x De E 04 H vw 02 0 0 0 0 2 0 4 0 6 0 8 l M IM bn br Figure D 1 Branching ratio u as a function of a and e for the Mass Method For this method the volumes should have a large region of overlap for an effective plot To use this method select the Mass method in the Branch ing properties view for each branched file See Branching on page 8 80 Branching Ratio Viscosity Method The branching ratio g M is formally defined as Equation 38 In M Di AM Uu where Na and lr are the intrinsic viscosities of branched and linear unbranched polymer samples to be compared Note that the ratio is taken at the same molar mass not at the same volume In general for a given molar mass the branched polymer will have a smal
338. of the cumulant analysis results particularly if they are to be used as the sole assay for polydispersity F 5 Appendix F QELS Theory Regularization Whereas the method of cumulants is one of the simplest approaches to analyzing QELS data from a polydisperse sample the regularization analysis is one of the most sophisticated There are many excellent refer ences for the regularization method and the theory is quite detailed Theory As opposed to the method of cumulants the regularization analysis makes far fewer assumptions about the underlying distribution of sizes that make up the polydisperse sample A simple predecessor of the regulariza tion method the histogram method demonstrates this nicely In the histogram method the distribution of decay times is not assumed to be Gaussian as it is for the cumulant method with only the first two cumu lants Instead the decay time distribution is divided into bins Consider for example the model correlation function in Figure F 3 for a bimodal distribution consisting of widely separated sizes 2 0 0 0 1e 006 1e 005 1e 004 0 001 0 01 0 1 1 10 tau sec Figure F 3 Bimodal correlation function for mixture of 10 nm and 1 um particles Clearly the correlation function in Figure F 3 would best be fit by a sum of two separate correlation functions one with a short decay time and one with a long decay time In terms of the histogram method the underlying distribut
339. of the experi ment You see data as it is collected and can use the checkboxes to turn on or off data traces from various sources Light scattering data is red RI data is blue UV data is green QELS data is magenta and viscosity data is black 5 During the experiment you are prompted for any information a proce dure needs in order to run For example for light scattering experi ments you will probably need to set baselines and peaks after the data is collected Security 6 If you are using ASTRA V with Security Pack you will likely be prompted to sign off on an experiment after it runs You can quickly create and start running an experiment using the default template by choosing File gt Begin Injection or clicking the toolbar icon The experiment is created and begins data collection automatically See page 6 19 for information on creating default templates Signing Off on an Experiment Security If you see a message that says some parameters are not set for the Sign off procedure follow these steps 1 Click OK You will see the Sign off procedure dialog E Procedures Sign off Category Comments User Id Password Domain BY OK St Cancel 6 12 M1000 Rev H Running an Experiment 2 In the Sign off dialog choose a sign off category from the list The cate gories are as follows Category Description Unsigned This is the initial state for a sign off You must select anoth
340. oft Excel exporting data to 11 18 Mie fit model 8 56 miniDAWN definition of 1 7 ISI installation for 5 2 profiles for 7 14 models data fitting 8 55 modules activating 2 5 demo period for 2 5 molar mass calculating 8 59 curve fitting model for 8 57 definition of 1 6 distribution D 18 light scattering theory and D 11 D 15 molecular standards profiles for 7 31 molecular weight compared to molar mass 1 6 see also molar mass N Name property experiment configuration 7 9 networked accounts 2 7 networked computers 2 10 New From Existing dialog 3 5 6 22 10 5 noise diagnostics 8 42 removing spikes from collected data 8 42 smoothing data 8 43 noise baseline 7 10 non fractionated samples 1 7 normalization coefficients 8 35 light scattering theory and D 8 standards 8 32 Normalization procedure 8 32 Number from LS Data procedure 8 69 number of particles per mL calculating 8 69 Number of Samples property sample set configuration 9 6 numeric instrument data 5 6 nwf file extension 6 8 6 9 O ODBC Open Database Connectivity 4 4 off line see batch mode Index 8 Online A2 procedure 8 65 online help using 3 15 online mode 1 7 1 8 Open command Sample Set menu 9 4 operating system requirements 2 2 operating tiers 1 3 Optilab 903 calibrating 8 21 connecting through AUX input 5 2 profiles for 7 18 Optilab DSP calibrating 8 21 connecting through AUX input 5 2 profiles
341. ollow these steps 1 Choose File gt Open Sample Set Shortcuts Press Ctrl Shift O Click the down arrow next to the DG icon 2 In the Open dialog navigate to the folder that contains the sample set you want to open 3 Select a file and click Open Importing an ASTRA Sample Set Security You must have at least Researcher access to import sample sets You can import ASTRA sample sets saved with ASTRA V and ASTRA 4 ASTRA V sample sets have a file extension of vsf ASTRA 4 sample sets have a file extension of ss Basic This item is disabled in ASTRA V Basic since it is identical to File gt Open Sample Set To import an ASTRA sample set follow these steps 1 Choose File gt Import Sample Set Shortcuts Press Ctrl Shift lI 9 4 2 In the Import dialog navigate to the folder that contains the sample set you want to import 3 In the Files of type field select the type of sample set file you want to import 4 Select a file and click Open The sample set is shown in your Sample Sets tab with the filename you imported 5 To save the sample set in your experiment database choose File Save M1000 Rev H Editing a Sample Set Importing an Empower Sample Set Security You must have at least Researcher access to import sample sets ASTRA can connect directly to the database for the Waters Empower chromatography software to read in Empower sample sets So you can set up your chromatog
342. ollowing e Compliance This may be a requirement for conducting business Compliance provides better preparation for FDA inspections e Improved Efficiency Electronic records can be searched quickly e Faster Time to Market Time delays in approval cycles can be reduced because records can be transferred electronically es Better Quality and Consistency Products may be improved and are manufactured in a consistent manner e Improved Research Data Compliant electronic records provide better data integration and allow trending information to be better examined e Reduced Cost Storage space for hardcopy records is more costly than electronic storage e Reduced Risk Compliant electronic records are less vulnerable to signature fraud and misfiling For details from the FDA about 21CFR Part 11 see http www fda gov ora compliance_ref part11 Making use of the 21 CFR Part 11 support in ASTRA V with Security Pack makes your experimental data collection analysis and storage compliant with the FDA ruling M1000 Rev H Connecting to a Database 21 CFR Part 11 Support in ASTRA V Security ASTRA 21 CFR Part 11 compliance features related to user accounts and logging activities are available only in ASTRA V with Security Pack The icon to the left identifies information that is specific to ASTRA V with Security Pack If you are using these features you must have an ASTRA administrator to manage 21 CFR Part 11 compliance
343. omatogram 3 none normalized Log Scale Abscissa Units SH cancel H Apply The EASI Graph has the following properties Table 11 1 EASI Graph Properties Field Description Display Choose the type of display you want This will be the main data set plotted The thicker solid line The options are chromatograms concentration molar mass RMS radius Rh RMS conformation plot Rh conformation plot intrinsic viscos ity Mark Houwink Sakurada plot geometric radius mean square radius trans lational diffusion branching ratio g and g branch units per molecule long chain branching protein conjugate and instrument voltages Distribution Type Choose the item you want to plot the display against The options are vs time or volume cumulative weight fraction differential weight fraction cumulative number fraction and differential number fraction If you choose vs time or vol ume that will be the x axis of the plot If you choose one of the fractional options your choice will be the y axis of the plot Chromatogram 1 Choose a data set you want to plot along with the main display data A thinner solid line is used for this data set The options are none Rayleigh ratio differ ential refractive index UV absorbance QELS count rate and specific viscosity 11 4 M1000 Rev H Using EASI Graphs Table 11 1 EASI Graph Properties Field Description
344. ome older experiments may not have a Data Set Definitions folder 3 See Chapter 11 Working With Graphs for details about specifying the data in the set Adding Reports and Graphs You can add reports only if you enable Experiment Builder mode by 6 28 choosing System Preferences gt Experiment Builder Mode In Run mode you can add graphs by choosing Experiment Graph Add Custom Plot or Experiment gt Graph Add Parametric Plot The results of an experiment are available through the reports and graphs you add to an experiment For details about customizing reports and graphs see Chapter 10 Working With Reports To add a report or graph to an experiment follow these steps 1 Choose Experiment Add to Experiment This opens the Add to Experiment dialog M1000 Rev H Adding Elements to an Experiment Shortcuts Press Ctrl Shift P Right click any folder in the experiment tree and choose Manage Add To Experiment 2 Open the Results folder and select Graph or Report E Add to experiment 1 Results SES SEN E Graph Cancel E Report B Dataset Definition 3 Click OK The graph or report is added to the Results folder of the experiment tree 4 See Chapter 10 Working With Reports for details about reports and Chapter 11 Working With Graphs for details about graphs M1000 Rev H 6 29 Chapter 6 Creating amp Running Experiments 6 30 M1000 Rev H Configuring E
345. ome procedures have a graph and properties some have only properties some have a message that says the procedure has no user configurable parameters 2 Set properties by typing selecting from a list checking a box or click ing a browse button Rows shaded in yellow are read only You cannot change the value You can expand or hide lists of related properties if there is a or sign next to a property name 3 Click Apply or OK to make the changes In Run mode you cannot open a dialog for a procedure unless that proce dure has already been run has the run icon or the procedure is the first one in the sequence that needs to be run In Run mode you can open only one procedure at a time When you open a procedure dialog any other procedure dialog you have opened closes auto matically This prevents you from relying on information that may no longer be true due to changes in the settings for other procedures Certain procedures are hidden in Run mode if you do not need to interact with them All procedures are shown in Experiment Builder mode Experiment Builder You can open any set of procedure dialogs if you enable Experiment 8 4 Builder mode by choosing System Preferences gt Experiment Builder Mode Remember that changes to the settings for one procedure affect other procedures The data shown for later procedures may be incor rect if you have changed earlier procedure settings M1000 Rev H Worki
346. ometric displayed depends upon the LS fit model specified for the peak This field is display only A2 Angle Fit Degree Shows the calculated second virial coefficient This field is display only The angular fit degree May range from 0 to 5 Concentration Fit Degree Percentage to Keep dn dc mL g The concentration fit degree May range from 0 to 5 Type the percentage of each peak to use for calculating the A2 mass and radius The default is 25 If the plateau is flat not drifting in the peak range using the default value is recommended Specify the dn dc value for the sample If the dn dc value is zero the default is taken from the sample in the configuration Band Broadening Correction LS Model If you are doing online Zimm plots specify the band broadening correction factor The corrupting effects of band broadening boil down to a single multiplicative cor rection factor You can typically measure this by performing the Plateau method for a well understood standard Once this factor is measured you can use this value on all subsequent analyses to correct for the effects of band broadening Choose the calculation model to use The units for the y axis in the graph change as appropriate for the model you select The options are Zimm the default Debye Berry Random Coil and Sphere Negative Scale Factor Put a checkmark in this box to use a negative scale factor for the Zimm plot Plate
347. on Chapter 1 About ASTRA V een 1 1 What iS ASTRA eege 1 2 How is ASTRA V Different from Earlier Versions ccccccesceeeeeeeeeeteeteeeeeeetteeeeeaes 1 2 Using TVS TC NEE 1 3 Usar MOGE S ee ee egEeegeg de a T 1 3 Operating Tiers sisisiieninareannri n aa a a a aE 1 3 User Account Levels 2 27egegugd neen a iiv enact wesinceetsisdic dees a A geed 1 4 How This Manual is Organized ccccceeceeeceeeeeeeeeeeeeeeeeeeeeeaeeeseaeeeeeaaeseeaeesetaaeeneaes 1 4 Manual Conventions o ccccceccccesecceceeeeeeeceeeeeeeeeeeeaaeeseaeeeeeaaeeegeneessaaeeeeeeeesseaeeeeeaeeeeaas 1 5 Ee E 1 6 Batch Mode vs Online Mode ccccccccseeeseceeeeeeeeeeeeeeeeaeceseaeeeseaeeeseaaesseeeeesaaeeeeaes 1 8 Getting More Help caso cies asec as eee eae esa 1 9 Contacting Technical Support ceeceeeeeeeeeeeeeeeeeeeeeeeeeeseaeeeeeeeeesaeeeeeeeeeesaeeeeeneeess 1 9 Where to Go dee 1 10 Chapter 2 Installing and Setting Up AGTRA eessen 2 1 EE E Wlan EE 2 2 Installing the ASTRA TE 2 2 Upgrading to a New Version of ASTRA ssssssesseessesesseessseesisssrisssinssirnssrnsssrnssrnnsen nnet 2 3 Migrating the System Database ccccceceeeeeeeseeeeeeeeeeeeeaeeeseaeeeeseaeseseneeeseaeeeeeaeeeeaas 2 3 Activating Optional ASTRA Features ssccctecstancdi eesti 2 5 Setting Up User Accounts ccececccceeeseceeeenseeeeeeseeeeeeeeaaeneeeesaeaeeeensaeeeesenees 2 6 Setting Up Grows detteeste eebe nresnani aanaeio earra iaaa iiaa 2 6 Creating Hleerg e
348. on the peak apex fit them to a 6th order polynomial of the form Equation 12 y a ber ce dz ex fx Lo Note that there must be at least seven points in the center half of the selected peak for the normalization calculation to run 3 The apex of the fit curve y in Eq 12 provides Ma Va paseline The maximum y is found iteratively by plugging into Eq 12 the x value for each slice used in the fit and selecting the largest resulting y This method is used due to the relatively small number of points typically involved and to preclude the chance of finding local maxima 4 Repeat steps 2 and 3 first for the 90 degree detector then for each light scattering detector for which a normalization coefficient is to be calculated Set any negative y values to 1 0 6 Finally divide the results of Eq 12 for all detectors by the result of Eq 12 for the 90 detector This yields the desired normalization coeffi cients D 9 Appendix D Light Scattering Theory Area Normalization Better normalization results were found when the results were calculated from the integration of the Rayleigh Ratio peak as follows rather than just using the peak apex Select a sample peak to use for normalization Integrate the Rayleigh Ratios over the entire peak Repeat the integration for each light scattering detector Any negative results are set to 1 0 Si Be ge BO E Finally set the normalization coefficients for all det
349. one using ASTRA V procedures CONTENTS PAGE About PrOCCCUICS iaievaisvesszieiacciadecdsa cera nesaictai doukateiaudeosdtavdieraubtavaincnatensiedad 8 2 Working With Procedures ceccceceeeeeeeeeeeeeeeeeeeeeeeseaeeeseaeeseeeesenaaeeeed 8 4 Advanced Procedure Editing cccccceccceeeeeeseeeeeeeseeaeeeeneeeeseaeeeeeneeee 8 7 Collection PrOCCCUICS ss viscsesssaisccvcssescuecatasercanh ssascvedastenatenaissauaeuesasadedcardeas 8 10 Configuration Procedures ccccceeeceeeeeeeeeeeeeeeeeeeeeeaeeeseeeeeetaeesenaeeeeed 8 14 Transformation PDrocedures ccccccccscsesesesseeessseeeceeeeeeececeeeeeeeeeeeeeneenenea 8 42 Analysis ProC Cures ssrin eaii eoa na aa a aaa 8 59 Administration Procedures ccccccccsscsssssssesceceeeeeeeceececeeeeeeeeeeeeeneenenea 8 100 Chapter 8 Editing Procedures About Procedures Data collection and analysis in an experiment are broken down into a logical sequence of units called procedures in ASTRA V The procedures are performed in sequence when the experiment is run There are configu ration collection transformation analysis and administrative procedures You must have at least Researcher access to add procedures and at least Security SC Technician access to modify existing procedures If you are a Guest you have read only access to procedures The Procedures node in the experiment tree shows actions ASTRA performs in sequence when you run the experiment
350. onfiguration ccececcececeeeeeeeeeeecaeeeeeeeeseaaeeeeaeeesecaeeeseaeeesaes 12 8 Replacing a Single Configuration Item ccceseeeeeeeeeeeeeeeeeeeeeeseaeeeseeeesecaeeesnaeeeees 12 8 Replacing an Entire Configuration ccccccecceeeeeseeceeeeeeeeeeeeeeeeeeeeaeseeeeeeesaeeeeeeeeees 12 9 Appendix A Menu Quick Reference 0s essseeseeees A 1 Modes and User ET A 2 File Menu srining EREECHEN A 2 Leg A 4 EE DATURA Ee Ee A 4 Experiment Men ssec peno ena aa a E eege Een A 5 Sample En EE A 6 System MENU rre vie A A 7 WiIRKGW MON siir aneniermeriene nna a oi ei A 8 le WGI NEE A 8 Appendix B System Templates sssseeeeeseeeeeeeeeeeees B 1 EIERE EE EE EE ee e B 2 General lee ed Eet B 3 Concentration Determination AT ceceeceeeeeeeeeeeeeeeeeaeeeeeeeeseaeeesaaeeseceeeseaaeeeenes B 3 Peak Areas AT reon aa E E TTE dbeedetei B 3 UV Extinction from RI Peak AT een B 3 Light e E due EE DA Baseline Subtraction sisian aiana ara aiani asidine B 4 Galibration CT zeien enean iarra an aaa a a ai aea aaa naaa i naaa B 4 lu WEE B 5 DIAQGMOSUICS Ausan sosie a cote sett cope beeteti E A A B 5 REE EEE EE B 6 Batch Debye plot EI aer ar ahaa hea enge CEA B 7 Batch Zimm plot GE EE B 7 Brane ning ATI EE B 7 ONNE O EE B 8 Online Zimm Plot CT aenieiai ea aia iaai aiaa eia B 8 Online A2 CT ei Manse ei ae Lalas eege EE B 8 Protein Conjugate AT rieure anii esac til
351. ons are Conventional Uni versal with Viscometer Data and Universal without Viscometer Data For conventional calibration the analyzed polymer must be the same as the polymer used for calibration For universal calibration the polymers may be different Universal calibration requires either viscometer data or known values for the Mark Houwink Sakurada K and a coefficients Fit Order Concentration Source Flow Marker mL You can choose linear regression up to 7th order If the fit order exceeds the number of degrees of freedom the graph and equation portions of the display indicate that no fit is possible If multiple concentration instruments are present select the one to use in determining the column calibration You can use this field to switch between multiple concentration sources when deciding which peak to use The setting here does not affect the setting in the Experiment Configuration on page 7 9 Use the drop down menu to select whether or not a flow marker is to be set and how Drop down options are as follows None no flow marker is to be used The flow marker value is set to zero Enter value enter the flow marker value in mL in the cell to the right of the drop down From positive peak the flow marker will be determined from the apex of the peak selected in the Flow Marker Peak drop down The resulting value is displayed in mL to the right of Flow Marker drop down From negative peak the fl
352. or the GPC measurement since the refractive index of the solvent changes the scattering angles and the geometrical factors for each detector We must use a sample that is an isotropic scatterer one which scatters equally in all directions so that we can be sure that the variations measured are due to detector geometry and not some interaction of the sample with the light The normalization coefficient for the 90 detector is assigned a value of 1 0 while the other detectors are adjusted by varying amounts to yield uniform results These coefficients must be determined using the same flow rate same pressure and the same solvent that will be used for the chromatography measurement since the refractive index of the solvent changes the scat tering angles for each detector The process of normalization is quite simple We assume that the 90 detector has already been calibrated as described above To normalize we introduce an isotropic scatterer i e Rg is independent of 0 and compute a set of coefficients so that each detector gives the same Rg as the 90 detector when its signal is multiplied by its coeffi cient Expressed algebraically we have Equation 10 EE as Vaos V laser laser dark Ra No m For Eq 3 and Eq 10 to agree when 0 90 Nog must be exactly unity Thus Eq 10 gives us a way to calculate Rayleigh ratios at any detector angle We recommend normalizing with a low molar mass sample which has a radius
353. or any high temperature third party viscometer for which data is collected through the AUX input of another instrument To create a generic viscometer profile choose File gt New gt System Profile and select Generic Viscometer and click OK Name your viscome ter profile Then you can double click the viscometer profile in the System Profiles tab to set its properties You can set the following properties for a generic viscometer Table 7 11 Generic Viscometer Profile Fields Field Description Name Name of the instrument If you have already created a system profile for this instrument click and select a profile to use Description Description of the instrument which typically contains more information than the Name Dilution Factor If you are using a UV detector plumbed before the ViscoStar in the flow sequence use the default dilution factor of 1 00 If you are using an RI detector or any other instrument plumbed after the ViscoStar see Measuring the Dilution Factor on page 7 22 to determine the value to enter here AUX input mode Specify the data provided by the AUX input from the viscometer The options are differential and inlet pressure default specific viscosity and differential pressure alone The Waters viscometer can be set to pro vide either of the first two types of data other viscometers may provide dif ferential data pressure alone Fixed
354. ort experiments and data from the earlier version ASTRA V rewritten from the ground up has numerous advantages over previous versions of ASTRA including the following e Easier to use You can get started simply by opening an experiment template connecting to your physical instruments and running the experiment Run mode simplifies the ASTRA interface e More powerful As you gain more experience with ASTRA you can begin to create and manage system profiles to extend the experiment templates Experiment Builder mode provides more power e 21 CFR Part 11 compliance You can store experimental data as required by the FDA if federal compliance is required in your lab e Less light scattering centric This new version of ASTRA is designed to work with a wide variety of instrument types without sac rificing functionality for light scattering data collection ASTRA V supports all Wyatt Technology instrumentation and analysis methods and adds new functionality 1 2 M1000 Rev H Using This Manual Using This Manual This manual describes how to install and configure the ASTRA software for collecting and processing data It is meant to be used in conjunction with the hardware manual that came with your Wyatt instrument for example a DAWN or miniDAWN instrument This manual assumes a basic knowledge of Microsoft Windows features and mouse operations User Modes You can use ASTRA in Run mode or Experiment
355. ou verify and or calibrate the absolute Refractive Index aRI measurement before operating the instrument in aRI mode for the first time and it is reeommended that you check these values for accuracy on a periodic basis General Information Optilab rEX aRI measurement performance should be checked against one or more standards For a complete aRI calibration at least 3 pure solvents with known aRI values which are specific for the operating wavelength of the Optilab rEX should be used See Table 8 7 for recommendations It is imperative that each solvent infused into the Optilab rEX must be miscible with the solvent that it replaces The Optilab rEX aRI is factory calibrated using the following four solvents in the following order L High Purity Water NANOpure water with a final 0 2 micron filter 2 Methanol HPLC Grade Fisher A452 4 3 Tetrahydrofuran GPC Grade w BHT Burdick amp Jackson Cat 341 4 4 Toluene HPLC Grade HPLC Grade Burdick amp Jackson Cat 347 4 Table 8 7 Known aR Values for Various Solvents at Various Wavelengths Note M1000 Rev H Solvent 685nm 658nm 633nm 532nm 514nm 486nm Water 3909 1 3309 1 3316 1 3347 1 3354 1 3364 _ Methanol 1 3242 1 3247 1 3253 1 3282 1 3289 1 3300 Tetrahydrofuran 1 4015 1 4022 1 4029 1 4069 1 4079 1 4094 Toluene 1 4882 1 4896 1 4910 1 4995 1 5017 1 5053 It is imperative that each solvent be mis
356. ow marker will be determined from the lowest point of the peak selected in the Flow Marker Peak drop down The result ing value is displayed in mL to the right of Flow Marker drop down Flow Marker Peak Mark Houwink Sakurada K Select the peak for the flow marker if you used one The known Mark Houwink Sakurada K parameter of the polymer used for cali bration This is only used if the Universal without Viscometer Data calibration technique is selected Mark Houwink The known Mark Houwink Sakurada a parameter of the polymer used for cali Sakurada a bration This is only used if the Universal without Viscometer Data calibration technique is selected Peak Expanding this row shows the peaks in the experiment The enabled checkbox determines which peaks to include or exclude from the fit The molar mass used for each peak comes from the Peaks dialog You can enter Intrinsic Viscosity for each peak here in the Calibrate Column dialog M1000 Rev H 8 41 Chapter 8 Editing Procedures Transformation Procedures The transformation procedures allow you to mark portions of the collected data or to convert the collected data in some way The following transformation procedure types are available e Despiking on page 8 42 e Smoothing on page 8 43 e Baselines on page 8 45 e Blank Baseline Subtraction on page 8 47 e Peaks on page 8 51 e Broaden on page 8 56 e Convert to
357. ows currently logged in user is ignored This username is stored with the collected data so that it is retained even if processing and report generation is performed by another user M1000 Rev H Changing a Report Format Changing a Report Format Experiment Builder You can choose a different report format only if you enable Experiment Builder mode by choosing System Preferences gt Experiment Builder Mode When you open a report while in Experiment Builder mode you see the following properties for the report You do not see this property list if you open the report while in Run mode Table 10 1 Report Properties Field Description Graphs gt Check the box or boxes for graphs you want to include in the report Template gt You can change the name of the report to be shown in the experiment tree list by Description modifying this name Template gt File Click the button then locate the XML file you want to use A large number of reports are provided with ASTRA in the Reports folder For example C Program Files WTC ASTRA 5 Reports Customizing Report Formats M1000 Rev H ASTRA provides about 50 report templates so you are likely to find tem plates that apply to your experiments However you may also need to modify a template to suit your needs The report templates are provided as XML files which are similar to HTML files However you should not edit these files with a we
358. ows the x axis coordinate of the left end of the baseline for this detector Y1 Shows the y axis coordinate of the left end of the baseline for this detector X2 Shows the x axis coordinate of the right end of the baseline for this detector Y2 Shows the y axis coordinate of the right end of the baseline for this detector 8 46 If your light scattering baseline drifts you may want to run one of the experiment templates provided with ASTRA for diagnostic purposes For an experiment template choose File New Experiment From Template to open a template in the System Templates gt Light Scattering gt Diagnostics folder The LS noise template reports baseline detector noise and drift M1000 Rev H Transformation Procedures Blank Baseline Subtraction While standard baseline subtraction is useful for combining instrument data during chromatography runs there are many cases where predeter mined changes in flow rate temperature and other effects can cause instrument baselines to drift such that the standard linear baseline sub traction feature cannot correct for the problem When the baseline changes are caused by a repeatable set of conditions such as Eclipse instrument flow adjustments a set of blank data can be collected that will model these effects By subtracting this blank run we can generate well behaved result data 1 Collect sample blank data for the experiment run by running an inj
359. oximation assumed in the preceding analy sis The simplest particles most frequently measured by particle sizing procedures are the polystyrene latex PSL spheres emulsions whose refractive index at wavelengths in the visible is about 1 59 Relative to water whose refractive index is about 1 33 these spheres have a relative refractive index m 1 59 1 33 1 2 Rigorous application of the RGD theory requires that m 1 lt lt 1 which is a slight stretch for these PSL spheres Perhaps more importantly the phase shift of a wave passing through the particle 27a m 1 no Ap where a is the sphere radius also must be lt lt 1 Even if we make the assumption that 0 2 lt lt 1 attempting to size larger submicron particles using this approximation will quickly lead us out of the range of RGD applicability M1000 Rev H Theory The saving grace of this approach is twofold first the theory happens to work significantly better than one might expect even when the RGD requirements are not strictly satisfied and second the pertinent values are calculated in the limit 0 0 as shown in Eq 20 a regime where the RGD requirements are much more easily satisfied As the scattering angle becomes very small the RGD approximation becomes more valid as was confirmed vividly by the analyses of Kerker et al The result is that values of R 0 may be generated directly from the measurements if the particle s structure is known or from the more gene
360. p dn de mL g Negative Scale Factor Plateau Method Enabled Detectors Enabled Peaks FOM SN OK X Cancel EP Apply By default the graph shows the results in a Zimm plot style although ASTRA V does not use a traditional Zimm plot analysis Instead a global fitting algorithm is used to present all concentration and angular data together In the presentation the grid represents the best fit results from the global fit The quality of the fit can be assessed by seeing how the measured data points lie with respect to the best fit grid 8 65 Chapter 8 Editing Procedures You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the mass If you place multiple methods that determine mass in a procedure only the first one will be valid Previously Ag measurements could only be done in batch plateau mode ASTRA now supports Ay measurement in online mode Use the online A2 experiment template in the System Templates gt Light Scattering folder for online Ap measurement A completed online A2 experiment is also available for importing The properties for this procedure are as follows Table 8 19 Online A2 Properties Field Description Mass Shows the calculated mass This field is display only Radius geometric or RMS Shows the calculated radius The type of radius RMS o
361. periment log See page 4 7 All Experiment Log Save As Save experiment log to a file See page 4 All 7 Sample Set Menu The Sample Set menu contains the following commands Command Keyboard Description Shortcut Sample Set gt Edit Open the property dialog for configuration of the All view currently selected sample set See page 9 5 only as Guest Sample Set Validate Validate the sample set procedure sequence T R A and instrument availability See page 9 9 Sample Set gt Run Start to run the sample set See page 9 9 T R All Sample Set Stop Halt the active sample set See page 9 10 T R A Sample Set gt Log gt Display sample set log See page 9 10 All Open Sample Set Log gt Save sample set log to a file See page 9 10 All Save As A 6 M1000 Rev H System Menu System Menu The System menu contains the following commands Command Keyboard Description Shortcut System Instruments View list of connected instruments See page 2 All 10 System Database Administration Connect to Database Connect to a different experiment database DB and See page 4 3 Security Delete Items Delete experiments sample sets profiles or DB and templates from database See page 4 8 Security page 6 19 page 9 11 page 12 7 R A Migrate Update system database with latest ASTRA DB and templates and profiles See page 2 3 Security A Automatic Pe
362. phs using ASTRA 11 2 Procedure Graphs Many procedure dialogs contain graphs that you can print See Working with Procedure Graphs on page 8 5 for infor mation about these graphs EASI Graphs These graphs are flexible and easy to create You make selections and see the resulting graph in the same dialog EASI graphs are not saved with the experiment See Using EASI Graphs on page 11 3 for details Custom Plots These graphs are more powerful and more compli cated to create They use a data set definition to identify the data to plot You can use custom plots to graph multiple types of data against any x axis values you choose See Creating Data Set Definitions on page 11 8 and Creating Custom Plots on page 11 10 for details Parametric Plots The parametric plot procedure generates a data set for two different types of x y data that share the same x axis For example you can use this procedure to create a plot of RMS radius vs molar mass See Parametric Plot on page 8 83 Surface Plots You can create a 3D surface plot of detector data See Creating Surface Plots on page 11 12 For all types of graphs see the following sections Viewing and Modifying Graphs on page 11 18 for information about how to zoom in and out and change the look of the graph Printing Graphs on page 11 15 for information about printing any type of graph Exporting Graphs on page 11 16 for informat
363. place All it on the clipboard This command is available only as appropriate Edit Copy Ctrl C Copy the currently selected item to the All clipboard This command is available only as appropriate Edit Paste Ctrl V Paste the contents of the clipboard to the All current loacation This command is avail able only as appropriate Edit Delete Delete the currently selected item This All command is available only as appropri ate Edit Select All Ctrl A Select everything in the active view This All command is available only as appropri ate View Menu The View menu contains the following commands Command Keyboard Description Shortcut View Standard Hide and show OK Apply Cancel toolbar at bot All Toolbar tom of views See page 3 14 View Status Bar Hide and show status bar at bottom of main win All dow See page 3 14 View Workspace Hide and show workspace with Experiment tree All Sample sets tree and System Profile tree See page 3 14 View Customize Open dialog to customize toolbars and keyboard All shortcuts See page 3 14 A 4 M1000 Rev H Experiment Menu Experiment Menu The Experiment menu contains the following commands Command Keyboard Description Shortcut Experiment Configuration gt Edit Open the property dialog for configuration All view of the currently selected experiment See only as page 7 5 Guest Rep
364. pply By default the graph shows the results in a Zimm plot style although ASTRA V does not use a traditional Zimm plot analysis Instead a global fitting algorithm is used to present all concentration and angular data together In the presentation the grid represents the best fit results from the global fit The quality of the fit can be assessed by seeing how the measured data points lie with respect to the best fit grid The data points for each peak are shown in a different color 8 63 Chapter 8 Editing Procedures You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the mass If you place multiple methods that determine mass in a procedure only the first one will be valid Previously Ay measurements could only be done in batch plateau mode ASTRA now supports Ay measurement in online mode through use of the Plateau calculation method Use the online A2 experiment template in the System Templates gt Light Scattering folder for online Ay measure ment A completed online A2 experiment is also available for importing The properties for this procedure are as follows Table 8 18 A2 Mass and Radius Properties Field Description Mass Shows the calculated mass This field is display only Radius geometric or RMS Shows the calculated radius The type of radius RMS or ge
365. pt ing for any values Experiment Builder This procedure is hidden in Run mode It is performed automatically as 8 74 part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode M1000 Rev H Analysis Procedures This procedure uses the injected mass value specified for the peak The procedure adjusts for viscometer dilution factor effects if the concentration detector is downstream from a viscometer RI Calibration from Peak This procedure calculates the refractive index calibration using the peak data from a batch mode run You can use data from a batch mode RI run to calculate either the calibration constant if dn dc is known or dn dc if the calibration constant is known See the manual for your RI instrument for details You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the dn dc or RI calibration If you place multiple methods that determine dn dc or RI calibration in a procedure only the first one will be valid There are no properties to set for this procedure It runs without prompt ing for any values This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences
366. pump which typically contains more information than the Name Flow Rate The rate at which the pump runs in mL min A pump configuration always has a solvent configuration associated with it in a profile See Solvent Profiles on page 7 32 M1000 Rev H 7 25 Chapter 7 Configuring Experiments Column Profiles Columns are used in size exclusion chromatography SEC to fractionate a mixture of polymer sizes In ASTRA both conventional and universal column calibration can be per formed In conventional calibration the analyzed polymer is the same as the polymer used for calibration In universal calibration the polymers may be different Universal calibration requires either a viscometer and concentration detector or known values for the Mark Houwink Sakurada K and a coefficients The dn dc value is required for universal calibration as it is necessary for intrinsic viscosity calculations but not for conven tional calibration ASTRA lets you store a profile for a generic column that contains coeffi cients obtained from column calibration experiments see page 8 37 You can set the following properties for a generic column Table 7 15 Generic Column Profile Fields Field Description Name Name of the column If you have already created a system profile for this instrument click and select a profile to use Description Description of the column which typically contains more information than the Name
367. q 16 obtained by simple algebra Equation 17 d R Ke 1 fi Bae 2 Eq 17 is not susceptible to round off error Note that the expression in Eq 17 reduces to Ra ZK ein the case A 0 as it must M To find the mean square radius lt r gt for the slice substitute the first two terms in the expansion for P into Eq 13 to obtain Equation 18 2 3m 2 167 M 1 4A Mc D 12 M1000 Rev H Determination of Molar Mass and Sizes Zimm Fit Method To perform calculations with the Zimm fit method which is a fit to Kc R vs sin 2 we need to expand the reciprocal of Eq 13 to first order in C D KE Equation 19 c 2A c R MP The appropriate results in this case are Equation 20 ae Se M 2A c Ry and Equation 21 2 7 Zum M IT ies where m d K c R d sin 6 2 Berry Fit Method To perform calculations with the Berry method which is a fit to JE ci R vs sin2 0 2 we must expand the square root of the reciprocal of Eq 18 to first order mc Equation 22 Ke 1 AC MP 6 VR Jung In this case the results are Equation 28 s 4 2 LE ei R dei 4A c and Equation 24 2 32 mp r E Sei M 1 M A c where d A v aka te2l M1000 Rev H D 13 Appendix D Light Scattering Theory Random Coil Fit Method To derive the Random Coil fit method we go back to Eq 138 but instead of fittin
368. quation 15 in where lt r gt is the mean squared end to end distance of the chain and do is a universal constant having the value 2 87x102 In practice this constant varies somewhat from polymer to polymer with an experimental value closer to 2 5x10 The Flory Fox relationship is valid for polymers in theta solvents Ptitsyn and Eizner considered the modification required to model other solvents They found the following relationship Equation 16 n w e r7 IM Equation 17 P e 1 2 63 2 86 where is now a function of the polymer solvent iteration parameter and du is the Flory Fox constant When 0 it reduces to the theta solvent result The parameter is experimentally measurable with a Mark Houwink analysis To perform a Mark Houwink analysis the data for a random coil polymer is fit to Equation 18 D KM where M is the molar mass The K and a are fit parameters which depend upon the polymer solvent and temperature Traditionally this data is also plotted as Log n vs Log a If the data is fit well this should be a straight line The slope parameter a is related to by Equation 19 E 2a 1 3 G 5 Appendix G Viscosity Theory G 6 M1000 Rev H Symbols plus sign between key names 1 5 Numbers 21 CFR Part 11 compliance benefits of 4 2 definition of 1 2 4 2 features enabled by 4 3 icons 1 4 installing module for 2 5 logging in for 2
369. r 3 5 Chapter 3 Getting Started 3 Open the Light Scattering folder 4 Select a batch template and click Create A new experiment called Experiment is created based on the tem plate you selected The Experiments tab in ASTRA shows the parts of the experiment Lis File Edit Yiew Experiment System Window Help D ts elal ixj Experiments E SampleSets E System Profiles lps Experiments ts Configuration EOS batch S DAWN EOS DAWN EOS d Solvent water Sample untitled CH Procedures Basic collection Despiking Procedure Define baselines Define peaks Determine mass and radius from LS data SQ Results EB Report summary EB Report detailed For Help press F1 You can expand or collapse the nodes in an experiment as desired by clicking on them Each experiment contains the following categories of items e Configuration The hardware devices and connections used in the experiment For online fractionated experiments this may include a pump injector solvent sample DAWN Optilab and data connec tions For details on all types of items that may be configured and their properties see Chapter 7 Configuring Experiments e Procedures The actions to be performed in sequence when the exper iment is run There are configuration collection transformation analysis and administrative procedures For details on all types of proc
370. r Item on page 7 7 M1000 Rev H 12 9 Chapter 12 Working with System Profiles 12 10 M1000 Rev H Menu Quick Reference This chapter contains a quick reference for ASTRA V menu commands and keyboard shortcuts CONTENTS PAGE Modes and User LeVels sisrate a a a A 2 Ee ME eege A Ee A 2 Edit MOI eege EENS A 4 KIT UE A 4 Experiment Mem A 5 Sample Set Men serres asias ninina reiini aariaa naana aaa eedeastecteead eteindiues A 6 System Mengt u mi a aaa E E NEA A 7 Window dE A 8 Help M n sis ses sirsiran eE e Ce A 8 M1000 Rev H A 1 Appendix A Menu Quick Reference Modes and User Levels The following tables list the commands in the ASTRA V menus The Description column includes a link to more information about the command The Modes column identifies when the command is available as follows Builder This command is inactive unless you enable System Preferences Experiment Builder Mode which is only available to researchers and administrators G Guest user level T Technician user level R Researcher user level A Administrator user level DB and Security This command exists only when using ASTRA V with Research Database or ASTRA V with Security Pack If a command is not limited to DB and Security it is always available in ASTRA V Basic even if user levels are listed The user levels apply only to ASTRA V with Research Database and ASTRA V with Security Pack
371. r geometric displayed depends upon the LS fit model specified for the peak This field is display only R44 Radius Shows the calculated Rf radius This is a physical quantity derived from the Zimm series expansion It is similar to the RMS or geometric radius except that it is the fourth root mean fourth power This field is display only A2 Shows the calculated second virial coefficient This field is display only A3 Shows the calculated third virial coefficient This field is display only Zimm Orientation Angle Fit Degree Toggle the display between the Zimm and Debye plotting formalisms The angular fit degree May range from 0 to 2 Concentration Fit Degree Percentage to Keep dn dc mL g The concentration fit degree May range from 0 to 2 When performing a batch analysis type the percentage of each peak to use for calculating the A2 mass and radius The default is 25 If the plateau is flat not drifting in the peak range using the default value is recommended For online analysis this field is unused Specify the dn dc value for the sample If the dn dc value is zero the default is taken from the sample in the configuration Negative Scale Factor Plateau Method Put a checkmark in this box to use a negative scale factor for the Zimm plot Put a checkmark in this box if this is a batch experiment or an online experiment with plateaus rather than peaks Enabled Detectors gt 1 18
372. r the resulting width of the distribution corresponds to an actual polydispersity For example applying the regularization analysis to a correlation function from a monodisperse sample often results in a dis tribution with some width In general the noisier the correlation function the lower the optimal resolution of the regularization algorithm and the broader the apparent width Therefore when interpreting distribution widths from regularization always consider the resolution obtainable given the level of noise in the correlation function Ideally correlation functions for a monodisperse sample and the sample of interest can be obtained with comparable levels of noise such that the regularization analysis resolution can be accurately assessed Finally the smoothing nature of the regularization algorithm can mask features in the true distribution even for correlation functions with very low noise Therefore if a very structured distribution in sizes is expected regularization typically returns a much smoother distribution In short it is prudent to compare regularization results with a quantitative method such as fractionation followed by light scattering to determine the true distribution In general regularization provides the most accurate analysis for samples that are broadly polydisperse over several orders of magnitude in size and that have intrinsically smooth distributions 1 A A Goldin Software for particle size distri
373. ra tion creating 3 5 6 5 6 6 data from see data data set definitions for 6 28 11 8 default template 6 18 6 19 definition of 6 2 deleting 4 8 6 20 example of running 3 5 exporting 6 17 importing 6 9 opening 6 7 6 8 6 9 9 4 opening multiple experiments 6 7 peaks for 3 10 8 51 printing configuration for 3 15 procedures for adding 6 26 8 7 administration procedures 8 100 analysis procedures 8 59 collection procedures 8 10 configuration procedures 8 14 data processing during 8 3 data sets produced by 11 7 definition of 1 7 8 2 deleting 8 8 editing 8 4 folder listing 3 6 6 2 8 2 graphs of 8 5 modifying 3 8 8 6 sequencing 6 27 8 8 status icons for 3 11 8 2 8 8 transformation procedures 8 42 validating 6 11 8 9 re running 6 13 8 6 results of see graphs reports running 3 9 6 11 saving 6 15 6 16 saving as template 6 18 M1000 Rev H Index sign offs for 4 7 6 12 8 100 stopping 6 13 templates for 3 5 6 5 Experiments tab Configuration folder 3 6 6 2 6 24 Data Set Definitions folder 6 28 definition of 3 6 6 2 Procedures folder 3 6 6 2 6 26 8 2 Results folder 3 6 6 2 6 28 10 4 11 11 Export command Sample Set menu 9 11 Export Experiment command File menu 6 17 Export tab Editing dialog 11 16 11 17 extensions see file extensions extinction calculating from peak data 8 77 calculating from RI data 8 77 Extinction from UV Data procedure 8
374. ral expansion of the form factor P Furthermore many of the calculated results for the analyses of distribu tions of PSL spheres may be checked with more exact LS theory to confirm the precision of the sizes measured using the RGD approximation Applying the Lorenz Mie theory confirms the results derived by the present treatment In addition average values measured by photon corre lation spectroscopy PCS at individual slices also confirm the average values generated by the present implementation of RGD theory 1 M Kerker W A Farone and E Matijevic J Opt Soc Am 53 758 1963 2 PJ Wyatt Analytica Chimica Acta 272 1 40 1993 M1000 Rev H E 9 Appendix E Particles Theory E 10 M1000 Rev H This appendix gives a quick overview of the theory behind cumulants and regularization which are analysis techniques used with QELS data This includes descriptions of the implementation in ASTRA V and interpreta tion of results CONTENTS PAGE GUMS eebe ege eege see eege e F 2 REQUIEM ZATION sosirii eniinn anaidai aama lances F 6 M1000 Rev H F 1 Appendix F QELS Theory Cumulants The analysis of QELS data is straightforward for a monodisperse sample For unfractionated polydisperse samples however the analysis becomes much more complicated The simplest approach to analyzing data from polydisperse samples is to assume that the sample is monodisperse apply the analysis from ASTRA and come up with som
375. raphy run in Empower and then import the data into ASTRA for light scattering and related data analysis To import an Empower data set follow these steps 1 Create the sample set definition in Empower 2 Choose File gt Import Empower Sample Set 3 Login to the Empower database using your Empower user ID and password Import Empower Sample Set Empower Login User technician Password wm ent 4 Find and select the sample set you want to import from the import dia log and click Open The sample set is shown in your Sample Sets tab with the name of the sample set you imported ASTRA converts the Empower sample set to a full featured ASTRA sample set Sample names and identifying information are all brought over from Empower 5 To save the sample set in your experiment database choose File Save For more about using ASTRA V with Empower go to http www wyatt com solutions software ASTRA cfm and follow the links to Connectivity gt Interoperability with Waters Empower Software Editing a Sample Set M1000 Rev H To edit a sample set you use the property dialog for the sample set To use this dialog follow these steps 1 Choose Sample Set Edit This opens the properties dialog for the sample set You will see the Sample Set property dialog which con tains the Configuration Samples and Collection tabs 9 5 Chapter 9 Using Sample Sets Shortcuts Double click Configuration
376. ration technique is selected Flow Marker Use the drop down menu to select whether or not a flow marker is set and how Drop down options are as follows None no flow marker is to be used The flow marker value is set to zero Enter value enter the flow marker value in mL in the cell to the right of the drop down From positive peak the flow marker will be determined from the apex of the peak selected in the Flow Marker Peak drop down The result ing value is displayed in mL to the right of Flow Marker drop down From negative peak the flow marker will be determined from the low est point of the peak selected in the Flow Marker Peak drop down The resulting value is displayed in mL to the right of Flow Marker drop down Flow Marker Peak Select the peak for the flow marker if you used one The error in the molar mass is estimated from the standard error of the calibration fit If the fit degree is equal to the degrees of freedom a default lower limit uncertainty of 0 001 is used in place of the standard error Using a Column Profile to Determine Mass To use a column calibration to measure the characteristics of an unknown sample follow these steps 1 Choose File New Experiment From Template and open the universal calibration or determine column calibration template in the System Templates gt Viscometry folder 2 Expand the configuration and double click on the Generic Colu
377. rform database maintenance See page 4 9 DB and Maintenance Security All Log Open View experiment database log See page 4 7 DB and Security All Log Save As Save experiment database log to a file See DB and page 4 7 Security All Properties View properties of experiment database See DB and page 4 3 Security System Feature Activate features by providing activation key DB and Activation See page 2 5 Security A System Preferences Experiment Builder Set to Experiment Builder mode See page 1 3 DB and Mode Security R A Auto hide Workspace Set the workspace to hide when not in use See All page 3 14 Set Default Template Set the default template to use when creating a T R A new experiment See page 6 19 System Security Specify a domain to use for user authentication DB and See page 2 8 Security A System System View system database log See page 4 7 All Log Open System System Save system database log to a file See page 4 All Log Save As 7 M1000 Rev H A 7 Appendix A Menu Quick Reference Window Menu The Window menu contains the following commands Command Keyboard Description Shortcut Window gt Close Ctrl F4 Close current view All Window gt Close All Close all views All Window Next Ctrl Tab Move next view to front All Window Previous Ctrl Shift Tab Move previous view to front All Window
378. ring instrument calibra tion It prompts the user to press Enter to continue once the instrument is ready lsInst LSInstrument Create lsInst LaserOn true lsInst SetCollectionInterval 0 125 lsInst StartCollection Collection PromptUser Press Enter to Start Calibration Collection Start Run 30 seconds with laser Collection SetDuration 30000 Run 30 more seconds without laser lsInst LaserOn false Collection SetDuration 30000 lsInst LaserOn true Collection Stop M1000 Rev H C 5 Appendix C Data Collection with Scripts The following script turns COMET features on and off as needed during the collection see the bold portions in the example lsInst LSInstrument Create lsInst LaserOn true lsInst SetCollectionInterval 0 125 lsInst SendCommand SetSwitch COMET T lsInst LaserOn true lsInst StartCollection Collection Start Collection SetDuration 600000 lsInst SendCommand SetSwitch COMET F Collection Stop Sample experiments with collection scripts for running the COMET cell cleaner and turning lasers on and off are provided in the System Templates gt Light Scattering gt Utilities folder of the system database For Optilab rEX users there are several experiment templates in the System Templates gt RI Measurement gt Optilab rEX Specific folder These templates have scripts for Purge On Purge Off and Zero dRI You may contact Wyatt T
379. risssrissttnssinssstnsstnnnstnnnnnnnennn nnt B 17 Appendix C Data Collection with Scripts 0000 C 1 eet Tee EE C 2 COIS CUON an a EEN EES C 3 Interacting with Instruments E C 3 Common Instrument Commande cecececeeeeeeeeneeceeeeeesaeeeeeneeeeeaaeeeeeneeeseaeeeseneees C 4 Static Light Scattering Instrument Commands ccccceeeeeereeeeeeeeeeeeeeeeaeeeeeeeeees C 4 Dynamic Light Scattering Refractometer and Viscometer Commands 0 1 0 C 4 Examples arie mieia meget a a E a nua Eamonn maaan eat C 5 Appendix D Light Scattering Theory esseeeeees D 1 tree Te EE D 2 lee Eu TEE D 3 Measured Quantities and Calibration AAA D 4 PUGH Ee Tu WEE D 8 lieu LE D 9 Determination of Molar Mass and Sizes ccceceeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeees D 11 Debye Fit Method evcciepecaiirie heed irnhs ee abled donde ie heats D 12 Zimin G lle WEEN D 13 Berry Fit Method sieua tege Edge Deeg ni pevte E tetas D 13 Random Coil Fit Method 0 0 2 ceecccceceeeeeeeeeeeeeeeeeeeee eee eeeeaaeseceeeeeeaeesecueeeenaeeeenneess D 14 Model Fit Method unaii aE ev ete eee dene D 14 Mi Fit Method eria e eege ebe ae Ree D 15 Molar Mass and RMS Radius Moments sssnneeeeneeeeeneeeenreesnnesserrrssrrrrnnrerene D 15 Uncertainties in Calculated Quantities 0 0 eect settee ee eeteeeteeeeeeeeteeenees D 17 CUM OT Range EE D 18 Differential Distribution Calculations ce
380. rmine the number density This template is normally used with online fractionated experiments E number density 100 nm Duke standard ProceduresWetermine number DER results graph control graph V detector 90 0 0 0 2 0 4 0 6 08 J J 20 0 30 0 40 0 sin theta 2 volume mL Value geometric radius 37 0 0 3 nm Humber Density 1 4716 0 0719 e 8 particles mL Peak Number 1 Slice Index 1276 Model Mie Fit Degree 5 Abscissa Position 15 950 mL Enabled Detectors lt 3 ok 2 Cancel Remarks e Model This field shows display only the light scattering model selected for this peak in the Define Peaks dialog s Fit Degree This field shows display only the fit degree selected for the peak B 10 M1000 Rev H RI Measurement RI Measurement The following templates and folders are provided in the RI Measurement folder 100 Mass Recovery Methods The following templates are provided in the RI Measurement gt 100 Mass Recovery Methods folder dn dc from Peak AT Keep in mind that this template works on the hypothesis that all the mass injected is retrieved under the peak This may not be a valid assumption for certain types of samples For each measurement slice of the peak the refractive index is known due to the refractometer You then need to enter the amount of injected sample in grams and the flow rate The software can calculate the dn dc value since it is proport
381. rocedure will start automatically If you downloaded an update double click on the downloaded ASTRA_5xxx_ Setup exe file If the setup procedure does not start automatically use Windows Explorer or the Run dialog to run setup exe in the CD s ASTRA folder Answer the prompts in the setup procedure Choose to perform a full installation so that all the components of ASTRA will be installed See Installing the ISI on Other Computers on page 5 4 for other types of installations After you install ASTRA the Windows Start menu contains a folder called Wyatt Technology M1000 Rev H Installing the ASTRA Software Upgrading to a New Version of ASTRA Note You can check for newer versions of ASTRA by choosing Help gt Check for Updates from the ASTRA menus You can also use this command to control how often ASTRA checks for updates automatically For recent versions of ASTRA you can install the new version without deinstalling the old version Installing a new version does not update the system database which is where experiment templates and system profiles are stored This is because you likely want to keep custom experiment templates and system profiles See Migrating the System Database on page 2 3 to update your system database so you have all the latest experiment templates and system profiles If you have an older version of ASTRA V you may be prompted to dein stall the old version of ASTRA before ins
382. rs sphere coated sphere rod we fit the Zimm equation to Rg K c vs sin 2 as in the Debye method we insert into Eq 13 the theoretical form factor P 6 for the desired model Form factor models have been derived for spheres coated spheres and rods and are covered in the text by van de Hulst Note that the sphere and coated sphere models yield geometric radius while the rod model produces a length Sphere Equation 26 P 0 sin ucosu where u 4m A r sin 0 2 Rods Equation 27 DER TE E 7 u 0 where u 2m A L sin 0 2 and L is the rod length L gt gt rod radius 1 Light Scattering by Small Particles Wiley New York 1957 D 14 M1000 Rev H Molar Mass and RMS Radius Moments Mie Fit Method In the Lorenz Mie Fit method Maxwell s equations for electromagnetic radiation are solved under the assumption of spherical particles The Mie solution does not require the particle to satisfy the Raleigh Gans Debye criteria and is therefore the most general method for analyzing spheres of any size To derive the fit method for various modeled form factors sphere coated sphere rod we fit the Lorenz Mie equation to Rg K c vs sin 2 The extended Lorenz Mie exact calculation for a coated sphere single layer is given in the text by Bohren and Huffman based on the paper of A L Aden and M Kerker Note that the Mie fit yields a geometric radius Molar Mass and RMS Radius Moments ASTR
383. rs of the experiment Shortcuts Press Ctrl Shift P E Add to experiment Graph Cancel Report B Dataset Definition Procedures Te 2 Gs Select Graph in the Results folder and click OK Double click the Graph untitled1 item that was added to the Results folder in the experiment tree 5 In the property list for the graph type a Name to appear at the top of the graph 6 For the Data Set Definition select a data set definition you have already created mean square radius vs volume Data Set Definition mean square radius vs volume sl Sa OK x Cancel Si Apply 7 Click OK 8 Choose Experiment Run to run or re run the experiment After the procedure runs successfully the graphs and reports are generated M1000 Rev H 11 11 Chapter 11 Working With Graphs Creating Surface Plots You can create a 3D surface plot of detector data To create a 3D surface plot follow these steps 1 Choose Experiment gt Graph Add Surface Plot from the menus This opens the Data for Surface Plot dialog Data for Surface Plot OK Name Processed Data in 3D A P wailable Included mes raw data despiked baseline subtrac instrument data in physical units des raw data despiked baseline subtrac Type a name for your plot Select one of the types of available data and click the gt gt button You can scroll the list to the right to read the full description of each
384. rsion V enhancements 1 2 ASTRA 4 exporting file 6 17 import wizard 6 9 importing sample sets 9 4 ASTRA Administrator group 1 4 2 6 3 3 3 13 4 6 ASTRA Guest group 1 4 2 6 3 3 3 13 4 6 ASTRA Researcher group 1 4 2 6 3 3 3 18 4 6 ASTRA Technician group 1 4 2 6 3 3 3 13 4 6 Auto hide Workspace 3 14 auto inject connections 7 28 autoinjectors 7 25 profiles for 7 29 AUX connections definition of 7 28 profiles for 7 29 axis settings for graphs 11 14 Index 1 Index B band broadening 8 14 baseline drift 8 46 Baseline Noise Region property 7 10 Baselines procedure 3 9 8 45 baselines setting 3 9 8 45 Basic collection dialog 3 8 Basic Collection procedure 8 10 Basic operating tier 1 4 Batch Apply Template command File menu 6 23 batch mode definition of 1 6 1 8 normalization and 8 33 performing experiment in 3 5 Begin Injection menu command 6 4 Berry fit method 8 62 D 13 Berry fit model 8 55 bitmap files exporting graphs to 11 17 Blank Sample Set menu command 9 3 Blank Experiment menu command 6 6 blinking red circle in Diagnostic Manager 5 7 branching calculations D 19 branching per molecule D 21 branching ratio mass method D 19 branching ratio radius method D 19 Browse for Computer dialog 2 12 buttons instruction conventions 1 5 C calculations see analysis procedures theo ry calibration calculating RI from peak data 8 75 calibration constant
385. rtcuts Right click the experiment name in the tree and choose Save As 6 16 M1000 Rev H Exporting an Experiment Exporting an Experiment Basic You can export experiment data to separate files These files can be imported by ASTRA or you can save experiments in formats that can be imported by spreadsheets If you are using ASTRA V Basic this command allows you to export exper iments as ASTRA 4 tab delimited and comma delimited files To save an experiment to a different vaf file in the current ASTRA format use the Save As command instead You must have at least Researcher access to export an experiment Security To export an experiment follow these steps 1 Choose File gt Export Experiment Shortcuts Right click the experiment name in the tree and choose Export M1000 Rev H 2 Inthe Export Experiment dialog navigate to the folder you want to contain the exported file Do not export the experiment to a folder that is a read only folder such as Sample Data or Analyzed Experiments In the File Name field type a file name for the experiment In the Save As Type field select a type The formats you can export are as follows Type Description vat ASTRA V file that can be imported by ASTRA V on this or another computer You can choose a version of ASTRA for compatibility with earlier versions Use Save As instead for ASTRA V Basic adf An ASTRA 4 format for use with
386. s Editing a Configuration Shortcuts Shortcuts To set properties of a configuration component follow these steps 1 Double click on a component in the Configuration node of the Experi ment tab in the workspace This opens the properties dialog and selects the tab for that component See Figure 7 2 for an example Choose Experiment Configuration Edit 2 Set properties by typing selecting from a list or checking a box You can expand or hide lists of related properties if there is a or sign next to a property 3 Alternately you may click the browse button to the right of the Name property and locate a system profile to use to replace the exist ing property values for this item 4 You can move to other tabs to view or set properties for other items Double click on an item in the Configuration tree to move to its tab Move to a tab using the Hit ri tab arrows 5 Click Apply or OK to make the changes The remaining sections of this chapter contain details about the properties you can set in the various tabs Adding Instruments and Connections Experiment Builder You can add items to a configuration only if you enable Experiment M1000 Rev H Builder mode by choosing System Preferences gt Experiment Builder Mode If you have already opened the Configuration properties dialog you need to close and reopen it after enabling Experiment Builder mode 7 5 Chapter 7 Configuring Experim
387. s For example size exclusion chromatography SEC and field flow fractionation FFF separate macromolecules based upon size intrinsic viscosity IV A measure of the capability of a polymer in solution to enhance the viscosity of the solution Derived using specific viscosity and concentration data M1000 Rev H M1000 Rev H Glossary light scattering A technique whereby the intensity of scattered light from a macromolecule in solution is measured at multiple angles to determine a molar mass RMS radius and second virial coefficient Also called classical Rayleigh or static light scattering miniDAWN A DAWN instrument with three detectors It is used pri marily for characterizing small less than 50 nm in RMS radius macromolecules non fractionated Samples that are typically polydisperse solutions containing a range of macromolecules with different weights sizes or conformations online Data collection with solution flowing through the instrument For example a pump pushes solvent through an injector and the resulting solution is passed through a fractionation device and then characterized by instruments downstream Online measurements are usually for fractionated samples but can also be for non fractionated samples Optilab The Optilab instruments rEX DSP and 908 are differen tial refractometers These instruments can be used to determine the concentration of a macromolecule in solution and to measure the
388. s If multiple detectors are used this procedure must also come after the Interdetector Delays and Band Broadening procedures Running a Column Calibration To calibrate a column follow these steps 1 2 8 38 Set up the equipment for an online experiment with your SEC column Choose File gt New Experiment From Template to open a new experiment For conventional calibration the template is in the System Templates gt RI Measurement folder For universal calibration the template is in the System Templates gt Viscometry folder Run the experiment using a set of known molecular standards If you are performing a universal calibration without viscosity data use a standard that is available in several known molar masses and for which the Mark Houwink Sakurada K and a coefficients are known for example polystyrene For each experiment define peaks Enter masses or select predefined molecular standards for each peak in the Peak view e For universal calibration supply intrinsic viscosity information in the Extended Parameters if viscometry data is not present in the experiment Choose Experiment Configuration Calibrate Column Exam ine the column calibration data fit For the Universal Calibration with Viscosity Data technique an additional column shows the intrin sic viscosity of the molecular standard e You can specify a flow marker peak to use in the calibration Select between positive and negative pe
389. s measurement If we set y 2 5 this can be used to define the equivalent spherical volume of a nonspherical molecule Similarly it can be used to define the hydrody namic volume ry as y 1 Equation 14 zy An When defined in this way r is the radius of a sphere with the same intrinsic viscosity as the molecule under study 1 A Einstein Ann Physik 19 289 1906 34 591 1911 and R Simha J Phys Chem 44 25 1940 Science 92 132 1940 G 4 M1000 Rev H Flory Fox Relation Flory Fox Relation M1000 Rev H While the Einstein Simha relation can be used to define the hydrodynamic radius for solid molecules with adsorbed solvation layers it not simply related to the molecular size of extended molecules such as random coil polymers Several models have been developed to consider the effect the hydrodynamic drag on the intrinsic viscosity One of the most successful models comes from Flory and Fox who modeled the random coil as a series of beads on a string or a jointed chain The string is flexible but beads are rigid Flory and Fox considered that hydro dynamic friction causes the solvent near the center of the molecule to move with the same velocity as the center of mass but solvent near the edges is free to flow into and out of the molecule This led them to a rela tionship between the intrinsic viscosity and the mean square radius of the polymer chain in a theta solvent Their model is E
390. s and or the Optilab rEX QELS Interval sec How often the QELS instrument if there is one should collect data The default is every 2 seconds This interval may be set to a multiple of 1 second The maxi mum interval is 1 hour M1000 Rev H Chapter 8 Editing Procedures Table 8 2 Basic Collection Properties Field Description Details gt Collection Recycle Controls how the Recycle valve is during the collection This may be set to waste or recycle The default is waste Details gt Injection Delay The delay in time or fluid volume between injection and the start of data collec tion The units are determined by the Abscissa Units property of the experiment configuration The default is zero Details gt Laser Saver Mode Turn the light scattering instrument s laser off after collection is finished If you will not collect more data for at least an hour or so after finishing this collec tion it is best to turn off the laser However you want to be sure not to cycle the laser frequently since this will shorten the life span of the laser If you are using this collection as part of a sample set do not check the Laser Saver Mode box Instead use one of the System Templates gt Light Scattering gt Utilities gt turn laser off templates as the last experiment template in the sample set Details gt COMET Run Duration min After data collection run the COMET cel
391. s and those that have been degassed It is possible to detect concentration differences in the 1 ppm range Consequently standard samples and solvent blanks must be prepared from the same solvent stock After preparing the standard solutions fill two or three extra containers with water from the same solvent flask to use for blank analyses M1000 Rev H M1000 Rev H Configuration Procedures Samples should be kept well sealed to avoid evaporation Some solvents such as water keep fairly well for a number of months but concentration changes may occur over time due to evaporation or contamination NaCl solutions may be infused into the dRI instrument directly via syringe pump or with an HPLC pump and an injector as described below HPLC Pump with Injector This arrangement is shown in Figure 8 1 Use 0 01 in ID tubing between the injector and the Optilab rEX and 0 02 in ID tubing elsewhere A flow rate of 0 5 1 0 mL minute is ideal Use a large sample loop 0 5 1 0 mL to ensure that each injection produces a clear plateau and not a peak as it passes through the Optilab rEX od NS HPLC pump Injector w loop RI detector waste flask Figure 8 1 Setting up an HPLC pump and injector for calibration The objective is to inject known concentrations into the detector If a peak with a rounded top and no clear plateau is obtained the concentration at the top of the peak will be unknown flat topped plateaus
392. s identified in this manual Lines above and below the Security icon in the left margin as shown here highlight such information Security information is specific to ASTRA V with Security Pack There are no access restrictions in other operating tiers Security M1000 Rev H 3 13 Chapter 3 Getting Started Customizing the Environment You can drag various parts of the ASTRA window to positions you like better The command menu bar the toolbars and the workspace with the Experiments and System Profiles tabs can all be repositioned ASTRA remembers the positions you chose the next time you start it The View menu allows you to hide or redisplay the following parts of the environment You might want to hide these items to allow you to make a graph display as large as possible on a small computer monitor View Standard Toolbar allows you to hide the standard toolbar This bar contains icon buttons for creating opening and saving exper iments using the clipboard printing and getting help View Status Bar allows you to hide the bar at the bottom of the window This bar shows messages about the experiment status the current user name and the access level this user has It shows graph coordinates when you hold down the Shift key and point your mouse at a graph View Workspace allows you to hide the workspace with its tree view of experiments and system profiles You can choose System Preferences Auto hide
393. s in a procedure only the first one will be valid The properties for this procedure are as follows Table 8 17 Mass and Radius Properties Field Description Molar Mass Shows the calculated molar mass This field is display only Radius geometric or Shows the calculated radius The type of radius RMS or geometric displayed RMS depends upon the LS fit model specified for the peak This field is display only Peak Number Type the number of the peak for which molar mass and RMS radius should be calculated Slice Index 8 60 Displays the selected slice number alternately you can enter the slice number for which to view results M1000 Rev H Analysis Procedures Table 8 17 Mass and Radius Properties Field Description Model This field shows the light scattering model selected for this peak in the Peaks dia log This field is display only Fit Degree This field shows the fit degree selected for this peak in the Peaks dialog This field is display only This information is valid only if the Zimm Debye or Berry model has been selected for the peak Abscissa Position Concentration This field shows the position on the x axis for the peak and slice selected This field is display only This field shows the concentration of this peak This field is display only dn dc Enabled Detectors gt 1 18 Shows the dn dc value for this peak This field is di
394. s the ViscoStar to intrinsic viscosity using concentration data See the ViscoStar User s Guide for details about the calculation that is performed You may place this procedure after the peaks are defined and before the analysis procedures You must also place the Convert to Physical Units procedure before this procedure If both RI and UV concentration data were collected for this experiment use the procedure Convert to Concentration on page 8 56 to specify which set of data to use for concentration calculations There are no properties to set for this procedure It runs without prompt ing for any values The procedure Peaks on page 8 51 lets you select a model to use for intrinsic viscosity calculations and any constants required by the selected model The model may be Huggins Kraemer or Solomon Gatesman Experiment Builder This procedure is hidden in Run mode It is performed automatically as part of certain procedures If you want to see this procedure enable Exper iment Builder mode by choosing System Preferences Experiment Builder Mode For more about viscometry data collection and analysis go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Viscometry Fit Mass or Radius M1000 Rev H You can use a curve fitting model to fit the mass and radius results You can set the properties for this procedure before running the experi ment or you can modi
395. s the initial resolution as documentation but this value changes over time You may enter the current resolution here as documentation when you perform an experiment The resolution quantifies the ability of a column to separate different spe cies This is typically measured for a column by injecting two different spe cies into the column and then measuring the distance between the peaks and the peak widths The relation is Rg 2 Vo V1 Ws W1 where V is the elution volume for each species and W is the width of each peak at the baseline 7 26 M1000 Rev H Column Profiles Table 7 15 Generic Column Profile Fields Field Description Calibration Technique The type of column calibration performed The options are none Conven tional Universal with Viscometer Data and Universal without Viscometer Data Flow Marker The elution volume of the flow marker which is used when combining peak data from multiple experiments If zero the flow marker correction is not used Mark Houwink Sakurada K The known Mark Houwink Sakurada K parameter of the polymer used for calibration This is only used if you select the Universal without Viscometer Data calibration technique In this case the equation used is 7 K M Mark Houwink Sakurada a The known Mark Houwink Sakurada a parameter of the polymer used for calibration This is only used if you select the Universal without Viscometer Data calibration technique
396. s to be used in batch mode Checking this box Band Broaden ing gt Enable associates a single sample and solvent configuration with the instrument configu ration For a description of the difference between batch mode and flow mode see Batch Mode vs Online Mode on page 1 8 Check this box to enable band broadening This box should be checked only if valid instrumental and mixing terms are entered for the band broadening parame ters These parameters are usually determined by running the Band Broadening procedure see page 8 14 If band broadening has been enabled you can disable it using this check box Band Broadening gt Instrumental Term Band Broadening gt Mixing Term See Band Broadening on page 8 14 for an explanation of the instrumental term The units are in microliters See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters Generic RI Instrument Profiles You can create a Generic RI Instrument profile for any third party refrac tive index instrument for which data is collected through the AUX input of another instrument You do not select a Physical Instrument for a Generic RI Instrument profile because ASTRA V does not support a direct data connection to such instruments Instead add an AUX connection to the experiment configu ration to indicate which AUX channel and instrument are to be used to read the signal See AUX Connection
397. s typically happens when you use the Debye model and have a lot of low angular cur vature in the data You must then visibly make sure that the low angle data is fit well otherwise the fitted molar mass and radius will be incorrect There are cases when no model accurately fits the angular curvature in the Debye plot Typically this is due not to a failure in the fit model but to incomplete fractionation the polydispersity in the sample cannot be repro duced by a model that assumes a monodisperse sample M1000 Rev H Analysis Procedures A2 Mass and Radius from LS Data M1000 Rev H This procedure calculates the second virial coefficient Ag mass and RMS radius of the sample based on light scattering data as a function of angle and concentration For Ay and Ag calculation see Online A2 on page 8 65 For more about determining the second virial coefficient go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt Second virial coefficient The procedure has the following dialog E online a2 200k ps Procedures A2 Mass amp Radius from LS ER Zimm plot Mass 1 985 0 002 e 5 g mol rms radius 17 540 3 nm A2 4 240 0 034 e 4 mol mio Angle Fit Degree Concentration Fit Degree Percentage To Keep dn de mL g Band Broadening Correction LS Model Negative Scale Factor Plateau Method Enabled Detectors Enabled Peaks FOM ep OK zx Cancel EM A
398. se You can set the following properties for an experiment configuration Table 7 1 Experiment Configuration Properties Field Description Name Name of the experiment configuration Make this name brief enough to be easily selected from your list of experiment configurations Description Description of the experiment configuration which typically contains more information than the Name Processing Operator The current user This changes each time you load the experiment It is the source for the Processing Operator field in reports In ASTRA V Basic you can edit this field to show the name you want listed in a report See Operator Names in Reports on page 10 2 for details Abscissa Units The x axis units for display The default units are milliliters for an online flow experiment and minutes for a batch experiment Also available are millisec onds seconds and hours This setting affects the units for a number of fields in the experiment procedures M1000 Rev H Chapter 7 Configuring Experiments Table 7 1 Experiment Configuration Properties Field Description Concentration Source Select the source of concentration data you want to use in this experiment The list shows concentration sources such as RI and UV currently in your experiment configuration if any Details gt Fwd Monitor If you selected Forward Monitor in the Divide by Laser Monitor field of the Basel
399. se to create experiments from a blank template however using a provided template saves time Creating Default Experiments If you have specified a default experiment template see page 6 19 you can choose File gt New Experiment from Default to quickly create a new experiment from this template You can quickly create and start running an experiment using the default template by choosing File gt Begin Injection Ctrl Shift V or clicking the toolbar icon The experiment is created and begins data collection automatically See page 6 19 for information on creating default templates 6 4 M1000 Rev H Creating New Experiments Creating Experiments from Templates Templates can be used to create new experiments or to re analyze data in a different way ASTRA comes with over three dozen system templates that allow you to start using ASTRA at its full potential immediately Experiment templates set properties to values you are likely to use There are just a few properties you typically need to modify Security You must have at least Technician access to create an experiment from a template You must have at least Researcher access to modify an experi ment that was created from a template To create an experiment from a template follow these steps 1 Choose File gt New Experiment From Template Shortcuts Press Ctrl Alt T Click the down arrow next to the L icon 2 In the New from Existing dialo
400. se of the viscometer and analyzes its level To do so a stable baseline needs to be measured Some criteria are already provided in the template they can be found in the Viscometer noise pro cedure The report indicates whether the measured noise levels are within specification Value shot Tex oe wera ni DP Short Term tloiae Limit pai 2 200e 005 IP Short Term Moise Limit pai 1 000e 002 Wader K er sat imin 5 000 DP Wader Limit paip 1 te Gi IP Wander Leet paip 5 c00e 002 DP Drift Limi paimin 6 000006 IP Orit Limit ipsi min 1 6700 003 Peak Humber fwhole range 1 M1000 Rev H Viscometry Utilities The Orbit on template places the Orbit device in Recycle mode for the ViscoStar Copolymer Analysis AT This template is for use with viscometer data It is the equivalent of the Protein Conjugate template but for viscometry Online CT This is the standard online template for use when you have only a viscom eter and no light scattering instrument Branching from VS Data AT This template is the equivalent of the branching template found in the Light Scattering folder However this one measures branching using vis cometer data To use this template you must first do a column calibration and set a column profile Mass from VS Data AT This template calculates the molar mass distribution all along the chro matogram based on the viscometer data Universal
401. se seed values of 20 ul for the instrumental term and 20 ul for the mixing term Click Perform Fit again If repeated attempts to obtain a good fit fail either verify that the baselines are set correctly or choose a different range for the fit When the fit between the two traces looks good click OK or Apply to re run the experiment with the band broadening correction This procedure has the following properties Table 8 3 Band Broadening Properties Field Description Reference Instrument The instrument with the largest degree of broadening in the instrument series This is typically the last instrument in the series unless a viscometer is present in which case the viscometer should be chosen as the reference instrument Perform Fit Click Perform Fit when you are ready to use the peak marker to calculate the band broadening terms The graph shows the corrected data Instrument Details gt Instrument Instrument Details gt Enabled The instrument trace to broaden This is set automatically when you choose a reference instrument Check this box to enable band broadening for specific instruments Instrument Details gt Delay mL Instrument Details gt Instrumental Term The interdetector volume or time between the reference instrument and the instrument to broaden The units are determined by the Abscissa Units field in Experiment Configuration You can set the initial seed value here or
402. sed absolute RI offset from the OptlabrEN Diff RI Calibration The differential RI calibration constant from which the absolute value was determined from the Optilab rEX Diff RI Offset The differential RI offset from which the absolute value was determined from the Optilab rEX Percentage to Keep The percent of the marked peak data to use for calibration If the plateau is flat not drifting in the peak range using the default value is recommended Enabled Peaks This list shows the peaks used in the fit to determine the calibration constant Checking or unchecking a peak adds or removes it from the fit to determine the calibration constant 8 30 The graph shows a fit to the absolute RI values using the previous absolute RI calibration data and the measured refractive index data For an example experiment that determines the absolute RI calibration choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the absolute RI calibration vaf experiment in the Sample Data gt Analyzed Experi ments folder You can place this procedure with other analysis procedures and after all the transformation procedures A procedure sequence can contain only one procedure that determines the dn dc or RI calibration If you place multiple methods that determine the dn dc or RI calibration in a proce dure only the first one is valid M1000 Rev
403. ser ID Electronic signatures can be executed for any data collected in ASTRA The electronic signature is saved in the experiment log associated with the data and shows up in the report associated with the experiment Multiple electronic signatures can be executed for an experiment In the reported results ASTRA flags any electronic signatures that were made before the last modification Failed electronic signature attempts are recorded in the system log with an alarm status for immediate notification of system administrators M1000 Rev H Using Sample Sets This chapter explains how to configure and run sample sets in ASTRA V CONTENTS PAGE About Sample Sets mirisi niia aiai ea 9 2 Creating New Sample Get 9 3 Opening a Sample Get 9 4 Editing a Sample Get 9 5 Running Sample Sets ccecccceseceeeeeneeesecaeeeeaeeeseaeeeesaaeeseeeeeessaeeesenees 9 9 Saving Sample Ges 9 10 Deleting a Sample Get 9 11 M1000 Rev H 9 1 Chapter 9 Using Sample Sets About Sample Sets ASTRA sample sets provide an easy way to manage experiment sequences with multiple samples A sample set can be configured with a default experiment template such that all samples are collected in the same fashion Or a different experiment template can be specified for each sample making it possible to collect different types of data for each sample For example you might do a QELS collection on two out of ten runs A sample set is used t
404. ses enter the known molar mass of the molecular standard for this peak Extended Parameters gt If you are performing a universal column calibration with viscosity data enter Intrinsic Viscosity ml g the known intrinsic viscosity of the molecular standard for this peak Extended Parameters gt If you are performing viscosity based calculations enter the K fit parameter Mark Houwink Sakurada K Extended Parameters gt If you are performing viscosity based calculations enter the a fit parameter Mark Houwink Sakurada a for the Mark Houwink Sakurada analysis for this peak for the Mark Houwink Sakurada analysis for this peak 8 Click OK to continue running the experiment Choosing a Fit Model The light scattering fit models are as follows M1000 Rev H Zimm model uses the K c R 8 formalism The Zimm model should be used for molecules that have RMS radii smaller than 50 nm and that do not conform to another standard model such as random coil or sphere The Zimm model has the advantage over the Debye model in that it often requires a lower fit degree for the same size molecule For large gt 50 nm molecules the Zimm model often produces a negative molar mass and should not be used Debye model Uses the R K c formalism It gives better results over a wider range of molar mass including the very large greater than 10 Daltons or 100 nm RMS radius But you may need to delete high
405. shed adding users to a group Note for Networked Accounts Security You can use a similar procedure to set ASTRA privileges for networked accounts Log in to the server used for the active directory Perform steps similar to those described in the previous sections However perform the steps for the active directory instead of the local computer Setting up accounts locally or for a networked account determines which domain name users need to type when logging in to ASTRA Add groups to either the corporate domain or the local machine depending on where the user logs in ASTRA expects to find the Group and User information in the same place So for example you cannot create a local group and add a domain user to this group M1000 Rev H 2 7 Chapter 2 Installing and Setting Up ASTRA Setting a Validation Domain for Security Security You can specify the domain to use for user authentication This allows you to prevent security problems where a user with a lower security group on the corporate domain could create a local group called ASTRA Adminis trator and use that local group to log into ASTRA Follow these steps 1 Choose System Security to open the following dialog Security Settings Validation Domain Enter the domain that will be used to authenticate users je Mr DOMAINE RND Validate No Domain Specified Local Authentication Allow authentication on this machine if the domain is not available
406. sity over hundreds of bins In fact for a standard correlator such as that in the WyattQELS instrument the largest number of bins that can be handled is only about ten The regularization method makes it possible to have a finer mesh of bins This is accomplished by constraining the types of distributions that can accurately reproduce the correlation function The most common con straint and the one employed in the DYNALS algorithm used in ASTRA V is that the distribution be smooth This is accomplished by adding a regularization term that penalizes solutions that are not smooth The magnitude of the regularization term determines how smooth the final result must be The trick of every regularization algorithm is to determine the optimal amount of regularization such that the final solution captures as many features of the true distribution as possible while balancing out the effects of noise in the correlation function Noise can add spurious com ponents to the calculated distribution hence as the noise increases the regularization term needs to increase to damp these spurious components Implementation of Regularization in ASTRA V The regularization algorithm in the ASTRA V software is the DYNALS 2 0 algorithm supplied by Alango Ltd The DYNALS algorithm sets the reg ularization level referred to as the resolution to the most appropriate M1000 Rev H F 7 Appendix F QELS Theory value for the level of noise in the correlation
407. splay only This list has a checkmark next to detectors whose data is used in the calculation You can disable individual detectors by removing the checkmark About Debye Plots The following procedure views show a Debye plot Mass and Radius from LS Data on page 8 59 Number from LS Data on page 8 69 Radius from LS Data on page 8 71 Debye plots let you view the light scattering data for each slice of the peak and see the weighted least squares fit to the data This plot is a good place to check the appropriateness of the polynomial used for fitting It also allows you to check visually the normalization coefficients for the DAWN When viewing a Debye plot there are two separate graphs On the right is the control graph On the control graph use the mouse to select a peak and slice for which the Debye plot on the left graph is to be viewed the selected peak is highlighted and a cursor shows the selected slice You can use the arrow keys to scroll through the various slices M1000 Rev H Debye Plot SD1250K 1 20x10 1 00x10 8 00x10 T o 6 00x10 ac 4 00x10 Lv o 2 00x10 0 0 sin theta 2 Peak Slice 1 663 Volume 2 727 mL Fitdegree 3 Conc 4 062 0 001 e 4 g mL Mw 1 864 0 008 e 6 g mol Radius 213 5 2 0 nm 90 amp AUX detectors Figure 8 5 Debye plot with Fit degree of 3 Chapter 8 Editing Procedures 8 62 Fitting the Light Scattering D
408. ss ASTRA to one of the following four groups e ASTRA Administrator Can change database settings and can create modify and delete experiment files Also has privileges of Researchers Technicians and Guests e ASTRA Researcher Can create and modify experiment files Can connect to networked computers and instruments Also has privileges of Technicians and Guests e ASTRA Technician Can run a given experiment procedure sequence and save the resulting data Also has privileges of Guests ASTRA Guest Has read only access to experiments and results With operating tiers other than ASTRA V with Security Pack users are not prompted to log in with a user name and password Chapter 2 Installing and Setting Up ASTRA contains a section on Setting Up User Accounts on page 2 6 since that portion of ASTRA administration needed to be done during initial setup That section provides steps for setting up the groups used by ASTRA creating user accounts and assigning users to groups You can modify and delete user accounts using the same Windows tools The status bar at the bottom of the ASTRA window shows the name of the user account that is currently logged in It also shows the ASTRA group to which that user is assigned A user should not be assigned to more than one ASTRA group M1000 Rev H Using the System and Experiment Logs Using the System and Experiment Logs Database ASTRA V with Security Pack and ASTRA V w
409. sssrrnesrrnnnenrennnasrnnnnnnnnnnnnsnnnnnnnnnennene 8 17 LS CAAM er eE eE aTa R AA I ALESE AATE AEAEE CAAA a 8 19 Differential RI Calibration cececcccceeeeeeeeeeeceeeeeeeeeeceeeeeeeaeeeseeeeeeeeaeseeeeeeesaeeeeeeeeee 8 21 Absolute Rl Calibration ek eebe nasa an aeaa eae ege 8 27 UV CaliDrAatiOM EE 8 31 Notmali ue EEN 8 32 Calibrate Column ou e a aa aa aaa e a aaa a a aaaea a aat 8 37 4 M1000 Rev H Contents Transformation Procedures ccccccsececcecesssecesaceeceecaeeeseesecenseeeensaaeeeensaestenes 8 42 Re e WEE 8 42 ele TI EE 8 43 ee EE ee Fee EEEE E dyeudad E TA 8 45 Blank Baseline Subtraction 8 47 ea RATTE E E E E E AE AA A A E E AEAEE ERER 8 51 Eet Dn WEE 8 56 Convert to Physical Units eate aaea a aaa aaa aae aaae iab E aai 8 56 Convert to Concentration oo cece ceeeeaeeeeeeeeeeeceececececeecececececeeeeeceeauusaaeaeaeneeseeees 8 56 Convert Specific to Intrinsic Viscosity eeeeeeeceeeeececeeeeeeeaeeceeeeeeeeaeeeeeneeetsaeeeseneees 8 57 Fit Mass or RAIUS 8 57 Analysis Procedures eege ee geg ee ee 8 59 Mass and Radius from LS Data 8 59 A2 Mass and Radius from LS Data cccccceeccececcececececcececeeeeeeecesueaeaeaeaaaeaaaeeeeaes 8 63 CMSA EE 8 65 EE el VS Datars E 8 69 Number trom ES Date 22 ee NENNEN NENNEN ENEE ENNEN 8 69 Raditis trom Lea is ici tags cectaedis Zeg savaiaes ida teda saves a a a sh dogidiacesatetiadaat eege 8 71 Distributi s and MOMENTS siiieiisssccdsadeadeis ceda
410. sure our samples in water and still obtain correct results The complication lies in the geometrical factors which describe the volume of scatterer seen by the 90 detector as well as the solid angle it subtends These factors depend on the refractive index of both the solvent and the glass of which the sample cell is made see the DAWN or miniDAWN Hardware Manual Thus the constant Acgcc is really dependent on the solvent type and cell type That is why we call it a configuration specific calibration con stant To allow users to calibrate with one solvent and or cell and make measurements with another we must have an instrument constant that is truly independent of these changing factors and is instead only a function of the particular instrument and the sample cell geometry This instrument constant Aa is related to Acgcc by Eq 4 EE Acsce Aing Reflection Correction Geometry Correction First the Reflection Correction will be considered The reflection correc tion represents the reflective losses at each interface in the sample cell For example the incident laser beam loses intensity at the air glass inter face of the sample cell and the glass solvent interface as well Similarly the scattered light that is to be detected also suffers from reflective losses at the solvent glass and glass air interfaces as it leaves the sample cell If the solvent is changed or a different cell is used these reflective losses w
411. t N Click the down arrow next to the D icon You see a sample set configuration in the Sample Sets tab of the workspace Experiments g Sample Sets pi System Profiles e Sample Sets H Sample Set 1 E Configuration ER Samples Sa Graph Collection If you have defined a default experiment template see page 6 18 and page 6 19 that template is the default for all samples in the new blank sample set Creating a Sample Set from a Template To create an experiment from a template you have created follow these steps 1 Choose File gt New Sample Set From Template Shortcuts Click the down arrow next to the D icon 2 In the New from Existing dialog open the folder that contains the sample set template you want to use You can choose from the follow ing folders My Templates These templates are ones you have saved as described in Saving Sample Sets as Templates on page 9 11 3 Select a template and click Create A new sample set is created based on the template you selected M1000 Rev H 9 3 Chapter 9 Using Sample Sets Opening a Sample Set You can open and work with any sample set you have saved If you are using ASTRA V with Research Database or ASTRA V with Security Pack experiments are stored in the experiment database and you open experi ments from that database If you are using ASTRA V Basic sample sets are stored in separate files with an extension of vsf To open an experiment f
412. t Support Center website www wyatt com support and follow the links to Downloads gt Databases Follow the link to the SQL Server 2005 Express Edition installation and connection instructions In addition the ReadMe Files directory of your ASTRA installation contains information about database and network issues Each experiment run with ASTRA uses an average of about 0 5 MB of storage so database size should be based on estimated number of experi ments to be saved Also a database user account and password must be created for the database The user account must have privileges to create delete and modify tables in the database M1000 Rev H Connecting to a Database Viewing the Current Experiment Database To see the current experiment database path follow these steps 1 Login to ASTRA using an account with ASTRA Administrator access 2 Choose System Database Administration Properties This opens the Database Properties dialog ASTRA V with Security Pack and ASTRA V with Research Database must be connected to an experiment database Only one database can be used at a time However it is possible to connect to different databases for differ ent types of experiments Creating a Microsoft Access Experiment Database Although SQL Server is recommended over Microsoft Access you can use Microsoft Access for your experiment database To create a new Microsoft Access experiment database and connect to it with ASTRA follo
413. t entry in the list Unless you changed the sorting this is the previous entry in time e Copy Icon Copy the text of the event to the clipboard You can then past the text into another application such as a word processor Deleting Experiments Security Only an ASTRA Administrator is permitted to delete experiments from the experiment database You should only delete an experiment if it is per mitted by your 21 CFR Part 11 policies and procedures To delete an experiment follow these steps 1 Choose System Database Administration Delete Items This opens the Select an item to delete dialog Experiments you have saves are in the TT folder This dialog also lists profiles templates sample sets and other items stored in the experiment and system databases WW Select an item to delete Close CI Example Configurations Import Templates CI My Profiles My Templates F collection only g empty sample set CI System Solvents System Templates 2 Highlight the experiment you wish to delete in the list 4 8 M1000 Rev H Performing Database Maintenance 3 Click Delete 4 Click Close when you have finished deleting experiments This command can also be used to delete sample sets system profiles and experiment templates For more information see Deleting a Sample Set on page 9 11 Deleting a Profile on page 12 7 and Deleting a Template on page 6 19 Performing Database Maintenance
414. t folder and finally open the RI calibration template In the experiment expand the Configuration node and verify that the proper experimental parameters have been entered Update the configuration if necessary Expand the Procedures node of the template Click on the Basic col lection procedure and enter appropriate values for each of the fields A typical Duration for the dRI calibration experiment is 20 minutes or more Close the dialog by clicking the OK button and run the experi ment Introduce pure solvent blank into the reference cell making sure the solvent flows through the system at a constant rate dRI detectors do not react favorably to sudden changes in flow as the detector drift would overwhelm the signal Thus it is important to maintain a nearly constant flow through the instrument while mea suring the solutions While ASTRA V collects data begin to introduce the series of prepared standards into the sample cell of the detector Each standard solution is introduced one at a time beginning with the lowest concentration Wait for the signal to stabilize allow signal to reach a plateau which may take several minutes while the previous solution is completely rinsed out of the cell After all the standards have been injected re inject a pure solvent sample blank to re establish the baseline Once a good baseline signal is acquired stop the ASTRA V data collec tion Data Processing After the data coll
415. talling the new version When you deinstall any files you have created or modified such as experiment files your experiment database and the system database are not deleted To deinstall an old version of ASTRA V follow these steps 1 Choose Start gt Control Panel from the Windows Start menu 2 Double click the Add or Remove Programs icon 3 Scroll down in the Add or Remove Programs list to Wyatt Technology ASTRA 4 Click the Remove button Install the new version of ASTRA as described in the previous section You can choose to install the new version of ASTRA in the same loca tion as the previous version This allows you to easily continue using the same experiment database and other files Migrating the System Database Security M1000 Rev H You must use an account with ASTRA Administrator access to follow the steps in this section The ASTRA system database stores experiment templates sample set templates system profiles system solvents and molecular standards These include items that are provided with ASTRA and items that you customize and save as templates or profiles All versions of ASTRA use a system database The installation does not overwrite an existing system database To update your system database so you have all the latest experiment tem plates and system profiles follow these steps 1 Choose System Database Administration Migrate 2 You see a message that says the procedure will cop
416. te the width of the fitted distribution They are asymmetric because the hydrodynamic radius is inversely proportional to the symmetric decay time distribution M1000 Rev H 8 99 Chapter 8 Editing Procedures Administration Procedures Administration procedures add an experiment log entry for the audit trail Sign Off Security In order to comply with 21 CFR Part 11 your operating procedures may require that one or more electronic sign offs be associated with each exper iment A Sign off procedure may be located at any position in the experiment sequence specified by your policies and procedures The user who runs the experiment is prompted to set parameters for the Sign off procedure The sign off dialog looks similar to the following WW linear sample vaf Procedures Sign off SEE Category Comments Run conditions changed to account for solvent change User Id bhzimm Password seo ook bk a oe Domain WYATT BY OK Si Cancel The properties to enter are as follows Table 8 37 Sign Off Properties 8 100 Field Description Category The type of sign off Options are Responsibility Approval and Review Comments Any comments about this experiment User Id A valid ASTRA user ID This need not be the same user ID used to log into this ASTRA session or this Microsoft Windows session Password The password for the specified user ID This is case sensitive Domain The domain for the specified u
417. the graph identify the flat no flow regions associated with each solvent Define a peak for each solvent by selecting a plateau of approximately 30 seconds located near the end of the flat region M1000 Rev H Configuration Procedures 2 Beneath the graph a table is shown with a column for each defined sol vent Expand the Refractive index node A row will now be visible titled Real In each solvent s column enter the known aRI value Make sure that it is the correct aRI value for the Optilab rEX s operat ing wavelength Table 8 7 lists aRI values for several solvents at vari ous wavelengths Click the OK button to close the window 3 Click on the Absolute RI Calibration procedure to open its settings pane Enter the dRI calibration constant dRI offset old aRI cal ibration constant and old aRI offset These are the numbers recorded in Step 2 on page 8 27 Click OK to close the window W absolute RI calibration Procedures Absolute RI calibration DER absolute RI calibration Iw data V fit 0 5 1 0 real refractive index RIU 2 EL e E T Q S e o 3 Value Hew Abs RI Calibration 5 1873 0 0442 e 3 RIU pixel New Abs RI Offset 2 6433 0 1373 RIU Old Abs RI Calibration RIU pixel 4 4700e 003 Old Abs RI Offset RIU 2 4767e 000 Diff RI Calibration RIU pixel 3 5000e 005 Diff RI Offset RIU 8 9037e 003 Percentage To Keep 100 Enabled Peaks Peak 1 Water E
418. the system database As such it is available to all users and can be imported into experiments or sample sets For example several common instrument configura tions might be saved as system profiles such that they can later be easily used in different experiments user mode You can choose to use ASTRA in Run mode or Experi ment Builder mode Run mode is easier to use Certain options are not available in Run mode workspace The portion of the ASTRA interface that shows the Experiments Sample Sets and System Profiles tabs and their contents Batch Mode vs Online Mode The distinction between batch mode and online mode is an important one for all types of instruments Batch Mode In a batch mode experiment the measurement instru ment stands alone and is not hooked up to a pump Samples are introduced into the instrument via vials or by injecting slugs of sample that completely fill the sample cell The concentration of the sample is known since the researcher has prepared it Also the solvent for the sample now needs to be associated with the instrument since it no longer comes from the pump Batch mode is also called non fractionated Online Mode In an online mode experiment a measuring instru ment such as the Optilab or DAWN EOS is connected to a pump and injector The solvent flow is controlled by the pump and the sample is added by the injector The solution continually flows through the system an
419. tion LS RI online S Generic pump pump d Solvent water Injector injector Sample untitled1 DAWN EOS DAWN EOS 3 Optilab rEX Optilab rEX a Fluid connection pump to injector Zu Fluid connection injector to LS ST Fluid connection LS to RI wi Auto inject connection O Figure 7 3 Configuration for an LS Online Experiment The hardware setup for the configuration in Figure 7 3 would be organized similar to Figure 7 4 data to computer injector DAWN Optilab flow rate volume Bess wave wave solvent sample f length length flow cell temp gains etc etc fluid dn dc connection refraction A2 flow cell volume viscosity concen glass RI Rayleigh tration geometry ratio etc Figure 7 4 Hardware Connections for LS Online Experiment Apparatus 7 4 M1000 Rev H Using Configurations Using Configurations Security Each experiment has a Configuration item that contains descriptions of all of the physical components used in the experiment The actions you can perform on a configuration are simple The complexity comes from the large number of physical components that can be used and the large number of properties some of these components have You must have at least Researcher access to work with configurations and system profiles If you are a Technician or Guest you have read only access to profile
420. tion has been calculated for each sample cell using computer ray tracing simulations based upon the exact geometry of the sample cell laser beam and detection optics for the 90 degree detector in the DAWN and miniDAWN For the K5 and F2 flow cell the resulting geometry correction goes as neng i e the index of refraction of the solvent times the index of refrac tion of the glass respectively The complete expression taking into account the reflection and geometry corrections is therefore Equation 7 nn Acsce Ainst TP Sg ga for the standard flow cell with an antireflection coated entrance window and uncoated exit surface and the transmission terms are calculated using Eq 5 For the scintillation vial both the geometry and solvent glass reflection corrections were folded into the ray tracing calculations so only the factor for the reflection correction due to the two uncoated glass air interfaces of the scintillation vial are in the final expression The resulting complete formula for the scintillation vial is Equation 8 nh Aesce Ainst T ga For the MicroCuvette both the geometry and solvent glass reflection cor rections were folded into the ray tracing calculations Both the entrance and exit windows for the MicroCuvette are anti reflection coated so there are no explicit reflection correction terms in the final equation The result ing complete formula for the MicroCuvette is Equation 9 _ 1 983 A A mn
421. tive Index Refractive Index Model gt Reference Refractive Index Specify the refractive index of the solvent at the wavelength and temperature you will use This property is required for both Fixed and Polynomial models Refractive Index Model gt Parameters 2 5 Refractive Index Model gt Reference Temp If the Refractive Index model is Polynomial set Parameters 2 5 using the following polynomial model which is used to compute the Refractive Index B REP n L P 3 4 4 PP L L L e P4 is the reference refractive index e P through Pz are solvent specific constants e Lis the wavelength in micrometers Um of the laser in the light scattering instrument This is taken from the light scattering instrument profile as part of the configuration so it does not need to be specified here If the Refractive Index model is Polynomial set the Reference temperature Pg in the previous equation C for which this fit is valid Rayleigh Ratio Model The model used to specify the Rayleigh ratio of the solvent May be Fixed or Corrected Lambda 4 Set the model type before setting the parameters required for that model 7 32 M1000 Rev H Solvent Profiles Table 7 21 Solvent Properties Field Description Rayleigh Ratio Model gt Reference Rayleigh Ratio If the Rayleigh Ratio Model is Fixed specify the Rayleigh ratio of the solvent in 1 cm at the wavelength and temperature you will
422. ton you can drag the data to view a different portion of the graph This is useful when you have zoomed in and want to scroll around the graph Press Ctrl right mouse button to restore the graph to its original position Axis Settings To control the values shown on each axis follow these steps 1 5 Double click on a graph to open the Edit Graph dialog Then click Advanced In the Chart tab choose the Axis tab The editing dialog has multiple levels of tabs In the Axis tab you can select an axis and then change many aspects of how that axis is displayed For example click Change under Mini mum or Maximum to change the range of values for that axis Also this tab has a checkbox to turn logarithmic scaling on and off If you drag the Edit Graph dialog to the side you can see the effects of changes in your graph as you make them Click Close when you are finished changing the display Other tabs of interest in the advanced Edit Graph dialog are as follows Chart Axis Title Modify axis title display Chart Axis Labels Modify axis label formats and font Chart Axis Ticks Modify major ticks on selected axis Chart Axis Minor Modify minor ticks on selected axis Chart Titles Modify graph title text location and format Chart Legend Modify graph legend location and format For help on settings in the advanced Edit Graph dialog move to a field and press F1 11 14 M1000 Rev H Printin
423. top Ctrl Shift S Halt the experiment See page 6 13 T R A Experiment EASI Graph Add a customizable graph to the results All See page 11 3 Experiment Report Export Export a report to a text file or a comma R A separated values file See page 10 6 Page Setup Set up page display for printing reports This command is not yet implemented Properties View the properties of the report This command is not yet implemented M1000 Rev H A 5 Appendix A Menu Quick Reference Command Keyboard Description Shortcut Experiment Graph Add Custom Plot Add a custom plot to the results See R A page 11 10 Add Parametric Plot Add a parametric plot to the results See R A page 8 83 Add Surface Plot Add a surface plot to the results See R A page 11 12 Auto Scale Auto scale the current graph This com R A mand is not yet implemented Scale Change graph scale manually This com R A mand is not yet implemented Export Export a graph to a text file or a comma R A separated values file See page 11 16 Page Setup Set up page display for printing graphs This command is not yet implemented Properties View the properties of a graph This com mand is not yet implemented Experiment Sign Off Allow user to sign off experiment for 21 TRA CFR Part 11 compliance See page 6 12 Experiment Log Open Display ex
424. tor You need to enter the dn dc and UV extinction values for the protein and modifier Total mass and protein fraction on a slice by slice basis are dis played ASTRA calculates the size of the complex mass of the complex and masses of the constituents displaying them with rigorous uncertain ties In addition the concentration and calculated dn dc values are displayed for that elution volume mass vs volume with correction 5 1 0x10 8 0x104 6 0x104 4 0x104 out Di ssew jow Rayleigh ratio 1 cm 2 0x104 7 0 75 8 0 volume mL Particles AT In particles mode it is possible to measure the size radius and number density of a sample using just a light scattering detector without any con centration detector Note that you won t be able to measure the molar mass using this template Number Density AT This template provides a procedure to calculate number density which means how many particles you have in your sample Particle measure ments are especially suited for use when a light scattering instrument is M1000 Rev H B 9 Appendix B System Templates coupled to a fractionation technique such as Field Flow Fractionation FFF or Capillary Hydrodynamic Fractionation CHDF but concentra tion is not measured This procedure calculates the radius and the number of particles per mL density in the sample You must specify the refractive index of the sample in the Define Peaks procedure to dete
425. trument Table 7 5 WyattQELS Instrument Properties Field Description Name Name of the instrument If you have already created a system profile for this instrument click and select a profile to use Description Description of the instrument which typically contains more information M1000 Rev H than the Name 7 15 Chapter 7 Configuring Experiments Table 7 5 WyattQELS Instrument Properties Field Description Physical Instrument Choose an instrument from the drop down list If your instrument is not listed choose Browse to open the Instruments dialog See Accessing and Viewing Hardware on page 2 10 Use QELS Temperature Check this box if the instrument is set to use a temperature probe signal Probe during collection Uncheck this box if you have a temperature controlled DAWN or miniDAWN If this box is unchecked the temperature is taken from the temperature set for the DAWN or miniDAWN instrument Model The instrument model Options are Wyatt QELS Flex 99 and Flex 99 ADN 7 16 M1000 Rev H Refractive Index Instrument Profiles Refractive Index Instrument Profiles A refractive index instrument measures the differential refractive index dRJ of a solution in order to calculate the concentration of the sample In order to calculate the concentration from the differential refractive index it is necessary to know the dn dc value for the sample Opt
426. trument profile you have saved as described in Creating Profiles on page 12 3 For example you might navigate to the My Profiles folder to select a profile you have created Select the instrument profile you want to add and click Open The instrument is added to your configuration and you can edit its proper ties by double clicking it in the configuration tree To add a connection follow the same steps but click the Browse but ton in the row to add connections M1000 Rev H Using Configurations Removing Instruments and Connections Experiment Builder You can remove items from a configuration only if you enable Experiment Builder mode by choosing System Preferences gt Experiment Builder Mode If you have already opened the Configuration properties dialog close and reopen it after enabling Experiment Builder mode 1 Choose Experiment Configuration Edit This opens the proper ties dialog for the configuration which has a tab for each item in the configuration tree 2 In the Experiment configuration tab click the Browse button in the row to remove instruments or the row to remove connections 3 In the Remove Instrument Profile or Remove Connection Profile dia log check the box next to the item you want to remove and click OK Replacing an Experiment Configuration or Item M1000 Rev H It is possible to replace an entire configuration with an experiment config uration stored as a system profile A
427. ts icon as follows Collection procedures have a special two page icon for all states Procedure has not been run since the procedure was last modified Procedure has been run successfully Procedure is currently running EN Eg liis i Procedure is in an invalid sequence location or does not have the nec essary data to run Starting a Data Collection Run M1000 Rev H To start the experiment run follow these steps 1 Begin by turning on warming up and stabilizing your experimental apparatus When everything is ready to go continue with the following steps in ASTRA 2 Choose Experiment Run 6 11 Chapter 6 Creating amp Running Experiments Shortcuts Press Ctrl Shift R Click the Run icon gt inthe experiment toolbar Right click any folder in the experiment tree and choose Manage Run You can alternately choose Experiment Run Indefinitely to run the experiment until you stop it This command ignores the Duration property for the collection The experiment will also stop collecting data if your disk or database runs out of storage space 3 For an experiment with a basic collection procedure as opposed to a custom script you will be prompted to click OK and then inject the sample 4 The spinning hourglass icon on the experiment node in the workspace shows that the experiment is running Alternately the drop down in the experiment status toolbar shows the current state
428. ts produced by a traditional Zimm plot analysis ASTRA The data collections analysis and lab control software for Wyatt Technology Corporation instruments batch mode Data collection performed in a stand alone fashion fluid injected into a cell not being pumped through plumbing For example using the MicroCuvette with the DAWN Batch measure ments are typically on unfractionated samples Batch mode is also called off line configuration ASTRA s description of the physical apparatus used to collect data It is an assembly of profile units describing sample solvent and instruments as well as the connections both fluid and signal between them data set The grouping of data produced by a procedure For example a typical light scattering measurement might produce a set of data including molar mass and RMS radius DAWN Multi angle light scattering detectors from Wyatt Technology Corporation They are used to determine the molar mass size and second virial coefficient for macromolecules in solution Versions include the DAWN EOS and miniDAWN dn dc The change in a solution s refractive index with a change in the solute concentration Measured in mL g experiment database The database in which ASTRA V with Research Database and ASTRA V with Security Pack store experiment information and data fractionation The separation of a polydisperse solution of macromol ecules by some physical property of the macromolecule
429. ty dialog If you created a profile type that has multiple components there will be a tab for each component For example a light scattering instru ment in batch mode has a tab for a solvent and a sample This infor mation is stored with and imported with the instrument profile 6 Edit the properties as needed See Chapter 7 Configuring Experi ments for details about the properties of all profile types 7 Click OK or Apply to save your changes 12 3 Chapter 12 Working with System Profiles Note 12 4 Exporting a System Profile You can create a system profile by saving it from an experiment To do this follow these steps 1 If you have more than one experiment open make sure the one you want to export from is selected in the experiment tree of the work space Select the item in the configuration you want to export Any items nested at a lower level will be exported along with the item you select For example in the following figure exporting the injector creates a system profile that contains the injector and the sample If you export the configuration item the entire configuration is saved as a system profile Choose Experiment Configuration Save Configuration As Or right click on an item and choose the Save As item from its right click menu In the Save As dialog choose the folder where you want to save the system profile Then type a name for the profile you are creating and click OK
430. u set the width of the line Some lines are made up of individual data points and some are drawn as a line Use the field that applies to your graph To change the color of a data set choose a data Series Then in the Color field select a defined color or Custom to choose other colors You can see the effects of your changes as you make them without closing this dialog If you click Advanced you have much more control over the graph display is provided than is described in this manual For help on settings in the Advanced dialog move to a field and press F1 Graph customizations such as line weight color marker style and title changes are saved for EASI graphs custom plots parametric plots and surface plots However such graph customizations are not saved when you close procedure dialogs that contain graphs See page 11 6 for details about the EASI Graph version of the Edit Graph dialog 11 13 Chapter 11 Working With Graphs Zooming In and Out Graphs Zooming in Hold down the Ctrl key and your left mouse button Drag a rectangle around the data you want to view larger Then release the mouse button Alternately you can press Ctrl F5 to zoom in one level Zooming out Hold down the Ctrl key and click your right mouse button Each click undoes one zoom in action Alternately you can press Ctrl Shift F5 to zoom out one level Repositioning Graphs When you hold down both the Shift key and the right mouse but
431. uests e ASTRA Guest Has read only access to experiments and results Where necessary the user level required to perform an action is identified Security i T in this manual Lines above and below the Security icon in the left margin as shown here highlight such information Security information is specific to ASTRA V with Security Pack There are no access restrictions in other operating tiers M1000 Rev H 3 3 Chapter 3 Getting Started ASTRA Tutorials on the Support Center Several tutorials are Welcome to the Wyatt provided for ASTRA on the Wyatt Tech Technology Support Center nology Support Be sure to let us know when you Center Log in and publish light scattering results 4s click the Tutorials from your Wyatt hardware Email e Ie youdon t us for a FREE t shirt mug or E gt NS laser pointer cis already have a P A support center account you can sign www wyatt com support up for one Currently the support center provides the following tutorials We strongly encourage you to use these tutorials to learn how to use ASTRA e ASTRA V Presentation This PowerPoint presentation provides an introduction to ASTRA V and the terminology it uses e ASTRA V Templates Exercise This tutorial shows you how to connect to instruments create experiments from a template adjust experiment configurations save experiment and profile templates and run experiments e ASTRA V S
432. use If the Rayleigh Ratio Model is Corrected Lambda 4 specify the Rayleigh ratio of the solvent at the reference wavelength in 1 cm Rayleigh Ratio Model gt Reference Wavelength If the Rayleigh Ratio Model is Corrected Lambda 4 set the reference wavelength in Um R 8 is the calculated solvent Rayleigh ratio using this formula 2 TETE APE a L H n 1 e P is the Reference Rayleigh Ratio of the solvent e Pais the Reference Wavelength in Um e Lis the wavelength in um of the laser in the light scattering instrument This is taken from the light scattering instrument profile as part of the con figuration so it does not need to be specified here e ny is the refractive index of the solvent at the wavelength of the laser in the light scattering instrument This is calculated using the formula speci fied for the Polynomial Refractive Index Model e nyo is the refractive index of the solvent at P the reference wavelength This is calculated using the formula specified for the Polynomial Refrac tive Index Model Viscosity Model The model used to specify the viscosity of the solvent May be Fixed Lin ear or Exponential Set the model type before setting the parameters required for that model Viscosity Model gt Reference Viscosity If the Viscosity Model is Fixed specify the viscosity of the solvent in P Poise at the temperature you will use If the Viscosity Model is Linear or Exponential
433. using ASTRA V with Research Database and open the branching ana lyzed vaf experiment in the Sample Data gt Practice Experiments gt Branching folder The procedure has the following dialog WW branching analyzed Procedures Branching branched conformation plot Iw branched conformation plot V branched conformation plot fit linear conformation plot Iw linear conformation plot fit rms radius nm 5 0x10 6 0x107 0x18 molar mass g mol Linear Conformation Slope 0 61 0 00 Branched Conformation Slope 0 45 0 00 Use Linear Model Imported Linear Data E Development ASTRA4_5 3 2 3 Data Sample D Plot conformation plot o Model trifunctional Ei Slice Type monodisperse Repeat Unit Molar Mass g mol 1 000e 002 Method radius Ei Linear Mark Houwink Sakurada K Parameter 1 000 Linear Mark Houwink Sakurada a Parameter Drainage Parameter e Linear Radius Model k Parameter Linear Radius Model b Parameter Sa OK Fa Cancel M1000 Rev H 8 81 Chapter 8 Editing Procedures The properties for this procedure are as follows Table 8 28 Branching Properties Field Description Linear Conformation Slope The slope of the linear conformation plot fit line This is calculated from the graph and is not settable Branched Conforma tion Slope The slope of the branched conformation plot fit line This is calculated from the graph and is not settable Use Linear Model
434. vent scattering This procedure is normally placed after any despiking or smoothing you want to perform and before conversion or analysis procedures When this procedure runs you see a message that says a baseline needs to be set Set a baseline by following these steps 1 Click OK to open the dialog for setting baselines W vs Is ri online ps standards in thf Procedures Baselines Define Baselines e NN w ono oN gt e S gt be S S 2 S 3 20 0 time min CDR Auto Baseline Source L5 1 Auto Baseline ll KH swe OK x Cancel EM Apply 2 In the list to the right of the graph select the detector for which you set the baseline For example detector 11 To check the quality of your data select different detectors from the detector list and examine the peaks 3 Click on the graph to add a baseline to the collected data You can press Delete to remove a baseline 4 Use your mouse to drag the baseline ends to appropriate locations Set the baseline ends far enough from the peak where the baseline is flat so they do not interfere with the signal M1000 Rev H 8 45 Chapter 8 Editing Procedures By default baseline ends snap to the voltage level for a particular time If you hold down the Shift key you can drag the end of a baseline to any location This may be useful if the collection was interrupted before the signal returned to the original baseline
435. w these steps 1 Login to ASTRA using an account with ASTRA Administrator access 2 Choose System Database Administration Connect to Data base This opens the Select Data Source dialog 3 Connect to an existing ODBC data source or create a new one using this dialog Remember that only Microsoft Access and SQL Server databases are supported For example you might follow these steps Move to the Machine Data Source tab in the Select Data Source dialog and click New Select System Data Source and click Next Alternately you could select User Data Source if you only want this account to access this database Select Microsoft Access Driver mdb and click Next Alter nately you could select SQL Server e Click Finish Type a Data Source Name For example astra_exp_db Type a Description For example ASTRA Experiment Database e Click Create Inthe New Database dialog type a database name For example astradb mdb Also browse for a location to store your database For example c Program Files WTC Astra V Database Click OK four times in the New Database dialog the success mes sage the ODBC Microsoft Access Setup dialog and the Select Data Source dialog M1000 Rev H 4 5 Chapter 4 ASTRA Administration Managing User Accounts Security 4 6 User accounts in ASTRA V with Security Pack are managed as Microsoft Windows user accounts You assign each user account that can acce
436. width of 127 48 125 55 1 93 min You should keep the other peaks to as close to the same width as possible while still capturing the leading and trailing edges of the peak 6 The Online A2 procedure is now ready for processing When you open the procedure be sure the dn dc value is set to the correct value for the sample being analyzed If you have previously set a dn dc value in the sample profile in the experiment configuration the number should already be filled in 7 By adjusting the fit degree from 0 2 for Angle Fit Degree and Con centration Fit Degree you can change the types of results produced The following table shows which results will be listed as n a depend ing on your fit degree settings Table 8 20 Online A2 Results for Various Fit Degrees Angle Fit Degree Sin2 T 2 Concentration Fit Degree Results Excluded 0 R 4 Radius RMS Radius 1 R44 Radius 2 None 0 A2 A3 1 A3 2 None 8 68 M1000 Rev H Analysis Procedures Mass from VS Data This procedure calculates the molar mass using viscosity data It uses a Mark Houwink Sakurada analysis to calculate the intrinsic viscosity See the Operating Principles and Theory appendix of the ViscoStar User s Guide for details You can use the Mark Houwink Sakurada procedure on page 8 83 to determine the appropriate Mark Houwink Sakurada parameter values for your polymer solvent and temperature comb
437. x E detector 3 detector 4 detector 5 3 52 x10 detector 6 2 52 x10 detector 7 4 52 x10 detector 8 j detector 9 1 9462 6 9462 11 9462 16 9462 i detector 10 time min detector 11 Smoothing Level Le ay ok RK Cancel SI Apply 5 52 x10 4 52 x10 64668648 4 v D S gt _ S Kan 5 2 S 5 If data has already been collected for this experiment the graph shows the data with the currently selected smoothing level applied After you change the smoothing level you should check the baselines and peaks to make sure no changes are needed because of the smoothing Smoothing always acts on the raw data You cannot increase smoothing by re smoothing The property you can set is as follows Table 8 13 Smoothing Properties Field Description Smoothing Level Choose the degree of smoothing The options are none normal or heavy The default is none Note Smoothing improves the appearance of the displayed data but not the pre cision To increase the precision of the calculated MM and radius you need to increase the signal to noise of the system by reducing the baseline noise and or increasing the signal from the polymer 8 44 M1000 Rev H Transformation Procedures Baselines Setting a baseline enables ASTRA to subtract the base signal from the col lected data For light scattering experiments the baseline level includes the photodiode dark offset and the sol
438. xing Term See Band Broadening on page 8 14 for an explanation of the mixing term The units are in microliters M1000 Rev H For Optilab rEX users there are several utility templates in the System Templates gt RI Measurement gt Optilab rEX Specific folder These experiment templates include Purge On Purge Off and Zero dRI We recommend that you purge the Optilab rEX when not running samples the Purge On template is a convenient way to automate this as part of a sample set In addition Optilab rEX templates for absolute RI calibration and RI calibration from a peak are included in this folder The purge valves on Optilab rEX instruments are automatically closed at the start of data collection The exception to this is when absolute RI analysis is conducted where the Optilab rEX purge valve must be left open 7 17 Chapter 7 Configuring Experiments Optilab DSP Profiles You do not select a Physical Instrument for the Optilab DSP because ASTRA V does not support a direct data connection to this instrument When using the Optilab DSP it is necessary to add an AUX connection to the experiment configuration to indicate which AUX channel and instru ment are to be used to read the Optilab DSP signal See AUX Connection Profiles on page 7 29 for details You can set the following properties for a Optilab DSP instrument Table 7 7 Optilab DSP Properties Field D
439. xperiment from the default tem TRA plate and start running it See page 6 4 File gt Import Experiment Import an experiment from a file See DB and page 6 9 Security R A Sample Set Import a sample set from an ASTRA 4 or DB and ASTRA 5 file See page 9 4 Security R A Empower Sample Import a sample set created with Waters R A Set Empower See page 9 5 File gt Export Experiment Save the selected experiment to a separate R A file See page 6 17 Sample Set Export a sample set from the database to a R A file See page 9 11 File Batch Apply Apply experiment template to multiple exper T R A Template iments See page 6 23 File gt Print Ctrl P Print the currently active view See page 3 All 15 File gt Print Preview Preview the currently active report See page All page 10 6 File Print Setup Set up page formatting for printing See All page 3 15 File gt Recent Open a recently used experiment See All Experiments page 6 7 and page 6 8 File gt Exit Close all windows and exit from ASTRA V All See page 3 15 M1000 Rev H A 3 Appendix A Menu Quick Reference Edit Menu The Edit menu contains the following commands Command Keyboard Description Shortcut Edit Undo Ctrl Alt Backspace Undo the previous action This command All is currently disabled Edit gt Cut Ctrl Shift NumDel Cut the currently selected item and
440. xperiments This chapter explains how to configure your experiments in ASTRA to reflect the instruments connections solvents and samples you use to collect and process data This is accomplished using ASTRA V configura tions and system profiles CONTENTS PAGE About Configurations and Profiles 7 2 Using Configurations eesseeeeeeeeessressiesetetsrissrissinssinsssinsstnnsnnssen nent 7 5 Experiment Configuration cccccccceesececeeeeeeeeeeeceeeeeeeeaeeseeeeessaeeseenees 7 9 Light Scattering Instrument Profiles cceeeeeeeeceeeeeeeeneeeeeeeeeesaeeeeaes 7 11 Refractive Index Instrument Profiles c cccceceeceeeeeeeeeeeeeeeeeeseeeeeseneees 7 17 Viscometry Instrument Profiles 7 21 UV Absorption Instrument Profiles cccceeeeeeeeeeeeeeeeeeeeseeeeeenaeeteaes 7 24 Injector lte UE 7 25 PUMP PHOflS geseit entegieedieeu gees ee idee cenmaeebdisteebaansans detgvendeaedeaaecee 7 25 Column ProtileSccas Anaahat nee ii lea ies 7 26 Connection Profiles A 7 28 Sample Profiles sisii aiaia a eaa Eaa aaia aaa 7 30 Solvent Profiles eu eeg gegeggegdeer dd ee anaana aa aE AEE aE 7 32 M1000 Rev H 7 1 Chapter 7 Configuring Experiments About Configurations and Profiles The configuration of an experiment reflects not only the physical appara tus used to collect the data but also elements such as the solvent and sample In describing the configuration ASTRA V breaks up the different parts of th
441. xperiments are in the top level folder As in standard file selection dialogs you can click the icon to change the way the list of experiments in the database is viewed In the detail view the last data and time the experiment was modified is shown 3 Select an experiment and click Open Tips You can open multiple experiments by holding down Shift key for a range or the Ctrl key for individual experiments while selecting experiments M1000 Rev H 6 7 Chapter 6 Creating amp Running Experiments Opening an Experiment from a File Basic Shortcuts 6 8 You can open and work with any experiment you have saved If you are using ASTRA V Basic experiments are stored in separate files with an extension of vaf To open an experiment follow these steps 1 Choose File gt Open Experiment Press Ctrl O Click the down arrow next to the DG icon Right click Experiments in the workspace and choose Open Drag and drop an experiment file from Windows Explorer or the desktop to the ASTRA window Open a recently used experiment from the list in the File menu 2 Inthe Open dialog navigate to the folder that contains the experiment you want to import 3 Select a file and click Open You can open any of the following types of files File Extension Description vaf Experiment file saved or exported by ASTRA V vrf ASTRA V crash recovery file See page 6 14 adf File saved by
442. y and delete experiment files Also has privileges of Researchers Technicians and Guests ASTRA Researcher Can create and modify experiment files Can connect to networked computers and instruments Also has privileges of Technicians and Guests ASTRA Technician Can run a given experiment procedure sequence and save the resulting data Also has privileges of Guests ASTRA Guest Has read only access to experiments and results In operating tiers other than ASTRA V with Security Pack users are not prompted to log in with a user name and password Where necessary the user level required to perform an action is identified in this manual Lines above and below the Security icon in the left margin as shown here highlight such information Security information is specific to ASTRA V with Security Pack There are no access restrictions in other operating tiers Setting Up Groups To create the groups you will use with ASTRA follow these steps Security 2 6 1 2 Log in using a Windows account that has administrator privileges Right click on My Computer in the Windows Start menu and select Manage This opens the Computer Management window In the tree on the left expand the Local Users and Groups item which is within the System Tools list Right click on Groups under Local Users and Groups and select New Group In the New Group dialog type the following e ASTRA Administrator in the Group name fi
443. y and use your mouse drag an outline around the area you want to see To zoom back out hold down the Ctrl key and click your right mouse button 6 If you selected Forward Monitor in the Divide by Laser Monitor field or the configuration for your light scattering instrument see page 7 11 you should create a peak for pure solvent and specify the number of this peak in the Experiment Configuration on page 7 9 This pure solvent peak acts as a baseline for the forward laser monitor signal If you do not specify such a peak the average of the first ten percent of the forward laser monitor signal range is used as a baseline 7 After setting the peaks enter the relevant information for each peak as needed in the property list Depending on the type of analysis to be performed different properties need to be specified See Table 8 15 to 8 52 M1000 Rev H Transformation Procedures determine which properties you need to specify the procedure lists for some properties may not be complete The list contains the following fields for each peak Table 8 15 Peak Properties Field Description Name A name you can give to the peak for use in reports Start The x axis starting point for the peak If you set peaks using the graph the Start and Stop values are set automatically Alternately you can type values in these fields The units are determined by the Abscissa Units property of the experiment configuratio
444. y custom templates and profiles to the new database Click Migrate to continue 2 3 Chapter 2 Installing and Setting Up ASTRA Hint 2 4 3 You see a warning that system log entries such as login attempts and database connections are not copied to the new database To perform the migration click Yes A System log entries login attempts database connections etc cannot be migrated but will be retained in the system database backup Continue anyway You can use database migration to purge system databases that contain a large number of system log records Note that the migration does copy 21 CFR log information related to templates and profiles it migrates 4 As your database is migrated you see progress information Messages identify any templates or profiles that are not updated because you have customized them Migrate System Database Database migration completed Your system database is now up to date Details System database migration successful System log entries were not migrated but exist in backup databas The following experiment templates exist in the destination databa System Templates Light Scattering Diagnostics HELEOS Noise System Templates Light Scattering Diagnostics TREOS Noise System Templates Light Scattering with QELS cumulants vj lt gt Show backup You can migrate an existing system database created with ASTRA v5 3 or higher Whenever you migrate
445. y procedure 8 17 intrinsic viscosity converting from specific 8 57 definition of 1 6 ISI Instrument Server Interface definition of 5 2 installing on other computers 5 4 integrated 5 2 network requirements for 5 4 ISI list adding computers to 2 11 removing computers from 2 12 isotropic scattering and normalization 8 32 J JPEG files exporting graphs to 11 17 jumper setting 8 19 M1000 Rev H K keyboard shortcuts Alt F4 3 15 assigning to menu commands 3 14 Ctrl 8 5 Ctrl Alt N 12 3 12 5 Ctrl Alt T 3 5 6 5 Ctrl I 6 9 Ctrl mouse button 3 11 Ctrl N 6 6 Ctrl O 6 7 6 8 Ctrl P 3 15 Ctrl right mouse button 3 11 Ctrl S 6 15 6 16 9 10 Ctrl Shift N 9 3 Ctrl Shift O 9 4 Ctrl Shift P 6 26 6 28 6 29 8 7 10 4 11 8 11 11 Ctrl Shift R 3 9 6 12 Ctrl Shift S 6 13 Ctrl Shift V 6 11 Shift key 3 10 Shift F5 8 5 keys for optional modules 2 5 keys instruction conventions 1 5 L laser turning off 8 12 Laser Monitor 7 12 7 138 7 15 level of user definition of 1 8 light scattering instruments calibrating 8 19 definition of 1 7 performing experiment with 3 5 profiles for 7 11 see also LS data light scattering theory Berry fit method D 13 branching calculations D 19 calibration D 4 Debye fit method D 12 differential distribution calculations D 18 molar mass and rms radius moments D 15 molar mass and sizes D 11 normalization D 8 out of range values D 18 Ran
446. ystem database connection pre lo y 3 You can save the system log to a text file by choosing System Log Save As Viewing an Experiment Log Database To view the entire experiment database log choose System Database Administration Log Open To view the log for the current experiment right click any folder in the experiment tree and choose Manage gt Log Or choose Experiment Log Open from the menu bar For information about experiments see Chapter 6 Creating amp Running Experiments You can save the experiment database log to a text file by choosing System Database Administration Log Save As M1000 Rev H 4 7 Chapter 4 ASTRA Administration Viewing a Sample Set Log Database To view a log for a sample set right click any item in the sample set tree and choose Manage Log Open Or choose SampleSet Log Open from the menu bar For more about sample sets see Chapter 9 Using Sample Sets Working with Logs Database In any log the most recent action is shown at the top by default You can click the column headings to sort the log in other ways such as by cate gory event or user You can double click an entry to view a dialog with more detailed informa tion about that log entry The buttons in this dialog act as follows s Up Arrow Go to the previous entry in the list Unless you changed the sorting this is the next entry in time e Down Arrow Go to the nex
447. ystem profile has no effect on experiments to which the system profile was previously copied In addition modifying portions of an experiment configuration that came from a system profile has no effect on the original system profile Saving a Profile Changes to profiles are automatically saved to the system database when you click OK or Apply in their property dialog If you attempt to close the dialog without saving you are asked whether to save the changes Duplicating a Profile with Save As To save a profile with another name follow these steps 1 Select a profile in the System Profiles tab of the workspace 2 Choose File gt Save As 12 6 M1000 Rev H Modifying Profiles Shortcuts Right click on any profile in the workspace and choose Save As 3 In the Save As dialog select the system database location where you want to save the new profile 4 Type a name for the new profile 5 Click Save You can use Save As and then delete the original system profile to rename a system profile Closing a Profile To close a profile select the profile you want to close and choose File Close The profile is still available for use in the system database it is simply not shown in the System Profiles tab Shortcuts Right click on any profile in the workspace and choose Close Deleting a Profile To delete an existing profile follow these steps 1 Select System Database Administration Delete Items 2 In the Select an
448. zation choose File gt Open Experiment or File gt Import Experiment if you are using ASTRA V with Research Database and open the QELS batch reg ularization experiment in the Sample Data gt Analyzed Experiments folder For an experiment template choose File New Experiment From Template to open the regularization template in the System Templates gt Light Scattering gt With QELS folder For more about analyzing batch QELS data go to http www wyatt com solutions software ASTRA cfm and follow the links to Data Analysis gt QELS batch You can place this procedure at the end of a QELS batch experiment procedure M1000 Rev H M1000 Rev H This procedure has the following dialog Analysis Procedures W gels batch regularization bsa insulin Procedures Regulariz SEE log differential hydrodynamic radius differential intensity fraction 1log nm count rate Hz 0 01 04 1 0 10 0100 Wmm 0 hydrodynamic radius nm Peak Number Plot Rh diff intensity Mass Model Subpeak Rh Mean nm Rh Peak nm Rh Std Deviation nm Peak Area El Processing Conditions Temperature C Viscosity g cm zech Refractive Index Resolution Min Rh Threshold nm Max Rh Threshold nm El Processing Parameters Min Fit Delay Time sec 0 000e 000 Max Fit Delay Time sec 1 000e 000 Suppress Peaks Below nm 1 00 Show Residuals Use Disabled Slices Prefilter SW OK Si Cancel EH Apply control

Download Pdf Manuals

Related Search

Related Contents

TEINTE BROU DE NOIX OCRINE  TC4015BP, TC4015BF  épuisé épuisé épuisé épuisé épuisé épuisé  Notice d'utilisation - Service  - Frank`s Hospital Workshop    i はじめに - メガソフト    

Copyright © All rights reserved. Failed to retrieve file