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PD Discover32 - The Molecular Materials Research Center

1. Molt Cal A130 Mol wt Cal RI Cal Solvent Index Cal Solvent Index Hun Uy Inter detectar Vol ml U OBET oo gt Mol wt Cal UV 15 lf Instrument Ly Inter detectar Wol ml 0 0446 Molwe Cal uvas 18628 Mal wt Cal 04 90 1471 6 Mal wt Cal LIV 890 0000 Inst Cal values gt Files Molt Cal UV 90 sf d rece Mol wit Cal LIV 5045 0889 Figure 5 15 Setup Calculation Dialog Box 5 2 4 4 Determine Cal Constants The Determine Cal Constants tab is used to enter calibration parameters calculate the cal constants and accept them A detailed discussion of the calibration process is described in Chapter 6 for monodisperse polymers and Chapter 7 for polydisperse polymers 5 2 5 Results The Results dialog box presents a series of tabs that present calculated information 5 2 5 1 Summary The Summary tab Figure 5 16 presents an overview of the analysis and the information is dependent on the nature of the analysis The analyst can edit the data in the Run Information fields 5 14 Discovery32 Chapter 5 Summary Run Params hy Dist Mw vg Elu Fh Dist Rh vs Elu Fig Dist Fig vs Elu HI Light Scatterng Calculation Made MMz 1 Dalton 4 5r bel Iz Daltons 2 44 72 03 Muy Daltons 1 840803 hin Daltons 1 5r0e 003 Mw a Mn Ir Mp Daltons 2 1888 03 Haz nm estat Area m ml RI 4 41 fe O02 1 003e 001 3 0 42 002
2. wavelength nm 200 0 E Temperature C 348 Refractive Indes 1 3310 Figure 5 23 Correlator Information Dialog Box Polynomial Fit Setup presents the Polynomial Setup dialog box Figure 5 24 which is used to define the fit Rg vs Elu tab only Polynomial Setup X v Fitting Enabled vw Show fit Fit Setup Polynornial Order E Left Fit Limit min 12 084 Right Fit Limit min 47 35 Last Computed L aefficients Coef 0 2 6949e 000 Coet 1 6 4032 e 002 END Cancel Figure 5 24 Polynomial Setup Dialog Box If you select Fitting Enabled fitting will be performed based on the parameters indicated for the Polynomial Order the Left Fit Limits and the Right Fit Limits If you select Show Fit the equation describing the Rg fit will be indicated on the tab 5 20 Discovery32 Chapter 5 5 2 5 5 GPC The GPC command is used to determine the GPC distribution and obtain the best results for the fit When the GPC icon is selected the GPC window Figure 5 25 is presented GPC xj Calibrate Conventional GPC Distributions No Calibration data loaded Figure 5 25 The GPC Window To establish a calibration curve a Right click to access the pop up menu shown in Figure 5 26 Plot Limits shift Print Polynomial Fit Setup Data Table Load File Save File Add Narrow Standard Information Narrow Standard Info Setup Clear Data Table Figure 5 26 The GPC Popup M
3. The Wait for Autostart Signal check box is used to indicate if the run should be started by an external signal i e a contact closure from the chromatograph If this box is not checked the run is initiated by pressing the OK button When the Run button is pressed data collection is initiated A typical chromatogram displayed in the workspace as shown in Figure 3 7 KE Precision amp cquire32 Acquisition Program amp COMI Ele Miew Acquire Beview setup Monitor Help Dag E i B ER al e r 5 M L BSA URI 15 Degree 8D Degree Rh RI Time Minutes Ready Idle 040201 06 e 5 F Figure 3 7 Typical Chromatogram At the end of the run the data will be stored and another run can be performed Discovery32 Chapter 3 3 7 3 2 3 2 Multiple Runs Multiple Runs accesses the Multiple Runs dialog box Figure 3 8 which is used to describe and program a series of automated data acquisition runs Multiple Runs i X Hun Base Hun Sample Prestore H Injection Set Name Length Interval Delay Injections Wolume E eed ALIE sp nen celled OLE Cancel mm I L l l 1 0 0 0 3 1 0 OOS 1 400 5 0 1 0 0 0 3 1 0 04 1 400 5 0 1 0 0 0 3 1 0 O05 1 00 5 0 1 0 0 0 3 1 0 Fill O06 b 00 5 0 1 0 0 0 3 1 0 DO DESEE 5 0 1 0 0 0 3 1 0 O06 o 00 5 0 1 0 0 0 3 1 0 Ed O03 3 00 5 0 1 0 0 0 3 1 0 O10 10 00 5 0 1 0 0 0 3 1 0 O11 11 400 5 0 1 0 0 0 3 1 0 O12 12 00 5 0 1 0 0 0 3
4. Discovery32 Chapter 3 3 19 The fields in this dialog box can be used to calculate molecular weight in real time the settings are not carried over to Discovery32 3 2 6 The Monitor Menu The Monitor menu Figure 3 23 is used to view control the status of the laser data display and other Monitor components of the system Dump Commands Monitor Photon Level Stripchart Laser Figure 3 23 The Monitor Menu 3 2 6 1 Dump Commands The Dump Commands command is reserved for service 3 2 6 2 Monitor Photon Level The Monitor Photon Level command presents the current laser output Figure 3 24 1 58e 004 Photons sec Press STOP button to end Figure 3 24 Laser Level The Laser Output level is dependent on the Rayleigh ratio and the degree of cleanliness of the solvent The range is typically between 20 000 and 100 000 Aqueous based solvents such as water or phosphate buffered saline will have background counts of approximately 30 000 methanol and THF will have background counts of 40 000 and high scattering solvents such as TCB will have even higher values If the level is outside that range contact Precision Detectors 3 2 6 3 Stripchart The Stripchart command accesses the Stripchart Setup dialog box Figure 3 25 which is used to set the size of the display for data collection 3 20 Discovery32 Chapter 3 xl xmas mv U 0 Type Y min m 0 00 Fixed period wicth min 0 00 C Scrolling
5. Instrument Al Inter detectar Wol ml iinan Molat Cal RL 15 4291 6 Molat Cal R1290 19048 Mol wt Cal RI 288975 1 3320 Cal Solvent Index Instrument LIV Inter detector Val ml U b46 Mol wt Cal 0 715 2D254 3 Molw Cal v90 453380 Mal wt Cal LIV 745 2000 Inst Cal values gt Files Figure 7 14 Setup Calculation Constants Tab To align the peaks change the value slightly typically a change of 1 1s used and press Apply on the Constants tab then view the chromatogram again This procedure may require a number of iterations until the chromatograms coincide 7 6 2 Determining the RI Constant To determine the RI Constant a Expand the integration region of the chromatogram to include the entire area of the peaks for the refractive index detector The area should include the monomer and the aggregates if any but the terminal peak should be excluded b Return to the Determine Cal Constants tab of the Setup Calculation dialog box and check the Accept box that corresponds to the Refractive Index Constant value then click Apply compatible with Discovery32 where the RI constant is typically around 2 to 3 7 16 Discovery32 Chapter 7 Ee Note Precision Acquire32 will report RI constants that are between 75000 and 85000 which are not 7 6 3 Determine Calibration Constants for 15 and 90 Channels To determine the Calibration Constants for 15
6. Section 4 3 1 e Sample Interval Single Run Info dialog box Section 4 3 2 e MW Calc Method Single Run Info and Calibration Setup dialog boxes Section 4 3 3 Discovery32 Chapter 4 4 1 The parameters in PrecisionAnalyze32 that affect the noise in the chromatogram when dynamic light scattering is employed include e Run Time Correlator Configuration dialog box Section 4 4 1 e Sample Time Correlator Configuration dialog box Section 4 4 2 e Tolerance 1 and Tolerance 2 Correlator Run Setup dialog box Section 4 4 3 The parameters in PrecisionAcquire32 are stored with the data and cannot be changed after data acquisition The operator can choose a smoothing factor to be used on stored data in Discovery32 Section 6 4 and readily determine the effect changing of the smoothing factor on the chromatogram This suggests that the user would be wise to select smoothing conditions in PrecisionAcquire32 that are good but less than ideal since additional smoothing can be performed in Discovery32 If ideal smoothing conditions are selected in PrecisionAcquire32 and the analyst wants to see a feature in the chromatogram in Discovery32 it might be necessary to re run the separation and collect data again Unless the methodology is fully characterized it is safer to select No Smoothing in PrecisionAcquire32 and post process the file later with Discovery32 The nature of the smoothing factor 1s dependent on the type of macromolecule that y
7. PrecisionAcquire32 and Discovery32 are application programs that are used with a broad range of light scattering detectors manufactured by Precision Detectors Inc These detectors are generally used with HPLC High Performance Liquid Chromatography GPC Gel Permeation Chromatography SEC Size Exclusion Chromatography and FPLC Fast Protein Liquid Chromatography systems for the determination of the molecular weight distribution the hydrodynamic radius and related parameters of proteins virons and liposomes as well as synthetic polymers 1 2 PrecisionAcquire32 PrecisionAcquire32 1s used to set up the various parameters that are used to collect light scattering data A detailed discussion of this program is presented in Chapters 3 and 4 The program is configured for use with the PD2000DLS A D interface box or the PDConnect A D interface box The program requires connection with one of the interface boxes in order to access all of the various parameters Two types of commands can be set via PrecisionAcquire32 e System parameters which refer to parameters that define the system configuration and other settings that are infrequently changed An example of a system parameter 1s the selection of the port used for communications between the computer and the analyzer module e Operating parameters which refer to parameters that are specifically set for a given experiment or series of experiments These commands are frequently changed during
8. Sg7 Note This discussion assumes that the user has optimized the chromatographic separation and the compounds of interest are well resolved The user is encouraged to experiment with the parameters described below to determine the effect of small changes in the analytical results These experiments should be performed on a controlled basis In these experiments parameters that describe physical properties of the sample e g temperature and refractive index should not be altered This chapter describes how to select the value for several parameters to assist in optimize the results from the detector and describe the various tradeoffs that are made 4 2 PARAMETERS THAT AFFECT THE NOISE Since light scattering is extremely sensitive to microparticulates sloughing off a column or dissolved in the eluant it is an inherently noisy measurement if the proper operating procedures are not followed PrecisionAcquire32 and Discovery32 include a number of parameters that can be used to minimize the noise in the chromatogram While data is being collected on a continuous basis the system integrates and then stores the data at the frequency indicated in the Sample Interval field on the Single Run Info dialog box Figure 3 5 a typical value for this parameter 1s sec The parameters in PrecisionAnalyze32 that affect the noise in the chromatogram when static light scattering is employed include e Collection Fraction Select Active Detectors dialog box
9. The PD2000 DLS is a dynamic light scattering system consisting of a detector positioned at 90 to the incident laser beam a photon counter and autocorrelator electronics This system is available in an ambient temperature bench top configuration or installed inside the temperature chamber of a Waters 410 or 2410 Differential Refractometer The detector can be used at temperatures up to 50 C and is normally used to determine the hydrodynamic radius R of molecules over a very wide range 1 5 nm to 1000 nm 1 2 Discovery32 Chapter 1 1 4 4 PD2040 The PD2040 is a high temperature light scattering detector that can operate at up to 250 C It includes detectors positioned at both 15 and 90 to the incident laser beam This configuration is installed inside the temperature chamber of any one of the following commercially available high temperature GPC systems Waters 150C Waters GPC2000 Polymer Labs GPC 110 or Polymer Labs GPC 210 high temperature system or inside a custom temperature chamber 1 5 CONVENTIONS USED IN THIS MANUAL PrecisionAnalyze32 and Discovery32 are Windows applications that conform to the general conventions of Microsoft Windows In this discussion we will assume that the user has a general understanding of the operating system and the user should note the following e tis assumed that the OK or Run button should be selected after all commands in a dialog box have been selected e Dialog boxes and commands th
10. Transfer You may not rent lease or sublicense the Software You may however transfer all your rights to use the Software to another person or entity provided that you transfer this Agreement with the Software Warranty The Software delivered to you is PDI s current standard version and performs as described in PDI s brochures For a period of one year from the date of delivery PDI agrees to correct defects that the user identifies as not performing as described in PDI s brochures PDI DOES NOT AND CANNOT WARRANT THE PERFORMANCE OR RESULTS YOU MAY OBTAIN BY USING THE SOFTWARE OR DOCUMENTATION PDI MAKES NO WARRANTIES EXPRESS OR IMPLIED AS TO MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE IN NO EVENT WILL PDI BE LIABLE TO YOU FOR ANY CONSEQUENTIAL INCIDENTAL OR SPECIAL DAMAGES INCLUDING ANY LOST PROFITS OR LOST SAVINGS EVEN IF A PDI REPRESENTATIVE HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES OR FOR ANY CLAIM BY ANY THIRD PARTY Some states or jurisdictions do not allow the exclusion or limitation of incidental consequential or special damages or the exclusion of implied warranties or limitations on how long an implied warranty may last so the above limitations may not apply to you Governing Law and General Provisions This Agreement will be governed by the laws of the State of Massachusetts United States of America excluding the application of its conflicts of law rules This Agreement will not be governed by the
11. and 90 channels a Set integration parameters for the peak if an envelope is observed set parameters for the entire envelope We suggest that the parameters be set approximately 10 percent by height above the lift off and the same on the landing b Return to the Determine Cal Constants tab of the Setup Calculation dialog box and place a check mark in the Accept box adjacent to the 90 and or 15 constant entry 7 7 CHECKING CALIBRATION RESULTS RI DETECTOR To Check Calibration Results a Access the Run Params tab of the Results dialog box Figure 7 15 Results a_ x Summary Fun Params Mus Dist iwi vg Elu Hh Dist Hh ve Elu Hg Dist Hg vg Elu hw Total Area Calculations HI LI 15 Deg ZO01E Moll Det 0 DIEI 3L Deg JUUIK No UY Det 2U 00K Sample Conditions Inj Wol 100 000 pl Flow Fate 1 0000 ml min Eluant Fil 1 4080 Fl units Sample Constants mla cae 01833 7 LIV ext 0 0000 calc Mo UV ml mg cm Concentration Detectors Al 1 5000 1 4996 mg ml calc Ly 1 5000 Mo LUV mg ml calc E Figure 7 15 Results Dialog Box Run Params Tab Discovery32 Chapter 7 7 17 The following items should be checked on this tab e The calibration of the peak should be correct for RI for 15 and 90 e The Calculated Value of dn dc in the Sample Constants field e The Calculated Value of RI in the Concentrati
12. 1 1306 002 15 Degree 90 Degree Ly Int Region Peak Time minutes HI SEN 15 Degree 12 050 90 Degree 11 382 Uv r650 Hun Information Aig Results Collected As bsalP Ag Renamed Ta bsalPOP 00 Fin Collection Date 8 08 1388 15 25 08 Figur Agn Age Operator Aig peak Shea pbs w azide ria 3n ij E esults HH S ample bsa Ili pbs c 1 0 ii T rw e AAA 0 1650 Delay 0 0 dnde t n T peak UW Est 0 6700 Flow Rate 0 7000 Coet ml min solvent 1 3310 Inj Vol 100 0000 Index ul Temp 35 0 Conc 2 2300 C mg ml norman corey SO00 8rm 1204 5u 5 T MC calumn Update File Header Figure 5 16 The Results Dialog Box Summary Sheet 5 2 5 2 Run Params Tab The Run Params tab Figure 5 16 is used to check the calibration results for RULS UV LS and batch LS MW calculations Mw for batch is entered manually and 1s selected by a check box in the concentration detector section It 1s used to cross check entered vs calculated dn dc UV extinction coefficients and concentration Discovery32 Chapter 5 5 15 5 2 5 3 Hun Params Miu Total Area Calculations HI LI Batch 15 Deg Mw Made OFF Mu Mode Off hw Made OFf 90 Deg Mw Made OFF Mu Made Off hw Made OFf Ha nm hw Made Off Sample Conditions Inj Val 190 000 pl Flow Rate 0 7000 ml min Eluant Fl 13710 IRI unital Sample Constants dn dc 0 186
13. Options Detectors Constants Determine Cal Constants Channel 1 A 3 4 F b T B g 10 De fg fps jw f w f dm fa DU o mu m mu d m Lr Er m Lr Detector Left y axis C v C C C C C C C C Right awis f CC C C c C C ic C Figure 7 5 Setup Calculations Dialog Box Detectors Tab Check the detector traces that you would like to view on the chromatogram display You can select one detector to be defined by the left Y axis and another detector to be defined by the right Y axis If you select both left and right access radio buttons for the same channel then only one axis will be displayed on the left axis This makes the chromatogram window larger and easier to process 387 Note Typically the UV and Rh data are not presented when you are defining the baseline and integrating the peak s for the RI detector and the RI and Rh data are not presented when you are defining the baseline and integrating the peak s for the UV detector This 1s done so that so that the data from the light scattering detectors is more readily viewed 7 6 Discovery32 Chapter 7 7 4 3 The Constants Tab The Constants tab Figure 7 6 is used to present a variety of instrumental constants that are required to calculate light scattering properties Setup Calculation x Options Detectors Constants Determine Cal Constants Hun Al Inter detector Wol ml 0 1003 Mol wt Cal RI 15 33534 Mal
14. Run Interval v Flow Rate mnm M Sample M Delay Iw Fluant Fil Iw Eluant M Cone lw Temprature Iw Aun Date MW dre M Sample Info 1 v Operator v LIV ext W Sample Ino 2 RunLengbh v Inj Val Iv Al Light Scattering Calculation Made Summary I Ag Results Iv Ah Results Iw Mz M Raz Iw Raw Iw Aw W Mz Iw Al Area W Ran W Ahn ve Mu Iw 15 Ares M Rgw Ran Iw Rhw Rhn Iw Mn Iw 90 Ares W Raz Jw Rhz Iv Mwn Iw LIV Area Iw Aig peak Iw Ah peak v Mp Figure 9 6 The Select Sample Information Dialog Box b Place a check mark in the items that you want included on the pane and select OK A series of entries will be placed on the pane as shown in Figure 9 7 Each line of the instrumental conditions information and sample information can be individually moved as desired The RI Light Scattering Cal Mode Summary as well as the Rg and Rh results can be moved as a block in the same manner as described above Discovery32 Chapter 9 If desired you can the move or resize the entire block using the Saved AS Fun Interval 360 ry ext iml mgq cm Inj Vol ul Flow Rate ml min emp RI Light Scattering Cal Mode Summary Mz 1 Daltons O 000e 000 Mz Daltons O 000e 000 Mu Daltons O 000e 000 Mn Daltons O 000e 000 Mir m z 0 000 Mp Daltons anger Rgz inm a nn RI Area mV ml O000e 000 15 Area m nli O00e 000 90 Area imV ml O000e 000 UY Area gt 000
15. ml mgeem CEU dnde mlg 0 1670 Meas Interval sec L0 Flow Rate ml min 1 0000 Injection Wol pu 100 0000 Eluant Al 1 4320 Mol Wt Standard e0000 0 sample Conc mg ml 1 7017 Figure 6 12 Setup Calculation Dialog Box Determine Cal Constants tab If the standard that you are using consists of a single peak and has no aggregation a Verify that all of the information in the fields in the lower half of the tab is correct and edit as appropriate The values should be completely indicated e g the molecular weight should be 66 230 D rather than 66 23 kD b Check the Apply All box to update the fields in red If the standard that you are using includes measurable dimer trimer and higher order aggregations as is the case with BSA it will be necessary to include the total area of these peaks so that an accurate concentration constant can be obtained 387 Note Check that the parameters indicated in the lower half of the dialog box are correct before proceeding It is especially important that the indicated flow rate corresponds to the value delivered by the solvent delivery module The Quality Factor should be between 0 98 and 1 00 If it is in this range place a check in the Accept box corresponding to Inter detector volume and then press Apply to implement the action At this point you should inspect the chromatogram to ensure that the peaks are properly aligned using the Operate on Chromatogram tab c
16. requires the RI and 90 channels to have baselines If 15 data is collected but is not being used for the calculation then baselines for that channel is not required the Low Angle Scatter check box refers to the 15 scatter channel and the High Angle Scatter check box refers to the 90 scatter channel For Point to Point baselines select that feature along with the channels you are going to establish baselines for and click OK The cursor will appear as a sign two vertical lines may be displayed on the plot indicating previously determined baseline regions based on Least squares techniques or there may be small rectangular boxes which indicate point to point baselines Y ou may start and stop the baseline selection going from left to right anywhere on the chromatogram and independent of any vertical lines that show previously determined regions As an alternative you may move the vertical lines by holding down the Ctrl button down and clicking while dragging the rectangular box Select a region of the chromatogram where the baseline is flat in front of the peak s of interest and then release the mouse button Repeat the process for the other traces if they require selection of different regions to obtain good baselines In Figure 7 10 we have set the baseline indicators to approximately 3 0 min and 11 0 min Discovery32 Chapter 6 Chromatograms I x Uperate Un Chromatograms Integration Method Autaintegration Haw Data T
17. will be indicated in red directly below the sort buttons e g C PDI Data 040201 06 is loaded and will be indicated in the Preview File field 6 2 Discovery32 Chapter 6 If desired you can select a file from a different directory than that indicated in the field to the right of the Change button If you choose a file in this manner the indicated directory remains the same and the new chromatogram will be loaded but there will be red indicated files in that list The file that is loaded will be indicated in green immediately above the file list If you do not select any files in the file list and close Discovery32 then reopen the program the directory that will be opened and listed in the field to the right of the change button will be the based on the last file accessed before closing When the file is selected the window appears as shown in Figure 6 2 EB Discovery32 Lightscattering Analysis Software File View Options Help Change gt gt gt e By Date D32101 5 12 BSAOPOPOPOP Z 07 Ready 0 Jqeemun e 000 Figure 6 2 The Main Discovery Window with 040201 06 loaded Discovery32 Chapter 6 CAPDAData CAPDAData 040201 506 is loaded No Default Report Loaded 2 8 ejg 4 S G3 N IF By Filename IF By Eluant IF By Sample Mame IF By DnDc O3 21 2001 087 0971999 0870971999 0870971999 087097 1999 08 09 1999 08 09 1999 08 09 1999 08 097 1999 087097 1999 0270571999 02 16 1
18. 004 Mw Mn 1 009 Mp Daltons 2 089e4004 Flow Rate ml min 1 0000 Rgz nm 0 000 RI Area mV ml 9 290e4001 15 Area mV m1l 1 020e 002 Eluant RI 1 4080 90 Area mV ml 3 080e 002 UV Area mV mi 0 000e 000 Temp 35 0 Sample Info 1 96 4K PS standard Figure 9 1 A Typical Report Discovery32 Chapter 9 9 1 Report layouts can be stored and retrieved The current report layout is indicated on the main window directly below the name of the loaded file To select the desired layout click Set Default Report Layout on the File menu and select the desired format from the Open dialog box 9 2 EXPORTING DATA The Export Data dialog box Figure 9 2 which is accessed via the Export command on the File menu used to select the desired export format Export Data E X v Ascii ASCII Output Directory Chromatogram C Mw Distribution CAPDINData Select C Mw vs Elu Time C Rgvs Mw PLPC Output Directory C APDINData PD Expert Select Iv PLGPC Single Channel File C RI Data VISCOTER Output Direct 15 Degree Data evidence rm Degies Date C PDID ata PD Expert Select C LM Data Select Files For Export PLGFPC Dual Channel File C RI 80 Degree Data C UV15 Degree Data C UV 80 Degree Data Cancel v Viscotek TRISEC FILE Figure 9 2 The Export Data Dialog Box To export data a Select the desired format for the data by checking the format type check box es b Se
19. 1 The file information region contains the following information accessed via the slide bar e File Name e Code indicating type of data collected R Rh L Low Angle 15 H high angle 90 S spare normally used for UV C Concentration RI e Date Time for file generation e Sample Name e Eluant e Information e Operator e Dn Dc e Flow rate e Injection Volume e Conc e RI e UV Ext e Temp You can sort the files by clicking on the appropriate button e g the Sample Name button will list the files alphabetically based on the sample name Discovery32 Chapter 5 5 29 This page intentionally left blank 5 30 Discovery32 Chapter 5 Chapter 6 Measuring Proteins and other Monodisperse Species 6 1 OVERVIEW A typical application of Discovery32 1s the determination of the molecular weight distribution the hydrodynamic radius and related parameters of proteins using data that was acquired via PrecisionAcquire32 In solution a protein will typically provide one or a few peaks corresponding to the monomer dimer trimer tetramer etc In contrast when a synthetic polymer is studied a large number of species 1s observed with a small molecular weight difference between the various species The discussion in this chapter also relates to other applications where one or a relatively few polymeric species are present A similar discussion for the analysis of synthetic polymers and other polydisp
20. 100K 1 Wt 410305 Fried 1125 01 1427 P Freck lon Deecer Ret ion 100 0 1000 to lI00n0003 Figure 5 31 Conventional GPC Distributions Tab 5 2 5 6 Branching The Branching command presents the Branching dialog box which includes a series of tabs for presentation of the data as Rg vs Mw Rh vs Mw g Mw Branching g Rg Branching and Rg Rh Branching plots The right mouse button presents a menu describing the actions on the displayed data The is similar to Figure 5 21 with the exception that the Polynomial Fit Setup command presents the Fit Data Setup dialog box Figure 5 32 Discovery32 Chapter 5 5 25 5 2 5 7 Fit Data Setup X FIER vg Mw Data Enabled Low Limit D alttona High Limit D altars Set both ta D for all data MW Replace experimental A_ Mw data by fit results from Hi Elu and Mua Elu plots Radius of Gyration Threshold U 1 o an T Cancel Figure 5 32 Fit Data Setup Dialog Box Overlay Select The Overlay Select command presents the Set Up Overlays dialog box Figure 5 33 which is used to chromatograms that should be displayed in the chromatogram field Set Up Overlays SelectAll Ar all f EAdata 1020 05 lt x Select Mw Distrib Angle v C data O0809990p 07 z Select Chromatagraph RI Iv C data 080999 07 4 Select C hramatagraph RI E CAUNZIPPEDXBOTHO 4 Select Chromatograph RI E CAUNZIPPEDSBO THO EE Select C
21. 9 ANALYZING ANALYTICAL DATA Data that is collected from PrecisionAcquire32 can be readily processed via Discovery32 using the following protocol a Open the file as described in Section 7 2 b Indicate the baseline and integration parameters as described in Section 7 4 If you are analyzing data from a number of samples it may be convenient to use the Autointegration tab of the Chromatograms dialog box Figure 7 17 This feature lets you apply the integration parameters of a given file to other files Discovery32 Chapter 7 7 19 Chromatograms ES i Operate On Chromatograms Integration Method amp uteintegration Raw Data Transformation Auta Integration File List 60 max No Files in List Add Files to List ear List Double click on item to remove tt from list amp uta ntegrate Using 22 SLASBUTSLABDatasTaddtUl7 RI 05 Figure 7 18 Chromatograms Dialog Box Autointegration Tab To use the Autointegration feature 1 Press the Add Files to List button to present a dialog box that is used to select the desired files ii When all files are selected press the Auto Integrate Using gt gt gt button The file that is used as a template is selected on the pop up menu on the Chromatogram window c View the Summary tab of the Results dialog box Figure 7 16 to view the desired data Similar plots for Rg and Rh can be obtained using the corresponding tabs d Click the Mw Dist
22. 96 4 kD polydispersity 1 01 is used TSKstandard Polystyrene F 10 Column Plgel um MIXED C 300x7 5 mm part no 1110 6500 Eluent THF Flow Rate 1 mL min Discovery32 Appendix A A 3 This page intentionally left blank Discovery32 Appendix A INDEX A Alignment of Peaks 6 15 Analog Board Setup Dialog Box 3 16 Analyzing Analytical Data 6 21 7 19 Area Calculation inserting in Report 9 9 ASCII File 9 3 Autocorrelator 8 6 Autointegration Discovery32 5 9 Autointegration Dialog Box 3 19 Baseline establishing a 6 9 7 9 Baseline Type 6 10 7 10 Subtraction 5 7 6 10 7 Branching 5 25 C Cal Constants 6 13 6 17 6 19 7 13 Calculation Parameters 6 5 7 5 Calibration Chromatography A 2 Experimental Conditions A 2 Standard A 1 Calibration File Location 2 7 Calibration LS Channel 7 19 Calibration Setup Dialog Box 3 14 Chromatogram Normalizing 6 11 Smoothing 5 11 Window 5 6 Collection Fraction 3 12 4 2 Command Types 1 1 Comm Port Selection 2 7 Configure System 3 15 Constants Tab 6 7 7 7 Contents of Manual 1 4 Conventions 1 3 Correlator Analysis Dialog Box 3 17 Correlator Information Dialog Box 5 20 Correlator Configuration Dialog Box 3 17 Create Layout Report 5 4 Data Directory Precision Acquire32 2 7 Detectors Menu 3 12 Detectors Tab 6 6 7 6 Discovery32 Index Determining Calibration Constants 6 8 7 8 RI Constant 6 16 Diffusion Constant 8 7 Discovery32 5 1 Display Detector
23. Chromatogram In a chromatogram detector outputs are indicated as normal traces and molecular weight outputs are indicated as step functions 3 24 Discovery32 Chapter 3 3 6 THE STATUS BAR The Status Bar Figure 3 31 presents the status of the system and provides information about the file that Is open Elapsed Time Laser Lock Status Ready le avert 00 mn e e Status Status Communication Status Laser Status Figure 3 31 The Status Bar The leftmost field indicates the present status of the system If you click on a command on the Menu bar the status field will indicate the role of the command i e if you click on Multiple Runs the message Setup and Start Multiple Runs 1s presented File Name Time elapsed in the chromatogram and overall time of the run Communication Status green icon indicates that the detector is communicating with the computer Laser Lock Status green is on Laser Status green is on Discovery32 Chapter 3 3 25 3 26 This page intentionally left blank Discovery32 Chapter 3 Chapter 4 Selecting Parameters for Data Acquisition with PrecisionAquire32 4 1 OVERVIEW PrecisionAcquire32 provides the user with a great deal of flexibility in the selection of parameters for light scattering measurements Many of these parameters e g the Collection Fraction and the Smoothing Option can be adjusted by the operator to optimize and automate the collection of the analytical results
24. Export as Single Channel File Export as Dual Channel File f RI Data f Al 15 Degree Data C 15 Degree Data C RI 80 Degree Data C 90 Degree Data C UV 15 Degree Data C LV Data C O 90 Degree Data Select Directory Pbi F LDER PDI PRGMS ACOLIIRE32 v0 99 Viscatek Esport Export as Viscotek TRISEC File Select Directory Pbi F LDER PDI PRGMS amp CBLIRE32 v0 98 ASCII Export Export as ASCII Chromatagram Append Correlator Data Select Directory Pbi F LDER PDI PRGMS ACOLIIRE32 v0 99 Enable Auto export at data storage time Cancel Figure 2 8 The Export Options Dialog Box For the purposes of the test experiment leave all square boxes unchecked Note If your system includes the Precision Detectors Connect Interface Box omit Sections 2 4 9 to 2 4 13 and proceed to Section 2 5 2 4 9 Correlator Configuration Dialog Box The Correlator Configuration dialog box Figure 2 9 1s used to describe a number of correlator settings Discovery32 Chapter 3 3 9 Correlator Configuration Figure 2 9 The Correlator Configuration Dialog Box For the purposes of this experiment a b c d 3 10 Ensure that the Dilation Factor Override Computed Slurp and Correlator Prescale Factors are as shown in Figure 2 9 Set the Rh Sampling Gate Level to 0 Set the Test Signal to off Set the Correlator type to 64 Chan Discovery32 Chapter 3 2 4 10 Correlator Analysis Setup Dialog B
25. JT JT c JT c mn 15 Deg Voltage mJ uM GI c az 280 TEC 2 4 B B 10 12 14 16 Time min 410 305 Printed 11 27 01 08 34 fa Precision Detectors Inc Figure 7 3 A Typical Chromatogram 387 Note The time date for data acquisition and the information on the bottom line can be automatically added to the chromatogram via the Display Options dialog box Section 5 2 1 6 The file name indicated on the bottom line will not match the file name that was loaded if the file was saved with a different name If desired you can expand the window to full frame by grabbing a corner and dragging it to the desired size In addition you can expand a region of the chromatogram by pointing the mouse at the upper left corner of the desired area and dragging to the desired right hand corner Some users prefer using a white background to the chromatogram to improve contrast and the background of the chromatogram dialog box can be changed using the Scheme field on the Appearance tab on the Display Properties dialog box accessed via the Display icon on the Control Panel This is a standard Windows function 7 4 Discovery32 Chapter 7 7 4 ESTABLISHING CALCULATION PARAMETERS The Setup Calculations dialog box Figure 7 4 is used to indicate the instrument configuration that was used to collect the data In addition this dialog box is used to coordinate the various activities that are performed to determine the calibration consta
26. Sample Name dn dc or Date by selecting the appropriate radio button A detailed discussion of the contents on the line describing a file is presented in Section 5 4 is8 x Be Discovery32 Lightscattering Analysis Software File View Options Help aje 2 4 SJ El 4 UAI Change gt gt gt Mo File Loaded Double Click File in List To Load No Default Report Loaded CAPDRData Preview File 0321 sspe SUL pulis cue 15400PO0P 00 16400P 00 1840 500 RAA 12 03 21 2001 08 09 1999 08 09 1999 08 09 1999 087 0971999 08 09 1999 08 09 1999 08 09 1999 08 09 1999 08 09 1999 02 18 1997 Dz 18 199 7 02 18 1997 32 22 1994 sumple B2o 3ample hsa i sSample bzsa i sample bsa i sSample hzsa i sSample bzsa i anp lepsa i sSample bzsa i sample bsa i anp lepsa i sSample ADE S3ample ADE s3ample ADE Zamn 1lme Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluanrzc rt ES SS SS SaaS Figure 6 1 The Discovery32 Lightscattering Analysis Software Window To access files in another directory press the Change button and select the desired directory in the Browse for Folder dialog box that 1s presented To open a file double click on the line that represents the desired file As an alternative you can open a file by selecting Open on the Fi e menu and choosing the file from the Open dialog box The selected line
27. Setup dialog box Figure 3 18 is used to enter parameters that control the analog board Analog Board Setup X S ample Rate f 100 Samples sec 90 Samples sec Request hardware to send a Start Signal now Ignore Autostart signals sent while acquisition is in progress Cancel Figure 3 18 Analog Board Setup Dialog Box The Sample Rate should be set to 100 Samples sec except in regions of Japan where the line frequency is 90 Hz The Request Hardware to send a start signal now check box is an unimplemented feature Uncheck this box The gnore Autostart signals sent while acquisition is in progress check box is used to prevent data acquisition from prematurely stopping if such a signal is sent 3 16 Discovery32 Chapter 3 3 2 5 6 Export Options Dialog Box The Export Options dialog box Figure 3 19 is used to select the format for data export Export Options X PLGPEC Esport Export as Single Channel File Export as Dual Channel File f RI Data f RI 15 Degree Data C 15 Degree Data C RI 80 Degree Data C 90 Degree Data C OV 15 Degree Data C LIV Data C UV 90 Degree Data Select Directory PD FOLDERSPOL PRGMS amp COGLIIRE32 v0 98 Vizcotek Export Export as Viscotek TRISEC File Select Directory CAPD F LDER PDI PRGMS ACQUIRE 3 0 98 ASCII Export Export as ASCII Chromatagram Append Correlator Data Select Directory PD F LDER PDI PRGMS amp COLIIRE32 v0 99
28. Single Run Info File Marne 24 800 Operator TH Hun Length min 15 PES 1 000 Sample BoA Prestore Delay min U H dn de 0 167 Flow Rate ml min 1 0000 0 675 ME UV Ext Loeff Injection Volume ul E e Sample Conc mg ml Temperature C 33 Mw Calc Type Sample Information EE Eluarit Sample Interval zec Hd 15 degree data only 90 degree data onl hw Calc Method Wal for Autostart Signal Low smoothing Medium smoothing LM eem rr ES L arrelatar Setup Hun Time seconds 5n Sample Time seconds H Cancel Figure 2 13 The Single Run Info Dialog Box For the purposes of this experiment a Enter the file name to be used The file name must include the character immediately after the period separating the two fields in the name e g test 00 2 Note When entering information for a new sample be sure to rename the run first if you do not do this first the software will ask 1f you want to overwrite the file b Enter as much information as possible in the fields on the left side of the dialog box c Deselect the Wait for Autostart Signal check box d Enter the Run Length for the chromatogram see Section 2 5 3 e Enter a Sample Interval of sec 3 14 Discovery32 Chapter 3 f Enter the Flow Rate mL min Injection Volume uL and Sample Conc mg mL as described in Section 2 5 3 g Selec
29. United Nations Convention on Contracts for the International Sale of Goods the application of which is expressly excluded If any part of this Agreement is found void and unenforceable it will not affect the validity of the balance of the Agreement which shall remain valid and enforceable according to its terms You agree that the Software will not be shipped transferred or exported into any country or used in any manner prohibited by the United States Export Administration Act or any other export laws restrictions or regulations This Agreement shall automatically terminate upon failure by you to comply with its terms This Agreement may only be modified in writing signed by the President of PDI Notice to Government End Users If this product is acquired under the terms of a GSA contract Use reproduction or disclosure is subject to the restrictions set forth in the applicable ADP Schedule contract a DOD contract Use duplication or disclosure by the Government is subject to restrictions as set forth in subparagraph c 1 11 of 252 227 7013 a Civilian agency contract Use reproduction or disclosure 1s subject to 52 227 19 a through d and restrictions set forth in the accompanying end user agreement Discovery 32 License Agreement 7 Only Terms and Conditions These Terms and Conditions are the only terms and conditions related to the use of this software they supercede any previous agreement with respect to the sof
30. coincident the molecular weight will be calculated incorrectly f The value of Mw Mn for the heart cut of the monomer peak should be very close to 1 00 Further checking can be performed by integrating the dimer trimer and tetramer The calculated molecular weight of the dimer should be approximately 2 times the calculated weight of the monomer that of the trimer should be approximately 3 times that of the monomer and that of the tetramer should be slightly higher than 4 times that of the monomer as there may be higher order aggregates coeluting within that region An additional check can be made by comparing the data from the UV and the RI measurements The UV should track closely with the RI data but shows a little less response in the tetramer region which leads to a higher calculation for the Mw compared to the RI 6 20 Discovery32 Chapter 6 6 10 ANALYZING ANALYTICAL DATA Data that is collected from Precision Acquire32 can be readily processed via Discovery32 using the following protocol a Open the file as described in Section 6 2 b Indicate the baseline and integration parameters as described in Section 6 4 If you are analyzing data from a number of samples it may be convenient to use the Autointegration tab of the Chromatograms dialog box Figure 6 17 This feature lets you apply the integration parameters of a given file to other files Chromatograms HM Xx Operate On Chromatograms Integration Method
31. continuous curve in the chromatogram e g Figure 3 7 and Rh data will be indicated as a step function in the bottom of the chromatogram The Collection Fraction field is used to indicate the fraction of the data that 1s collected that will actually be used This feature is used to minimize shot or random noise as an example if 0 50 1s selected the lowest 50 of the data that is collected will be used For typical aqueous applications a value of 0 10 is used since the particles normally are large and for typical applications using organic solvents e g THF 0 75 1s recommended The remaining data will be discarded The Use as Temp Sensor check box is used if Temperature Rising Elution Fractionation 1s employed In this mode the temperature will be input into the spare channel The Coefficients for Temp Conv fields are used to enter the coefficients for the temperature sensor that are provided by the sensor manufacturer The Collect Correlator data check box should be used if you want to collect data from the correlator The Compute Mw check box should be selected if you want the molecular weight to be calculated The Show Rh Correlator data during acquisition check box should be checked if you want the Rh data presented when you are collecting data 3 2 5 2 Select Detectors for Display The Select Detectors for Display dialog box Figure 3 15 is used to indicate the detectors for which output should be displayed during data acqui
32. detailed discussion of the export format is presented in Chapter 9 5 2 1 4 Create Report Layout Create Report Layout presents the ReportLayout window Figure 5 4 which is used to define the general layout of the report A detailed discussion of the report feature is presented in Chapter 9 Ex ReportLayout ReportE dit OF x File Font View Window s Dee alee fe i BE J 11 6 2000 at For Help press F1 Figure 5 4 Report Layout Window 5 4 Discovery32 Chapter 5 5 2 1 5 Set Default Report Layout Set Default Report Layout presents an Open dialog box to allow the selection of the desired report layout Report layouts are of the form rlo and are normally stored in the same directory as the data When you open a layout the name of the selected layout is indicated directly below the loaded file name see Figure 5 1 5 2 1 6 Display Options Display Options presents the More Report Options dialog box Figure 5 4 which allows the operator to indicate the company name and if the information indicated by the check boxes should be indicated on the report Display Options x Company Name Tour Company Mame AddRun Date Time to Graph Title Add File Name to Footer Add Curent Date Time to Graph Footer Add Company Name to Graph Footer Cancel Figure 5 4 The More Report Options Dialog Box 5 2 1 7 Print Print presents the Print Setup dialog box which is a standard W
33. kD the sample time should be 10 usec 4 4 3 Tolerance Tolerance 1 requires that the slope of the linear fit to the log of the correlation function to be less than 0 001 If the correlation function has a slope greater than what is set for Tolerance 1 that value is not used in the hydrodynamic radius calculation 1 e noisy data are thrown out Tolerance 2 compares the absolute value of the error of the measurement to the absolute value of the slope if the ratio of the two values is less than 0 100 the data is accepted for the calculation 4 4 Discovery32 Chapter 4 Chapter 5 Introduction to Discovery32 5 1 OVERVIEW Discovery32 1s used to process data collected from Precision Detectors Light Scattering Detectors using PrecisionAcquire32 The program is used to display chromatograms determine the molecular weight molecular weight average of polymers generate reports and overlay data from multiple runs In addition it is used to determine the molecular size by dynamic light scattering and determining the radius of gyration by 2 angle light scattering This chapter describes the Main window of Discovery32 Figure 5 1 which is used to coordinate all activities of the application program access a variety of tools to process and present data A discussion of the use of these commands in a typical scenario for protein analysis is presented in Chapter 6 and a typical scenario for synthetic polymer analysis is presented in
34. molecular weight for the sample corresponding to the slice of the chromatogram that was integrated The calibration 1s based on the total area of the light scattering signal and the Refractive index detector signal If the curves are not perfectly coincident the molecular weight will be calculated incorrectly e The value of Mw Mn for the monomer peak should be very close to 1 000 Further checking can be performed by integrating the dimer trimer and tetramer The calculated molecular weight of the dimer should be approximately 1 97 times the calculated weight of the monomer that of the trimer should be approximately 2 96 that of the monomer and that of the tetramer should be approximately 3 95 that of the monomer To verify operation for the large aggregation of BSA integrate the full RI chromatogram The average MW should be 73 kD with a polydispersity of 1 062 6 8 CALIBRATING THE UV AND LS CHANNELS To calibrate the UV detector a Return to the Detectors page of the Setup Calculation dialog box Figure 6 5 and deselect the RI channel Select the UV channel by activating the check box In addition check the left and right Y Axis buttons for UV b Establish the baseline for the UV detector C Zoom in on the monomer dimer region and reintegrate the monomer region The UV trace may slightly precede the light scattering channels d Return to the Determine Cal Constants tab of the Setup Calculations dialog box and accept the Inte
35. tab to present a molecular weight distribution Figure 7 19 7 20 Discovery32 Chapter 7 Results SM x Summary Run Params Mw Dist Mus v Elu Hh Dist Hh ve Elu Hg Dist Hg vg Elu 96 4 K PS 02 05 1999 15 04 19 1 0 1 0 1 981e4004 0 35 1 573 amp 64004 0 35 1 003 EE 7 08984004 et 0 7 0 7 n 7 E 0 6 t 0 6 E D iB ct E o U 5 r 0 5 m E i 2 i m d 0 4 K r d 0 3 0 3 r 0 2 0 2 0 1 0 1 0 0 0 0 1E 3K 10 30K 100K 4103065 Fried 1127 01 1326 4p Preck be Detectors lnc Not e ion 100 0 1000 to LOooooOoO Figure 7 19 Molecular Weight Distribution Plot e Click the Mw vs Elu tab to present a chromatogram that presents the Mw as the ordinate Figure 7 20 Similar chromatograms for Rh and Rg can be viewed via the corresponding plots Discovery32 Chapter 7 7 21 Results eee x Summary Hun Params Mw Dist Mw vs Elu Rh Dist Hh ve Elu Hg Dist Hg vg Elu 96 4 K PS 02 05 1999 13 04 19 LOOK L 3991le 004 Ll 373e 004 l 0 03 2e 08393er004 20E Wh g a p m LOK D 4 m E aK 1E E 5 4 5 5 5 8 6 0 E 6 4 6 5 Elution Time mir HOFS Prec 1127 01 11357 p Peck be Detectors fic Figure 7 20 Mw Chromatogram To view hydrodynamic data select Rh on the Detector tab and select the Y Axis Left and Y Axis Right boxes The results will be seen with signs Figure 7 21 with the left y axis showing the Rh scale 7 22 Discovery32 Chapter 7 Chrom
36. the Chromatogram Smoothing dialog box Figure 5 12 is presented Chromatogram Smoothing X Select Detectors ta Smooth S m RI Wrr Ir m ee E E E p E p E ps Smoothing Degree f None Low C Medium C High Cancel Figure 5 12 The Chromatogram Smoothing Dialog Box The check boxes are to indicate the detector traces that are to be smoothed and the Smoothing Degree radio buttons indicate the desired level of smoothing It is possible to change the smoothing in several formats and process each file individually Invert RI Chromatogram The Invert RI Chromatogram command is used to change the format of each data point in the RI chromatogram Points which are above the baseline are below it and vice versa The numerical deviation from the baseline remains constant 1 e if the baseline is 150 mV and a peak is at 200 mV it will be at 100 mV after the command is performed Save Operated Chromatogram When a chromatogram that has been altered via any command is to be saved it will be saved with the letters OP after the name e g If PDI 07 is altered and saved it will be saved as PDIOP 07 If additional operations are performed the operated file is this process will continue e g PDIOP 07 becomes PDIOPOP 07 The original raw data file is never lost in this process Discovery32 Chapter 5 5 11 5 2 4 Setup Calculation The Setup Calculation dialog box Figure 5 13 is used to ente
37. wt Cal R1290 11102 m Mol w t Cal RI 33670 Cal Solvent Indes 1 4080 Hun Ly Inter detector Wol ml 0 0000 0 3 Mol wt Cal LIV 15 0 0 Instrument RI Inter detector Wol ml U O82 Molat Cal RL 15 4291 6 Mol wt Cal RI 80 1904 8 Mol wt Cal RI 28975 1 3320 Cal Solvent Indes Instrument Uy Inter detector Wol ml 0 0646 Molwe Cal uweis 202643 Molwit Cal 04 90 45336 0 Mol wt Cal LIV 745 2000 Inst Cal Values gt Files Mol wt Cal LI 80 0 0 qe Molt Cal LIV 0000 Figure 7 6 Setup Calculations Dialog Box Constants Tab The Run RI and Run UV constants shown on the left side of the Constants tab are the current values that are being applied to the chromatogram and are stored with the data file while the constants shown on the right side are the instrument values which are currently stored in Discovery32 You can think of these constants as being analogous to the memory function of a calculator They are available to update any files with these constants When a calibration routine is performed both the Run and Instrument side constants are updated with latest determined values If you choose to update the constants in PrecisionAcquire32 they can be copied automatically by selecting Update Acquire32 Constants from the drop down menu options found in the main browsing window of Discovery32 Calibration co
38. 0 0 0798 r calc E UV es 6670 qu 03866 m ml mg cm Concentration Detectors Al 2 2090 1 0442 mg ml calc E Ly 2 2090 1 2805 mg ml calc v Apply Figure 5 16 The Results Dialog Box Run Params Tab Distribution Tabs The Results dialog box presents a series of tabs that describe the distributions Mw Distribution Rh Distribution Rg Distribution The format of these distribution tabs is similar and those tabs that are active depends on the selection s that were made on the Options tab of the Setup Calculations dialog box Section 5 2 4 1 5 16 Discovery32 Chapter 5 Summary Run Params Mw Dist Mw WE Elu Hh Dist Hh vs Elu Hg Dist Hg vs Elu ESA UV BRI O4 02 2001 l2 24 53 issu J a i 0 9 0 9 0 8 0 8 0 7 DoT Amount nm tn o in ABTASAg APATIE THOM 0 3 0 3 2 2 Br 1 0 0 LOE ZOE 30K 40K SOE 60E OR 30K 90K aag gs Pried 1123 01 Ce Preck bw Detector Tee eed ein E 1000 to l OO 0 Figure 5 17 The Results Dialog Box MW Dist Tab The menu shown in Figure 5 18 is accessed by right clicking on the MW Dist tab Plot Limits shirk Print Cumulative OFF Data Table Figure 5 18 MW Dist Tab Commands Plot Limits presents the Set Plot Limits dialog box Figure 5 19 which is used to indicate the desired limits for the plot Discovery32 Chapter 5 5 17 Set Plot Limits X Define Plot Region Let E Ri
39. 00 Calculation Option Settings Main Calculation Type Mw Smoothing Mw Calculations Hh Calculations Rg Calculations Laser Wavelength nm sample Condition Inj vol ful Flow Rate ml min Eluant RI RI unite Concentration Detectors RI ug ml Uv fmo ml i Cal Parameters Cal RI RI units Cal Inj Vol ful Cal Solvent Index Hw Total Areas 15 Degree an Degree Faq inm EI OO 00 00K 00 00K OOO ann OO0 0000 000 0000 OOO 0000 Integration and baseline getting regions mirn Region l Region 2 Region 3 RI Baseline 15 deg Baseline spare Baseline Mw Calc Region 0 00 0 00 0 0070 00 0 0070 00 0 000 00 0 0070 00 0 0070 00 0 0070 00 0 0070 uc 0 0070 00 0 0070 00 0 0070 00 0 0070 ud Figure 9 9 Information Fields Region 4 0 unu 0 0070 o onu 00 Ju ua t Discovery32 Appendix A 9 3 7 Placing a Line in the Pane If desired you can place a line on the pane 1 e to connect annotation with a chromatogram To place a line in the pane a Press the icon on the menu bar then move the cursor to the approximate position in the pane and click A line outlined in red will be placed at the indicated position b Move and or enlarge the line to the desired size and position in the pane These activities are done in the same manner as when you move or change the size of a window 1 e to expand the rectangle you can click on a cor
40. 1 0 Stark O13 13 30 5 0 1 0 0 0 3 1071 Use External Synch Load Multiple Run File Save Multiple Run File Import trom Millennium Figure 3 8 The Multiple Runs Dialog Box A run describes the collection of one or more chromatograms using a common set of conditions To start a run highlight the desired run set and press Start As an example if you selected the second run set three injections would be made using the conditions indicated for the run set and would be named 1 00 1 01 and 1 02 Multiple Run Files can be stored and loaded by the Save Multiple Run File and Retrieve Multiple Run File buttons The Run Set parameters can be edited by the Multiple Run Information dialog box Figure 3 9 which is accessed by clicking Edit 3 8 Discovery32 Chapter 3 Multiple Hun Information Base File Name 9m 00 EE H of Injections Operator Performers Name mui Eluant THF Run Length min 2 S ample Interval sec 1 000 Prestore Delay min 0 0 Sample 36 4 K PS ee 0 1840 LI Ext Caef 0 1670 Flow Rate ml min 1 0000 Injection S alume ul 100 Sample Core mg ml 1 0000 solvent Index 14080 L arrelatar Setup Run Time seconds 5 0 5 0 Temperature C 35 Sample Information no comment na comment Cancel S ample Time Figure 3 9 Multiple Run Information Dialog Box The Base File Name 1s used to indicate the file name that should be used for the dat
41. 10 Forge Park Franklin Massachusetts 02038 USA Tel 508 520 8765 Fax 508 520 8772 e mail PDI precisiondetectors com Web site www precisiondetectors com Copyright 1997 1998 1999 2000 2001 by Precision Detectors Inc Printed in the United States of America Precision Detectors Inc Electronic End User License Agreement NOTICE TO USER THIS IS A CONTRACT BY INDICATING YOUR ACCEPTANCE DURING INSTALLATION YOU WILL BE ASKED TO ACCEPT ALL THE TERMS AND CONDITIONS OF THIS AGREEMENT This Precision Detectors Inc PDI End User License Agreement accompanies a Precision Detectors software product and related explanatory materials The term Software shall include all software packages delivered to you by PDI and any upgrades modified versions or updates of the Software licensed to you by PDI This copy of the Software is licensed to you as the end user for use by you and other users of a specific PDI hardware system purchased leased or rented by you Please read this Agreement carefully PDI grants to you a non exclusive license to use the Software provided that you agree to the following l Use of the Software a You may install the Software in a single location on a hard disk or other storage device install and use the Software on a file server for local execution over your network but not for the purpose of copying onto a local disk or other storage device for use only with the specific system b You may ma
42. 2 13 25 POStIEXDELIIICHEZ oc otn a ae ancien UMeUi cnet Maui 2 13 254 Introduction to the Test x periment uu educi en o oe RE Rege RU Sp tR CORE DAE SEL DNE C identities 2 13 2 9 2 The smele Run Into Dialog BOX lt 29 IE ert a n aie eth mA Mam ERI 2 14 2 Chromatocrapnic Conditions eet ES eee ee ibam Ms iex etu AS EDU C 2 15 Chapter 3 Introduction to PrecisionAcquire32 eere Leere eee eee eerte eee eene eese sette tette eese asset eee eeee 3 1 MES cau cere EE 3 S TOE Me eri e 3 2 AMEN DIS c er cxT cem 3 2 DI V MERO RT x xx RD 3 4 E imme Ul aaa eonun x M x EP 3 5 MNME dde Eccc TP 3 5 3 29 25 NMIUOBIDIC RIS roo aam RR Rea nM RM A tM DN E EA LL uM LI ED ME 3 8 Discovery 32 Table of Contents ix EVEN MEN Dresd A a mmm 3 9 SPN ME diuum RU c 3 9 BA RC VAC We IVI CIM ERREUR Tc 3 10 SOL eun TOMA OI essor E ERE bac ove E bu uei detis Bet oce o ben nuns ieee 3 10 IA OAL DISCOV EL Y NEHME 3 10 3 24 3 Display Detector dala eee uto eo telae anuo ctos beat chee oowiebens ci tcp decli ins 3 10 Spr e M IMPR 3 11 ILo The Se WI doni PR RR c 3 11 SM MERI DITS URN OO 3 12 32 922 ClCH Detectors Jor DISDIAV descente te Cotes petes utente tu cutie iS eabeter indu eei n EE eds 3 13 CL Atl rerne ute osedeeti niinted bad tut teu me e tnt u idees Eat cios 3 14 oe COM MOU CS SIC Ibo cie tesbeten tse E E neds teas eiue ned iet escena steterat e asada 3 15 3 2 5 9 A
43. 3 Wiewine the CroratoS EAE sucie da des m e uta un es om uu du lutea am pu NM PEE d d TuS 6 4 64 Establishing Calculation Pardmelterseseo ennt UH HE IN Hebe bid dde IU HMM UE 6 5 OAL Te Options zio ET T 6 5 OAD The Detectors M slo rcc rr 6 6 O45 The Comstamts Mio EeePC x exc 6 7 644 Determining Calibration Constants iota Pale a akc desea Unas 6 8 6 5 Preparing the Chromatogram for Data Processing sse eene 6 9 GS I Establishing A Baseline ausos ORI MORI eh dina deutet ui E t de 6 9 6 5 2 Normalizing the Heights and Zooming on the Peaks eeeeeeeeeesesseseeeeeeeeeeeerernrs 6 11 6 5 2 Defining the Integration Re910fi eene ERR UR eri ee pe ND be BM IRI peu 6 13 6 6 Determine Cal Constants usce RR RED SHARM eee 6 13 6 6 1 Determining the T ter Detector Volume aeter e D tees dedi ere diatedul 6 13 6 62 Detemining the RIC Onstant stone tbee e diae et bobo eb ammi atur sbetis 6 16 6 6 3 Determine Calibration Constants for 15 and 90 Channels sse 6 17 6 7 Checking Calibration Results RI Detector sssrinin nnne 6 17 6 5 Calibratine the UV and ES Came stoi utu eva e had ib e edo e to 6 19 6 9 Checking Calibration Constants with UV Detection sssssssssseennneeennes 6 20 6 10 Analyzing Analytical Datasheet e doma dade ente bu Dd 6 21 Chapter 7 Measuring Synthetic Polymers e ee
44. 42 mV for the 90 trace in Figure 6 8 To establish a baseline a Open the Operate on Chromatograms tab of the Chromatograms dialog box Figure 6 8 Chromatograms B Xx Operate On Chromatograms Integration Method Autombegration Haw Data Transformation BSA UWRI 3 02 2001 12 24 53 BUD LT p c LT r 3 c 48 440 15 Deg Voltage mv 400 Time min 040201 06 Printed 11425401 19 04 i Precision Detectors Inc Figure 6 8 The Chromatograms Dialog Box Operate on Chromatograms Tab b Right click on the chromatogram to present a pop up selection box and select Set Baseline to present the Select Detectors dialog box Figure 6 9 Discovery32 Chapter 6 6 9 c d 6 10 Select Detectors X Baseline Type Least Squares Fil C Point to Point Fit Subtract Baseline For jw Refractive Index Iv Low Angle Scatter Iv High Angle Scatter M UY Detector Cancel Figure 6 9 Select Detectors Dialog Box There are two options for selecting baselines Least Squares Fit or Point to Point Fit If the baseline is flat before and after the peak of interest we recommend that the Point to Point Fit radio button be selected If there 1s a flat region of baseline only on one side of the peak s then the least squares fit is appropriate as it can extrapolate baselines The baseline must be established for all detector channels for the selected calculation types e g RI LS 90
45. 7 is used to generate the calibration constants for the system When you first open up the screen all constants are blank if you have not yet established baselines Setup Calculation Xx Options Detectors Constants Determine Cal Constants Mew Accept Inter detectar volume ml nase27 M Quality Factor 15 Degree Constant 42982 90 Degree Constant 13053 Oy Lionetart E Refractive Index Constant 30 4402 28877 Apply All UV Extinct ml mg cm CESU dn dc rolg 0 1670 Meas Interval sec IU Flow Rate ml min 1 0000 Injection Val pl 100 QUU Eluant Fil 1 3320 Mal wt Standard ouu Sample Conc mg ml Aur Figure 6 7 Setup Calculations Dialog Box Determine Cal Constants Tab This dialog box is used to review the calibration process as described in Section 6 7 6 8 Discovery32 Chapter 6 6 5 PREPARING THE CHROMATOGRAM FOR DATA PROCESSING Preparing the chromatogram refers to determining the baseline normalizing the chromatogram and selecting the region to be integrated 6 5 1 Establishing A Baseline The baseline is used to indicate the zero signal level for the sample so that the area of the peak can be properly determined The baseline is normally selected by defining two points in the chromatogram that are on a flat part of the trace one before and one after the peak s of interest The baseline is indicated on the chromatogram as a black line e g see the line at 3
46. 997 12 1m7 149027 sSample B2 sSample hasa sample hzasa sSample hasa sSample hzsa sSample hzsa 3ample hbsa 3ample hbsa s3ample hbsa 3ample hbsa Preview File 040201 50B5 in in in in in in in in in sSample 25 43 E sSample ADpE 1640 zLamnle nk 140 pha pha pha pha pha pha phas pha pha PS l x Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluantz 6 3 6 3 VIEWING THE CHROMATOGRAM The chromatogram for the active file Figure 6 3 can be viewed by selecting Chromatogram on the Option menu or pressing the button on the toolbar The status bar will indicate the name of the file that was loaded Chromatograms BE x Operate Un Chromatograms Integration Method Autointegration Haw Data Transformation BSA UWR 4 02 2001 12 24 53 BOO 560 520 460 440 15 Deg voltage m 400 Time min 040201 06 Printed 11725411 17 23 amp amp Precision Detectors Inc Figure 6 3 A Typical Chromatogram 387 Note The time date for data acquisition and the information on the bottom line can be automatically added to the chromatogram via the Display Options dialog box Section 5 2 1 6 The file name indicated on the bottom line will not match the file name that was loaded if the file was saved with a different name If desired you can expand the window to full frame by grabbing a corner and dragg
47. Chapter 7 EBDiscovery32 Lightscattering Analysis Software E x File View Options Help a e s 4 NI EJ A Change gt gt gt CAPDAData 6S4 507 is loaded Default Report 101001 rlo CAPDKData Preview File BSA 507 Discovery32 Chapter 5 O3 21 2001 08 09 1999 08r 09 1999 08 09 1999 08709 1999 021871997 02 16 1997 02 16 1997 O3 22 1994 sSample BZ2 i sSample bhsa i Sample bhsa i sample bzsa i sSample bzsa i sample ADE sSample ADE sample ADE 3ample Ready BOF Figure 5 1 The Main Window Eluant pH Eluant pH Eluant pH Eluant pnpH Eluant pnpH Eluant T Eluant T Eluant Tq The Main window includes the following regions e the Menu Bar which is used to access a broad range of commands Section 5 2 e the Tool Bar which provides shortcuts to a number of commonly used commands Section 5 3 e the File Access and File Selection regions which is used to open a file Section 5 4 la Note The commands in a menu the icons on the tool bar and the options in various dialog boxes that are active at a given point depend on the present status of the program e g the Save command is not active unless one or more files are open In this chapter we will describe each command and indicate when it 1s active At a given instant commands options that are selectable will indicated in the menu in black while inactive commands options will be indicated in gray In this man
48. Data Command 3 10 Display Options Dialog Box 5 5 Display Setup Dialog Box 5 20 Dynamic Light Scattering 8 1 Elution Tabs 5 18 End Run Command 3 9 Establishing a Baseline 6 6 Export Command Discovery32 5 3 PrecisionAcquire32 3 3 Exporting Data 9 2 F File Access Region Discovery32 5 27 Fonts in Report 9 11 Form Factor 8 5 G General Information in reports 9 12 GPC 5 21 Generating Calibration Curve 5 21 Obtaining Data 5 24 High Angle Light Scattering 8 5 Hydrodynamic Data 6 21 7 22 Information Region Discovery 32 5 28 Inserting Text in Report 9 6 Instrumentation 1 2 Installation 2 1 Integration Region 5 8 6 13 7 13 Integration Template 5 8 Interdetector Volume 6 13 Introduction 1 1 Invert IR Chromatogram 5 11 IR Chromatogram 5 11r L Laser Command 3 22 Layout Report 5 4 Light Scattering Theory 8 1 Line in Report 9 11 Low Angle Light Scattering 8 5 Monitor Photon Level Dialog Box 3 21 Multiple Runs Command 3 8 Dialog Box 3 8 MW Calc Method 3 14 4 3 MW Calc Type Field 3 14 MW Distribution Plot inserting in Report 9 6 MW Distribution Tab 5 17 MW vs Elu Tab 5 19 Normalizing the Chromatogram 6 11 7 11 Narrow Standard Information Table 5 22 Narrow Standard Table Options 5 22 Natural Polymers 6 1 Noise 4 1 o Open Command PrecisionAcquire32 3 3 Discovery32 6 2 7 2 Operate on Chromatogram Tab 5 6 Overlay Select 5 25 P Pages in Report 9 11 Photon Level 3 20 Polyn
49. Discovery 32 Application Software for Light Scattering Detectors for High Performance Liquid Chromatography Gel Permeation Chromatography and Fast Protein Liquid Chromatography User Manual Created by Precision Detectors Inc Notices This product is covered by a limited warranty A copy of the warranty is included in this manual No part of this document may be reproduced in any form or by any means electronic or mechanical including photocopying without written permission from Precision Detectors Inc Information in this document is subject to change without notice and does not represent a commitment on the part of Precision Detectors Inc No responsibility is assumed by Precision Detectors for the use of this detector or other rights of third parties resulting from its use The software described in this document is furnished under a license agreement and may be used or copied only in accordance with the terms of the agreement The user may make a single copy of the software for archival purposes Precision Detectors products are covered by US Patents 5 305 073 and 5 701 176 Additional patents applied for Precision Detectors PrecisionDeconvolve PrecisionDeconview Discovery32 PrecisionAcquire 32 Discovery32 PDDLS Batch and PDDLS CoolBatch are trademarks of Precision Detectors Inc All other brands and products mentioned are trademarks or registered trademarks of their respective holders Precision Detectors Inc
50. ER EE 4 3 WSs MW ale NICU OG 25 eee reas chen ia Routen Hot ou os Pets val OU Laud TOi vou deu S o TE RUE c Us 4 3 24 Parameters dor PI22000 Sy SUCHIS dn oes sadossts pause aaa NER 4 3 AAL RON Nd Em 4 3 241 Sample JT I6 oops Ui bsb Eu RIEN LI Gi EeP pe p a ste ees 4 3 AAD TOET uique iuba des ote estaba teca SPEI EU DN echo Pape ad e Rp ua Sa Eu De nta an Suc E DUREE nate eee 4 4 Chapters Introduction fo DISCOVGE VO iib UI ERES YOU Eo E eas baeo eae Ee Ee VERUS Ve Qoo ved ae a 5 1 S MERDA RP EE 5 3 TheM MNB uice NEM nM EIE 5 2 3o The FE Me S oed A spends ac el LED e eat 5 2 S2 ET SO C EH EET 5 3 SUE PM CL NS NER EET ET 5 3 SV BE Eque DR MEN 5 3 5 2 1 4 Create Report Way Out oie iieri Ee tu Ee heit eter ene i o tei eee N 5 4 S215 Set DDelaulb Report E A Olio oii p neon rend dbi dedu nA SER Uds EE 5 5 3 2 1 6 Display OODHOLDS enc teda hd iR ah dot on tedn dcc emi sen dL is Loue bein d URS MM Ps 5 5 SALI oiu 5 5 SALS PRINT SCUD sissies terre el GA teas E 5 5 Dre NC cp pc ae eae E ud cea aaomanehelen E E E A aces eames 5 5 2 mes 8 iP c XT D 5 6 Discovery 32 Table of Contents 320 GDrTombd3lopt AIMS eese tbt tctetemtend uote uc etui SM De due Bed ee uid 5 6 35 2 9 2 Afite prato Method sme rtr roten S rato e io boue EE e se rop pe Ro sabes UE IER TEE 5 0 312 9 95 AMOI OPA OMe dois toe oe eire o oe dune tella cite ec aside sim atu petuo totam ecd en Mc s m a 5 9 S234 Raw Data Trans tormaO ERES
51. Enable Auto export at data storage time Cancel Figure 3 19 Export Options Dialog Box If you want the data exported into a format that can be employed with PLGPC Viscotek software or in ASCII format place an x in the appropriate check boxes and indicate the format for PLGPC format The data will be stored in the appropriate directory 3 2 5 7 Correlator Configuration The parameters on the Correlator Configuration dialog box should not be changed by the user 3 2 5 8 Correlator Analysis The Correlator Analysis Setup dialog box Figure 3 20 is used to set a variety of run parameters Discovery32 Chapter 3 3 17 Correlator Analysis Setup EJ Special v Store correlator raw data in Znn file Analysis Viscosity 0 0040 Pu EU verage Retr Index T4050 m1 ea Wavelength m jf 3 ft 4 fnm Tolerance 1 0 0010 Base Type Teea 0 1000 f P P M C Base Value C Linear Fit Cancel Figure 3 20 Correlator Analysis Setup Dialog Box For typical operation a Deselect the Special Store correlator raw data in znn box These files are not necessary for saving as the Rh data is saved in the xx files The Znn files are large and waste unnecessary disk space b Setthe Viscosity and Refractive Index to that for the temperature and solvent that will be used for the measurement c Set Lambda to 800 0 Tolerance 1 to 0 001 and Tolerance 2 to 0 1 d Select Corr should be set to 1 e Base Type shoul
52. Mw as the ordinate Figure 6 20 Similar chromatograms for Rh and Rg can be viewed via the corresponding plots 6 22 Discovery32 Chapter 6 Summary Hun Params Mw Dist Mw vs Elu Rh Dist Hh ve Elu Hg Dist Hg vg Elu 100E ESA UV BRI 04 02 2001 l2 24 53 SUE 6 6676 004 SOK E58e 004 70E 1 0 l 6 76ler u4 60K SUR 40K Daltons 3UR Tic Mol EUR LOE S 9 2 2 3 2 4 SPTE 29 6 Elution Time mir Ou FDS Prid 112901 07 X6 p Preck low Detectors lic Figure 6 20 Mw Chromatogram To view hydrodynamic data select Rh on the Detector tab and select the Y Axis Left and Y Axis Right boxes The results will be seen with signs Figure 6 21 with the left y axis showing the Rh scale Discovery32 Chapter 6 6 23 Chromatograms E x Operate On Chromatograms Integration Method Aukombegration Haw Data Transformatian 3 00 smi 3 60 3 90 3 40 040201 085 Printed 11 26 01 07 14 me Precision Detectors Inc Figure 6 21 Hydrodynamic Data 6 24 Discovery32 Chapter 6 Chapter 7 Measuring Synthetic Polymers 7 1 OVERVIEW A typical application of Discovery32 1s the determination of the molecular weight distribution the hydrodynamic radius and related parameters of synthetic polymers using data that was acquired via PrecisionAcquire32 A synthetic polymer sample may contain a single polymeric species which would correspond to a single peak in the chromat
53. Program Files sPrecision D etectorssPrecisian Disable Calibration File Loading iM PD2a 00 Compatibility Mode Use DTR DSR M Carelator daughterboard present Cancel Figure 3 17 System Calibration Dialog Box Discovery32 Chapter 3 3 15 The Comm Port Baud Rate and Stop Bits field should be set as described in Section 2 4 6 The desired Data Directory and Calibration File should be selected by pressing the Select button and then choosing the directory The Calibration file provided with the system should normally be used This file optimizes the interface box not the optics bench and its file name matches the serial number on the PD2000 DS box If an external interface box is used the Disable Calibration File Loading box should be checked The PD2000 Compatibility Mode check box is used to allow for backward compatibility in data acquisition The Use DTR DSR check box is used to enforce more rigorous communication between the computer and the A D box The Correlator daughter board present check box is used to indicate whether a PD2000DLS box which has a correlator board or a PDConnect box which does not have a correlator board 1s used This prevents PrecisionAcquire32 from posting erroneous dialog boxes or searching unnecessarily for correlator boards that do not exist Typically all three check boxes at the bottom of the dialog box should be selected 3 2 5 5 Analog Board Setup Dialog Box The Analog Board
54. S Calibration the UV and ESC hanne ls eed c D RM o DEOR UR S M aS tdt 7 19 TO Analyzime Amalytical Data supe DH NEMINEM MM EM dE 7 19 Chapter S Theory Of Light Scatterlig ooatecie iiec e Hed evo dae eee Ree Va ee cue Fa UR EB reed e NT a EU Eae oaa Ne aer U A PRA ea 8 1 MES sa E 8 8 2 he Physical Basis Of Licht Scattering eode one Sedet eid tenete tst 8 2 BS MEE rated Birednie res iocos EE REN NM 8 3 8 3 1 Measuring the Molecular Weight eseueeseseeeeseeseeseeeeeee nennen nnn nnnnnnnnnnnnnnnnnnnn hann nnns sns nis 8 3 8 3 2 Using Refractive Index Measurements to Measure Concentration eesesseeeeeere 8 5 io ETO AC LOR E E EET TEE 8 5 8 3 4 Characteristics of Low Angle 15 Light Scattering seen 8 5 B50 EHiaeh Angle Light SOatiet IB oisi tto HR ER decane meas Mri REPE pube t PER A 8 5 8 4 Measuring Rh via Dynamic Light Scattering eese 8 6 SAIL Basicsof Dynamic Light Scatterin S sooo decet postice ub ac ep aseo ipei cu tate Dips Pp DAD ERR 8 6 OZ TheoXUtOCOLLelatOE inti un eb pbeseo dn Mai qoom uelut bese audaces cutn obeunda putes N 8 6 8 4 3 Using Rh to Determine the Diffusion Constant ccccccccccccccccccceeeeeeceeeeeeeeeeeeeeeeeeseseesseeeeseeeess 8 7 09 2 Jeelerences Tor Further Readlflp ust oon Dried tod ento ene D matu dae ale dix dont RU dus 8 8 8 5 1 Static Eie ME Se Atletico ceca cca clr ato atttem Mam AR iu e
55. S ample Interval sec 0 00 Cancel Figure 3 25 The Stripchart Setup Dialog Box Enter the Y range and the X range as well as the Sample Interval The Fixed period selection will present a recording for the time indicated in the Xwidth field and will not present data collected after the indicated time The data will continue to be collected and saved The Scrolling selection will present a recording for the time indicated in the Xwidth field and will redefine the X axis on the strip chart when the Xwidth time is reached so that a continuous presentation is obtained The Sample Interval field 1s used to indicate the frequency that the plot is updated The data will be saved using the value entered in the Single Run Info dialog box Figure 3 5 When you press OK the monitor presents a display of the grid that will be used to present data Figure 3 26 and a dialog box that you can use to edit the box Discovery32 Chapter 3 3 21 RE IPrecisionAcquire32 Acquisition Program COoM1 l m x File Edit View Acquire Review Setup Monitor Help D n mmm mm llel amp lv e Tr do mI i 15 Degree i 0 J Degree i Eh i RBI L4 dtriprhart Display Laser Om Stripchart axis Limits Pause Lm Z Top m E d Bottom Faz i 4 i li lb zu 4 ze ae ET au Time min Ready Idle Mo File oe gt Figure 3 26 Strip Chart Display The Stripchart Y axis Limits dialog box is us
56. Select the number of Stop bits in most cases select 1 d Indicate the directory into which data files should be stored e Indicate the file that is should be used for calibration The default file that was sent with the software should be used This file optimizes the interface box and the filename matches the serial number on the PD2000 DLS module The Disable Calibration File Loading check box should not be checked f Place checkmarks in the three check boxes at the bottom of the dialog box Discovery32 Chapter 3 3 7 2 4 7 The Analog Board Setup Dialog Box The Analog Board Setup dialog box Figure 2 7 1s used to establish communication parameters between the computer and the Analog Board on the analyzer module Analog Board Setup X Sample Hate f 100 Samples sec C 90 Samples sec Request hardware to send a Start Signal now ignore Autostart signals sent while acquisition is in progress Cancel Figure 2 7 Analog Board Setup Dialog Box For the purposes of the test experiment a Set the Sample Rate to 100 Samples sec except in Japan where the 90 samples sec rate should be chosen b Deselect the two check boxes 2 4 8 Export Options Dialog Box The Export Options dialog box Figure 2 8 is used to indicate the format that the data should be exported in to meet the requirements of third party programs for data processing 3 8 Discovery32 Chapter 3 Export Options X PLGPE Esport
57. TET 5 10 224 Cui Cale Wl AMON uso concen etti ate T sede eotw nue sd tu eoete ET ea pede ds 5 12 IAr GUI EL MERERETUR TREE CRI CORR CERT MSN 5 12 20 20 SICH CONS tactus abes apes etu paesi enn oap cim saves ccd alpen none a cr ort aa cap cultes 5 12 2024 9 CONG ANS enue coactanh ood ds cio Deme tdv beim a md aad been cce dias copa cultes 5 13 3 242 Determine Cal OS ais pesci Fee ne os Pad cet tte e pest aoreet dass tad patet uM DS bas Cae oh 5 14 TR SUN sais ae cs rane teeta Nm NT stadt atlas 5 14 22 aR U1 o N E A en ae ee DN E N on een ne Per E 5 14 23 2512 RAE AS a accedi ases ni a Utente ea Cap Us 5 15 D225 gt SIS OMIM PADS sconto as estime tas baie biatnnste astaterstaeecaasoodtetieteasMaanaiecintaieesbantuties 5 16 5 2 5 4 Elution Tabs in the Results Dialog BOX i b ttt edes 5 18 Uu is a o EE 5 21 D220 Ban D oeil ai eee E edd quema Motel asa 5 25 3 22 95 Overlay SIC CLs erick Gin do eat ieee ce ie ean ciara ae ce atonal teeta unio baud 5 26 S020 Update Acquires 2 Constans use tb a a bed pu edet dk een te as 5 27 9 280r MN Dee th iat a ete eect E ea ae ee a end rl steep ea a end ea d 5 27 SE K LITE UTR 5 27 5 4 The File Access and File Information Region eee e eterne en 5 28 Chapter 6 Measuring Proteins and other Monodisperse Species eee ee eee eee eee eee enne 6 1 MEE uil fU 6 1 6 2 Opening a E16 onse ae En ob itte he pa De MER n tetur d uta pM E S ORI tese ub IIR dU 6 2 6
58. a files In the example shown in Figure 3 8 the data files would be collected would be testmix 00 testmix 01 and testmix 02 All other parameters in this dialog box are identical to that for a single run Section 3 2 4 1 The multiple run feature is commonly used in conjunction with an autosampler In this mode of operation the program will automatically collect save and process the data To initiate runs from the autosampler place a check mark in the Use External Synch box on the Multiple Runs dialog box Figure 3 8 The Fill Figure 3 8 button is used to copy an edited line to other lines To use this feature highlight the line to be used as the template then highlight the line s to which the edited line should be copied The file which is used as the template must be above the line to be edited 3 2 3 3 End Run End Run stops the present run When the run is stopped all data collected up to that point 1n the run will be saved using the indicated file name 3 2 3 4 Silent Toggle The Silent Toggle button is used to display the most recently collected data file Discovery32 Chapter 3 3 9 3 2 4 Review Menu The Review menu Figure 3 10 is used to review collected data and start data collection Review Run Information Start Discoverys2 Display Detector Data Show Mw Calculations Figure 3 10 The Review Menu 3 2 4 1 Run information The Run Information command is used to present a dialog box that include
59. a i sSample bzsa i sample bsa i anp lepsa i sSample ADE S3ample ADE s3ample ADE Zamn 1lme Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluant Eluanrzc rt Ready E Figure 7 1 The Discovery32 Lightscattering Analysis Software Window To access files in another directory press the Change button and select the desired directory in the Browse for Folder dialog box that is presented To open a file double click on the line that represents the desired file As an alternative you can open a file by selecting Open on the File menu and choosing the file from the Open dialog box The selected line will be indicated in red directly below the sort buttons e g C PDI Data 040201 06 is loaded in Figure 7 and will be indicated in the Preview File field 7 2 Discovery32 Chapter 7 If desired you can select a file from a different directory than that indicated in the field to the right of the Change button If you choose a file in this manner the indicated directory remains the same and the new chromatogram will be loaded but there will be red indicated files in that list The file that is loaded will be indicated in green immediately above the file list If you do not select any files in the file list and close Discovery32 then reopen the program the directory that will be opened and listed in the field to the right of the Change gt gt gt button will be the one
60. a let ashes ata a eee fedes ace bU UE 1 4 Chapter 2 Installation of PrecisionAcquire32 and Discovery32 eee e eee e eee eee eee erento 2 1 PCMES Au TU T TEM 2 2 2 Interfacing the Detector to the Personal Comrpu ter en ec tte etie ds 2 2 3 Installing the Application Programs on the Personal Computer seee 2 2 4 Setting Parameters for the Test Experiment sessssssssssessssseeeeeeee nennen nnnnnnnnnnn n nnns anna 2 3 Zack Parameter hit RO TUUM 2 3 24 2 Setting Parameters for the Fest Experiment ish endete dett t edu eddiqus s iet ENS Ee E Nix 2 3 2 43 elect Active Detectors Dialog BOX ei utt oo me pd ten tr ee snas TE 2 4 244 Setup Display Dialog BOxGsi cee een E Roe alee ed abus E nan P DE 2 5 245 The Calibration Setup Dials BOK ir E FUR ea E Rte id bs RENE 2 6 2 4 6 The System Configuration Dialog Box seen nennen nennen nennen nennen 2 7 24 7 The Analoe Board Setup Dialog BOX ood contain a eva i eRe Ae 2 8 ZAS Export Options Diadot Ox insana uiis este viste aisuot eode a a osi me DELIS D D OE 2 8 249 Corelator Conftpuration Dialog BOX eee Uribe broche tede evo e Ores Desisto eee oO bec sS beh 2 9 24 10 Cottelator Analysis Setup Dialog BOX enit eb aae nna nee 2 11 DAV Readback status DraloP BOX sci raa to eeta d a ee 2 12 24 12 Autointecration Setup Dralos BOX 5 5 i oH anne Ue eei e e eon eo reb Duae oe see SOS Ub c sU UE
61. ab This dialog box is used to review the calibration process as described in Section 7 7 7 8 Discovery32 Chapter 7 7 5 PREPARING THE CHROMATOGRAM FOR DATA PROCESSING Preparing the chromatogram refers to determining the baseline normalizing the chromatogram and selecting the region to be integrated 7 5 1 Establishing A Baseline The baseline is used to indicate the zero signal level for the sample so that the area of the peak can be properly determined The baseline is normally selected by defining two points in the chromatogram that are on a flat part of the trace one before and one after the peak s of interest The baseline is indicated on the chromatogram as a black line e g see the line at approximately 275 mV for the 15 trace in Figure 7 8 To establish a baseline a Open the Operate on Chromatograms tab of the Chromatograms dialog box Figure 7 8 x Operate On Chromatograms Integration Method Auteintegratian Haw Data Transformation Time mini 410 305 Printed 1127901 09 55 i Precision Detectors Inc Figure 7 8 The Chromatograms Dialog Box Operate on Chromatograms Tab b Right click on the chromatogram to present a pop up selection box and select Set Baseline to present the Select Detectors dialog box Figure 7 9 Discovery32 Chapter 7 7 9 d 7 10 Select Detectors X Baseline Type Least Squares Fil C Point to Point Fit Subtract Baseline For jw Refractive In
62. ae 8 8 SR Dynamic Uy Inseln er eee ne ee 8 8 Chapter 9 Printing and Exporting Dataisiicscsssccccsssvicecosdssccosssnsdeveusvsecsassnddevesdeccsenssnecensvoncecoasevedecevandess 9 1 LE 018 9 dtd Heat eee ditat eee re dtum bid tuc ein 9 D2 EPONA DES Rm 9 2 9 9 Jreneratmie ARENON dy OUL coche et tu tao tasa taste etos huie dans 9 4 Do she Report Layout Dialog BOX uod at dba tti te Sac et b eng PE M 9 4 03 The ChrOndtOSEdmi osos ae eontra tide he Cere ea EU NL tached pce 9 5 DG ANS CHUNG Tex sige es onn t nte t moenium I ce 9 6 934 Inser nsaMW Distribution Plot carinii pt Pee m PG P Et ER RE 9 6 9 5 5 Addie Sample InfOtinadblOnsss oie o e E exe iem k 9 7 9 5 6 dame Total Arer Cale ul at Ons sies petisse esty ceeds tedeeend Ie te oie atu em Ea b aie sles 9 9 9 5 7 JPlacfhe a Lie 1n the Pale eo do e aaa eb e hear ete eU 9 11 9 555 Adna Pa5es to a REPO ui eese pt SSH toe etai ten t bete sue e os poU bt ue Ru hd Rer SLM e EE 9 11 DAI MNA CERTO 9 11 9 3 10 General MOnato coucou te De ATH ovo tos dL eR ap SERM TM tana rah oe e Mas S Nea das OE dii un 9 12 9 3 LE Saving and Bditine Report Layouts eet ER ARIS E RATS IRE Erbe DAS ER NUR AR A E RUEE 9 13 Appendix A Calibrating the System Experimental Considerations e eeee creer eene A 1 IG Ioq I 1 Discovery 32 Table of Contents Chapter 1 Introduction 1 1 OVERVIEW
63. amp utointegration Haw Data Transformation Auta Integration File List 60 max No Files in List Add Files ta List lear List gt Double click on item to remove it from list amp uta ntegrate Using SSLABOTSLABD ata odd 01 SAl 06 Figure 6 18 Chromatograms Dialog Box Autointegration Tab To use the Autointegration feature 1 Press the Add Files to List button to present a dialog box that is used to select the desired files ii When all files are selected press the Auto Integrate Using gt gt gt button The file that is used as a template is selected on the pop up menu on the Chromatogram window c View the Summary tab of the Results dialog box Figure 6 16 to view the desired data Similar plots for Rg and Rh can be obtained using the corresponding tabs Discovery32 Chapter 6 6 21 d Click the Mw Dist tab to present a molecular weight distribution Figure 6 19 Result x Summary Run Params Mw Dist Mu We Elu Hh Dist Hh vs Elu Hg Dist Rg vs Elu BSA UV RI O4 02 2001 12 24 53 l1 JE IH 0 3 n 3 0 2 0 2 B Amount Mm tn o in AmI iaea AATJE TAMA 0 3 3 0 2 2 0 1 Ba 5 0 0 0 10E ZO 20K 40E 10E 60K VOK 20E 90E O01 FDS Fred 112901 OF 34 m P reck los mar hen Fa t ionz100 0 1000 to l non on Figure 6 19 Molecular Weight Distribution Plot e Click the Mw vs Elu tab to present a chromatogram which presents the
64. an be expressed by Na c V M Now if we solve for M we obtain equation 8 4 M Na R I 4 r M sin c dn dc I V 8 4 Collecting all the constants and instrumental parameters into an overall instrumental constant A we obtain equation 8 5 M I A c dn dc I 8 5 Equation 8 5 can be used to measure the molecular weight Mw of small molecules at any scattering angle 0 It should be noted however that larger molecules scatter less light at high values of 0 than at low angles because of interference effects caused by the fact that light scattered from one part of the molecule travels a different distance from another part of the molecule and is not in phase with light scattered This phenomenon can be quantified by defining the light scattering form factor equation 8 6 A more detailed discussion of the form factor 1s presented in Section 8 3 3 P 0 scattered intensity at angle 0 scattered intensity at angle 8 6 It should be noted that P 0 can be written as a series as shown in equation 8 7 P 0 21 1 3 q R 8 7 where q 4z n sin 0 2 A R is the radius of gyration of the molecule n is the index of refraction of the liquid is the wavelength of light in a vacuum Discovery32 Chapter 8 8 3 For scattering at 15 and 90 equation 8 7 can be expressed as equations 8 8 and 8 9 respectively P 0 1 26 3 Rz n 1 8 8 P 0 1 0 0897 R n 4 8 9 We present a table of P values for 15 and 90 d
65. apter will be slightly different than those presented in discussions of light scattering when non polarized sources are used The electric dipole moment that is induced is shown in equation 8 1 2 gt p ak 8 1 where p is the dipole moment a is the polarizability E is the electric field 8 2 Discovery32 Chapter 8 The polarizability can be related to measurable parameters via equation 8 2 A M dn dc 2zN4 8 2 where Mw is the molecular weight Na is Avogadro s number 6 02 x 10 molecules per mole dn dc is the change in the index of refraction as a function of the change in concentration It is considered to be a constant for any specified solvent solute pair under constant operating conditions The oscillating dipole will radiate light in all directions at the oscillating frequency This is the origin of ERN scattered light If a single molecule has dimensions that are small with respect to the wavelength of the incident light the intensity of the light can be defined by equation 8 3 I 4 n M sin dn dc L NA 4 R 8 3 where I is the intensity of the radiated light I 1s the intensity of the incident light is the wavelength of light in a vacuum and R are as defined in Figure 8 1 If we collect light from a volume V of a solution with a concentration c gm mL the intensity of scattered light can be found by multiplying equation 8 3 by the number of molecules in the volume V The number of molecules c
66. at Cal UV 24534493 57 Figure 6 14 Setup Calculation Constants Tab To align the peaks change the value slightly typically a change of 1 1s used and press Apply on the Constants tab then view the chromatogram again This procedure may require a number of iterations until the chromatograms coincide 6 6 2 Determining the RI Constant To determine the RI Constant a Expand the integration region of the chromatogram to include the entire area of the peaks for the refractive index detector The area should include the monomer and the aggregates if any but the terminal peak should be excluded b Return to the Determine Cal Constants tab of the Setup Calculation dialog box and check the Accept box that corresponds to the Refractive Index Constant value then click Apply SEPT Note Precision Acquire32 will report RI constants that are between 75000 and 85000 which are not compatible with Discovery32 where the RI constant 1s typically around 2 to 3 6 16 Discovery32 Chapter 6 6 6 3 Determine Calibration Constants for 15 and 90 Channels To determine the Calibration Constants for 15 and 90 channels a Set integration parameters for the monomer peak Approximately 10 percent by height above the lift off and the same on the landing This is to avoid possible coelutions with dimer and polypeptide fragments b Return to the Determine Cal Constants tab of the Setup Calculation dialog box and place a che
67. at are common to most Windows application programs will not be discussed As an example the Open command on the File menu presents a dialog box that is used to open a file This dialog box 1s identical to the Open dialog box found in other Windows applications such as Microsoft Word and Microsoft Excel e In many instances there is more than one way to perform a specific activity As an example in Discovery32 you can access the Setup Calculation dialog box by selecting Setup Calculation on the Options menu or by pressing the corresponding icon the Wrench icon We will describe the use of the commands and indicate the role of each icon as we describe the program e The user can obtain a pop up menu containing commands that are commonly used for processing data in various windows that contain data by right clicking on the data As an example when the Chromatogram window is presented a pop up menu allows for commands such as setting the baseline or normalizing a user selectable region Discovery32 Chapter 1 1 3 1 6 CONTENTS OF THIS MANUAL This manual includes the following 1 7 Chapter 2 Installation describes how to load and configure the program for the specific system that you are using It includes a sample experiment that can be used to demonstrate that the computer and the detector module are functioning in an acceptable manner Chapter 3 Introduction to PrecisionAcquire32 describes the details of the data acquisition prog
68. at show previously determined regions As an alternative you may move the vertical lines by holding down the Ctrl button down and clicking while dragging the rectangular box Select a region of the chromatogram where the baseline is flat in front of the peak s of interest and then release the mouse button Repeat the process for the other traces if they require selection of different regions to obtain good baselines In Figure 7 10 we have set the baseline indicators to approximately 4 2 min and 5 1 min Discovery32 Chapter 7 Chromatograms Xx Operate Un Chromatograms Integration Method Autombegratian Haw Data Transformation 640 RR c 15 Deg voltage mv DI DI E TI F LT LT hd D Cc JI co hd Cid c c c c c c c HJ oo c Time min 410 305 Printed 11 27 01 10 16 g Precision Detectors Inc Figure 7 10 Setting the Baseline e Once you have set both vertical lines to the desired times click in the region between the two lines to save the settings 1f you want to erase the settings and start over click outside the region defined by the vertical lines After you have accepted the settings the vertical lines disappear and a small square represents where the baseline is set for each detector trace f When selecting baselines by Least squares click and drag on a flat region of baseline before or after the peak of interest A baseline will be extrapolated based on the region you have selected You can also have the l
69. atograms E x Operate Un Chromatograms Integration Method Auteintegration Haw Data Transformation gB 4 kK PS 04001998 13 04 18 9 06 9 04 0 96 0 B 4 h z 10 12 14 1B Time min 410 305 Printed 11 27 01 14 18 fe Precision Detectors Inc Figure 7 21 Hydrodynamic Data Discovery32 Chapter 7 7 23 7 24 This page intentionally left blank Discovery32 Chapter 7 Chapter 8 Theory of Light Scattering 8 1 OVERVIEW Light Scattering refers to the process in which light from an incident polarized laser beam is scattered in all directions when it interacts with a molecule or particle Light scattering 1s an everyday occurrence and was first described by Lord Rayleigh in the late 1800 s An example of light scattering is the scattering of sunlight by particles in the atmosphere the sky is blue because shorter visible wavelength radiation blue light is scattered more strongly by the gas molecules in air than light of longer wavelengths red light There are two general techniques for the measurement of physical properties of polymers synthetic polymers and natural polymers such as proteins and polysaccharides virons liposomes and particles e Static Light Scattering also known as Rayleigh scattering or classical light scattering The intensity of the scattered light from the sample dissolved in the solvent This difference between the two measurements can be used to determine the average molecular weigh
70. ck mark in the Accept box adjacent to the 90 and or 15 constant entry 6 7 CHECKING CALIBRATION RESULTS RI DETECTOR To Check Calibration Results a Access the Run Params tab of the Results dialog box Figure 6 15 Summary Fun Params hw Dist Min vg Elu Hh Dist Hh vs Elu Hg Dist Hg vg Elu hw Total amp rea Calculations Hl Ly Batch 15 Deg bz 17K bb 32K BF SBE 90 Deg b 17E bb 3K by Sek Hg nm lt 10 0 Sample Conditions Inj Val 100 000 id Flow Aate 1 0000 rl rin Eluant HI 1 3320 IRI units Sample Constants dn dc 0 1670 0 1690 rni calc T RS 0 6750 cac 06814 7 ml mag cm Concentration Detectors Al 1 7017 1 7224 mg ml calc i Lv 1 7017 1 7178 mam calc E Figure 6 15 Results Dialog Box Run Params Tab Discovery32 Chapter 6 6 17 The following items should be checked on this tab e The calibration of the monomer peak for BSA should be correct for RI for 15 and 90 e The Calculated Value of dn dc in the Sample Constants field If BSA is used the calculated value is 0 1355 mg ml and the calculated value should be 0 1355 if the area of the monomer peak was exactly 2 mg mL The entered value was 0 1670 It should be noted that since BSA forms dimers trimers etc the value of 2 1s not expected e The Calculated Value of RI in the Concentration Detectors field If BSA is used the calculated concentration
71. column correspond to the values that were entered on the previous dialog boxes 3 12 Discovery32 Chapter 3 2 4 12 Autointegration Setup Dialog Box The Autointegration Setup dialog box Figure 2 12 is used to describe how the initial calculations are to be performed Auto Integration 5etup Fa Fitting Regions Decimal Minutes Stark Baseline 1 Baseline 2 Stop of o me D 0 0 iw Graph Limits 2 area rejection hd ax Cony Factor Show plot at run end Min Figure 2 12 The AutoIntegration Dialog Box 2 5 TEST EXPERIMENT 2 5 1 Introduction to the Test Experiment The test experiments provided in this section are designed to demonstrate that the system is operating in a reasonable manner While these experiments are not meant to formally qualify the system they are very useful in checking that the computer is communicating with the analyzer module and the analyzer module is acquiring data in a reliable fashion 297 Note Precision Detectors is capable of providing Instrument Qualification to verify that the system is capable of providing data to meet the needs of regulatory agencies or in house requirements For additional information contact Precision Detectors or your local representative Discovery32 Chapter 3 3 13 2 5 2 The Single Run Info Dialog Box The Single Run Info dialog box Figure 2 13 is used to enter a broad range of parameters to describe the run
72. ctors the arrows indicate the integration region which is set as described below The chromatogram can be zoomed by clicking on a corner and dragging the mouse There are four tabs in this dialog box Chromatograms Operate Un Chromatograms Integration Method Autointegralian Haw Data Transformation 03241994 17 39 20 5B 440 ae 400 E 380 5 r3 E 44 s 40 320 fs lt 5 3B 280 eu 32 240 0 io 28 5 200 24 180 1B 120 Time min BEA 12 Printed 11 06 00 15 11 amp PDI Version 32 Bit Figure 5 6 The Chromatogram Dialog Box 5 6 Discovery32 Chapter 5 a The Operate on Chromatogram tab 1s used to perform a number of basic chromatographic data processing operations such as setting the baseline normalize the chromatogram etc which are accessed by right clicking on the chromatogram Figure 5 7 Set Baseline Set Integration Region Set As Integration Template Show Chromatograms Shaw Raw Chromatograms Normalize Chromatogram Normalize Region shirk Print Raw DataTable Reduced Data Table Figure 5 7 Chromatogram Operations Menu Set Baseline The baseline start and finish are indicated by open and close brackets on the chromatogram e g at 2 0 and 2 5 min on Figure 5 6 To set the baseline a Click on Set Baseline to present the Select Detectors dialog box Figure 5 8 Select Detectors X Baseline Type Least Squares Fil C Point to Point Fit Subtract Bas
73. d be set to Linear Fit 3 2 5 9 Readback Status Dialog Box The Readback Status dialog box Figure 3 21 is used to ensure that the settings on the Setup Correlator and Correlator Analysis Setup dialog boxes have been successfully downloaded to the box 3 18 Discovery32 Chapter 3 Headback Status ES Haeadback Command Slurp Size 1024 1024 Huntime secs 15 4 15 4 Sample Time uzec sli sil Prescale Settings Corr 1 1 1 1 1 SA Reset OFF OFF Dil 4 4 Mem Size B B Dips witches hex OOO Contig Register hex oggi Interrupt Mask hex O14 Version hex EI Figure 3 21 Readback Status Dialog box When this dialog box is opened check that the parameters in the Readback column correspond to the values that were entered on the previous dialog boxes If the values are identical click OK Note It is important to open this box and verify that the parameters that were made as changes in Figure 3 19 are not saved until the OK button on the Readback Status dialog box has been pressed 3 2 5 10 Autolntegration The Autointegration dialog box Figure 3 22 is used to indicate the desired integration parameters Auto Integration 5etup EJ Fitting Regions Decimal Minutes Start Baseline 1 Cancel Baseline 2 Stop E Integration a ro 5 hw Graph Limits area rejection hax Cony Factor Show plot at run end Min Figure 3 22 The Auto Integration Setup Dialog Box
74. dex Iv Low Angle Scatter Iv High Angle Scatter M UY Detector Cancel Figure 7 9 Select Detectors Dialog Box There are two options for selecting baselines Least Squares Fit or Point to Point Fit If the baseline is flat before and after the peaks of interest we recommend that the Point to Point Fit radio button be selected If there 1s a flat region of baseline only on one side of the peak s then the least squares fit 1s appropriate as it can extrapolate baselines The baseline must be established for all detector channels for which data has been collected for the selected calculation types e g RI LS 90 requires the RI and 90 channels to have baselines if 15 deg is collected but is not being used for the calculation then baselines for that channel is not required the Low Angle Scatter check box refers to the 15 scatter channel and the High Angle Scatter check box refers to the 90 scatter channel For Point to Point baselines select that feature along with the channels you are going to establish baselines for and click OK The cursor will appear as a sign two vertical lines may be displayed on the plot indicating previously determined baseline regions based on Least squares techniques or there may be small rectangular boxes which indicate point to point baselines You may start and stop the baseline selection going from left to right anywhere on the chromatogram and independent of any vertical lines th
75. dialog box is accessed via the Display Options command on the File menu main window x Delete Current Page Add Company Name to Page Head Cancel Company Name vw Add Date to Page Head Date Format f 07 20 2000 C Jan 21 2000 w Print Page Number Harz Aliqnment Lef C Right Center Y er Alignmernt f Top C Bottom Figure 9 11 Page Dialog Box x Company Name Precision Detectors Ine MW Add Run Date Time to Graph Title M Add File Name to Footer i Add Current Date Time to Graph Footer v Add Company Mame to Graph Footer Figure 9 12 Display Options Dialog Box A 12 Discovery32 Appendix A 9 3 11 Saving and Editing Report Layouts A report layout is saved by selecting Save or Save as on the File menu A dialog box will be presented to indicate the file name Report Layouts have the file suffix rlo e g xxxxx rlo If desired you can edit an existing report layout by a Select Create Report Layout on the File menu of the main Discovery32 window to present the Report Layout dialog box b Select Open on the File menu of the ReportLayout dialog box and choose the file that you want to edit The file will be loaded and can be edited Discovery32 Chapter 9 9 13 This page intentionally left blank Discovery32 Appendix A Appendix A Calibrating the System Experimental Considerations A 1 OVERVIEW Selection of the standa
76. e 75 points will lead to one point per second if you set the interval to 2 point every second then we get fewer data points The latter case will give a more stable baseline however it should be noted that if the interval were too large the rise time of the chromatogram would be affected 4 3 3 MW Calc Method The MW Calc method field on the Single Run Info and the Calibration Setup dialog boxes is used to select the degree of smoothing This selection refers only to the smoothing of the molecular weight trace on the chromatogram as the data is presented in the workspace The actual raw data is stored in the data file and is not affected by the smoothing It is recommended that this field be set to No Smoothing for most cases especially when polymers are used and polynomials should be used for synthetic polymers where there is a true distribution Discovery32 Chapter 4 4 3 4 4 PARAMETERS FOR PD2000 SYSTEMS 4 4 1 Run Time The Run Time is the amount of time over which the correlation function calculation is averaged Typically a run time of 10 sec is used This parameter is used only with DLS systems If the Run time is decreased the correlation may become noisier and if the run time is increased fewer points for the correlation will be collected 4 4 2 Sample Time The sample time is the time for collection of a single data point for use in the correlation function Typically a value of 5 usec is used if the weight is greater than 1000
77. e 000 lier lieu 0 000 000e 000 000e 000 000e 000 000e 000 0 000 000e 000 000e 000 Figure 9 7 Information Fields be 3g gg fh E E E keys These keys are active only when the information fields are first opened once you have moved any field they will be deactivated Discovery32 Appendix A 9 3 6 Adding Total Area Calculation To add total area calculation a Click the icon and click the cursor on the desired position in the pane to present the Mw total area calculation dialog box Figure 9 8 Mw total area calculation X Sample Constants Sample Condition pU Concentration Detectors Cal Parameter Mw Total Areas R PF uv Batch 15 Degree 90 Degree Rafnm Integration and Baseline Setting Regions Run Calibration Constants Calculation Option Settings Figure 9 8 The Mw Total Area Calculation Dialog Box b Place a check mark in the items that you want included on the pane and select OK A series of entries will be placed on the pane as shown in Figure 9 9 Discovery32 Chapter 9 9 9 A 10 sample Constants dun dc idl qi Uv ext ml mu cm Run Constants RI Inter detector Volume iml Mol Wt Mol Wt Mol Wt Lal RI 145 Cal RI 90 Cal RI Cal Solvent Index IT Inter detector Volume iml Mol Wt Mol Wt Mol Wt Cal UV 15 Cal Uv 9nj Cal Uv QOOO 0000 Cale OOO 0000 OOO 0000 Calc OOO 00
78. e Run Info Dialog Box Discovery32 Chapter 3 e File Name is used to enter the file name that is to be used to for data that 1s to be collected in this run to be collected The format of the File Name must be xxxxxxxx xx 1 e the first character after the period must be a Sg Note When you are preparing to analyze a fresh sample make certain that you have renamed the run before entering other parameters If you do not rename the run the program will ask if you want to overwrite the existing file If you indicate that you do not want to overwrite the existing file any parameters that you have already entered will be lost and it will be necessary to re enter them e The Operator Eluant and Sample fields are used to enter the relevant information This data is stored with the sample data file e The dn dc UV Ext Coeff Solvent Index Refractive Index and Temperature fields are used to enter the relevant parameters for the appropriate calculations in Discovery32 and should be entered Note If these parameters are not available before the collection of data an approximation or dummy value can be used The correct values can be entered during the data processing procedure If an approximate or dummy value is used do not select 0 e The Run Length min field is used to enter the time period for the separation This value cannot be changed after the run has started e The Sample Interval sec field is used to indicate the
79. e baseline for 10 15 minutes Once you have established a satisfactory baseline inject 100 uL of sample aqueous system BSA 2 mg mL organic system Polystyrene MW 96 4 kD dissolved in THF 2 mg ML and monitor the signal A sharp peak should be observed after a few minutes 397 Note In light scattering measurements cleanliness is critical The mobile phase and the sample must be filtered before use Discovery32 Chapter 3 3 15 3 16 This page intentionally left blank Discovery32 Chapter 3 Chapter 3 Introduction to PrecisionAcquire32 3 1 OVERVIEW PrecisionAcquire32 1s used to control PDI Light Scattering detectors used in high performance liquid chromatography size exclusion chromatography Fast Protein Liquid Chromatography and gel permeation chromatography Data collected via PrecisionAcquire32 can be ported to Discovery32 for the determination of the molecular weight distribution the hydrodynamic radius and related parameters of proteins virons and liposomes and the generation of reports This chapter describes the Main window of PrecisionAcquire32 Figure 3 1 and includes e Menu bar Section 3 2 e Tool bar Section 3 3 e Workspace Section 3 4 e Status Bar Section 3 5 A detailed discussion about the selection of parameters for data acquisition is presented in Chapter 4 Discovery32 Chapter 3 3 1 is P IPrecisionAcquire32 Acquisition Programiet 1 l E n x Fie View Acqu
80. e displayed on the left axis This makes the chromatogram window larger and easier to process 387 Note Typically the UV and Rh data are not presented when you are defining the baseline and integrating the peak s for the RI detector and the RI and Rh data are not presented when you are defining the baseline and integrating the peak s for the UV detector This 1s done so that so that the data from the light scattering detectors is more readily viewed 6 6 Discovery32 Chapter 6 6 4 3 The Constants Tab The Constants tab Figure 6 6 is used to present a variety of instrumental constants that are required to calculate light scattering properties Setup Calculation x Options Detectors Constants Determine Cal Constants Hun AI Instrument Al Inter detector Wol ml 0 0373 olw Cal Rias 5031 Mol wt Cal R1790 15488 Mol wt Cal RI 304402 1 3310 Molt Cal A1715 Molwt Cal A130 Mol wt Cal RI Cal Solvent Index Cal Solvent Index Instrument LY Inter detector Val rl 0 0646 Molwe Cal uvas 202643 Molwt Cal uv go 45338n Mol wt Cal UY 745 2000 Inst Cal Values gt Files Hun Lv Inter detectar Val ral 0 0646 gt Mole Cal LI 15 20124 4 Mol wft Cal U90 45394 2 feos Mal wt Cal LIV 745 3491 ER RN Figure 6 6 Setup Calculations Dialog Box Constants Tab The Run RI and Run UV constants shown on th
81. e following Precision Detectors Light Scattering systems 1 4 1 PD2010 The PD2010 is a static light scattering system with the detector positioned at 90 to the incident laser beam This system is available as an ambient temperature bench top configuration a high temperature bench top configuration or installed inside the temperature chamber of a Waters 410 or 2410 Differential Refractometer The detector can be used at temperatures up to 50 C This configuration is typically used to determine the molecular weight of smaller molecules such as proteins and low molecular weight polymers It is frequently installed with the PD2000 DLS a dynamic light scattering detector as an accessory to provide information about the size of the molecules 1 4 2 PD2020 The PD2020 is a static light scattering system with detectors positioned at both 15 and 90 to the incident laser beam This system is available in an ambient temperature bench top configuration or can be installed inside the temperature chamber of a Waters 2410 or 410 Differential Refractometer The detector can be used at temperatures up to 50 C This configuration is used to determine the molecular weight radius of gyration and other parameters for a wide range of molecular sizes typically from 100 to 10 000 000 daltons It 1s frequently installed with the PD2000 DLS dynamic light scattering detector as an accessory to provide information about the size of the molecules 1 4 3 PD2000 DLS
82. e left side of the Constants tab are the current values that are being applied to the chromatogram and are stored with the data file while the constants shown on the right side are the instrument values which are currently stored in Discovery32 You can think of these constants as being analogous to the memory function of a calculator They are available to update any files with these constants When a calibration routine is performed both the Run and Instrument side constants are updated with latest determined values If you choose to update the constants in PrecisionAcquire32 they can be copied and pasted individually into the Calibration dialog box of that program from the Discovery32 program Calibration constants can be manually entered into the appropriate fields When the data has been entered to a Run field e g Inter detector Vol ml it can be copied into the corresponding Instrument field using the gt key or vice versa using the corresponding key If values are moved using the key or key a dialog box will pop up asking you to confirm the change The Update key becomes active after a change has been made To accept the changes made the update button must be clicked If you want to apply the newly determined values to stored files press Inst Cal Values key It will then be necessary to recalculate the values Discovery32 Chapter 6 6 7 6 4 4 Determining Calibration Constants The Determine Cal Constants tab Figure 6
83. east squares method behave like point to point by selecting a baseline region as discussed above then select a second region on the other side of the peak to repeat the process 327 Note When the baseline selected in the above manner the same points are selected for each detector trace If you want to change the baseline for a specific detector click on the square and drag it to the desired point 7 5 2 Normalizing the Heights and Zooming on the Peaks Selecting the integration region is simplified if the peak s of interest are sufficiently large so that you can see where the trace begins to deviate from the baseline Typically the operator will normalize the peaks so that the largest peak in the region of interest is full scale Zooming on the peaks is used to have the region of interest to be full scale on the display Discovery32 Chapter 7 7 11 To Normalize the Height of the Peaks a Right click on the chromatogram to present the pop up menu b Select Normalize Region The cursor will appear as a sign c Select the region that you would like to normalize in the same way you selected the baseline points To Zoom the Normalized Region a Take the cursor and place it on one of the corners of the desired area to be zoomed b Click on the left mouse button and drag to the opposite corner then release the mouse button In this example we will start at 4 0 minutes by the lower baseline and drag the mouse to 7 2 by the upper bou
84. ed to change the display while data is being collected 3 2 6 4 Laser The Laser command turns the laser on off When the laser is on a checkmark appears adjacent to the legend and a small green light on the status line is illuminated 387 Note If you turn the laser off and on again it will take approximately 15 to 30 minutes for the system to stabilize 3 2 7 Help The Help menu Figure 3 27 accesses information to assist in the operation of the system Help About Precision amp cquire3z aet Board versions Help Figure 3 27 The Help Menu 3 22 Discovery32 Chapter 3 About PrecisionAcquire presents a dialog box that includes the version number and copyright information for the program Get Board Version accesses a dialog box that indicates the version of various printed circuit boards inside the connector box The information in this dialog box is not related to routine use of the system but may be of assistance in troubleshooting Help accesses the PrecisionAcquire help file which provides on line assistance 3 3 THE TOOL BAR The Tool Bar Figure 3 28 provides rapid access to a variety of commands that are commonly used in PrecisionAcquire32 Das ms mmm en n Sle e o v eice ea ca Figure 3 28 The Tool Bar is equivalent to New on the File menu which is inactive accesses the Open dialog box is equivalent to Open on the Fi e menu equivalent to Start Single Run on the Sing
85. egins to deviate from the baseline Typically the operator will normalize the peaks so that the largest peak in the region of interest is full scale Zooming on the peaks is used to have the region of interest to be full scale on the display Discovery32 Chapter 6 6 11 To Normalize the Height of the Peaks a Right click on the chromatogram to present the pop up menu b Select Normalize Region The cursor will appear as a sign c Select the region that you would like to normalize in the same way you selected the baseline points To Zoom the Normalized Region a Take the cursor and place it on one of the corners of the desired area to be zoomed b Click on the left mouse button and drag to the opposite corner then release the mouse button In this example we will start at 4 0 minutes by the lower baseline and drag the mouse to 13 0 by the upper boundary This zoom will present the display shown in Figure 6 11 Avoid any baseline perturbations such as large negative dips near the terminal region of the chromatogram This is more prominent with RI detectors than observed for UV Chromatograms Operate On Chromatograms Integration Method Autointegration Raw Data Transformation 360 320 280 KJ les e 200 c D amp 160 gt Ta s M LI co 9 0 92 Time min Figure 6 11 Normalized Zoomed Display 6 12 Discovery32 Chapter 6 6 5 3 Defining the Integration Region The integration re
86. egrees as a function of molecular weight for molecules with molecular weight from 10 to 10 Daltons in Table 8 1 These values assume that the molecules are random coils Table 8 1 Value of P 90 and P 15 for 4 7 685 nm n 1 5 R Approx nm P 90 P 15 x10 J23 0993 1 0000 5x10 7 0993 0 9998 0 9993 0 9976 0 9780 0 7622 values depend on shape If the value of P O found in equation 8 7 is below 0 7 higher order components become important In this case P 0 depends on R and also on the shaper of the molecule as shown in Figure 8 2 1 2 pig q Rg Figure 8 2 Values of p 0 as a function of q XRg for various particle shapes 1 Sphere 2 Gaussian Coils 3 Rods It is clear that all three molecular shapes yield the same value of P 0 when q lt Rg gt is less than approximately 1 As the value of P 0 increases the shape of the molecule clearly influences the light scattering intensity 8 4 Discovery32 Chapter 8 8 3 2 Using Refractive Index Measurements to Measure Concentration Refractive index measurements can be used in conjunction with static light scattering measurements to determine the concentration of the compound in the chromatographic slice The difference in refractive index between the solution under study and the pure solvent is measured by passing a light beam thru two cells as shown in Figure 7 3 One of the cells contains pure solvent and the other contai
87. elected the intensity of the data point will be higher but the chromatogram will be noisy Similarly a small value for the Collection Fraction will lead to less noise and a smaller signal In Figure 4 1 we present chromatograms that were taken with the same sample using collection fractions of 0 1 and 0 75 Typically a collection fraction of 0 10 is used for aqueous samples and 0 75 is used for samples dissolved in organic solvents such as THF 4 3 2 Sample Interval The Sample Interval field on the Single Run Info dialog box 1s used to indicate the frequency of collection of data points Typically a 1 sec value is selected if the chromatogram is long e g gt 45 min a value of 2 sec is acceptable if you want to reduce the number of points A small value will provide better chromatographic resolution but may increase the noise in the chromatogram and will significantly increase the size of the data file and increase the time for processing If desired you can reduce the number of data points by indicating a Prestore Delay When a Prestore Delay is indicated data will not be collected for the indicated period of time 1 e if the peaks of interest elute in the region from 25 00 min to 32 00 min you can enter a 14 00 min prestore and reduce the file size thus allowing for a smaller sample interval By increasing the time for each point we can get better signal to noise As an example if you select 100 points per second at 75 collection rat
88. eline For jw Refractive Index Iv Low Angle Scatter Iv High Angle Scatter M UY Detector Cancel Figure 5 8 Select Detectors Baseline Correction Dialog Box b Select the desired Baseline Type and indicate the detectors for which the baseline should be subtracted After you click OK a pair of vertical lines may appear on the chromatogram if the chromatogram was processed previously Figure 5 9 Discovery32 Chapter 5 5 7 Chromatograms E bi Operate On Chromatograms Integration Method Autointegration Haw Data Transformation 24D bsa in obs agn B4D 22D anit 760 co a 0 180 B80 2 mm B40 lt dn B0D D 140 SAO S 520 3 SEP 480 5 os 12 16 Time min Figure 5 9 Chromatogram with Baseline Markers c Indicate the position of the baseline by clicking on the vertical line and dragging it to the desired start finish of the baseline The black lines on the chromatogram indicate the selected baseline Note There are a number of ways of defining the baseline The above example involves the use of a baseline drawn by exponential skim which is useful good for extrapolating a baseline if the front or tail end of the chromatogram lacks a flat region for selection As an alternative you can select a portion of the baseline before and after the peak For a point to point baseline simply select a start and finishing point for baseline determination You can define the baseline by click
89. en opened are indicated immediately above the Exit command e Exit closes the program 3 2 2 View Menu The View menu Figure 3 4 1s used to enable disable the Toolbar and Status bar on the main window When these features are activated a check mark is indicated to the left of the command View v Toolbar w Status Bar Figure 3 4 The View Menu 3 4 Discovery32 Chapter 3 3 2 3 The Acquire Menu The Acquire menu Figure 3 5 accesses a series of dialog boxes that are used to set conditions for and initiate a run Acquire Single Run Multiple Runs End Run Silent Toggle Figure 3 5 The Acquire Menu 3 2 3 1 Single Run Single Run accesses the Single Run Info dialog box Figure 3 6 which is used to set parameters for data acquisition Single Run Info 24 500 File Mame Operator TH Run Lenath min 15 Eluant PES Sample Interval zec 1 000 sample BoA Prestore Delay miri 0 0 dn dc 0 167 Flow Aate ml min 1 0000 UV Ext Coett mm Injection Malum ul 100 Solvent Index S Sample Conc mg l Temperature C 3 S ample Information oo o Wal for Autostart Signal Cancel Mw Cale Type 15 degree data only 90 degree data onl hw Cale Method Low smoothing Medium smoothing ll l xxx mb L arrelatar Setup Hun Time seconds 5n Sample Time seconds H Figure 3 6 The Singl
90. enu b Select Narrow Standard Info Setup to present the Narrow Standard Table Options dialog box which is used to select the data to be used for the calculation Discovery32 Chapter 5 5 21 Narrow Standard Table Options Xx v Add 90 degree data to table Iw Add 15 degree data to table Cancel Iv Add Ag data to table Figure 5 27 Narrow Standard Table Options Dialog Box c Select Add Narrow Standard Information from the pop up menu to present a table that presents the calibration data obtained from the chromatogram Figure 5 28 A point will be presented for each peak in the chromatogram Narrow Standard Information Time min Mw exp 17 065 19 250 21 733 24266 Figure 5 28 Narrow Standard Information d Close the Narrow Standard Information dialog box to present a plot containing the points indicated in the table Figure 5 29 Drag the blue line from the left axis to set the polynomial fit If you drag the line all the way to the right all data points will be included The plot will include the equation for the calibration line as shown in Figure 5 30 5 22 Discovery32 Chapter 5 No Cal Data Loaded ul n j ir am Wt ol M i Elution Time rin Figure 5 29 Calibration Plot Discovery32 Chapter 5 5 23 GPC i x Calibrate Conventional GPC Distributions Loox No Cal Data Loaded Log Mas 4 5941e 000 41 82435e D02 t Sigma 1 3109e 004 30K Da
91. erse applications is presented in Chapter 7 This chapter describes e Opening a file Section 6 2 e Viewing the Chromatogram Section 6 3 e Establishing Calculation Parameters Section 6 4 e Preparing the Chromatogram Section 6 5 e Calibrating the System with RI Detection Section 6 6 e Checking Calibration Constants with RI Detection Section 6 7 e Calibrating the System with UV Detection Section 6 8 e Checking Calibration Constants with UV Detection Section 6 9 e Analyzing Analytical Data on a Routine Basis Section 6 10 2Note The calibration data calibration guidelines and sample data presented in this chapter refer to the use of a 2 00 mg mL solution of BSA Bovine Serum Albumin A detailed discussion of the analytical conditions used to collect the data is presented in Appendix A It is not unlikely that data that you collect may be slightly different from that presented in this chapter e g retention times will differ if a different column is used Discovery32 Chapter 6 6 1 6 2 OPENING A FILE When Discovery32 1s opened the Main window 1s presented as shown in Figure 6 1 The Preview File chromatogram that 1s presented in the upper right corner represents the file that was open when the application was closed and the bottom half of the display describes files that are in the directory that is indicated in the field adjacent to the Change button The files in the listing can be sorted by the Filename Eluant
92. es via the Open dialog box that is presented Discovery32 Chapter 5 5 9 Chromatograms X Operate Un Chromatograms Integration Method amp utointegration Haw Data Transformation Auta Integratian File List BU max iNo Files in List Add Files to List ear List Double click on item to remove it from list Auta dntegrate Weng ss C SPDISD ata B5A 07 Figure 5 10 The Autointegration Tab After you have selected the desired files press the Auto Integrate Using gt gt gt button The data in each file will be integrated using the template indicated adjacent to the button this file was selected in the Operate On Chromatogram tab Section 5 2 3 1 5 2 3 4 Raw Data Transformation The Raw Data Transformation tab is identical in format to the Chromatogram dialog box Figure 5 6 and can be used to smooth the chromatogram and invert the RI chromatogram When the right mouse button is pressed the menu shown in Figure 5 11 is presented Chromatogran Smoothing Invert RI Chromatograrn Save Operated Chromatogram Figure 5 11 Raw Data Transformation Menu 5 10 Discovery32 Chapter 5 Chromatogram Smoothing 32 Note Chromatogram smoothing is used to reduce noise and random variations in the chromatogram The analyst should note that small peaks which may be of analytical use may also be reduced so some caution should be used with this function When Chromatogram Smoothing 1s selected
93. es from the column The photon counting measurement is performed over a very short period of time to record the very rapid diffusion that 1s taking place Small particles or biomolecules diffuse quickly with the scattered light showing rapid small fluctuations while larger particles such as protein aggregates nanoparticles polymers etc diffuse more slowly resulting in lower frequency fluctuations 8 4 2 The Autocorrelator The autocorrelation function of these short interval counts is computed by an autocorrelator which is a special purpose proprietary parallel computer that has been specially designed by Precision Detectors The correlator uses 256 channels that can be distributed over a maximum channel space of 1 024 channels Each channel can be considered as a separate bucket in which the emitted photons are counted during the sample period which is in the order of a few usec The 1 024 channel spaces occupy a total time space of 1 024 equal sampling times that can be set by the operator according to the size and shape of the molecule of interest Counts are collected in up to 256 of these channel spaces which are arranged logarithmically throughout the total channel space by the software the higher density being at the start The autocorrelator function over the 1 024 channel space 1s computed by correlating the counts in each of the channels The results are presented on the monitor Figure 8 3 and the value of Rh is auto
94. f it is necessary to remove the cover make certain that the power is off to both modules Do not apply power to either module unless the fiber optic cable is connected to both modules If stray light room light enters the system the high light level may destroy the photomultiplier and or its power supply This failure is specifically excluded from the warranty The instrument must be plugged into a grounded power line It is strongly recommended that both modules are connected to a common ground The detector is designed for an operating environment of 10 40 C In many cases the toxicological nature of samples that are measured by the detector is not known The user should take proper precautions are taken to avoid personal contact with the sample If an organic solvent is used make certain that the laboratory is well ventilated Do not use an open flame and do not install any equipment that can cause sparks in the laboratory Discard all samples in a manner that is consistent with the safety and environmental regulations of your organization REQUIRED STATEMENTS The United States Department of Agriculture Department of Health and Human Services and the NIPH require that the following information be supplied e The following controls and adjustments are available An ON OFF switch on the front panel controls main power to the system e Location of NIPH Label The NIPH laser warning is located on the laser housing e Cauti
95. f your specific system for example the solid collection angle of each detector the power of the laser etc and are expressed as a single number for each calibration constant that is used in the calculation of molecular weight and radius of gyration If the calibration values for the detectors are known they should be entered If not you can ignore this box for the time being and run the calibration using a calibration standard and calculating the calibration constants via Discovery32 The Instrument Conc Source and Run Conc Source radio buttons are used to indicate the detector that is used to collect the data used for generating calibration data If a Refractive Index detector is used peaks can be both positive and negative If both positive and negative peaks are observed in the chromatogram the Invert Signal check box should be checked The Mw Calc Type field is used to indicate the data that should be used for the calc 75 degree data only 90 degree data only and all data Select the calculation type that will provide the best representation of the data for your particular application The three calculation methods present a graph of the differential distribution of molecular weight Note Low and High Angle detectors must be in place for the all data option to be valid 3 14 Discovery32 Chapter 3 The Mw Calc Method field 1s used to indicate how the data should be processed There are five MW Calc Methods No smoothing Low s
96. for the Collection Fraction if a BSA sample is to be analyzed If a polystyrene sample is to be analyzed select 0 75 c Deselect the check box adjacent to Use as Temp Sensor d The Coefs for Temp Conv field should be set to 0 e Deselect the three check boxes at the bottom of the dialog box 2 4 4 Setup Display Dialog Box The Setup Display dialog box Figure 2 4 is used to indicate the desired display format Select Detectors for Display X Display Channel ati 2 MA Iw 15 Degree Iw 90 Degree y Iv Rh E L 0 H 4 C s r C C C C C C Cancel Figure 2 4 The Setup Display Dialog Box For the purposes of the test experiment a Check the detectors that were selected on the Select Active Detectors dialog box Figure 2 3 b Select one of the radio buttons in the Use for Y Axis Label column Discovery32 Chapter 3 3 5 2 4 5 The Calibration Setup Dialog Box The Calibration Setup dialog box Figure 2 5 is used to enter edit calibration data that is obtained from the Certificate of Calibration supplied with the detector Calibration setup x Instrument Cone Source Fl Rn t uv Inter detectar V alume frl 0 0354 m t Signal nvert Signa Mal wt Cal Constant Low 20655 2 Fun Conc Source AL C Uy Invert Signal Mol wt Cal Constant High 55713 Mol wt Cal Constant RT fb4P1 5 hw Calc Type May Calc Method 90 degree data only Low sm
97. frequency for which data points should be averaged in the Collection Fraction field of the Select Active Detectors dialog box see Section 3 2 5 1 Each averaged data point will be stored in the data file In most instances 1 sec is satisfactory If the run is very long e g gt 45 min a sample interval of 2 sec is used to reduce the number of points This value cannot be changed after the run has started e The Prestore Delay field is used to indicate the period of time after the start of the run before data should be collected This feature is useful if there is a considerable period of time after injection before data of analytical utility 1s presented This value cannot be changed after the run has started e The Sample Conc mg ml field is used to indicate the concentration of the sample e The Sample Information fields are used to enter any information that the analyst would like to store with the data e The Flow Rate Injection Volume and Sample Concentration fields are used to enter the relevant parameters and are stored with the file e The Mw Calc Type field is used to indicate if 15 data 90 data or all data should be employed in the calculation If desired you can change the selection in Discovery32 3 6 Discovery32 Chapter 3 e The Mw Calc Method field is used to indicate the degree of smoothing none low medium or high that should be used in the calculation If desired you can change the selection in Discovery32
98. ght fo 0 Top Bottom Set bath Left and Right limits to 0 for amp uta scaling Set both Top and Bottom limits to D For Auto scaling Clear All Limits M Show Result values Iw Show polynomial fit coefficients if applicable v Connect points with lines Cancel Figure 5 19 The Set Plot Limits Dialog Box If Show Result Values 1s checked the results values will appear in the upper right corner of the plot as shown in Figure 5 18 If Show polynomial fit coefficients 1s checked a curve fit can be generated along with the polynomial fit equation If Connect points with lines 1s checked the data points will appear as shown in Figure 5 18 If it is not checked only the data points will be presented Shift Print is used to generate a hard copy of the plot Cumulative Off is used to turn off the cumulative Mw trace on the plot When the cumulative trace 1s off the command will indicate Cumulative on Data Table presents a table containing the molecular weight the corresponding ordinate value and the cumulative ordinate value for each data point The table can be copied or saved as desired 5 2 5 4 Elution Tabs in the Results Dialog Box The Results dialog box presents a series of tabs that describe the calculated parameter as a function of the elution time distributions Mw Distribution Rh Distribution Rg Distribution The format of these distribution tabs is similar and those tabs that are active depends
99. ght Scattering Applications of Photon Correlation Spectroscopy R Pecora ed Plenum Press New York 1985 Chapter 2 Katime and J R Quintana in Comprehensive Polymer Science Booth and Price eds Pergamon Press Oxford 1989 Chapter 5 Discovery32 Chapter 1 Chapter 2 Installation of PrecisionAcquire32 and Discovery32 2 4 OVERVIEW NEM This chapter describes the installation of the application software and interfacing the personal computer with the system In addition a short experiment is provided to allow the user to demonstrate that the system 1s functioning in an acceptable manner 2 2 INTERFACING THE DETECTOR TO THE PERSONAL COMPUTER Note Do not turn on the power to the Detector until you are instructed to Connect the detector to the personal computer as described in the User s Manual supplied with the detector Note The personal computer and printer should be connected to the same ground as the HPLC GPC SEC system 2 3 INSTALLING THE APPLICATION PROGRAMS ON THE PERSONAL COMPUTER Two programs are provided on the Discovery32 CD e PrecisionAcquire32 which is used to establish the data acquisition protocol and collect data e Discovery32 which is used to process data and generate reports To install PrecisionAcquire32 a Place the distribution disk in the CD drive The Precision Detectors Software installation screen Figure 2 1 will be presented Discovery32 Chapter 3 3 1 Precision De
100. gion 1s defined in the same manner as the selection of the baseline To Define the Baseline a Right click on the chromatogram and select Set Integration Region on the pop up menu b Move the vertical lines on the chromatogram to indicate the region to be integrated When you have selected the appropriate limits press inside the vertical lines The arrows on the baseline indicate the region for which the area will be determined e g between 9 2 and 9 8 minutes on Figure 6 11 You may also move previously determined baseline arrows by holding down the Ctrl button and clicking the arrow to be moved Hold the left mouse button down and drag the arrow to where you want to relocate it 6 6 DETERMINING CAL CONSTANTS It is necessary to determine the following calibration parameters e The Inter Detector Volume Section 6 6 1 e Determining the RI Constant Section 6 6 2 6 6 1 Determining the Inter Detector Volume In Setup Calculation Options select RI Light Scattering and the calculation type To determine the Inter detector volume access the Determine Cal Constants tab of the Setup Calculation dialog box Figure 6 12 Discovery32 Chapter 6 6 13 Setup Calculation E x Options Detectors Constants Determine Cal Constants Mew Accept Inter detectar volume ml oosa Quality Factor 15 Degree Constant 4291 6 F 90 Degree Constant 19048 Ut arstant m Refractive Indes Constant 30 4402 28975 Apply All LV Extinct
101. he molecular weight approaches 1 x 10 daltons e If possible the standard should be free of aggregations e The dn dc should be well defined e The extinction coefficient of the standard at the monitoring wavelength is required if a UV detector is used as the concentration source Discovery32 Appendix A A 1 A 3 THE CHROMATOGRAPHY e The standard should chromatograph as a single peak e Recovery of standard should be 100 e Enough material should be injected to obtain good signals from the light scattering detectors and the concentration source detectors e If aggregates are present they should be well resolved from the monomer e The monomer peak must be well resolved from the terminal peak e The Refractive Index of the Eluent at the temperature of the detector cell must be known when the 15 light scattering detector is used e The Viscosity in poise of the eluent at the temperature of the detector cell is required for Rh determinations A 4 EXPERIMENTAL CONDITIONS FOR SEC CALIBRATION WITH BSA The conditions presented below are typical conditions for the calibration of the system with BSA and were used to generate the figures presented in the manual These conditions are presented as advisory to the user who should develop and perfect the separation using the instrumentation column etc that is available in the facility It is not unlikely that there may be small differences in the retention time if other columns buffe
102. hromatogram presents all traces on a common set of axes with the largest peak full scale Baselines need to be established to use this feature effectively Normalize Region Once baselines have been determined the normalize region is a useful feature that will height normalize based on apex and baseline of a peak that are found in a selected region The lines are similar to those used to define the baseline and can be dragged to define a region to be normalized The largest peak in the selected region will be presented full scale and all points in the region will be normalized to it 297 Note Show Chromatograms Show Raw Chromatograms Normalize Chromatogram and Normalize Region are toggle commands when one is selected it replaces the existing selection Shift Print prints out the chromatogram Raw Data Table presents a list of points with the time and intensity for each detector which can be copied or saved This table includes the entire range of the chromatogram Reduced Data Table presents a list of points that lie within the integration region up and down arrow and includes the value of Ai The list can be saved or printed 5 2 3 2 Integration Method The ntegration Method tab 1s not active 5 2 3 3 Autointegration The Autointegration tab Figure 5 10 is used to list the files that are to be integrated on an automated basis You can include files by selecting the Add Files to List button and then choosing the desired fil
103. hromatograph HI E CAUNZIPFEDSBOTHO E Select Chromatograph RI Cancel Display M ow Figure 5 33 The Set Up Overlays Dialog Box You can select the chromatograms to be displayed by clicking on the select box then selecting the chromatogram what you want to show 5 26 Discovery32 Chapter 5 5 2 5 8 Update Acquire32 Constants This command which is on the Options menu is used to transfer the calibration constants that you have developed via Discovery32 to PrecisionAcquire so that they can be applied as data is acquired 5 2 6 Help The Help menu Figure 5 34 includes two commands Help Help About Discovery 3z Figure 5 34 The Help Menu e Help accesses an on line help system e About Discovery32 presents a dialog box that includes copyright information and the version number 5 3 THE TOOL BAR The Tool bar Figure 5 35 1s used to present a series of shortcuts for some commonly used commands ajaj e X X S 3 oe AJ Figure 5 35 The Tool Bar a equivalent to Open on the File menu mi equivalent to Save on the File menu E equivalent to Print on the File menu 2 A accesses the Data Acquisition program equivalent to Chromatogram on the Options menu Ca tal equivalent to Set Up Analysis on the Options menu la ee equivalent to Results on the Options menu C Discovery32 Chapter 5 5 27 GPC accesses the GPC dialog box BH accesses
104. ime during operation The top six commands are active for all systems and the bottom four are active only if the PD2000 DLS A D Interface box is used Discovery32 Chapter 3 3 11 Setup Detectors Display Calibration Configure System Analog Board Export Advanced Correlator Configuration Correlator Analysis Correlator Board Readback Auto Integration Figure 3 13 The Setup Menu 3 2 5 1 Detectors The Select Active Detectors dialog box Figure 3 14 is used to indicate the detectors that are used for the measurement the collection fraction and temperature related data Channel 2 MAI A 1 wv 15 Degree e v 90 Degree zu 5 uy ze Use as Temp Sensor 4 Rh c Coefs for Temp Cony tsafta tad v vera e 5 E T 0 0000e 000 ia a 3 E 31 0 0000e 000 E c 32 0 0000e 000 a3 O 0000e 000 Collect Correlator Data even if Rh is not selected Show AY Gorelator Data during acquisition Compute Mw Cancel Figure 3 14 The Select Active Detectors Dialog Box Select the detector s that you want to use in this measurement by checking the check box es adjacent to the desired channels the arrows are not valid in this release 3 12 Discovery32 Chapter 3 If you select Rh in the channel column the Show Rh Correlator data during acquisition check box will be activated When a chromatogram is being presented detector data will be indicated as a
105. includes a number of dialog boxes that are used to enter a broad range of parameters that relate to data acquisition These parameters can be generally classified into two general categories e System parameters which refer to settings that are changed infrequently In many cases these parameters are dependent on the configuration of the system or relate to user preferences As an example once the COM port for communication between the computer and the analyzer module has been selected it is not normally changed e Operating parameters which refer to parameters that are specifically set for a given experiment or series of experiments These parameters are frequently changed during operation of the system and are sample dependent 2 4 2 Setting Parameters for the Test Experiment This section describes setting system parameters and operating parameters for the test experiment provided in Section 2 7 The Setup dialog boxes described below Section 2 4 3 2 4 12 are accessed via the Setup menu The first six dialog boxes Section 2 4 3 2 4 8 are common to all systems and the last four entries Sections 2 4 9 2 4 12 are used only with systems that include the PD2000 Interface box In addition the user should enter information in the Single Run Info dialog box which is described in Section 2 5 The approach used in this section is to describe the appropriate settings that should be set so that the user can demonstrate that the system 1s functi
106. indows dialog box to select the printer paper size and orientation for printings The format of this dialog box is dependent on the selected printer 5 2 1 8 Print Setup Print Setup presents the Print dialog box which is used to select the printer the print range and the number of copies to be printed The format of this dialog box depends on the printer attached to the computer Recently open files are indicated at the bottom of the File menu You can open a file by clicking on the desired entry 5 2 2 View The View Menu includes the following commands e Toolbar presents the tool bar Section 5 3 e Status Bar presents information about the present state of the program on the bottom line of the display see Figure 5 1 To select deselect either both option click on the command if the options is selected a check mark will be indicated to the left of the command Discovery32 Chapter 5 5 5 5 2 3 Options The Options menu Figure 5 5 includes a number of commands that relate collection and presentation of data Options Chromatograms Setup Calculation Results aPt_ Branch Overlay Select Update Acquire3z Constants Figure 5 5 The Options Menu 5 2 3 1 Chromatograms Chromatograms presents the Chromatograms dialog box Figure 5 6 which displays the chromatogram s in the selected file Figure 5 6 includes data from an RI detector as well as from 15 and a 90 angle light scattering dete
107. ing it to the desired size In addition you can expand a region of the chromatogram by pointing the mouse at the upper left corner of the desired area and dragging to the desired right hand corner Some users prefer using a white background to the chromatogram to improve contrast and the background of the chromatogram dialog box can be changed using the Scheme field on the Appearance tab on the Display Properties dialog box accessed via the Display icon on the Control Panel This is a standard Windows function 6 4 Discovery32 Chapter 6 6 4 ESTABLISHING CALCULATION PARAMETERS The Setup Calculations dialog box Figure 6 4 is used to indicate the instrument configuration that was used to collect the data In addition this dialog box is used to coordinate the various activities that are performed to determine the calibration constants for the system 6 4 1 The Options Tab Setup Calculation l E x Options Detectors Constants Determine Cal Constants Calculation Types IY Mw Calculations C 90 Degree only PD2010 15 Degree only P2020 Detector Calculations HI Light S catering Calculations Lv Light Seattering Calculations v Rh Calculations PD 2000 4 DL5 v Ag Calculations PD 2020 hw Smoothing None C Low C Medium C High Laser Wavelength Polynomial C 680 nm 809 nm Figure 6 4 Setup Calculations Dialog Box Options Tab At this point we will select the desired field
108. ing on the vertical line and dragging it to the desired start finish of the baseline The black lines on the chromatogram indicate the selected baseline A least squares baseline can be made to perform like a point to point baseline by selecting a baseline region before and after the peak Set Integration Region The Set Integration Region start and finish are indicated by up and down arrows on the chromatogram e g at 12 0 and 23 5 min on Figure 5 9 These can be moved in the same manner as the baseline markers by holding the Ctrl button down and dragging the up and or the down arrows on the chromatogram using the left mouse button If the integration region has not been previously selected as would be the case for a unprocessed data file the start and stop positions are determined by selecting integration and then dragging the left mouse from the start to the stop positions 5 8 Discovery32 Chapter 5 Set as Integration Template When you set the integration region as described above the region will be used only with the active chromatogram If you want to use this region for other chromatograms in the present study select Set as Integration Template to avoid having to set the integration region for each chromatogram A message box will be presented indicating that the template was established Show Chromatograms displays the active chromatogram Show Raw Chromatograms displays the chromatogram before smoothing etc Normalize C
109. ire Review Setup Monitor Help Tool E mm m gr a r amp l 6 7 p mT Bar Work Space ia bhsady o o ye No File ete 7 Figure 3 1 The Main PrecisionAcquire32 Window 3 2 THE MENU BAR The Menu Bar accesses a series of drop down menus that allow the operator to open various dialog boxes and select other operating conditions 3 2 1 File The File menu Figure 3 2 is used to access a variety of data storage retrieval commands most of which are common to most Windows applications 3 2 Discovery32 Chapter 3 File New Ctril h Open Chrl 0 Save Chrl 5 Save AS Export Print Ctrl P Print Preview Print Setup 1 A840 00 2 AALB4O0OP 00 3 AbsaoP 00 4 4 1080999 07 Exit Figure 3 2 The File Menu ag7 Note The commands that are active at a given instant are dependent on the status of the program As an example the Export command will not be active if no files are open For the sake of clarity we present the menu as if all commands are active e New inactive e Open presents a dialog box to open an existing data file The mode of operation is similar to that of other Windows application All PrecisionAcquire files are of the form e Save inactive e Save As inactive e Export presents the Export Options dialog box Figure 3 3 which is used to indicate the export format type of data and directory for the data to be stored in if the data
110. ke backup copies of the Software c You may transfer the Software from one computer to another over your network or relocate the Software on your site but you may not copy it to additional sites over the network or make additional copies for use on additional networks or sites for use with other hardware d You may copy the Software to the personal computer of Users and such Users may use the software to examine recompute and print out files collected in conjunction with the System e You may obtain additional electronic copies of the Software directly from PDI for the cost of media handling and shipping 2 Copyright The Software 1s owned by PDI and its suppliers and its structure organization and code are valuable trade secrets of PDI and its suppliers The Software is also protected by United States Copyright Law and International Treaty provisions You agree not to modify adapt translate reverse engineer decompile disassemble or otherwise attempt to discover the source code of the Software You may use trademarks only to identify printed output produced by the Software in accordance with accepted trademark practice including identification of trademark owner s name Such use of any trademark does not give you any rights of ownership in that trademark Except as stated above this Agreement does not grant you any intellectual property rights in the Software Discovery 32 License Agreement iii 3 6 1 Gi Gii
111. le Run Info dialog box Figure 3 5 accesses the Multiple Runs dialog box starts equivalent to Multiple Runs on the Acquire menu accesses the Select Detectors for Display dialog box equivalent to Display on the Setup menu accesses the Calibration Setup dialog box equivalent to Calibration on the Setup menu accesses the Select Active Detectors dialog box equivalent to Detectors on the Setup menu accesses Discovery32 equivalent to Run Discover32 on the Review menu p m E s B B je o accesses Discovery32 so that you can work with stored data during a run equivalent to Silent Mode on the Acquire menu prints the current display in the workspace equivalent to Print on the File menu o G presents the About PrecisionAcquire dialog box which includes the version number equivalent to About PrecisionAcquire on the Help menu Discovery32 Chapter 3 3 23 stops the present run equivalent to End Run on the Acquire menu terminates the restore process and lets you collect data shows the present detector data shows molecular weight data I je 2 ls le turns laser on off 3 5 WORKSPACE The Workspace 1s used to display the chromatogram as it is being collected A typical chromatogram 1s shown in Figure 3 30 PrecisionAcquire32 Acquisition Program 0M 1 Ele Edit View Acquire Review Setup Monitor Help Time Minutes Red Idle avemtis 00 00 09 1 00 e 9 Figure 3 30 A Typical
112. lect the radio button that corresponds to the data type to be exported c Select the directory for each data file type d Click the Select files for export button to access an Open dialog box and select the file to be exported If the PLGPC or Viscotec format is selected the file will be converted into a format that is directly compatible with the appropriate GPC software A 2 Discovery32 Appendix A If ASCII format is selected the chromatogram header and data will be converted into a format that can be opened by a broad range of data processing programs In some cases e g Microsoft Excel the format of the input data must be specified via a wizard When the data 1s used with Excel the default options presented by the wizard should be chosen A portion of a typical ASCII file is presented in Figure 9 3 4 Sample Information 14 Filename C PDI Data 1840 00 Operator iE Date gt 02 18 1997 Time gt 0320 05 Bie S gt TCB Polymer gt ADE 1840 dandos gt O23 Temperature gt 140 0 Ln Ole oS LNTO 2 Run Information Run Length min gt 40 Measurement Interval sec gt 2 000000 Delay Time Before Storing Readings gt O Low Angle MW Cal Constant gt 202039 296875 High Angle MW Cal Constant gt 43656 398438 Flow Rate ml min gt 1 000000 Samp Mass Inject Vode Uls gt 200 000000 Sample Concentration mg ml gt 2 000000 f Raw Data 4 00 32 103 276 364 369 54 505 a
113. lecular weights of large molecules This measurement angle is especially useful for the study of proteins with molecular weight of greater than 1 000 000 Daltons and for random coils with molecular weight between 200 000 Daltons and about 10 000 000 Daltons In addition 15 data is also used with 90 data to measure Rg the radius of gyration of molecules over a limited range of sizes 12 150 nm using static light scattering analysis 8 3 5 High Angle Light Scattering Low angle light scattering data is collected at a 90 angle to the incident beam and is typically used with static light scattering analysis to measure the molecular weight of smaller molecules such as proteins with a molecular weight below 1 000 000 Daltons random coils with molecular weight below 200 000 Daltons and for lower molecular weight non spherical coil polymers In addition it is used with dynamic light scattering analysis to measure Rh the hydrodynamic radius of molecules and particles from 1 to 1 000 nm Discovery32 Chapter 8 8 5 8 4 MEASURING RH VIA DYNAMIC LIGHT SCATTERING 8 4 1 Basics of Dynamic Light Scattering The fundamental measurement for dynamic light scattering is the fluctuation of the intensity of the scattered light This data 1s analyzed as described to yield the diffusion of the molecules or particles moving under Brownian movement When polarized laser light is scattered the scattered light 1s at the same wavelength as the incident beam Howe
114. ltonsa 10K Mol Wt 3K 5 6 Y B g 10 11 12 13 14 15 Elution Time mir Figure 5 30 Calibration Plot with Equation e Ifdesired you can add more data points to the calibration curve by selecting Data Table on the pop up menu and adding Time Molecular weight points manually When you have completed adding points close the data table to view the updated calibration table f When the calibration curve is acceptable save the table by selecting Save Table and assigning a file name files are saved as opc The legend above the calibration plot will indicate the complete path and file name e g PDI samples M 23 gpc To employ the calibration curve to obtain molecular weight data Once the calibration curve has been generated or recalled you can obtain analytical data from the selected data file by opening the Conventional GPC Distributions tab A typical presentation 1s shown in Figure 5 31 the calculated molecular weight data is presented in the upper right hand corner A popup menu 1s provided which can be used to set Plot Limits Print the plot present the Cumulative plot and present the data table in the normal manner 5 24 Discovery32 Chapter 5 GPC l x Calibrate Conventional GPC Distributions a0 96 4 K PS 02 05 1399 15 04 19 s37et n04a 7O O36e t004 1 000 60 _ O42e 004 037e 004 037e 004 g 40 Wnilog Hj 3 20 lu l E ZUR 20K 40K 50K 60K FOESOEKE
115. malogam Freon s TEE S suus ae P Mu vs Elu Time v pce Rav vE Mw ER PLGPC Output Directory LE s cei ms Qmm FILESNPREC See T rs PLGPC Singe C Channel Fie Cis ee ore ies S9 o 0 cee ian m Po MISCOTEK D it 4 Di ste EEE ee 15Deges Dala EM e l C PROGRAM FILES PREC Select US 30 Degree Data E Spare Data ra PLGPC Dual C Dual Channel File B pou IRE Eo RIAES Degree Data z VERDI ET RI s Degree Data e Voss ep DINI Et iut ET Spare 15 Degree Data x elc c ss mcn um 2 Spare 30 Degree iius AE OEE EEEN maine CHEE esto LIEN AA BT Viscotek TRISEC FILE Figure 5 3 The Export Window Discovery32 Chapter 5 5 3 To export files a Press the Select Files for Export button to present a dialog box similar to the Open command b Select the desired file s c Select the desired format s by placing a check in the desired checkbox d Select the desired file type by clicking on the appropriate radio button e Indicate the directory in which the file should be stored by pressing the Select button for the desired file type f Press OK The files will be exported to the indicated folder with the following suffixes ASCII xxxxxxxx Axx PLGPC this feature is not yet operational Viscotek xxxxxxxx vdt A file can contain up to 1600 data points If the file contains more than 1600 data points the operator will be given the option to truncate the file A
116. matically calculated Observation of the correlation function provides information that is useful to optimize the analysis In addition the display indicates the fraction of the sample with the indicated particle size 8 6 Discovery32 Chapter 8 fates e TE Dad Size Dist ibution octiS 002 a Precision Deconvolve Sample Polystyrene Radius 45nm If only one particle species is present the decay time of the autocorrelation curve provides Rh When a distribution of particles is present the correlation curve is a sum of the exponentials and the deconvolution algorithm in PrecisionDeconvolve 1s used to separate the exponentials 8 4 3 Using Rh to Determine the Diffusion Constant D the diffusion constant of the compound of interest can be determined using the Stokes Einstein equation equation 8 11 Rh kT 6r v D 8 11 where k is Boltzman s constant T is the temperature of the eluant D 1s the Diffusion coefficient calculated from the autocorrelation function Discovery32 Chapter 8 8 7 8 5 REFERENCES FOR FURTHER READING 8 5 1 Static Light Scattering C Tanford Physical Chemistry of Macromolecules John Wiley and Sons Inc New York 1961 Chapter 5 M Kerker Electromagnetic Scattering Pergamon Press Oxford 1963 I A Katime and J R Quintana Comprehensive Polymer Science Booth and Price eds Pergamon Press Oxford 1989 Chapter 5 8 5 2 Dynamic Light Scattering B J Berne R Pecora Dyna
117. mic Light Scattering John Wiley and Sons Inc New York 1976 K S Schmitz Dynamic Light Scattering by Macromolecules Academic Press Inc New York 1990 S E Harding D B Satelle V A Bloomfield Eds Laser Light Scattering in Biochemistry The Royal Society of Chemistry Cambridge UK J P Helfrich Dynamic Laser Light Scattering Technology for the Molecular Weight and Hydrodynamic Radius Characterization of Proteins Pharmaceutical Laboratory August 1998 Page 34 J P Helfrich Flow mode Dynamic Laser Light Scattering Technology for 21 Century Bimolecular Characterization American Biotechnology Laboratory October 1998 page 64 8 8 Discovery32 Chapter 8 Chapter 9 Printing and Exporting Data 9 1 PRINTING DATA Discovery32 includes a routine to establish a report layout for printing chromatographic data The report can include the chromatogram the molecular weight distribution sample information data acquisition information and user generated text A detailed discussion on the generation of a report layout is presented in Section 9 3 A typical report is shown in Figure 9 1 1 12 2 2001 Polystyrene Standard 100K Seun m Se ARRE 1 991e 004 1 973e 004 1 009 2 089e 004 30K 10K 3K 1K RI Light Scattering Cal Mode Summary dn de di g 0 1840 Mz 1 Daltons 2 021e 004 Mz Daltons 2 007e 004 Mw Daltons 1 991e 004 Inj Vol ul 100 0000 Mn Daltons 1 973e
118. moothing Medium smoothing High smoothing and GPC polynomial smoothing 297 Note Low and High Angle detectors must be in place for this option to be valid If you have any questions about the best treatment of your particular sample please consult Precision Detectors Precision Detectors software determines the molecular weight distribution by first calculating the molecular weight at each slice From these results a column calibration curve is created that plots the molecular weight as a function of the elution time The molecular weight distribution is then determined using this curve and the concentration chromatogram The smoothing options are provided to reduce the noise that is sometimes observed at the extremities of the Mw vs Elution time curve where the chromatographic signals are relatively weak The smoothing function takes the Fourier Transform of the signal multiplies it by the high frequency cutoff smoothing function and then takes the inverse Fourier transform 3 2 5 4 Configure System The System Configuration dialog box Figure 3 17 is used to establish the communication parameters between the detector and the computer and indicate the location of the data directory as well as the calibration file System Configuration X Baud Rate Stop Bits 79200 7 v 38400 E Comm Port COMI COM C COM2 COM4 Data Directory Select CADATAN Calibration File Select C A
119. nalog Board Setup Dialog BOX epe e eee IRE pese beer te bet 3 16 5 2 5 0 Epon Opttons DialoP BOXc ais tab Go ein abe Get te nete abends 3 17 Bud Corelator C Oil OU CAN OM Sod oett eich seta boast seals ace Maa Sed naa ace 3 17 35 2 5990 CO clar AMAL SIS ost ooo bob a call aaa arenes ubl a ted 3 17 3 2 5 9 ISeadback Status Dialog BOX 2 5 b tbi Rem EE E etta dpa 3 18 3 2510 AWO IC P PAU ON oes a bep ad ia a ened ia ates oe d cM asse Mi eee tt ads 3 19 eZ MAS Montor MeN oce oa rr odd tease add i E Nee aide Cad ake eae ae 3 20 306 1 Dump Command S cocotte t o re laches etd alten inu sese deacon 3 20 320 2 Monitor Photon Deyel se ioo ete tein Podio totes uisus Donee E 3 20 tI DMPC 1 N ee 3 20 SEIN RE UU mE Ce T 3 22 S MEN SURE Tc 3 22 3 he Pool BE teen tm totu use a Eu Tp I M EE 3 23 34A WOrkSpaCe E m m m 3 24 3 5 Ehe Slats BE soe ette dots tetto aluet asd intents cet dtes eu edes t ne Md aum 3 25 Chapter 4 Selecting Parameters for Data Acquisition with PrecisionAquire32 4 1 NES Unas PNE 4 4 2 Patametersthiabaffect tbe NoiSe ao rt ierra etu ot iro Pu ode Utt o edil oes 4 AS Parameters Tor all SySIeTIIS ierant tuo detta at tate ati edens tod tan dust etd albe dtu oti nd ons tod tua dps asd e eaa 4 2 AS The CoHecuon Fraction ssi eti e ti e o entes e e d du is SE ei Dude 4 2 AD 2 3Sdrmple TCC ry a secus bates puoi eu M c A OA LOU
120. ndary This zoom will present the display shown in Figure 7 11 Avoid any baseline perturbations such as large negative dips near the terminal region of the chromatogram This is more prominent with RI detectors than observed for UV Chromatograms 7 x dB 4 K PS 0A051998 13 04 138 320 200 240 200 160 120 15 Deg Voltage mv ca E 40 2 4 b D 10 12 14 16 Time min 410 805 Printed 12 02 01 15 57 ft Precision Detectors Inc Figure 7 11 Normalized Zoomed Display 7 12 Discovery32 Chapter 7 7 5 3 Defining the Integration Region The integration region 1s defined in the same manner as the selection of the baseline To Define the Baseline a Right click on the chromatogram and select Set Integration Region on the pop up menu b Move the vertical lines on the chromatogram to indicate the region to be integrated When you have selected the appropriate limits press inside the vertical lines The arrows on the baseline indicate the region for which the area will be determined e g between 9 2 and 9 8 minutes on Figure 7 11 You may also move previously determined baseline arrows by holding down the Ctrl button and clicking the arrow to be moved Hold the left mouse button down and drag the arrow to where you want to relocate it 7 6 DETERMINING CAL CONSTANTS It is necessary to determine the following calibration parameters e The Inter Detector Volume Section 7 7 1 e Determining the RI Constan
121. ndicular to the direction of propagation of the light as shown in Figure 8 1 When light strikes a molecule the electrons will experience a force due to the electric field and will move slightly This movement will induce an oscillating dipole moment that will radiate light in all directions at the oscillating frequency This radiated light 1s the scattered light that 1s detected and processed as described below Z scattered light incident light Y molecule X Figure 8 1 Light Scattered by a Molecule Figure 8 1 describes the spatial arrangement of the incident light and scattered light in the light scattering experiment The light is polarized in the vertical direction thus the electric field will oscillate in the Z direction and the magnetic field will oscillate in the X direction A diode laser which 1s a monochromatic source of light that can be focused to a very small point in the center of the sample cell is used in PDI light scattering systems Typical sources include a semiconductor laser that can provide 20 to 30 mW at 685 nm and another which provides 100 to 150 mW at 800 nm The scattered light is collected at a given angle and orientation e g 15 or 90 from the incident radiation and is used to deduce the desired molecular properties 8 3 STATIC LIGHT SCATTERING 8 3 1 Measuring the Molecular Weight Note Since Precision Detector systems include polarized laser light sources the equations presented in this ch
122. ner and drag it 9 3 8 Adding Pages to a Report If desired you can add pages to a report by clicking the B icon Once pages have been added to a report they can be accessed via the navigation icons no e CA cu The first icon presents the first page in the report the second icon presents the previous page the third icon presents the next page and the fourth icon presents the last page in the report 9 3 9 Font The font font style and size of the alphanumeric characters in the report can be changed by selecting an object that contains text e g user annotation Section 9 3 2 or data Section 9 3 4 and selecting Font from the menu bar The Font dialog box Figure 9 10 will be presented this is a standard Windows dialog box Font LLL 321 x Font Font style Size Cancel Franklin Gothic Book Italic Franklin Gothic Demi Bald Franklin Gothic Demi on Bald Italic Franklin Gothic Heavy Franklin Gothic Mediur O Franklin Gothic Madiur Y Effects Strikeout Underline Color mm Black Script wester Figure 9 10 Font Dialog Box Discovery32 Chapter 9 9 11 9 3 10 General Information The Page dialog box Figure 9 11 and Display Options dialog box Figure 9 11 are used to enter a variety of general information can be added to the report The Page dialog box is accessed by right clicking in the pane in a region that does not contain an object and the Display Options
123. ns the eluant as it passes thru the cell after the column The cell with the pure solvent should be filled with the solvent prior to the separation and should be at the same temperature as the eluant Em The observed signal RI which corresponds to the deviation of the light beam is proportional to the difference in the refractive index of the fluid in the two cells as shown in equation 8 10 RI B dn dc c 8 10 where B is an instrumental constant c is the concentration of the sample in the solvent dn dc is the change in the index of refraction of the solution as a function of concentration It is considered as a constant for any specified solvent solute pair under constant operating conditions 8 3 3 The Form Factor The form factor at a particular angle 1s the ratio of the signal at that angle when compared to the signal expected at the theoretical angle of 0 where there is not form factor as indicated by equation 8 6 The importance of the form factor is that small molecules e g those which have a radius that is lt 10 nm which is small when compared to the wavelength of the incident light are studied generate comparable signals at all angles while large molecules generate signals that are smaller at higher angles and larger at small angles 8 3 4 Characteristics of Low Angle 15 Light Scattering Low Angle light scattering data is collected at a 15 angle to the incident beam and is typically used for determination of the mo
124. nstants can be manually entered into the appropriate fields When the data has been entered to a Run field e g Inter detector Vol ml it can be copied into the corresponding Instrument field using the key or vice versa using the corresponding amp key The Update key becomes active after a change has been made to accept the changes made the update button must be clicked If you want to apply the newly determined values to stored files press Inst Cal Values key It will then be necessary to recalculate the values If values are moved using the key or key a dialog box will pop up to confirm the change Discovery32 Chapter 7 7 7 7 4 4 Determining Calibration Constants The Determine Cal Constants tab Figure 7 7 is used to generate the calibration constants for the system When you first open up the screen all constants are blank if you have not yet established baselines Setup Calculation Xx Options Detectors Constants Determine Cal Constants Mew Accept Inter detector volume ml 0 1003 Quality Factor 15 Degree Constant 3352 5 7 90 Degree Constant 1105 9 F Oy Eonstant E Refractive Index Constant 3 3661 7 Apply All UV Extinct ml mg cm COOU dide mg 0 1840 Meas Interval sec IU Flow Rate ml min 1 0000 Injection Val pl 100 QUU Eluant Fil 1 4080 Mal wt Standard ouu Sample Conc mg ml 1 3000 Figure 7 7 Setup Calculations Dialog Box Determine Cal Constants T
125. nt delivery module The Quality Factor should be between 0 98 and 1 00 If it is in this range place a check in the Accept box corresponding to Inter detector volume and then press Apply to implement the action At this point you should inspect the chromatogram to ensure that the peaks are properly aligned using the Operate on Chromatogram tab command on the Chromatograms dialog box A typical chromatogram is presented in Figure 7 13 7 14 Discovery32 Chapter 7 Chromatograms gt x Operate On Chromatograms Integration Method Auteintegratian Haw Data Transformation db 4 K PS UZl 1388 13 04 18 JA JT c JT um c 15 Deg Voltage mv DI D mn aU Zul 2 4 h z 10 12 14 1B Time min 410 305 Printed 11 27 01 16 56 i amp Precision Detectors Inc Figure 7 13 Alignment of Peaks The three traces should be coincident If they are not coincident it will be necessary to change the value of the Interdetector Vol field on the Constants tab Figure 7 14 slightly This procedure is very infrequently required Discovery32 Chapter 7 7 15 Setup Calculation Options Detectors Constants Determine Cal Constants Hun Al Inter detector val ml 0 1003 Molt Cal RL 15 33534 Molat Cal RI 80 11102 3 3670 1 4080 Mol Mt Cal Al Cal Solvent Index Hun U Inter detectar Val ml LU UD Mol Mt Cal U1 5 Molt Cal 030 Molt Cal 0
126. nts for the system 7 4 1 The Options Tab Setup Calculation l E x Uptions Detectors Constants Determine Cal Constants Calculation Types IY Mw Calculations C 90 Degree only PD2010 15 Degree only P2020 Detector Calculations HI Light S catering Calculations Lv Light Seattering Calculations v Rh Calculations PD 2000 4 DL5 v Ag Calculations PD 2020 hw Smoothing None C Low C Medium C High Laser Wavelength Polynomial C 680 nm 809 nm Figure 7 4 Setup Calculations Dialog Box Options Tab At this point we will select the desired fields which should correspond to the configuration of your system In Figure 7 4 the selections are e Detector Calculations RI Light Scattering e Calculation Types Mw Calculations 15 amp 90 degree Rh Calculations Rg MW MW Smoothing None e Laser Wavelength 809 nm If your system is capable of measuring Rh an 809 nm laser is definitely included in your system For other systems please refer to your CalCert document for the actual wavelength of the laser in your system If you are uncertain or believe that your unit has been upgraded please contact Precision Detectors to verify the laser that installed in your unit Discovery32 Chapter 7 7 5 7 4 2 The Detectors Tab The Detectors tab Figure 7 5 1s provided to indicate the detector traces that you want to display on the chromatogram Setup Calculation E X
127. of the monomer peak is 1 6223 mg mL which is correct due to the presence of dimers and other aggregates b Access the Summary tab of the Results dialog box Figure 6 16 Summary Run Params Mw Dist Mw vs Elu Hh Dist Hh vs Elu Fig Dist Fig vs Elu HI Light 5cattering Calculation Mode Hun Information Collected As Renamed To hz 1 Daltons Mz Daltons hw Draltans 6 683e 004 6 675e 004 5 66 7e 004 6 658e 004 1 001 6 761e 004 hn D altanz Mw Mn hp Daltons Figz nm z 10 0 uri l Operator Eluant pbs Sample BSA UV RI Area mV ml RI B 2 34e 001 cid 0 1670 dda 15 Degree 6 399e 001 LIV Ext 0 6750 90 Degree 1 443e 002 Coet Lv B b42e Lu C alvent 1 33220 Index Int Region Peak Time minutes Temp 4650 RI 3 389 C 15 Degree 9 400 9 400 3 338 90 Degree Lh dem4 040701 06 040701 06 Collection Date O4 02 2001 12 24 53 peekeep T MC new TAD 280 LaFor Update File Header 1 528e n 1 528e 001 1 000 1 526e 001 1 100e 001 i E 20 0 Hh Results uma TT gm anes up oUm 7 nea pea baec EM Ea qi pem o Ea Figure 6 16 Results Dialog Box Summary Tab Discovery32 Chapter 6 The following items should be checked on this tab e The molecular weight which should be in the order of 66 230 daltons for BSA This is the
128. ogram In many cases a synthetic polymer sample includes a number of species that are fairly similar in nature but differ in the number of the repeating polymer units In this latter scenario a chromatogram with a large number of closely spaced peaks if the column is able to resolve the various species or a broad envelope which corresponds to the elution of the various species if the column is not able to resolve the various species would be observed The discussion in this chapter also relates to other applications where one or a relatively few polymeric species are present A similar discussion for the analysis of natural polymers virons and other monodisperse applications is presented in Chapter 6 This chapter describes e Opening a file Section 7 2 e Viewing the Chromatogram Section 7 3 e Establishing Calculation Parameters Section 7 4 e Preparing the Chromatogram Section 7 5 e Calibrating the System with RI Detection Section 7 6 e Checking Calibration Constants with RI Detection Section 7 7 e Calibrating the System with UV Detection Section 7 8 e Checking Calibration Constants with UV Detection Section 7 9 e Analyzing Analytical Data on a Routine Basis Section 7 10 2g Note The calibration data calibration guidelines and sample data presented in this chapter refer to the use of a polystyrene standard with a molecular weight of 96 4 kD A detailed discussion of the analytical conditions used to collect the data 1s
129. omial Setup Dialog Box 5 20 PrecisionA cquire32 File menu 3 2 Overview 3 1 Print Setup Dialog Box 5 5 Printing Data 9 1 Raw Data Transformation 5 10 Readback Status Dialog Box 3 18 References 8 8 Refractive Index Measurements use of 8 5 Report Layout 5 4 9 4 Results Dialog Box 5 14 Run Params Tab 5 15 summary Tab 5 14 Review Menu 3 10 Rg Distribution Tab 5 16 Rg vs Elution Tab 5 19 Rh Distribution Tab 5 17 Rh vs Elution Tab 5 19 RI Constant determining 6 16 7 16 Run Information Dialog Box 3 10 Run Params Tab Results Dialog Box 5 15 Run Time 4 4 S sample Information inserting in Report 9 7 Sample Interval 4 2 sample Rate 3 16 sample Time 4 4 saving Report Format 9 13 Select Active Detectors PD2000 Compatible Mode Dialog Box 3 12 Select Detectors for Display Dialog box 3 12 Set Baseline 5 7 Set Integration Region 5 8 Set Integration Template 5 8 setting Parameters Test Experiment 2 3 set Plot Limits Dialog Box 5 18 Setup Calculation Dialog Box 5 12 Constants Tab 5 14 Detectors Tab 5 13 Options Tab 5 12 setup Menu 3 13 Setup Overlays Dialog Box 5 26 Show MW Calculations 3 11 single Run Info Command 3 5 Silent Toggle Command 3 9 Smoothing 5 11 static Light Scattering 8 1 8 2 Start Discovery32 Command PrecisionA cquire32 3 10 status Bar PrecisionA cquire32 3 25 Stripchart Command 3 21 Subtract Baseline 5 7 Summary Tab Results Dialog Box 5 14 Synthetic Polymers 7 1 System Configuration Dialog B
130. ommand on the Chromatograms dialog box A typical chromatogram is presented in Figure 6 13 6 14 Discovery32 Chapter 6 Chromatograms i Xx Operate Un Chromatograms Integration Method Auteintegration Haw Data Transformation BSA LViIRI UROA 00I 12 24 53 be r 3 ba Go c I ca c c c c 15 Deg oltage m 3 E H g2 Ha H B HB 10 0 10 2 10 4 Time min 040201 06 Printed 11 25 01 19 48 i Precision Detectors Inc Figure 6 13 Alignment of Peaks The three traces should be coincident If they are not coincident it will be necessary to change the value of the Interdetector Vol field on the Constants tab Figure 6 14 slightly This 1s very rarely required Discovery32 Chapter 6 6 15 Setup Calculation El x Options Detectors Constants Determine Cal Constants Hun Al Inter detectar Wol rnl 0 0635 o 0 Molt Cal RIZ15 144233 Molt Cal F130 64003 m Molat Cal RI 28827 Cal Solvent Index 1 3320 Hun Uy Inter detector Vol ral 0 0646 gt Mol wt Cal LIv 15 20124 4 Instrument Al Inter detector Val ral 0 027 Molt Cal RIZ15 42816 Molat Cal F190 f 19048 Molwt Cal RI 28875 1 3320 Cal Solvent Index Instrument Ly Inter detector Yol ml 0 0646 Molwe Cal uvas 202643 Molwt Cal uweag 453380 Molat Cal LIV 745 2000 Inst Cal Values gt Files Mol wt Cal U90 453042 E ooo Mol
131. on Use of controls or adjustments or performance of procedures other than those described herein may result in hazardous radiation exposure Discovery 32 Warnings and Safety vii LABELS The following labels are affixed to the detector Electrical Requirements 100 120 230 240 V 50 60 Hz 15 W This unit is to be serviced by factory authorized personnel only Protective conductor terminal Earth Ground Terminal cep Danger Laser radiation when open Avoid direct exposure to beam viii Discovery 32 Warnings and Safety Table of Contents Precision Detectors Inc Electronic End User License Agreemennt eeee eee eee eere eene enun iii Warnings and Safety InIormoatlon ooi iceoes ehe etd do dee eaaa EaR vii Chapter T Infroductionoudi iere eode sens cceusdoasaceaswnadeuss aaa aa a a aa ia a 1 1 NES Ua PRU cm 1 1 BS Precision ACUE 32 ercac cccnes aces incain ta eiie cause dcudentaginealastia adnanteinena Ei EA 1 1 S MMIBIE VO Tn 1 2 1 4 Instrumentation used with PrecisionAcquire32 and Discovery32 eeeseeeeeeeeeeel 1 2 E NNLLA T 1 2 IN SN PD ISP EP 1 2 WAS PI DOOO MIE S erg 1 2 ee PTD OA RET 1 3 L5 Conventions used m this Manual nic ere E e be tonio to eepe eee ens 1 3 LO Contents oft this Manual aeo tr pa actes tap ut mutua eue a eu a metals ci Ea neta 1 4 L7 Por Further HiiobmatiODs 2 ete eain Pelest e t
132. on Detectors field b Access the Summary tab of the Results dialog box Figure 7 16 Results LASS Summa Run Params hwy Dist Mi ve Elu Hh Dist Hh vs Elu Rg Dist Hg vg Elu HI Light Scattering Calculation Mode 2 021e 004 2 00 e 004 1 991e 004 1 973e 004 1 005 2 089e 004 hz Daltons Wiz Daltons hw Daltons hn D altans hw e n Mp Daltons Figz nm lt 10 0 Area rV ml RI 8 2308001 1 020802 3 080e 007 0 000e 000 15 Degree 90 Degree Lv Int Region Peak Time minutes Al 5 783 15 Degree 5 ra3 90 Degree 8 a3 NM 2 2L LL ALII Hun Information Collected As Renamed To 410 05 LE i Operator Eluant HF Sample 56 4 EPS dn dc 01840 EN om We m ia 35 0 96 46 PS standard 410TO001 00 Collection Date 02 05 1338 13 04 19 Update File Header 1 418e 001 1 41 7e 001 1 000 1 418e 001 1 023e 001 is E 15 0 Rh Results min Rh 1 241 001 ii dc 1 0 Abr 1 2472 0071 sec Rhw Ahr 1 000 Eod 0 0 Rhz 1 242e 001 oe Rh peak 8 37 1e 000 ow Hate 1 0000 rl rin Inj ol 100 0000 ul Cone 1 5000 mg ml Figure 7 16 Results Dialog Box Summary Tab The following items should be checked on this tab e The molecular weight which should be in the order of 96 400 Daltons for the Polystyrene sample This is the molecular weight for the sample corresponding to the
133. on the selection s that were made on the Options tab of the SetupCalculation dialog box Section 5 2 4 1 A typical plot for Mw vs Elu is shown in Figure 5 20 5 18 Discovery32 Chapter 5 Summary Hun Params Mw Dist Mw vs Elu Rh Dist Hh ve Elu Hg Dist Hg vg Elu BSA UV RBI 04 02 2001 12 24 53 100E SUE 6 6676 004 SOK ee oeetood Sie 1 001 6 7561e4004 EOE Sor th E 40E p Ji A F 30E m E EUR LOE S 9 2 2 3 2 4 SPTE 29 6 Elution Time mir Quo FDS Prid 112501 15 19 4 Preck boa Detectors lic Figure 5 20 Results Dialog Box Mw vs Elu Tab The menu shown in Figure 5 21 is accessed by right clicking on the MW Dist Tab not all commands are present for all tabs Plat Limits shift Print Display Setup Polynomial Fit Setup Correlator Info Data Table Figure 5 21 Elution Tabs Commands Plot Limits and Shift Print are the same as in Section 5 2 5 3 Display Setup presents the Display Setup dialog box Figure 5 22 which is used to indicate which traces should be presented Discovery32 Chapter 5 5 19 Display Setup x MW Show Al UY detector MW Show 15 degree detector Cancel __ Cancel Figure 5 22 Display Setup Dialog Box Correlator Info presents the Correlator Information dialog box Figure 5 23 which is used to enter information about the sample in the correlator Rh vs Elu tab only Correlator Information X Viscosity 0 007
134. oning in an acceptable manner it should be recognized that this section 1s not meant to provide optimum parameters for a given sample M Note A detailed discussion about the role of each parameter and how it affects the measurement is presented in Chapter 4 Discovery32 Chapter 3 3 3 Parameters for two distinct test experiments are provided e Analysis of BSA Analysts who expect to be studying polymers of a biological nature should use this experiment e Analysis of Polystyrene Analysts who expect to be studying synthetic polymers should use this experiment 2 4 3 Select Active Detectors Dialog Box The Select Active Detectors dialog box Figure 2 3 which is accessed by selecting Detectors on the Setup menu is used to indicate the detectors that are connected to the system and to select a variety of operating parameters Channel 2 v Al 1 wv 15 Degree MW 90 Degree EE Uv F Use as Temp Sensor v Rh Coets for Temp Care E uu c Izall al vray yeaa yyy 0 0000e 000 at 0 0000e 000 al 0 0000e 000 az aa 0 0000e 000 Collect Corelator Data even if Ah is nat selected el 333377 Show Rh Correlator Data during acquisition Compute Mw Cancel Figure 2 3 The Setup Active Detectors Dialog Box 3 4 Discovery32 Chapter 3 For the purposes of the test experiment a Select the detector channel s that that are present in your system b Select 0 10
135. oothing Medium smoothing High smoothing Figure 2 5 The Calibration Setup Dialog Box For the purposes of the test experiment a Enter the calibration values if known They are usually supplied on the Certificate Of Calibration provided with the system b Select the Mw Calc Type that corresponds to your system c Select the Instrument Concentration Source used with your system it is not necessary to edit the Run Conc Source Field d Select No Smoothing in the MW Calc Method field 3 6 Discovery32 Chapter 3 2 4 6 The System Configuration Dialog Box The System Configuration dialog box Figure 2 6 is used to define the communications protocol between the personal computer and the analyzer module In addition it is used to indicate where data files and the calibration file should be stored System Configuration X Baud Hate Stop Bits C 19200 38400 Comm Fart f COMI DCOM3 C COM C COM4 2 Data Director Select C DA TAS Calibration File Select C AProgram Files Precisian D etectarssPrecisian Disable Calibration File Loading i PD2000 Compatibility Mode Use DTR DSR Correlator daughterboard present Cancel Figure 2 6 The System Configuration Dialog Box For the purposes of the test experiment a Indicate the Comm Port to which the Analyzer Module is connected in most cases COMI is used b Select the Baud Rate to be used 38400 is normally used c
136. operation and the user will likely vary them to determine the optimum conditions for the measurement The user should note that the above division of parameter types is not absolute In some facilities the detector is used for a variety of applications with more than one system and it may be necessary to change System Parameters from time to time Similarly if the system is dedicated to a single application it 1s possible that once the Operating Parameters have been determined they are rarely altered Raw data collected via PrecisionAcquire32 can be processed via Discovery32 If desired raw data can be exported as an ASCII file or directly to third party software Discovery32 Chapter 1 1 1 1 3 Discovery32 Discovery32 1s used to present process view report and export light scattering data that has been collected via PrecisionAnalyze32 and other detectors e g Refractive Index and UV absorbance The program is used with for HPLC GPC FPLC and SEC systems and allows the analyst to view chromatograms calculate molecular weight and size distributions determine the hydrodynamic radius etc In addition this program allows the user to view data from several runs at a given time Discovery32 can be used to generate calibration factors for the detector A detailed discussion of this program is presented in Chapters 4 9 1 4 INSTRUMENTATION USED WITH PrecisionAcquire32 AND Discovery32 PrecisionAcquire32 and Discovery32 are used with th
137. ou are investigating e Discontinuous distributions e g Proteins involve cases where the main peaks or the multiple peaks contain single size macromolecules with impurities of other sizes Smoothing routines other than the polynomial fit are typically used to study these distributions e Continuous distributions involve a broad range of macromolecular species e g polymers synthesized via a Condensation polymerization or free radical polymerization The polynomial fit is aimed to study these 2 distributions 4 3 PARAMETERS FOR ALL SYSTEMS 4 3 1 The Collection Fraction During the sample interval a large number of readings are made The difference in the intensity between individual readings is fairly large due to the randomness of the measurement The Collection Fraction field on the Select Active Detectors dialog box Figure 3 12 is used to indicate the fraction of the data points to be used to generate a data point In each case the readings with the lowest intensity in the time interval are used to minimize signals that are especially large As an example if 75 is selected the system uses 75 of the readings and selects those readings that are the lowest in intensity to generate the data point as shown in Figure 4 1 4 2 Discovery32 Chapter 4 All data averaged to 1 Hz 10 lowest values averaged to 1 Hz Figure 4 1 Use of Collection Fraction There is a tradeoff in the selection of the Collection Fraction if a large value is s
138. ox The Correlator Analysis Setup dialog box Figure 2 10 is used to set a variety of run parameters Correlator Analysis Setup Special v Store correlator raw data in 2nn file Analysis Viscosity 0 0040 Pu EE verage Retr Index 1 4050 7 oe Wavelength Bo 0 nm Tolerance 1 0 0010 Tolerance 2 0 1000 C Base Value C Linear Fit Figure 2 10 The Correlator Analysis Setup Dialog Box For the purposes of this experiment a Deselect the Special Store correlator raw data in znn box b Setthe Viscosity and Refractive Index to that for the temperature and solvent that will be used for the measurement c Set Wavelength to 800 0 Tolerance 1 to 0 001 and Tolerance 2 to 0 1 d Select Corr should be set to 1 e Base Type should be set to Linear fit Discovery32 Chapter 3 3 11 2 4 11 Readback Status Dialog Box The Readback Status dialog box Figure 2 11 is used to ensure that the settings on the Setup Correlator and Correlator Analysis Setup dialog boxes have been successfully downloaded to the box Version hex Headback 5tatus Headback Command Slurp Size 1024 1024 Huntime secs 153 15 4 Sample Time usec 5 0 5 0 Prescale Settings Corr 1 1 1 1 SH Reset OFF OFF Dil 4 4 Mem Size B B DipSwitches hex DOO Contig Register hex DOO Interrupt Mask hex 0147 Figure 2 11 Readback Status When this dialog box is opened check that the parameters in the Readback
139. ox 3 15 Discovery32 Index T Temperature Sensor Coefficients 3 12 Test Experiment 2 3 Text 1n Report 9 6 Theory 8 1 Tolerance 3 18 4 4 Tool Bar Discover 5 27 PrecisionA cquire32 3 23 Total Area Calculation in Report 9 9 Vv View Chromatogram 6 4 7 4 View Menu PrecisionAcquire32 3 4 Ww Workspace PrecisionAcquire32 3 24 Z Zooming 6 11 7 11 Discovery32 Index This page intentionally left blank Discovery32 Index
140. presented in Appendix A It is not unlikely that data that you collect may be slightly different from that presented in this chapter e g retention times will differ if a different column is used Discovery32 Chapter 7 7 1 7 2 OPENING A FILE When Discovery32 1s opened the Main window 1s presented as shown in Figure 7 1 The Preview File chromatogram that 1s presented in the upper right corner represents the file that was open when the application was closed and the bottom half of the display describes files that are in the directory that is indicated in the field adjacent to the Change gt gt gt button The files in the listing can be sorted by the Filename Eluant Sample Name dn dc or Date by selecting the appropriate radio button A detailed discussion of the contents on the line describing a file is presented in Section 5 4 is8 x Be Discovery32 Lightscattering Analysis Software File View Options Help aje 2 4 SJ El 4 UAI Change gt gt gt Mo File Loaded Double Click File in List To Load No Default Report Loaded pazil as ale 03721 2001 16 26 Sample BSA CAPDRData Preview File 0321 15400PO0P 00 16400P 00 1840 500 RAA 12 087 0971999 087 0971999 087 0971999 087 0971999 0870971999 087 0971999 087 0971999 0870971999 087 0971999 027 1871997 02 16 1997 02 16 1997 13 22 1934 3ample hsa i sSample bzsa i sample bsa i sSample hzsa i sSample bzsa i anp leps
141. r various parameters for calibrating the system Setup Calculation p x Options Detectors Constants Determine Cal Constants Detector Calculations Calculation Types Mw Calculations f 90 Degree only PD 201 0 15 Degree only PD 2021 75 90 Degree PD 2020 v Rh Calculations PD 2000 DL5 Ag Calculations PD 2020 HI Light Scattering Calculations Lv Light Scattering Calculations hw Smoothing Mone C Low C Medium C High Laser Wavelength C Polynomial C BB nm 809 nm Figure 5 13 The Setup Calculation Dialog Box Options Tab 5 2 4 1 Options Tab The Options tab Figure 5 13 is used to indicate the type of calculations to be used the degree of smoothing and the laser used 5 2 4 2 Detectors The Detectors tab Figure 5 14 1s used to indicate the detectors for which data should be displayed and radio buttons to indicate the axes legend 5 12 Discovery32 Chapter 5 Setup Calculation ERE ou I Eu LL Figure 5 14 The Setup Calculation Dialog Box Detectors Tab 5 2 4 3 Constants The Constants tab Figure 5 15 1s used to indicate a series of instrumental constants Discovery32 Chapter 5 5 13 Setup Calculation E x Options Detectors Constants Determine Cal Constants Run Al Instrument RI Inter detector Val rl 0 0664 Molwe Cal AIS 1742511 Molwe Cal Rl90 21720 Molt Cal RI 47406 1 3310 Molt Cal Al 15
142. ram and explains the role of each command Chapter 4 Selecting Parameters for Data Acquisition with PrecisionAquire32 discusses how PrecisionAcquire 32 1s used to establish an analytical method and collect data Chapter 5 Introduction to Discovery32 describes the details of the data processing program discusses the features that are accessed via the main window and explains how files are selected for processing Chapter 6 The Chromatogram Window discusses the processing functions that are provided via the chromatogram window Chapter 7 Using Discovery32 to Study Proteins provides a tutorial to explain how light scattering data is used to obtain information about proteins Chapter 8 Using Discovery32 to Study Synthetic Polymers provides a tutorial to explain how light scattering data 1s used to obtain information about synthetic proteins Chapter 9 Exporting Data explains how the data can be formatted for use with external programs FOR FURTHER INFORMATION For further information please refer to The User Manual provided with your Precision Detectors Light Scattering System On line help files which are accessed via the help command on the help menu General references on Light Scattering include l 1 4 Tanford C Physical Chemistry of Macromolecules John Wiley amp Sons Inc New York 1961 Chapter 5 Kerker M Electromagnetic Scattering Pergamon Press Oxford 1963 Ford N C Jr in Dynamic Li
143. ransformation BoA UviEIGSUZA2001 12 24 53 BU LT Dl c LT r 3 c I Oo c 44D 15 Deg voltage im T um c 2 4 B 10 12 14 16 Time min 040201 05 Printed 11 25 01 19 13 ta Precision Detectors Inc Figure 6 10 Setting the Baseline e Once you have set both vertical lines to the desired times click in the region between the two lines to save the settings 1f you want to erase the settings and start over click outside the region defined by the vertical lines After you have accepted the settings the vertical lines disappear and a small square represents where the baseline is set for each detector trace f When selecting baselines by Least squares click and drag on a flat region of baseline before or after the peak of interest A baseline will be extrapolated based on the region you have selected You can also have the least squares method behave like point to point by selecting a baseline region as discussed above then select a second region on the other side of the peak to repeat the process 327 Note When the baseline selected in the above manner the same points are selected for each detector trace If you want to change the baseline for a specific detector click on the square and drag it to the desired point 6 5 2 Normalizing the Heights and Zooming on the Peaks Selecting the integration region is simplified if the peak s of interest are sufficiently large so that you can see where the trace b
144. rd and the chromatographic separation is based on a broad range of criteria and a wide variety of standards are commonly used Section A 2 describes the general criteria for standards and analytical conditions It should be recognized that the selection of the appropriate standard and set of conditions for a given application is a compromise some workers like to use an extremely well characterized standard e g BSA while others like to use a standard that is extremely similar to the polymer that is being studied For aqueous SEC 2 mg mL bovine serum albumin Sigma P 0834 is commonly used For room temperature GPC using THF polystyrene Molecular weight 96 4 kD polydispersity 1 01 is used TSKstandard Polystyrene F 10 Experimental conditions for SEC calibration with BSA is presented in Section A 4 and experimental conditions for GPC calibration with Polystyrene is presented in Section A 4 A 2 THE STANDARD A good standard for calibrating the system should meet the following criteria e The material should be of known molecular weight and well characterized e The solution should be of known concentration e The sample should be monodisperse Mw Mn should be between 1 00 and 1 01 e The compound should be an isotropic scatterer The molecular weight should be large enough to generate a sufficient light scattering signal and the signals at 15 and 90 should be of the same size for globular proteins non isotropic scattering begins as t
145. rdetector volume which is now negative e Return to the chromatogram and see if the UV peak and the light scattering peaks are coincident If they are not access the Constants tab of the Setup Calculations dialog box and make a small change in the Inter detector Vol field of the Constants tab as we did earlier f Repeat the above process until the chromatograms are coincident then accept the constant and press Apply g Reintegrate the monomer region then accept the 15 and 90 constants and finally press Apply Discovery32 Chapter 6 6 19 6 9 CHECKING CALIBRATION CONSTANTS WITH UV DETECTION To check calibration using UV Detection a Access the Run Params tab of the Results dialog box b Verify that the monomer BSA peak is correct 66 50 kD for UV 15 and UV 90 c The entered UV extinction coefficient Sample Constants was 0 6750 and the calculated values should be 0 5453 if the monomer concentration was 2 mg mL d The entered value for the UV concentration was 2 0000 and the calculated value for the monomer peak is 1 6158 mg mL since some of the BSA is aggregated e The average Mw for BSA should be close to 66 23kD While the calibration is based upon the total area of the light scattering signal and the UV signal the calculations are performed on the peak on a slice by slice approach The calibration is based on the total area of the light scattering signal and the UV detector signal If the curves are not perfectly
146. re eee eene eee e ertet reete eee essa s eate tete eese s ss stet teste 7 1 CE ORE I UU E 7 1 1 525 ODE ale eso pU rer re me d MeN ee 7 2 Psd AGW tho COTA OT ANN ones etr EH C nne eee 7 4 74 Establishing Calculation Parametets o ertt n po ed det n MUR 7 5 TL The ODUOHSD3Di 2 odit epa ditat oic dia apu adu besar P diei os etum DUE 7 5 TAD MCI LC CLORS D Dosusesnestes cuesta a terc uestes ad Bedec bid edat o bed ud onitacdt edenda i Lodo E 7 6 749 JTheConstdntsT3D uisseenesatu a ras bed tu ben tesdt o fondation estes uo aside 7 7 JT Determining Calibration Constants nei vinci be utei atau Gated a 7 8 Discovery 32 Table of Contents xi xii 7 5 Preparing the Chromatogram for Data Processing sse nennen 7 9 JL 3Hetabhshinp A Baseline i sss act cass IPEA a tu elis Msc sLn LE rM EDD 7 9 7 5 2 Normalizing the Heights and Zooming on the Peaks esssseseseeeeeeeel 7 11 7 5 9 Denn the Inteeratior Re9IOD emi pr ob ss Urtbe stu r E Deum bte beu Pad oM UPS 7 13 76 Determining CaL Constants uiii aerei b e debba au oe cham E eui od eu UE 7 13 7 6 1 Determining the Inter D etector Volume ose toet i Aa AEEA camara eee c EE EUN HdUS 7 13 7 6 2 Determining the RTI Constatit cue a a I Dum Nm n AR M MU EE 7 16 7 6 3 Determine Calibration Constants for 15 and 90 Channels sse 7 17 Tor Checking Calibration Results RI DeteCtOEs uo eo ERR VE TUI SIN UR VN E v eo 7 17 T
147. rs etc are employed Standard BSA 2 mg mL Sigma Cat No P 8034 Column YMC Pack Diol 300 3000 x 8 mm ID 5 um particles YMC Cat No DL30S05 3008WT Eluent Phosphate Buffered Saline PBS 120 mM NaCl 2 7 mM KCI 10 mM phosphate buffer Sigma Diagnostics 1000 3 Flow Rate 1 mL min Injection Vol 100 uL Order of detection UV 280nm LS and RI last The outlet from the UV to the inlet of either a Benchtop LS detector or a PDI inside Refractive index detector should be the shortest length of 0 007 ID PEEK tubing or 0 009 ID stainless steel The UV channel was assigned to the spare channel an acquisition rate of 1 Hz sample interval of 1 sec was used with a collection fraction of 0 1 A Precision Detectors low dispersion inline filter was placed before the UV detector this filter was fitted with a 13 mm diameter 0 2 um Supor membrane A 2 Discovery32 Appendix A A 5 EXPERIMENTAL CONDITIONS FOR SEC CALIBRATION WITH POLYSTYRENE The conditions presented below are typical conditions for the calibration of the system with polystyrene and were used to generate the figures presented in the manual These conditions are presented as advisory to the user who should develop and perfect the separation using the instrumentation column etc that is available in the facility It is not unlikely that there may be small differences in the retention time if other columns etc are employed Standard polystyrene Molecular weight
148. s which should correspond to the configuration of your system In Figure 6 4 the selections are e Detector Calculations RI Light Scattering e Calculation Types Mw Calculations 15 amp 90 degree Rh Calculations Rg MW MW Smoothing None e Laser Wavelength 809 nm If your system is capable of measuring Rh an 809 nm laser is definitely included in your system For other systems please refer to your CalCert document for the actual wavelength of the laser in your system If you are uncertain or believe that your unit has been upgraded please contact Precision Detectors to verify the laser that installed in your unit Discovery32 Chapter 6 6 5 6 4 2 The Detectors Tab The Detectors tab Figure 6 5 is provided to indicate the detectors that you want to display on the chromatogram Setup Calculation E X Options Detectors Constants Determine Cal Constants Channel 1 A 3 4 F b T B g 10 he fg fps jw f w f dm fa DU o mu m mu d m Lr Er m Lr Detector Left y axis C v C C C C C C C C Right awis f CC C C c C C ic C Figure 6 5 Setup Calculations Dialog Box Detectors Tab Check the detectors that you would like to view on the chromatogram display You can select one detector to be defined by the left Y axis and another detector to be defined by the right Y axis If you select both left and right access radio buttons for the same channel then only one axis will b
149. s the conditions for the last file that was opened Figure 3 11 The information in this dialog box cannot be edited Previous Run Information X 080999 4 07 06 09 1999 15 26 08 Detectors 15 307 Iv RAH RI Operator bill Hun Length min 3L Eluarit pbs w azide Heading Intl sec 1 0 Sample ba in pbs Delay min 0 0 dnde 0 6700 Flow Rate ml min D OC LIV Ext Coeff 0 1650 Inj Vol ul 100 0000 Salv Index 1 3310 Sam Conc 2 2300 Temperature C 35 0 1800 points Info norman corey Bits rmm 1204 5u 5 T MC column Figure 3 11 Previous Run Information Dialog Box 3 2 4 2 Start Discovery32 Start Discovery32 transfers control of the system to Discovery32 A detailed discussion of Discovery32 is presented in Chapters 5 8 3 2 4 3 Display Detector Data Display Detector Data presents the chromatogram Figure 3 7 3 10 Discovery32 Chapter 3 3 2 4 4 Show MW Calculations Show MW Calculations presents a plot of molecular weight data as a function of time Figure 3 12 HE PrecisionAcquire32 Acquisition Program amp COM 1 Elle Miew Acquire Rewiew Setup Monitor Help ia P3 es Bi EN amp e r p u Figure 3 12 Typical MW Chromatogram 3 2 5 The Setup Menu The Setup menu Figure 3 13 includes a number of commands that are used to establish instrument settings In most instances these are established when the unit is installed but some settings can be set from time to t
150. should be analyzed via an external program The operator should select the desired format via the check box es and if either of the PLGPC formats 1s selected the appropriate radio button should be selected The directory buttons are used to select the desired folder in which the data is to be stored as an alternative the directory name can be typed in the field Additional information about the Export Options dialog box is presented in Section 9 2 Discovery32 Chapter 3 3 3 Export Options l X PLGPE Export Export as Single Channel File Export as Dual Channel File Al Data Al 15 Degree Data C 15 Degree Data C RI 80 Degree Data C 90 Degree Data UV A15 Degree Data C LV Data C UV 90 Degree Data Select Directory Pbi F LDER PDI PRGMS ACGUIRE 32 v0 38 Viscatek Export Export as Viscotek TRISEC File Select Directory Pbi F LDER PDI PRGMS ACOLIIRE32 VvD 38 ASCII Export Export as ASCII Chromatogram Append Corelator Data Select Directory Pbi F LDER PDI PRGMS ACOLIIRE32 VTD 38 Enable Auto export at data storage time Cancel Figure 3 3 The Export Options Dialog Box If the data should be exported to a file as soon as the run is complete select Enable Auto Export at data storage time e Print Print Preview and Print Setup are standard Windows commands and the four lines immediately above Exit indicate the most recently opened files The last few files that have be
151. sition and the detector for which the Y axis 1s defined If the data is not displayed it is still being collected if the detector is selected in Figure 3 15 Select Detectors for Display x Display Channel EE 2 I Al 1 I 15 Degree i 90 Degree UV Rh I cc E z f E Figure 3 15 The Select Detectors for Display Dialog Box Discovery32 Chapter 3 3 13 3 2 5 3 Calibration The Calibration Setup dialog box Figure 3 16 is used to enter calibration data and indicates how various calculations are to be performed Calibration Setup X Instrument Cone Source Lh Rn f uv HI Inter deteckar V alume rl 0 1001 1 0446 m Signal nyert Signa Mol wt Cal Constant Low 1658 5 186286 0 Mal wt Cal Constant High 4 5 Mol wt Cal Constant RIT 330 0 hw Cale Method Hun Cane Source f Al C Uy Invert Signal Low smoothing Medium smoothing High smoothing Figure 3 16 The Calibration Setup Dialog Box 2 Note Low and High Angle detectors must be in place for this option to be valid The calibration constants are the calibration values associated with your particular detector and are calculated after running an appropriate narrow standard of known molecular weight These constants are then used in the molecular weight calculations of other samples in subsequent runs These constants describe the physical parameters that o
152. slice of the chromatogram that was integrated The calibration 1s based on the total area of the light scattering signal and the Refractive index detector signal If the curves are not perfectly coincident the molecular weight will be calculated incorrectly e The value of Mw Mn for the peak should be very close to 1 000 7 18 Discovery32 Chapter 7 7 8 CALIBRATING THE LS CHANNELS To calibrate the light scattering channels a Return to the Detectors page of the Setup Calculation dialog box Figure 7 5 and deselect the RI channel Select the each LS channel by activating the check box In addition check the left and right Y Axis buttons for UV if employed b Establish the baseline for the UV detector c Zoom in on the peak and reintegrate The UV trace may slightly precede the light scattering channels d Return to the Determine Cal Constants tab of the Setup Calculations dialog box and accept the Interdetector volume which is now negative e Return to the chromatogram and see if the UV peak and the light scattering peaks are coincident If they are not access the Constants tab of the Setup Calculations dialog box and make a small change in the Inter detector Vol field of the Constants tab as we did earlier f Repeat the above process until the chromatograms are coincident then accept the constant and press Apply g Reintegrate the monomer region then accept the 15 and 90 constants and finally press Apply 7
153. t Section 7 7 2 7 6 1 Determining the Inter Detector Volume In Setup Calculation Options select RI Light Scattering and the calculation type To determine the Inter detector volume access the Determine Cal Constants tab of the Setup Calculation dialog box Figure 7 11 Discovery32 Chapter 7 7 13 Setup Calculation XJ Options Detectors Constants Determine Cal Constants Mew Accept Inter detector volume rl l 0 1003 Gualty Factor 15 Degree Constant l 3952 5 7 90 Degree Constant l 11033 Uy Eonstant Refractive Index Constant l 3 3661 Apply All UV Extinct ml mgem COU dede mlg 0 1840 Meas Interval sec V0 Flow Fate ml min 1 0000 Injection Vol pl 100 0000 Eluant HI 1 4080 Mal wt Standard 2000 0 Sample Lonc mg ml 1 3000 Figure 7 12 Setup Calculation Dialog Box Determine Cal Constants Tab If the standard that you are using consists of a single peak and has no aggregation a Verify that all of the information in the fields in the lower half of the tab is correct and edit as appropriate The values should be completely indicated e g the molecular weight should be 96 400 D rather than 96 4 kD b Check the Apply All box to update the fields in red S Note Check that the parameters indicated in the lower half of the dialog box are correct before proceeding It is especially important that the indicated flow rate corresponds to the value delivered by the solve
154. t and Rg radius of gyration To complete the molecular weight determination the concentration of the compound of interest 1s required typically obtained from a refractive index detector or an absorbance detector Static Light Scattering 1s described in Section 8 3 e Dynamic Light Scattering also known as Quasi elastic scattering Photocorrelation Spectroscopy or Beat Spectroscopy The fluctuations of the intensity of the scattered light is used to determine the diffusion coefficient of the molecules as they move in solution Brownian motion The Stokes Einstein equation is used to determine Rh the hydrodynamic radius of the molecule Dynamic Light Scattering 1s described in Section 8 4 Static and Dynamic measurements can be made on a sample in a cuvette or in a flowing stream such as that found in high performance liquid chromatography HPLC size exclusion chromatography SEC or gel permeation chromatography GPC In flowing streams measurements are made on each elution slice using a unique light scattering design and very fast digital signal processors which are essentially special purpose computers This chapter describes the fundamentals of the theory of light scattering A short list of references for additional reading is presented in Section 8 5 Discovery32 Chapter 8 8 1 8 2 THE PHYSICAL BASIS OF LIGHT SCATTERING Light consists of perpendicular electric and magnetic fields that oscillate in a direction that is perpe
155. t the MW Calc Type to correspond to the system that you have h Select No Smoothing for Mw Calc Method 2 5 3 Chromatographic Conditions short piece of narrow bore tubing 10 ft x 0 010 should be placed in the system in place of the column and a Precision Detectors Inline Filter part number SP2915 should be placed between the end of the tube and the flow cell The test experiment is designed to determine if an eluting compound can be detected by the system A NEM The mobile phase to be used for the test is dependent on the use of the system e Ifyou are working intend to work with an aqueous system a phosphate buffered saline buffer pH 7 0 to 7 4 should the used as the mobile phase This should be filtered through a 47 mm 0 2 um Supor filter Pall Gelman e Ifyou are working intend to work with an organic solvent THF should be used as the mobile phase If the solvent bottle has not been used before filtration is usually not necessary If the solvent is used and has been recirculated then it should be filtered through a 47 mm 0 2 um PTFE TF 200 filter Pall Gelman Allow the mobile phase to flow through the system for a few minutes to establish a baseline To start data collection press OK on the Single Run Info dialog box Figure 2 12 and monitor the chromatograms The signal should be 500 mV if an aqueous system is used and less than 300 mV of THF is used The baseline should be stable and should not drift Monitor th
156. te the desired format and layout to meet his her specific needs A typical report layout window is shown in Figure 9 4 A 4 Discovery32 Appendix A Precision Detectors Inc 11 30 2001 j Bavedas 7 Raw Data Run Interval sec Uv ext ml mq cm Flow Rate ml min Fluent RE Vevey RI Light Scattering Cal Mode Summary Mz 1 Daltons 0 000e 000 Daltons 000e 000 0 Daltons 0 000e 000 Daltons 0 000e 000 0 000 5 Daltons 0 000e 000 nm 0 000 mV ml 000e 000 mV ml 000e 000 mV ml 000e 000 mV ml 000e 000 Mw vs Elu 6 000e 000 000e 000 0 000 For Help press F1 Figure 9 4 Typical Report Layout 9 3 2 The Chromatogram To indicate the position and size of the chromatogram on the pane a Press the L icon on the menu bar then move the cursor to the approximate position in the pane and click A rectangle outlined in red will be placed at the indicated position b Move and or enlarge the rectangle to the desired size and position in the pane These activities are done in the same manner as when you move or change the size of a window 1 e to expand the rectangle you can click on a corner and drag it Discovery32 Chapter 9 9 5 9 3 3 Inserting Text To enter text into a page a Press the AE placed on the pane with the message Right Click Mouse to Enter Text icon and move the cursor to the desired position for
157. tectors Install Software Manuals and Tutorials Laser Safety Sheet Explore CD ROM Browse our Web Site Support 6000006 Exit Figure 2 1 The Precision Detectors Software Installation Screen b Select Install Software to present the Software Installation screen Figure 2 2 Aa WM OP ES COEUR NES AC EN ME xA E vaca TE UC bey Figure 2 2 Software Installation Screen 3 2 Discovery32 Chapter 3 c Select PrecisionAcquire32 and press Enter The PrecisionAcquire32 installation program will present a series of screens that describe the installation process Follow the instructions presented on each screen During the installation you will be prompted for the software license key which is included in the documentation your name and organization the type of A D box which is to be used PD2000DLS or PDConnect and the serial number of the A D box d After the installation is complete a dialog box will be presented to prompt you for the calibration file disk which is provided with the system Place the disk in drive A and click OK to download the calibration file To install Discovery32 return to the Precision Detectors Software Installation screen Figure 2 2 and NNNM select Discovery32 The install program presents a series of screens that describe the installation process Follow the instructions presented on each screen 2 4 SETTING PARAMETERS FOR THE TEST EXPERIMENT 2 4 1 Parameter Types PrecisionAcquire32
158. that contains the last file that was accessed before closing When the file is selected the window appears as shown in Figure 7 2 EB Discovery32 Lightscattering Analysis Software File View Options Help Sie ej amp amp S GJ Al Change gt gt gt CAPDKData 410TODD1 00 is loaded No Default Report Loaded C APDI Data Preview File 410TOUD BSAOPOPOPOP Z 07 hsacPorP 00 BSA0P 07 0870971999 087 0971999 0870971999 08 097 1999 08 09 1999 08 09 1999 camp lesbsa i camp lesbsa sample hzasa sSample hzsa sSample hzsa sSample hzsa in in in in in pha pha pha pha pha pha Eluant a Eluant Eluant Eluant Eluant Eluant BSAOPOPOP SZ 07 06 o9 1999 Sample bsa in pbs Eluant Samplede 00 02r 0571999 18400POP 00 02718 1997 1640 00 02718 1997 1 amp 400F 00 02718 1997 ESA 12 03 227 1994 Sample Eluant 3ample 96 23 E P amp S Eluant 3ample ADE 15640 Eluant 3ample ADE 1640 Eluant 3ample ADE 15640 Eluant 4 ER Ready TODD OD Figure 7 2 The Main Discovery Window with 040201 06 loaded Discovery32 Chapter 7 7 3 7 3 VIEWING THE CHROMATOGRAM The chromatogram for the active file Figure 7 3 can be viewed by selecting Chromatogram on the button on the toolbar The status bar will indicate the name of the file Option menu or pressing the that was loaded Chromatograms XJ 96 4 K PS 04001999 13 04 18
159. the Branching and Structure dialog box J equivalent to Overlay Select on the Options menu 5 4 THE FILE ACCESS AND FILE INFORMATION REGION The File Access region Figure 5 36 is used to present information about the various files that are in a given directory Change gt gt gt C By Eluant i By Filename C By Sample Name By DnDc imu En m rm Figure 5 36 The File Access Region The File Access region 1s used to access all of the chromatograms in a given folder and sort the file presentation list Section 5 6 To select the folder that contains the files of interest a Click on Change gt gt gt to present Figure 5 37 Browse for Folder ki E3 Select Browse Directory Desktop Eg My Documents emm Begg 33 Floppy A Fy Local Disk C Fl Compact Disc D H Removable Disk E Fis Compact Disc F Cancel Figure 5 37 The Browse for Folder Directory 5 28 Discovery32 Chapter 5 b Select the desired folder in the standard Windows approach The files in the folder will be presented in the File Selection region Figure 5 38 EIE camp le 06 09 1999 Sample hsa i 06 09 1999 Sample hsa i 06 09 1999 Sample hsa i 06 09 1999 Samp le hsa O2 16 1997 sSample ADE Figure 5 38 The File Information Region You can select a file to be the active file by clicking on it e g BSA 12 The chromatogram for that file will appear on the right side e g Figure 5
160. the text A rectangle will be b Move the mouse so that the cursor is inside of the text box and right click A Text Input dialog box will be presented enter the desired text and press OK when the desired message has been entered c Ifdesired you can move the text box by clicking on it 9 3 4 Inserting a MW Distribution Plot To insert a molecular weight distribution plot a Press the NM icon and move the cursor to the desired position for the molecular weight distribution plot and click A rectangle with the title MW Dist will be presented b Move and or enlarge the rectangle to the desired size and position in the pane These activities are done in the same manner as when you move or change the size of a window 1 e to expand the rectangle you can click on a corner and drag it c Click inside the rectangle to access the Grap dialog box Figure 9 5 and select the graph to be displayed A 6 Result Graphs 7 Mi Dust C Raw Data C Mw ve Elu C Rgws Mw Cancel C Bh Dist C Rhvs Mw C Rh vs Elu C GPC Dist C Rg Dist Calibrate C Ag vs Elu Figure 9 5 The Graph Dialog Box Discovery32 Appendix A 9 3 5 Adding Sample Information To add sample information Ll a Press the icon in the tool bar and click the cursor on the desired position in the pane to present the Select Sample Information dialog box Figure 9 6 Select Sample Information i X S ample Information v Filename
161. tware and may only be altered in a written agreement signed by PDI and you Unpublished rights reserved under the copyright laws of the United States Precision Detectors Inc 10 Forge Park Franklin MA 02038 Your acceptance or decline of the foregoing Agreement was or will be indicated during installation Special Additional Warranty Terms In addition to the Warranty Terms and Conditions under which the System is purchased the following items are excluded 1 Breakage of glass cuvettes and flow cells 11 Breakdown of the photon detector by turning the PD2000DLS electronics module power ON without the fiber optic input connector correctly capped to prevent the input of ambient room light Correct capping of the connector is achieved by correctly fitting a light tight cap or by connecting the correct fiber optic with its far end correctly attached to a fully operational PDI light scattering platform Discovery 32 License Agreement v vi This page intentionally left blank Discovery 32 License Agreement Warnings and Safety Information Precision Detectors instrumentation is designed to meet all applicable safety standards The following precautions should be followed to minimize the possibility of personal injury or damage to property during operation l There are no user serviceable components inside the detector modules Do not remove the cover of either module without authorization from Precision Detectors I
162. u RO o OU 0 065 eri 76 38 389 945 3 ENS 0 000 Figure 9 3 Typical ASCII Output Format The information in the raw data columns is dependent on the type of file that is opened and the data that was collected The file extension for the exported files are indicated in Table 9 1 nn is replace by the run number Table 9 1 File Format Extensions ASCII PLGPC Single Channel File 90 1 Gm PLGPC Dual Channel File Visctek o oo Vd O Discovery32 Chapter 9 9 3 9 3 GENERATING A REPORT LAYOUT 9 3 1 The Report Layout Dialog Box The ReportLayout dialog box Figure 9 3 is used to design the template for the generation of a printed report The dialog box is accessed by selecting Create Default Layout on the File menu pe ReportLayout ReportEdit ol x File Font View Window s D W 5 gt 5 ae lS i Be Sel pe B E kw E 11 7 2001 w 4 Ld For Help press F1 Figure 9 3 The ReportLayout Dialog Box The user can customize the report to meet the needs of the facility or for publication A broad variety of data can be placed in the report and the various objects such as the block that contains analytical report as examples you can annotate the report indicate the integration and baseline setting regions etc The general approach of this section will be to describe each of the activities that can be performed to generate a part of a report The operator can genera
163. ual all options will be indicated in black for the sake of clarity 5 2 THE MENU BAR The Menu bar includes the following drop down menus the menu bar is active after a file has been opened 5 2 1 The File Menu The File menu Figure 5 2 includes a number of commands that relate primarily to archival activities and printing of files File Open CErI O Save S Export Create Report Layout Set Default Report Layout Display Options Print Ctrl F Frink Setup 1 CAPDIIDatalBsA 07 2 CPD Datalbsaor 00 3 CPD Datalbsa 00 4 O ibsa 00 5 AAD3z101 12 6 A 1032101 02 Exit Figure 5 2 The File Menu 5 2 Discovery32 Chapter 5 5 2 1 1 Open Open presents a dialog box that lists the files in the indicated folder Discovery32 uses binary files from the appropriate data acquisition program and have the format xxxxxxxx xx e g ABCD1234 33 5 2 1 2 Save As Save as presents a dialog box that is used to assign a new name to the file Files are saved in the binary file format with the name xxxxxxxx xx 5 2 1 3 Export Export presents the Export dialog box Figure 5 3 which is used to export data in ASCII format Polymer Laboratory Single Channel or Double Channel format or Viscotek format so that the data can be employed with the corresponding data processing programs iie Data pee ee eee ee ee eee x Ascii RA ASCII Output Bu Ge AEN s B meson c Cho
164. ver it should be noted that when the light is monitored over extremely short time increments in the microsecond time regime a Doppler shift occurs and the frequency of the light appears to be changing As the particle moves toward the detector the frequency increases and as it moves away the frequency decreases The amount of the increase decrease of the change in the frequency is related to the diffusion rate of the scattering molecule in the solvent While the diffusion coefficient could be obtained from the frequency spectrum it is easier to measure the small intensity fluctuations and then compute the autocorrelation function The autocorrelation function is related to the frequency spectrum by the Fourier transform 1 e the data processing 1s performed in the time domain rather than the frequency domain From an experimental perspective the intensity of the scattered light is detected by counting the photons scattered via an avalanche photodiode an electronic device that emits a pulse every time a photon passes thru its detector This high sensitivity detector sees a time varying signal rather than a relatively constant in batch mode or slowly varying total intensity signal It should be noted that even when measurements are taken in flow mode SEC or GPC the sample can be considered as slowly varying This is due to the time frame of the scattered light measurement relative to the rate of elution of the light scattering molecul

PD Discover32 - The Molecular Materials Research Center

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