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Precision Deconvolve - The Molecular Materials Research Center

1. 06 015 Iof x 0 9 100 R 10 65nm 07 0 5 Figure 3 20 Typical Distribution Window Contribution of intensity fluctuations that are so slow that their correlation function does not decay notably at the delay times measured is not included into distribution Instead it is reported as fluctuations parameter in the Fit dialog box Adjacent columns of the distribution differ in relaxation times by a factor of 1 2 PrecisionDeconvolve 2 Chapter 3 15 Part of the distribution can be selected by dragging mouse cursor across the distribution window while holding the left mouse button down Selected part of the distribution is shown as dark columns see what fraction of the distribution is highlighted and the average abscissa radius diameter diffusion coefficient or molecular weight of the highlighted part of the distribution check Show average box in Distribution dialog box The total area of the distribution is normalized to 1 unless Mass normalization is in effect Section 6 2 2 When mass normalization is in effect the distribution represents the weight fraction of the particles of a particular size To compute the weight fraction from scattering intensity it is assumed that the scattering intensity by a particle is proportional to its mass squared The relationship between the molecular mass and the hydrodynamic radius defined in the Distribution dialog box or the Normalization dialog box is used Only contributions
2. Print Preview Printer setup Apply Figure 7 4 Printer Output Properties Dialog Box Print Tab The options for the Choose what to are e Active Data Window includes the correlation function distribution window and the selected parameters Printed on Thu Nov 21 07 48 17 2002 Y LUN DE NC 7 074 f o 0 01 um 4 0 14 12 si uU and 7 2 10 10 msec 10 10 10 cm sec Date May 03 10 35 03 Distribution over diffusion coefficient D 10 c m sec D 0 447 0 536 0 644 0 773 0 927 1 113 96 2 4 4 196 293 294 194 03 Figure 7 5 Active Data Record Report 7 4 PrecisionDeconvolve3 Chapter 7 e Panels Window includes the contents of the panel window Printed on Thu Nov 21 07 50 04 2002 1st Cumula 15651 10 108 107 1st Cumula 16383 1st Cumula 16967 FIER T rr rro rrmmm Pu TUTO cm sec 10 108 107 2 10 104 107 cm sec Figure 7 6 Panels Window e Plot Window the present plot e Summary for Selected data Summary report defined by Figure 7 4 but limited to the files that were selected e Selected records Data records that were selected Print presents a hard copy of the formatted report Preview provides a display of the printer image on the monitor Printer setup provides to the Printer Setup dialog
3. k k Vector 4 k k iscalled the scattering vector A 2 PrecisionDeconvolve3 Appendix A A 4 METHOD OF QUASIELASTIC LIGHT SCATTERING SPECTROSCOPY 1 5 A 4 1 The Motion of Particles in Solution When light is scattered from a collection of N solute molecules at the observation point we also have a sum of waves scattered by individual particles Figure 1 Each particle could be at any random location within the scattering volume the intersection of the illuminated volume and the volume from which the scattered light is collected Since the size of the scattering volume is much bigger than q with the exception of nearly forward scattering where q the phases of the waves scattered by different particles will vary dramatically As a result the average amplitude of the scattered wave is proportional to JN and the average intensity of the scattered light is simply N times the intensity scattered by an individual particle as expected The local intensity however fluctuates from one point to another around its average value The spatial pattern of these fluctuations in light intensity called an interference pattern or speckles is determined by the positions of the scattering particles As the scattering particles move the interference pattern changes in time resulting in temporal fluctuations in the intensity of light detected at the observation point The essence of the QLS technique is to measure t
4. Index
5. measurements the smaller the smoothing parameter can be without loss of stability This will yield finer resolution in the reconstructed distribution D Second narrow distributions generally require much less smoothing and can be reconstructed much better than can wide distributions This is because oscillations in narrow distributions are effectively suppressed by non negativity conditions The moments of the distribution reconstructed by the regularization procedure coincide closely with those obtained by other methods However the regularization procedure in addition gives unbiased apart from smoothing information on the shape of the distribution This shape cannot be extracted through use of the direct fit method nor from cumulant analysis In a typical QLS experiment regularization analysis can resolve a bimodal distribution with two narrow peaks of equal intensity if the diffusion coefficients corresponding to these peaks differ by more than a factor of 2 5 PrecisionDeconvolve2 Appendix A A 9 5 1 Pecora Dynamic Light Scattering Applications of Photon Correlation Spectroscopy Plenum Press New York 1985 2 S Schmitz Introduction to Dynamic Light Scattering by Macromolecules Academic Press Boston 1990 3 H C van de Hulst Light Scattering by Small Particles Dover New York 1981 4 A N Tikhonov and V Y Arsenin Solution of Problems Halsted Press Wash
6. Correlation Function window the distribution of the scattering particles will be displayed in the Distribution window and the average intensity will be shown in the ntensity window Figure 5 1 When the measurement is initiated the Start command on the menu bar will be changed to Stop HET Ee Meme Daa Senp Window Hdp H6 SE Correlation Function 102 Rel4dom For Hep pess F1 Figure 5 1 The Main Screen PrecisionDeconvolve 2 Chapter 5 5 1 The sample name which is based on the file name and extension on the Measurement Settings dialog box will be assigned when the run is initiated and indicated on the title bar of the Distribution window The data in the Correlation Function window is updated after every run Section 4 2 2 1 The correlation function is constantly re analyzed by the deconvolution procedure and the distribution is updated as soon as the computations are completed At first the correlation function will be quite noisy and the distribution will change fairly dramatically between each update after a while the results will be more stable The ntensity window displays the average intensity of the scattered light for the previous runs each point represents one run up to 2000 points can be displayed and the average intensity of the last run is indicated in the right top corner of the window If desired you can change the vertical scale in the intensity w
7. Data Analysis A 7 Data Display Window 3 11 Data Input Field 2 2 Data Menu 3 4 6 12 Data Presentation Dialog Box 6 2 Correlation Tab 6 5 Data Tab 6 9 Distribution Tab 6 3 PrecisionDeconvolve Index Intensity Record 6 8 Panels Tab 6 7 Text File Content Tab 6 10 Data Tab 6 9 Data View Command 3 6 Decay Constant 3 18 Deconvolution A 8 Delete Active Command 3 5 6 12 Diffusion Coefficient A 5 Direct Fit Method A 7 Distribution Tab 6 3 Distribution Window 3 15 F File Menu 3 2 Find Command 3 5 Find Data Dialog Box 6 14 Fit Tab 3 16 Fluctuations 3 17 G General Conventions 1 3 General Principles A 1 Hardware Settings Tab 2 3 Hardware Command 3 7 Help Menu 3 9 Help Topics Command 3 9 Intensity Record 6 8 Intensity Tab 4 7 Intensity Window 3 13 Introduction to Light Scattering 1 1 Insert Blank Command 3 5 6 12 Inverse Selection Command 3 5 6 12 L Laser Switch 2 3 Last field 4 4 Layouts Command 3 8 License Agreement iii Loading Software 2 1 Load Settings Command 3 6 Load with Main Peak 6 3 Log CF 6 5 Main Window 3 1 Make Active Command 3 4 Measurement Settings Dialog Box 4 2 Measurement Tab 4 2 Measure Menu 3 3 Menu Bar 3 2 Method of Cumulants A 8 Motor 4 10 M M 3 18 MW Normalization 6 3 Next File 4 9 Normalization Tab 3 19 Operating Parameters 3 1 Overlay Selected Data 6 6 Overview 1 1 P Panels Tab 6 7 Panels window Command 3 8 Parameter Lists 6 1
8. Introduction to PrecisionDeconvolve 2OVeEVIe Ws oo ect Deere ette eere d fe ee 3 2 Man Window teer poene ee eure ene drea enu eoo erra reo Pe green eras creta repete terr re 3 2 2 1 3 2 22 3 2 2 3 3 224 3 225 3 2 2 6 39 3 TRE TOO Bar een tete er tO 3 24 The Data Display Window 3 2 5 The Status Bar en tette e RUP rep Qa Ud e re TO URDU 3 3 Data Acquisition Windows esee eene nennen 3 3 T 3 32 The Intensity Window sse ettet 3 33 The Correlation Window 3 4 The Distribution nene ene e n nennen nnn nennen nnne nennen 3 5 ACUVE D ta Dialog BOX eere ed eere ertet dene 3 6 The Smoothness Dialog BOX te re Hee eh emet ee ELE eene shee Chapter 4 Selecting Operating Parameters 4 1 4 T OVOIVIe W xi ice eiecti nt n er uc shag ee ec e ges deu abe Asa sha ne Ed e Wha PLI PRA ERU 4 4 2 The Measurement Setting Dialog Box enne enne enne nenne 4 2 42 T The Measurement Tab sicot set et PO He Te PREX EEUU 4 2 42 101 The Sample Due eR EDI IER ERA 4 2 4 2 1 2 Channels 4 2 1 3 4 2 1 4 4 2 1 5 4 2 1 6 4 2 1 7 PrecisionDec onvolve Table of Contents vii 42 2 Th Intensity EaD rerit erre deiner tede toi vrl m idee 4 7 42 3 The Sample REOR IEEE AMO ENERO 4 8 4 2 4 The Sample Record
9. Move to button when you highlight a position the horizontal bar is colored green e g 0 994 The Stop button will halt the movement of the stirrer at the present position and the Home button is used to move the stirrer to the bottom most position If desired you can cycle the position of the stirrer The stirrer will move from one position to the next at the end of the measurement 4 5 SELECTING THE APPROPRIATE CONCENTRATION The concentration of the sample required for successful measurement is dependent on the size of the scattering particles Since a large particle will scatter more light than a smaller particle a lower concentration will be required If the concentration is too high multiple scattering can be a problem In addition inter particle interactions makes analysis of the results obtained at high concentration difficult A few important guidelines to determine an acceptable concentration are e Compare the count rate reported in top right corner of the Intensity window for your sample and for the cuvette filled with pure buffer Scattering from your sample should be at least 5076 more than from the blank e Use the count rate reported in top right corner of Intensity window to determine how many photon counts you get from your sample during the correlation time The correlation time is the time at which the correlation function minus baseline decreases by a factor of approximately 3 times If there are more then 2 cou
10. The command is active only if some data are selected a Sort presents the Sort by dialog box Figure 6 12 which is used to select the parameter on which the files are to be sorted The parameters that can be used are selected via the Configure parameters list dialog box Section 6 3 x Chose parameter to sort by 1st Cumulant Inverse order x Figure 6 12 Sort by Dialog Box b Find presents the Find data dialog box which is used to find files which meet certain user specified information The parameters that can be used are selected via the Configure parameters list dialog box Section 6 3 PrecisionDeconvolve2 Chapter 6 6 13 x Find data with v that Has value and OR Contains Select from e DK Select from selected C Add to selected Figure 6 13 Find Data Dialog Box For some parameters e g 1 cumula you can select the value range to be used as the criteria The radio buttons are used to select the set of files to be used for the search C Plot presents the Plot dialog box Figure 6 14 which is used to create plots for the data Creation of plots is presented in Section 6 4 1 Abscissa X Ordinate Y Y st Cumulant no fit Logx linear fit LogY quadratic fit x mm Figure 6 14 Plot Dialog Box 6 4 1 Plots The Plot dialog box Figure 6 14 is used to indicate the format of the plot to be generated using the se
11. box for your printer The Data Printout tab Figure 7 7 is used to format the printer output Print options E x Print Data Printout Margins 2nd Cumulant Date Concentration Parameters in columns 1st 2nd 3d Print distribution values Cancel Apply Print parameters Do not show units Tab 110 characters Parameter names no than characters Print distribution plot Print corfunction plo Print intensity plot Figure 7 7 Printer Output Properties Dialog Box Data Output Tab PrecisionDeconvolve2 Chapter 7 The items to be included on the report are selected in the square in the upper left corner Items to be included in this box are selected on the Configuring Parameters List dialog box Section 6 3 using the Printout radio button The Parameters in columns radio buttons is used to indicate how many columns you want to use for the report The Print check boxes are used to indicate what should be included The Tab field is used to indicate the number of spaces between a parameter name and the value The Parameter Names field is used to indicate the length of the Parameter name The Margins tab Figure 7 8 is used to indicate the size of the report Print options Print Data Printout Margins ED Top 30 Left 30 Bottom 30 Right Figure 7 8 Printer Output Properties Dialog Box Margins Tab A typical
12. can be changed for completed runs and on data that has been stored to recalculate the distribution To change the smoothness value on stored data change the value and press the Apply button 4 6 PrecisionDeconvolve3 Chapter 4 4 2 2 The Intensity Tab The ntensity tab Figure 4 3 is used to set a number of parameters on the Intensity plot Figure 4 4 Measurement Setup x Measurement Intensity Sample data Sample record m Cutoff intensity level Absolute Relative 100000 7711 Counts sec of average Average over 10 points Trace average IV Reset on start nl Track average Auto scale Cancel Apply Figure 4 3 The Intensity Tab The Cutoff intensity level fields are used to select the maximum level of the data that will be presented in the ntensity dialog box The level can be set at some absolute value by selecting the absolute button and entering the appropriate value or by indicating that the present intensity is some percentage of full scale i e the present signal is 34 56 of full scale e Absolute runs with intensity above specified cutoff intensity level are dropped The Cutoff level can also be set by right button of the mouse if you click in the ntensity window e Off no intensity management is performed e Relative runs with intensity that is a specified percentage above the averaged intensity are dropped In this mode a right button mouse cl
13. light the description of the effects of orientational motion and deformation of the solute particles is much more straightforward when these particles are treated as individual scatterers PrecisionDec onvolve Appendix A A 1 Intensity of the light scattered by a single particle is dependent on the mass and the shape of the particle In this discussion we will consider an aggregate composed of m monomers and the amplitude of the electromagnetic wave scattered by an individual monomer is E atthe point of observation If the size of the aggregate is small compared to the wavelength of light all waves scattered by individual monomers interfere constructively and the resulting wave has an amplitude E mE Since the intensity of a light wave is proportional to its amplitude squared the intensity of the light scattered by the aggregate is proportional to the aggregation number squared m I p gt Where 7 is the intensity of scattering by a monomer The quadratic dependency of scattering intensity on the mass of the scatterer is the basis for optical determination of the molecular weight of macromolecules It is this dependency which is accounted for by the Mass Normalization function of PrecisionDeconvolve If the size of an aggregate particle is not small compared to the interference of the electromagnetic waves scattered by the constituent monomers is not all constructive and the phases of these waves must be taken into accoun
14. on the Setup menu Print distribution plot Click this button to print the distribution plot as it shown in the Distribution window Print corfunction plot Check this box to print the plot of the correlation function as it is shown in the Correlation function window Print intensity plot Check this box to print the plot of the intensity record if it is available Note that new data does not have intensity record until it is saved and therefore not new anymore Print distribution values Check this box to print the numerical values for the distribution shown in the Distribution window 6 2 7 Text File Content Tab The Text File Content tab Figure 6 9 is used to select the information which is stored as a text file Data presentation Distribution Correlation Panels Intensity record Data Printout Test file content Parameters vjRadius v Correlation function Polydispersity D i 1st Cumulant File name v Distribution wi Operator Intensity record OK Apply i Figure 6 9 The Text File Content Tab This tab is similar to that for Panels Section 6 2 4 Parameters are selected via the Parameter lists dialog box Section 6 3 and then by checking the desired parameters in the panel on the left side of the tab The correlation function distribution and intensity record can be saved via the check boxes 6 10 PrecisionDeconvolve3 Chapter 6 Parameter
15. parameters is discussed in Appendix A PrecisionDeconvolve2 Chapter 6 6 3 The check boxes provide the following roles a Show Y axis places a scaling on the Y axis on the distribution window see Figure 6 1 b Show average indicates the average for the parameter in the upper right corner of the plot see Figure 6 1 c Load with Main Peak the peak with maximum area is automatically selected when data is loaded When the distribution is stored in the data file see the description of the Input Output dialog box Section 2 2 the selection with which the data was saved will be restored unless this box is checked When the MW Normalization box is checked a mass normalization function is employed to display the data the default is an intensity distribution function The objective of MW normalization is to obtain a concentration normalized plot Since the intensity of scattering is proportional to the mass of the particle squared large particles scatter much more than small particles even when they are present in very small concentration In most cases the intensity distribution is satisfactory but if a small quantity of a high molecular weight aggregate is present the peaks on the distribution plot for the high molecular weight species could be very large An example of the use of the intensity distribution function and the MW normalized concentration distribution function is presented in Figure 6 3 015 09 10096 R 4
16. time e 2nd cumulant square of dispersion of relaxation times e Average error Average deviation between measured correlation function and the best fit calculated from distribution computed with zero smoothness parameter The Results tab Figure 3 23 presents the analytical results and cannot be edited Data record review EE x Sample Fit Results Conditions Measurement Normalization selected Relax time usec Radius nm 297 0607 Polydispersity R Diffusion em 2 124 007 0 497 Polydispersity D Mol weight kDa 978 1 81 MwMz Figure 3 23 Active Data Dialog Box Results Tab 3 18 PrecisionDeconvolve3 Chapter e 69 selected fraction of distribution that is selected e Radius average hydrodynamic radius of the selected part of the distribution in nanometers e Diffusion average diffusion coefficient of the selected part of the distribution in cm2 sec e Mol weight average molecular weight of the selected part of the distribution in KiloDaltons Molecular weight is computed using a relationship between particle size and its molecular weight supplied by user in Normalization dialog e Relaxation Time average relaxation time of the contribution of the selected part of the distribution into the correlation function e Polydispersity R relative dispersion the ratio of the mean square width to the average value of the selected part of the distribution over radiu
17. 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 is 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 PrecisionDec onvolve License Agreement iii 3 Transfer You may not rent lease or sublicense the Softwa
18. 1 Parameters List Command 3 7 Path 4 9 PD Expert Tab 2 7 Plot Dialog Box 6 14 Plot window Command 3 8 6 14 Polydispersity 3 18 A 7 Positioning the Stirrer 4 10 Processing Raw Data 6 1 Print 3 3 Printer Output Properties Dialog Box 7 4 Printing Data 7 4 Q Quasielastic Light Scattering A 3 Regularization A 9 Relaxation Time Distribution 6 3 Remove Active Command 3 5 6 12 Remove Duplicates Command 3 5 6 12 Remove Selected Command 3 5 Repeat Parameter 4 5 Reset Command 3 5 6 12 Reset Parameters Dialog Box 6 13 Results Tab 3 17 Run Time 4 5 S Sample Data Tab 4 8 Sample Parameters 4 9 Sample Record Tab 4 9 Sample Tab 3 15 Sample Time 4 2 Save Command 3 3 7 2 Save Data Command 3 2 Save Settings Command 3 7 Scale Factor 6 6 Scaling Law 6 4 Select AII Command 3 5 6 12 Selecting Concentrations 4 11 Setup Command 3 3 Setup Menu 3 6 Show Average 6 3 Show Deviation 6 5 Show Fit 6 6 Show Y Axis 6 3 6 5 Smoothness Command 3 5 3 17 Smoothness Parameter 4 6 Smoothness Dialog Box 3 20 Sort Command 3 6 Start Command 3 4 5 1 Status Bar Command 3 7 Stop Command 3 4 Storing Data 7 1 Subtract Base 6 5 Summary Dialog Box 7 3 System Type 2 2 T Test Mode 2 2 Test Run 2 8 Text File Content Tab 6 10 Time Command 3 8 Tool Bar 3 9 Toolbar Command 3 7 Trace Average 4 7 Tracking the Average 4 7 v Viewing Processing Stored Data 6 1 Wavelength 2 3 Window Menu 3 8 PrecisionDeconvolve3
19. 14907counts sec Intensity 14859counts sec Intensity 59453counts sec Operator with samir Operator with samir Operator with samir 79 Mw 2 95 10 KDa 8096 Mw 3 09 10f KDa 7496 Mw 484 109 KDa File name Dec 4 15 pdi File name Dec 4 8 pdi File name Decl4 9 pdi Intensity 58952counts sec Intensity 14988counts sec Intensity 14370counts sec Operator with samir Operator with samir Operator with samir 2x 4 6 8 40 EN Dec04_9 pdi 7496 Mw 4 84 105 KDa Intensity Accepted 150 of 155 1 0 sec runs with average intensity 14370 counts sec For Help press F1 Figure 6 1 A Multifile Display Window PrecisionDeconvolve2 Chapter 6 6 1 The upper left portion of the display window presents a series of panels that show the distribution for each file that has been opened The panels are shown in the order in which they were loaded and can be rearranged as described in Section 6 4 In addition the general format of the window can be altered by standard Windows techniques and the Data Presentation dialog box Section 6 2 One of the panels is outlined in red and is the active file The Correlation Function window upper right corner and the Distribution window lower right corner refer to the active file To select a file as the active file point the mouse to it and click If you want to remove a file from the window point the mouse to it and press the Delete key As an alternative select the file as the active file a
20. 5 collected 4 4 POSITIONING THE STIRRER The position of the stirrer in the cuvette can be selected via the Positioning dialog box Figure 4 6 x 1000 Range 694 Current Home Edit selected E Move to Stop 3 Cycle Spread Positions Figure 4 6 The Positioning Dialog Box The present position of the stirrer is indicated in the Current field and by the red indicator in the vertical bar on the right side of the dialog box e g 0 694 The position value that is indicated is related to the full scale movement of the stirrer table To define a position for the stirrer enter the desired value e g 0 222 and press the Set button When the value is accepted the value will be listed in the Positions field and a horizontal bar will be placed in the vertical bar on the right side of the dialog box The Spread button is used to re set the positions so that there is an equal distance between each intermediate position that is checked using the highest checked position and the lowest checked position as reference points As an example if there are three positions with check marks 0 1 0 5 and 0 7 and the Spread button is pressed the middle position will be set to 0 4 Positions that are not checked have a white bar inside the vertical bar e g 0 100 4 10 PrecisionDeconvolve3 Chapter 4 To manually move the stirrer to a given position highlight that position by clicking on it then press the
21. 7 44nm i 100 R 23 24nm M norm 3 00 a b Figure 6 3 a Intensity Normalization b MW Normalization To perform MW normalization the program assumes the power law for the dependency of the MW on the size of the particle as defined by equation 6 1 MW 6 1 Appropriate values for for several typical cases are listed in Table 6 1 This parameter cannot be less then 1 for a linear system or more then 3 for a spheroid 6 4 PrecisionDeconvolve3 Chapter 6 Table 6 1 Scaling Law Exponent Selection Molecular Type Rigid Rod Polymer 1 Random or Disc Proteins Globular When you start an experiment begin without the use of this function i e the MW normalization box is not checked and is set to 0 by default so that you can see if there are small quantities of high molecular weight species Once you have identified that these species are present select the appropriate exponent When you select the exponent a red line on the X axis will indicate the range over which mass normalization is applied When mass normalization is used only contributions from particles above a certain size are shown and used for normalization This domain is marked by a red segment on the abscissa and can be reset by right clicking the mouse The contributions outside the marked domain are indicated by small black triangles 6 2 3 Correlation Tab The Correlation tab Figure 6 4 is used to indicate the features desi
22. 8 Intensity Tab e Open the Sample tab to access the Sample dialog box Figure 2 9 and enter the Viscosity Temperature and Refraction Refractive Index values indicated in Figure 2 9 If desired you can enter the information in the left column PrecisionDeconvolve 2 Chapter 2 2 9 Measurement Setup x Measurement Intensity Sample data Sample record optional required 1 Concentration 0 0089 Viscosity 70 pH 25 Temperature Refraction 10 Molarity 0 002 dn de 0 Dther Cancel Apply Figure 2 9 The Sample Data Tab f Open the Sample record tab Figure 2 10 and enter the information as described in the figure The operator and info fields need not be edited Measurement Setup Measurement Intensity Sample data Sample record file name Dec31 f Browse path C APDIdatas operator Smith info Samples from ED Cancel Apply Figure 2 10 The Sample Record Tab 2 10 PrecisionDeconvolve3 Chapter 2 g Prepare sample containing 2 00 mg ml of BSA centrifuge it for 10 minutes at 50002 to remove any particulate matter place it in the cuvette and insert the cuvette in the instrument or inject it into the flow cell h Access the Layout command on the Window menu and select Measurement The window should appear as shown in Figure 2 11 The specific windows that are presented in the main window when the program is
23. A 4 5 Brownian Motion Temporal fluctuations in the intensity of the scattered light are caused by the Brownian motion of the scattering particles The speed of the particles is related to the size small particles move faster than large particles Though each particle moves randomly in a unit time more particles leave regions of high concentration than leave regions of low concentration This results in a net flux of particles along the concentration gradient Brownian motion is thus responsible for the diffusion of the solute and is quantitatively characterized by the diffusion coefficient D The laws of diffusive motion stipulate that over time amp the displacement of a Brownian particle in a given direction is characterized by the relationship 2p A 4 6 Determination of the Diffusion Coefficient D As explained earlier temporal fluctuations in scattered light intensity are caused by the relative motions of particles in solution Two spherical waves scattered by a pair of individual particles have at the observation point a phase difference of q 9r where r is the vector distance between particles As the 4 scattering particles move over distance q along the vector q the phases for all pairs of particles change significantly and the intensity of the scattered light becomes completely independent of its initial value The correlation time T is thus the time required for a Brownian particle to move a dista
24. Find utility Cancel Apply Figure 3 10 Configure Parameter Lists Dialog Box PrecisionDeconvolve 2 Chapter 3 7 e Hardware presents the Hardware configuration dialog box Figure 3 11 which is used to set communication parameters and indicate the nature of the detector It is normally used only when the instrument and software are installed and is described in Section 2 2 Hardware configuration Hardware settings Calibration Communication diagnostics Input Output Laser switch PD Expert Main laser power 2 E 00 Align laser Set temperature 25 Enable T Record temperature 25 T Record Sample temperature aet Temp Shutter G ain 4 d 45 Cancel Apply Help Figure 3 11 Hardware Configuration Dialog Box e Toolbar used to indicate if the Toolbar Section 3 3 should be presented on the display e Status Bar used to indicate if the status bar Section 3 4 should be presented on the display 3 2 2 5 Window The Window menu Figure 3 12 is used to indicate what data should be displayed on the monitor PrecisionDeconvolve provides exceptional flexibility to allow you to format the display to optimize the viewing of data The commands that are indicated on the menu depends on the present activity when a window is open the command does not appear on the list If a specific window is not open e g the Intensity window it will appear on the Window
25. MAIN WINDOW 3 2 1 Introduction to the Main Window When PrecisionDeconvolve is opened the Main window Figure 3 1 is presented The specific windows that are presented in the main window when the program is opened will depend on the configuration that was present when the program was previously used If a different configuration is presented the configuration shown in Figure 3 1 can be presented by selecting the Layout command on the Window menu and then choosing the Measurement option Poy PrecisionDeconvolve E ini x File Measure Data Setup Window Menu Bar NE t E E Tool Bar Lm B For Help press F1 Status Bar Figure 3 1 The Main Window PrecisionDeconvolve 2 Chapter 3 1 There are four regions of the Main window The Menu bar Section 3 2 2 The Tool bar Section 3 2 3 e The Data Display region Section 3 2 4 The Status bar Section 3 2 5 3 2 2 The Menu Bar The Menu bar includes a series of menus that are used to access various functions of the program 3 2 2 1 File The File menu Figure 3 2 includes the following commands File Open Save data Save data As Save 4 Print Ctrl P Exit Figure 3 2 The File Menu Note The commands that are active at a given instant depends on the status of the program As an example the Save data command will not be active unless a data file is open Inactive commands are indicated in g
26. PrecisionDeconvolve Application Software for Collecting Processing Storing and Printing Dynamic Light Scattering Data for use in Research Laboratories QC and Production Installations 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 software 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 PDDLS Batch and PDDLS CoolBatch are trademarks of Precision Detectors Inc other brands and products mentioned are trademarks or registered trademarks of their respective holders Precision Detectors Inc 34 Williams Way Bellingham Massachusetts 02019 USA T
27. 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 aGSA contract Use reproduction or disclosure is subject to the restrictions set forth in the applicable ADP Schedule contract 1 a DOD contract Use duplication or disclosure by the Government is subject to restrictions as set forth in subparagraph 1 ii of 252 227 7013 iii a Civilian agency contract Use reproduction or disclosure is subject to 52 227 19 a through d and restrictions set forth in the accompanying end user agreement PrecisionDeconvolve3 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 software and may only be altered in a writte
28. a from other detectors These systems are designed to collect and process dynamic light scattering data for macromolecules and particles greater than 1 nm in diameter The PDDLS Batch and PDDLS CoolBatch Systems use a cuvette to contain the sample and a static measurement is made These systems provide batch measurements of the diffusion coefficient and related parameters The CoolBatch system includes temperature control The PD Expert Laser Light Scattering DLS Workstation provides molecular size and conformation data from the autocorrelation of dynamic light scattering signals at any user selectable angle in 5 degree increments a 360 platform The angular choice scattering capabilities provide exceptionally accurate measurements for hydrodynamic radius Rh and hydrodynamic radius distributions from any type of sample ranging from molecules protein and antibody to nanoparticles such as liposomes sols magnetic particles emulsions etc The 360 degree platform is a new concept of DLS measurement in a goniometer like instrument and provides ease of use and flexibility for all applications Many manually placed detectors can be multiplexed and with the unique shuttering mechanism measurements can be obtained at different angles in sequence The DLS detectors are interfaced with a single APD avalanche photodiode detector for fast efficient and economical operation ALS3000 Automated DLS System incorporates an automated samp
29. abi s eee eere d adi ipee ovr beue e Red 4 8 4 2 4 1 Path 4242 Next File32 2 rre e ER RARE RENE UA 4 3 Hhe Sampl Parameters Karierne enna e a e ae a Eno ea de ere e noe ane ena 43 1 regii A 4 32 Temperature nate it ee en OST Ee I n I e OR PO rire 4 3 3 Refraction 4 34 Annotative Information 4 4 Positioning the Stirrer eniti 4 5 Selecting the Appropriate Concentration enne ennt 4 11 Chapter 5 Collecting Data 5 e ee ue e ee no a ee e a e e e oo eu e e oo e e n Pe e e 5 1 SEMESTRE 5 1 5 2 Star m the Measurement a ette rcl eee ie REI te ei dor 5 1 5 3 Monitoring Data Collection 5 3 Chapter 6 Viewing and Processing Stored 6 1 DEMO Tau ERR 6 1 6 2 Selecting the Desired Data Presentation Format eese nennen 6 2 6 2 1 Data Presentation Dialog Box 6 2 2 Distribution Tab sets 6 2 3 Correlation Fb a eee tti DO AR ERI ERR eal dale alae 6 24 Panels Tabore iri aa o tr e n EE EORR ons shh EGER ER e eee etie tee perpe EP 6 25 Intensity Record 6 2 6 Data tree 62 7 Text File Content Tabs eret cre ER ERU OP e DR HO re Co Poet REX Set 6 3 Parameter Lis
30. ameters listed in the Distribution View dialog box Sections 4 4 and 6 2 2 are not part of the data and are not saved in the data file as they define the mode in which data are presented These settings are however saved in the configuration file when you exit the program When you restart the program the mode of presentation that was last used is restored When you reopen the file you may change the Smoothness parameter in the Record dialog box If you change the smoothness parameter the correlation function will be re analyzed and the new distribution will be displayed In addition you also can select highlight another fraction in the distribution If you change the smoothness and or the fraction in the distribution and then save the data the new information will overwrite the information in the existing file As an alternative you can change the file name and or path to create another file in different location PrecisionDeconvolve2 Chapter 7 7 1 7 2 2 The Save Command The Save command on the File menu presents a variety of options e Save as a Text file Saves the data in a format that can be imported directly into a word processor e Update all Files Saves all open files e Selected Data Used to save the selected files with gray A file can be selected by depressing the Shift button and right clicking with the mouse or as described in Section 6 4 e Intensity The Save Intensity command on the File menu is used to
31. an be done via the Save command on the File menu Note When the program is opened the Run time Accumulate and Repeat parameters are set to their default values shown in Figure 4 1 PrecisionDeconvolve Chapter 4 4 5 4 2 1 7 Smoothness Parameter The Smoothness parameter is used to eliminate false spikes or transients in the distribution We recommend that a small value e g 10 should be used at first If you see bimodality spikes or transients raise the value until the bimodality disappears and then back off slightly If the smoothness factor is too large the resulting distribution will be broad and stable reproducible but will lack detail which may contain important information On the other hand if the smoothness factor is too small the deconvolution procedure will be less stable and in polydisperse system may produce set of narrow spikes instead of one wide distribution We recommend that the value be kept below 20 Table 4 2 presents suggested starting smoothness values for different situations Table 4 2 Suggested Starting Points for Smoothness Values Smoothness Value lt 200 8 Proteins MW gt 20 Samples with particulate matter The optimal choice of smoothness parameter is to a great degree a matter of experience The following recommendations may be useful e If repetitive measurements on the same sample are not very reproducible even though the correlation function looks identical
32. and the deviations between the experimental data and the fit do not have systematic errors the smoothing parameter is probably too small For this reason we recommended that the user make several measurement of every sample when possible e Narrow distributions generally require less smoothing than wide distributions If the correlation function is measured with about 196 accuracy the appropriate smoothness parameter for a narrow distribution e g well purified protein solution could be as small as 6 8 At the same accuracy with a wide distribution a smoothness parameter of about 15 20 might be needed e More accurate data allows better resolution and allows the use of a smaller smoothness parameter but the effect is small The accuracy needs to be approximately doubled time of measurements quadrupled to allow reduction of the smoothness parameter by 1 e The test mode provides a simulated bimodal distribution with two peaks of the same scattering intensity but with a three fold difference in correlation times It is worthwhile to see how the smoothness parameter affects reconstruction of such distribution e fthe intensity window shows little noise a smaller smoothing parameter can be used without loss of stability this will provide a distribution with finer detail e Narrow distributions generally require less smoothing than wider distributions age Note The Smoothness parameter is applied to data as it is being collected It
33. and then pressing the Delete key the fitting is updated page Note If X or Y parameters of Active Data are changed as a result of editing in the Record dialog box changing the Smoothness parameter or changing a selection in the distribution diagram the plot is not updated automatically and the user should re plot the data PrecisionDeconvolve2 Chapter 6 6 15 Note The Plot utility is designed for data evaluation For publication quality graphics we suggest that you export data in text format using Save Summary and plot it using specialized software The plot shows selected data only and it is interactive You may click the point in the plot to make the corresponding data active You can then review the data or remove it from the plot by de selecting it in Panels window or using Remove Active command Active data point has a red border around it It is only present in the plot if it is selected pag Note When the Plot window is active the Remove Active command and the Del key will deselect active data and thus remove it from the plot Active data will remain active and will not be removed from computer memory or Panels window Use the Plot dialog box to choose what parameters to plot on X and Y axes and to indicate whether to plot them in linear or logarithmic scale There is no way to change the scale or set the range for the axes You can choose to fit the data with a linear or quadratic polynomial A mean square fit is use
34. ated below e Sample Time Section 4 2 1 1 e Total Channels Section 4 2 1 2 e Last Channel Section 4 2 1 3 PrecisionDeconvolve2 Chapter 7 7 7 Duration is the overall time used to record the data This is the product of Run Time and Accumulate Sections 4 2 1 5 6 Smoothness Section 4 2 1 7 Fluctuations is the baseline after the statistical baseline has been subtracted This baseline is due to small fluctuations in laser intensity non stationary particulate matter in the sample and dust particles drifting trough the scattering volume The size of the fluctuation is related to the goodness of the data data with a large fluctuation more then 2096 should not be trusted a discussion of the statistical baseline is presented in Appendix A Accuracy is the average of the deviation between the experimental correlation function and the fit This value is affected by the Smoothness parameter It is minimal with lowest smoothness 1 and should not increase by more then several percent for the appropriately chosen smoothness parameter If Smoothness parameter is too large the accuracy parameter will start to increase noticeably If the accuracy parameter is below 0 03 the data will be very precise Temperature Viscosity and Refractive Index are required parameters that are entered via the Sample dialog box Section 4 3 Optional parameters from this dialog box will also appear if defined 587 Note Optional parameters are cons
35. ature field 1s the temperature used in calculations i e the expected temperature The Sample Temperature field indicates the present temperature of the sample cell The Laser ON check box is used to indicate that power should be supplied to la ser The Align laser check box is used to provide power to the alignment laser when the sample platform is being aligned This procedure is done during manufacturing and is not normally performed by the user It is recommended that you contact PDI service before you attempt to align re align the system The Enable T enables temperature control The Shutter Gain check boxes are used to indicate which shutters should be opened to collect data The Get Temp button measures the present temperature and displays it in the Sample temperature field PrecisionDeconvolve 2 Chapter 2 2 7 2 3 A TEST RUN While you may begin to collect data at this point we suggest that you make a few runs in Test Mode to verify that your software is working properly and to familiarize yourself with the various parameters to be set For this exercise make certain that the Test Mode check box in the Input Output tab of the Hardware settings dialog box is checked After you have run a few test runs to familiarize yourself with the program we recommend that you make a few test measurements of a well defined standard A good sample to use for this purpose is Bovine Serum Albumin BSA e g Sigma Chemical Co St Louis MO part
36. butor The Laser Switch field is used to turn the laser on off This switch is provided for used with other Precision Detectors systems Model 2000 DLS Model 2010 DLS Model 2020 DLS Model 2030 DLS To avoid receiving the Laser is OFF message this box should not be checked c The Laser switch Tab The Laser Switch tab Figure 6 3 is used to set a variety of laser protection features Hardware configuration x Hardware settings Calibration Communication diagnostics Input Output Laser switch PDExpet LASER C ON C OFF Overload shutoff Switch laser OFF on exit at 600000 counts sec a i Cancel Figure 2 3 The Hardware Settings Dialog Box Laser switch Tab The LASER radio buttons are used to indicate the status of the laser and should be set to ON The Overload shutoff check box is used to indicate if the laser should automatically be turned off if the detector observes the number of counts entered below This option is used to protect the detector from being damaged from too much light The Switch laser OFF on exit check box is used if it is desired that power to the laser is turned off when the application program is terminated The Switch Laser ON on start field is used to indicate that the laser should be automatically powered up when the unit is powered up 2 4 PrecisionDeconvolve3 Chapter 2 d The Calibration Tab The Calibration tab Figure 2 4 is used to set a number of fun
37. considered as a continuous rescattering of the incident electromagnetic wave from every point of the illuminated medium The amplitude of each secondary wave is proportional to the polarizability at the point from which this wave originates if the medium is uniform rescattered waves will have the same amplitude and interfere destructively in all directions except in the direction of the incident beam If however at some location the index of refraction differs from the average value the wave that is rescattered at this location is not compensated for and some light will be observed in directions other than the direction of incidence and light scattering occurs Scattering of light can be viewed as a result of microscopic heterogeneities within the illuminated volume and macromolecules and supramolecular assemblies are examples of such heterogeneities A 2 LIGHT SCATTERING TECHNIQUES Static light scattering probes concentration molecular weight size shape orientation and interactions among scattering particles by measuring the average intensity and polarization of the scattered light Static light scattering measurements which are performed at different scattering angles provide information on the molecular weight size and shape of the scattering particles Measurements of the intensity of light scattering as a function of concentration yield the second virial coefficient which is the key characteristic of the strength of attractive or repulsiv
38. creen will be presented if your computer is not configured for Autorun select Setup exe on the CD to access the Welcome screen b The Install program presents a series of dialog boxes that are self explanatory When you access the dialog box that presents the programs to load select PrecisionDeconvolve The password that is provided with the system will allow you to load the program Once you have loaded the software start PrecisionDeconvolve and select Hardware on the Configuration drop down menu to open the Hardware configuration dialog box Figure 2 1 PrecisionDeconvolve2 Chapter 2 2 1 The Input Output Tab Hardware configuration E Hardware settings Calibration Communication diagnostics Input Output Laser switch PDEwpet Data input PD2000DLS Do not save distribution recompute while loading Do not save intensity Allow duplicates of the same measurements Testmode V Cancel Apply Figure 2 1 The Hardware Configuration Dialog Box Input Output Tab Indicate the system type in the Data Input field The options are e PD200DLS is used when the Precision Detectors PD2000DLS detector is employed e From File Only The option From File Only is used for analysis of data from third party correlators In this mode functions of PrecisionDeconvolve that are related to the collection of data and saving data are disabled For additional information on what data
39. ction from the theoretical correlation function that corresponds to the distribution shown in the Distribution window is plotted The deviation is indicated in red and has a scale that is three times larger than the correlation function see Figure 6 5 The deviation decreases as you accumulate the correlation function and the Show Deviation feature is used to determine the quality of the fit obtained by the deconvolution procedure Systematic non random deviations that do not decrease when you increase the Accumulate or Run Time parameters may indicate that the smoothness parameter is too large If systematic deviations persist even when smoothness parameter is less then 8 dust in the sample a dirty or scratched cuvette instability of the laser or misalignment of the optics may be the cause of the deviations Figure 6 5 Correlation Function with Fitting Function Red and Residuals Green ag Note If the intensity of scattered light is high it is possible that more than one photon is counted per Sample Time This deviation will look as if it is systematic but it will not be reproducible from one measurement to another and it will decrease in magnitude if you increase the measurement time f Scale Factor The Scale Factor command is used to select the magnitude of the residuals green line on plot presented on the display 6 6 PrecisionDeconvolve3 Chapter 6 g Show Fit When the Show fit command is selected red line is p
40. d Comments can be added 3 16 PrecisionDeconvolve3 Chapter Deci15 33 Properties C APDIdatasDec15 33 pdi Samples from ED Figure 3 21 Active Data Dialog Box Sample Tab The Fit tab Figure 3 22 presents general information about the fit The smoothness parameter can be edited Section 3 6 Dec15 33 Properties Figure 3 22 Active Data Dialog Box Fit Tab PrecisionDeconvolve 2 Chapter 3 17 e Smoothness the smoothness parameter with which the distribution of active data was computed It is the only parameter in this dialog that can be changed If it is changed the distribution will be re computed The smoothness parameter is described in Section 4 2 1 6 e Background the ratio of the square of the average count rate to the correlation function at zero time in percent Ideally if light is collected from less then one coherence area background is 5090 If this parameter exceeds 50 by a significant amount it may be an indication of misalignment e Intensity shows average count rate during accepted runs in counts second e Fluctuations the difference between the value of the normalized correlation function at infinite delay time and Background If this parameter exceeds few percent the shape of reconstructed distribution is not reliable This parameter is indicative of intensity fluctuations due to dust particles and or use of too small a sample time e 1st cumulant average relaxation
41. d Cumula 43200sec Und Cumula 1 4710 sec 100 Mw 8 08 10 KDa 10096 Mw 7 75 109 100 Mw 8 45 109 1st Cumula 16383sec 1st Cumula 16967sec 1st Cumula 15651sec Rnd Cumula 18610 5sec Bnd Cumula 2 4010 sec Bnd Cumula 22410 sec ni For Help press F1 lcu Figure 3 16 Display Multichannel Data Processing 3 2 5 The Status Bar The Status bar contains information about the operation of the system If you select a command on a menu or move the pointer to a button on the toolbar the status line will present a short discussion of the role of that tool 3 12 PrecisionDeconvolve3 Chapter 3 3 THE DATA ACQUISITION WINDOWS 3 3 1 Overview When data acquisition is initiated the display presents the correlation function window distribution window and the intensity window unless you have customized the display as described in Section 2 2 4 3 3 2 The Intensity Window The ntensity window Figure 3 17 serves two roles e show the intensity of individual data e support intensity fluctuations management during measurements RE Intensity 9225 8565 Figure 3 17 The Intensity Window Three types of information are presented in three different colors e Black photo count rate in counts per second averaged over each individual run The black horizontal line is provided as a reference guide If the Intensity window is the active window pressing the space ba
42. d and no errors in parameter values are taken into account The results of the fit are shown at the top of the window Parameters of the quadratic fit are shown in a little unusual way to facilitate estimation of the second virial coefficient from the intensity concentration plot 6 16 PrecisionDeconvolve3 Chapter 6 Chapter 7 Storing and Printing Data 7 1 OVERVIEW PrecisionDeconvolve allow the user to save and print data in a variety of formats e Saving collected and or processed data is described in Section 7 2 e Printing reports is described in Section 7 3 7 2 SAVING DATA 7 2 1 Saving Data as a Binary File When a measurement is completed data is automatically saved in a binary file named xxxxxx_yyy pdi where xxxxxx is the name indicated on the Sample record tab of the Setup dialog box Section 4 2 4 and yyy is the number assigned the numbers are assigned in consecutive order A file is typically approximately 2 5 kb in length If desired you can save the data using a different file name using the Save data command on the File menu This command presents a standard Windows Save Data As dialog box For consecutive runs the name will be same and the number after the underscore will be updated automatically A binary file contains e the parameters used to collect the data e the date e the sample description including information entered via the Record dialog box e the correlation curve Note The par
43. d not contain numerous spikes and it should not drift If a few spikes in intensity are present set the cutoff level below these spikes so as to exclude such runs from the measurement Set the Deviation to be displayed Section 4 4 2 and make sure that it does not contain systematic features The deviation should decreases as the accumulation proceeds PrecisionDeconvolve2 Chapter 5 5 3 This page intentionally left blank PrecisionDeconvolve3 Chapter 5 Chapter 6 Viewing and Processing Stored Data 6 1 OVERVIEW PrecisionDeconvolve allows the analyst to view and process stored data files A typical multi file display is presented in Figure 6 1 The format of the main window can be selected via the standard Windows tools e g dragging the boundary of the windows or via the Data Presentation dialog box as described in Section 6 2 In addition the commands on the Windows menu can be used to select the windows that are presented gage Note If the display is in the Measurement format and more than one file is selected only the last file will be presented although all files will be opened To view all files select Panels window on the Window menu PrecisionDeconvolve File Measure Data Setup Window SHE ED o und 7496 Mw 4 23 105 KDa 296 Mw 2 46 105 KDa 396 Mw 1 38 105 KDa dl File name Dec 4 10 pdi File name 04 11 pdi File name Dec 4 14 pdi ntensity
44. damental instrument parameters The parameters are set during manufacture and testing and should not be changed by the user Hardware configuration Figure 2 4 The Hardware Settings Dialog Box Calibration Tab PrecisionDeconvolve 2 Chapter 2 2 5 The Communications diagnostics Tab The Communication diagnostics tab Figure 2 5 is used to set a number of parameters for communication between the computer and the detector The parameters are set during manufacture and testing and should not be changed by the user Hardware configuration Figure 2 5 The Hardware Settings Dialog Box Communication diagnostics Tab 2 6 PrecisionDeconvolve3 Chapter 2 f The PD Expert Tab The PD Expert tab Figure 2 6 is used if the PD Expert multi angle detector system is incorporated into the detector Hardware configuration x Hardware settings Calibration Communication diagnostics Input Output Laser switch PD Expert Main laser power 2 100 Align laser Set temperature 25 EnableT Record temperature C 25 T gt Record Sample temperature C N A Get Temp Shutter G ain 2 1 11 Apply Figure 2 6 The Hardware Settings Dialog Box PD Expert Tab The Main laser power field indicates the fraction of the laser power that should be employed The Set temperature field indicates the present temperature of the sample cell The Record temper
45. data from display use the Remove Active command PrecisionDeconvolve 2 Chapter 3 5 e Insert Blank inserts a blank space after active data in the Panels window This command is useful for proper alignment of data panes e Select All selects all files for the generation of a plot Section 6 4 e Inverse Selection reverses selection of the files in a plot e g files which are presently selected for a plot are deselected and vice versa e Remove Selected removes the indicated file e Remove Duplicates removes duplicate files This command removes all but one duplicate data files from display and from computer memory but does not delete them from hard drive Duplicate files are modified versions of one and the same original data file saved under different names e Reset resets selected parameters to that of the active file see Section 6 4h e Sort sorts files based on the selected parameters see Section 6 41 e Find presents the Find Data dialog box see Section 6 41 e Smoothness presents a dialog box to smooth correlation data see Section 3 6 3 2 2 4 Setup The Setup menu Figure 3 8 presents the following commands Setup Data View Ctrl V Load Settings Save Settings Parameter Lists Hardware Ctrl H Toolbar wv Status Bar Figure 3 8 Setup Menu e Data View presents the Data Presentation dialog box Figure 3 9 which is used to indicate the informati
46. e interactions between solute particles Quasielastic dynamic light scattering L2 probes the relatively slow fluctuations in concentration shape orientation and other particle characteristics by measuring the correlation function of the scattered light intensity Fast vibrations of small chemical groups which lead to significant changes in the frequency of the scattered light is the domain of Raman spectroscopy These latter two methods which probe the dynamics of the particles which cause light scattering are intrinsically more complicated than static light scattering since they involve measurements of spectral characteristics or related correlation properties of the scattered light A 3 LIGHT SCATTERING FROM MACROMOLECULES IN SOLUTION One may consider the solution as a homogeneous medium and ascribe light scattering to the spatial fluctuations in the concentration of a solute An alternative way is to consider each individual solute particle as a heterogeneity and therefore as a source of light scattering The first approach is more appropriate for solutions of small molecules in which the average distance between the center of the scatterers is small compared to the wavelength of light The second approach is more appropriate for solutions of large macromolecules and colloids when the average distance between particle centers is comparable to the wavelength of light When the size of the solute particles becomes comparable to the wavelength of
47. e overall quality of the data An additional advantage of tweaking is that the analyst will get a better understanding of data acquisition and data processing In addition a Test Mode is provided via the Input Output tab of the Hardware Settings dialog box to allow the user to determine the effect of each parameter on the collection processing of data gage Note This chapter describes the role of each parameter from an experimental perspective A discussion of the fundamental concepts of dynamic light scattering and the analysis of the correlation function is presented in Appendix A gar Note Most of the parameters described in this chapter are saved in the Config file upon normal exit from the program The Config file is located in the same directory as the operating file Decon exe When the program is started the values for these parameters are restored If the Config file is absent all parameters are set to the default values PrecisionDeconvolve Chapter 4 4 1 4 2 THE MEASUREMENT SETTING DIALOG BOX The Measurement Setup dialog box Figure 4 1 is used to select data collection parameters as well as the name and location of the data file to be stored Measurement Setup X Measurement intensity Sample data Sample record Sample Time 1 Run Time 256 Channels 150 Accumulate 1024 5 Repeat Dontsave 10 Smoothness Keep in memory Cancel Figure 4 1 The Measurement Setting Dia
48. ea e dirty cuvette Whenever possible we suggest that these effects be remedied so that a large Last channel number can be used 4 4 PrecisionDeconvolve32 Chapter 4 4 2 1 4 The Run time is the frequency at which the operator is updated on the current analysis i e the duration of an individual correlator run If for example the Sample Time is 10 usec and the Run Time is 1 sec measurement will take the Sample time multiplied by the number of channels 1024 or 10 24 ms Since the run time is 1 sec the instrument will perform 97 measurements to generate the results At the end of each run the data is transferred from the correlator board to the personal computer for further analysis and display If the sample is clean i e no particulate matter a longer run time could be used because it will provide better signal to noise ratio When a long run time is selected the effect of particulate matter e g a dust filter is minimized Longer Run times minimize overhead on communications between the correlator board and the computer However if the sample contains particulate matter a short run time is important so that the intensity can be observed more often the intensity is averaged over each run time This will allow the operator to then set the threshold and reject runs when a large particle was in the scattering volume A short Run time is also convenient for preliminary assessment of t
49. each dialog box To access help files follow standard Windows procedure i e right click the item for which help is desired and then click the What s This button or move the cursor to the item and press F1 the In addition a complete help file can be accessed by clicking Content on the Help menu 3 2 3 The Tool Bar The Tool Bar Figure 3 14 includes the following Ar E Figure 3 14 Tool Bar PrecisionDeconvolve 2 Chapter 3 9 Behe m H mx ma E Open equivalent to Open on the File menu Save equivalent to Update all files Save command on the File dialog box Print print the data using the settings indicated in the Print dialog box File menu Measurement Settings presents the Measurement tab of the Settings dialog box to allow for settings for the correlator board and the sample Start Measurements initiates the collection of data using the settings in the Measurement tab of the Settings dialog box Shows the Accumulating Data makes the data that is being collected the active data to show the data which is currently being measured This command is enabled only during measurements Stop equivalent to Stop on the Measure menu active when a run has started Show Parameters presents parameters of the active data equivalent to Active Data on the Data menu Remove Active Data file from Display equivalent to Remove Active command o
50. el 508 966 3847 Fax 508 966 3758 e mail info precisiondetectors com Web site www precisiondetectors com Copyright 1997 1998 1999 2000 2002 2003 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 1 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 make backup copies of the Software c You may
51. er directly The maximum number of channels is 256 this number is available when the sample time greater than 14 usec If you increase the Sample Time above 14 the number of channels will remain constant at 256 which is the maximum number of physical channels provided by the correlator in the system par Note Although the chip used to collect data has 1024 memory spaces a maximum of 256 channels are used by the correlator to process data to optimize the data processing PrecisionDeconvolve 2 Chapter 4 4 3 Table 4 1 Available Channels TIME Channels Used 14 29 29 NER METRI 3 4 2 1 3 Last Time delays in correlator channels can be set between 1 and 1024 sample times The Last field is used to set the last channel of the correlation curve All other channels are then allocated automatically to maximize the resolution of the subsequent deconvolution procedure In most cases set Last to 4 6 times the total number of channels The Last value cannot be less than number of channels or more than 1024 Note If the correlation function looks too broad the sample time should be increased rather than the Last parameter A reduction in the Last channel number should be made if there are significant long term fluctuations in the intensity which may be due to e thermal gradients e large particles in the sample are slowly drifting through the light scattering volume the coherence ar
52. er hand if the smoothness factor is too small the deconvolution procedure will be less stable and in polydisperse system may produce set of narrow spikes instead of one wide distribution the value should normally be kept below 20 Table 4 2 presents suggested starting smoothness values for different situations The Smoothness parameter can range from 1 to 40 and the Relative increase in mean square difference between the data and the fit due to the smoothing requirement can range from 1 3 A detailed discussion of this parameter is presented in Section 4 2 1 7 PrecisionDeconvolve 2 Chapter 3 21 Chapter 4 Selecting Operating Parameters 4 1 OVERVIEW This chapter describes how to e choose appropriate values for data collection parameters Section 4 2 e enter sample parameters Section 4 3 e determine the appropriate sample concentration Section 4 4 Parameters that are essential for the measurement process are found in the Measurement Settings dialog box Section 4 2 which is accessed by selecting Setup on the Measure menu The user is strongly encouraged to determine the effect of making small changes in the various parameters to see the effect of each parameter The guidelines described below will generally provide good results but it should be recognized that no one set of parameters will be appropriate for all samples and experimentation is required Changing each parameter on a controlled basis may improve th
53. es the average diffusion coefficient for these particles Particles of a size comparable to or larger than q i essentially preserve their orientation as they travel a distance smaller than their size For these particles the single exponential expression of equation 5 for the field correlation function is not strictly applicable For particles that are small compared to 4 the hydrodynamic radius is calculated numerically and in some cases analytically for a variety of particles shapes The important analytical formula for the prolate ellipsoid with the long axis a and the ratio of lengths of the short axis to the long axis p is 2 1 1 2f fy Eat 2 p A 8 The above formulae connecting the diffusion coefficient or hydrodynamic radius to particle geometry are strictly applicable only for infinitely dilute solutions At finite concentrations two additional factors significantly affect the diffusion of particles viscosity and interparticle interactions Viscosity generally increases with the concentration of macromolecular solute According to equation A 6 this leads to a lower diffusion coefficient and therefore to an increase in the apparent hydrodynamic radius Interactions between particles can act in either direction If the effective interaction is repulsive which is usually the case for soluble molecules otherwise they would not be soluble local fluctuations in concentration tend to dissipate faster meaning higher apparent di
54. ffusion coefficients and lower apparent hydrodynamic radii If the interaction is attractive fluctuations in concentration dissipate slower and the apparent diffusion coefficients are lower Thus depending on whether the effect of repulsion between particles is strong enough to overcome the effect of increased viscosity both increasing and decreasing types of concentration dependence of the hydrodynamic radius are observed In this context it should be noted that the interaction between large particles as compared to q generally leads to a non exponential correlation function that does not take the form of equation A 4 and therefore cannot be completely described by a single parameter D A 6 PrecisionDeconvolve3 Appendix A A 5 DATA ANALYSIS A 5 1Polydispersity and the Mathematical Analysis of QLS Data Polydispersity can be an inherent property of the sample for instance when polymer solutions or protein aggregation are studied or it can be a consequence of impurities or deterioration of the sample In the first case the polydispersity itself is often an object of interest while in the second case it is an obstacle In both instances polydispersity significantly complicates data analysis For polydisperse solutions equation 5 for the normalized field correlation function must be replaced with yu exp D ad o i A 9 In this expression D is the diffusion coefficient of particles of the i th kind and 7 i
55. formats are acceptable contact Precision Detectors e Langley Ford 1096 which should be used when that correlator is used The Do not save distribution recompute while loading check box is used to indicate that the raw data is to be saved but the distribution should not be saved If this box is selected the program will re compute the distribution whenever the file is loaded reloaded This option is used to save disk space when you store data for archival purposes after you have finished analyzing it If this option is selected it may take some time to load stored files on a slow computer It should be noted that when you save data without distribution information on what part of distribution was highlighted is lost 2 2 PrecisionDeconvolve3 Chapter 2 The Do not save intensity check box is used to indicate that the raw data points should not be saved in the data file The intensity record is useful if you want to see if dust particles or large aggregates were present in the scattering volume and if they were whether the intensity fluctuations management was adequate However if there are no intensity fluctuations in the data there is little reason to store intensity record The average intensity is always stored The Allow duplicates of the same measurements check box is used to indicate duplicate data files can be loaded Duplicate files are those that contain the same measurements but have been saved under different names If
56. from particles above certain size are shown and used for normalization This domain is marked by red segment of the abscissa and can be changed by right click of the mouse The contributions outside marked domain are indicated by small black triangles Selection of the part of the distribution is effective only within marked domain It is updated when a new data file is selected as the active file or if the distribution of the current active file is recalculated e g when the Smoothness parameter is changed either in the Fit dialog box or as a result of executing the Smoothness command in the Data menu In addition new data is in a constant re calculation loop as it accumulates If new data is active data every time the calculation of its distribution is completed the distribution window is updated During measurements the distribution function is recalculated after every update of the correlation function To highlight a part of the distribution Hold left button and drag to highlight part of the distribution Double left click to remove all highlighting Right click to set the lower size cutoff of the mass normalization domain To change the scale of the distribution use the up down arrows to change the scale of the distribution 3 5 ACTIVE DATA DIALOG BOX The Active Data dialog box Figure 3 21 presents a complete description of the active file The Sample tab describes the file name data collected and path If desired the Operator name an
57. ge 2 Pixels 200 perun C persec Show axis Figure 6 7 The Intensity Record Parameters Show active data intensity Select the desired format e Never if you always want to see the intensity history of the current measurement This is the only option that allows you to see this history when no measurements is underway f not measuring if you always want to see the intensity history of the current measurement when the correlator is running e Always you will see the intensity record of active data You will see the intensity history of the current measurement only if new data is active Use the Make Active command to make new data active 6 8 PrecisionDeconvolve3 Chapter 6 Show description click to show number of accepted and rejected runs run duration and average intensity of active data Show cutoff trace click to show in red color the cutoff level that had been in effect during measurements Central alignment when clicked the intensity plot is shifted to the center of the Intensity window Time scale choose the horizontal scale of the intensity plot in pixels of the computer display per one run or per one second each run can be several seconds see Measurement setup dialog box Intensity range choose the vertical scale of the intensity plot in percents above and below the average level If you check the Show axis box the vertical axis with these percents and the ave
58. he temporal correlations in the fluctuations in the scattered light intensity and to reconstruct from these data the physical characteristics of the scatterers A 4 2 Coherence Area There is a characteristic size for speckles in the interference pattern If the intensity of the scattered light is above average at a certain point it will also be above the average within an area around this point where phases of the scattered waves do not change significantly this area is called the coherence area Within different coherence areas the fluctuations in intensity of light collected are statistically independent Increasing the size of the light collecting aperture beyond the size of a coherence area does not lead to improvement of the signalto noise ratio because the temporal fluctuations in the intensity are averaged out For a monochromatic source the scattered light is coherent within a solid angle of the order of NIA where A is the cross sectional area of the scattered volume perpendicular to the direction of the scattering Because the coherence angle is fairly small powerful 100 mW and well focused laser illumination and photon counting techniques are used in the PDI BATCH instrument A 4 3 The Correlation Function While the photodetector signal in QLS is random noise information is contained in the correlation function of this random signal The correlation function of the signal i t which in the particular case of QLS is the photocurren
59. he quality of measurements and for the validation of the settings used 4 2 1 5 Accumulate The Accumulate parameter is the number of runs the system will accumulate to obtain a single measurement and the total time of a single measurement is the product of the Accumulate and Run time parameters After the completion of data acquisition the results will be saved using the indicated file name and extension e g 7120 001 You can change this parameter during measurements If it is set to the value less then the current accumulation current measurement will end and next measurement will be started 4 2 1 6 Repeat The Repeat parameter is the number of accumulation processes that should be performed before the system stops If one measurement is desired set the repeat value to 1 At the conclusion of the accumulation process the data will be stored e g Jul20 001 using the name and sequence number the Sample Record tab Section 4 2 4 and the system will wait for another Start command If the value is greater than 1 the data will be stored the file name extension will be incremented e g Jul20 001 Jul20 002 etc the Repeat parameter will be reduced by one and another measurement will be initiated This process will be repeated for the indicated number of data accumulation processes Note If Repeat is set to zero the measurement will be performed but the results will not be saved If the user decides to save the results that c
60. hen all items have been programmed press OK g Note Selecting items for Panel display and Data Printout does not directly lead to the parameters being included in the indicated activity It is necessary to select them on the appropriate tab If desired a set of parameter settings can be saved by selecting Load Settings on the Setup menu and assigning a file name in the Save dialog box The stored set can be recalled using the Load settings command Note You can select any parameter for any list in this dialog However not all parameters are valid for all operations For example you cannot plot operator name or date and you can reset only modifiable parameters In most cases illegal parameters simply will not be included into the list even if you check them here Still you are advised to exercise common sense PrecisionDeconvolve2 Chapter 6 6 11 6 4 THE ROLE OF THE DATA MENU WITH STORED DATA DISPLAY The Data menu Figure 6 11 includes a number of commands that are very useful in viewing stored data a b 9 h Data Active Data Ctrl D Remove Active DEL Delete Active Ctrl x Insert blank Ctrl Space Select All Inverse selection Remove selected Ctrl DEL Remove duplicates Reset Sort Find Plot Smoothness Ctrl R Figure 6 11 The Data Menu Active Data presents a dialog box that contains data about the active file A detailed discussion is presented
61. ick in the Intensity window sets the cutoff level relative to the current averaged intensity Average xxxx points specifies the number of runs over which an exponential averaging of intensity is performed Dropped runs with intensity above the cutoff level are also included into averaging process but with 10 times reduced weight Trace average is selected if the analyst wants to use to display the average over the number of points indicated in the field The tracing will be presented as a green line on the plot Tracking the average the intensity curves in Intensity window are periodically offset so as the current averaged intensity would be at 4 of window height level shown by the black horizontal line PrecisionDeconvolve2 Chapter 4 4 7 Reset on start used if a fresh intensity plot should be presented when a run is started previous data is erased you want to get an intensity at the start Auto scale when this box is checked the intensity curves in the ntensity window are periodically automatically re scaled to fit the window 4 2 3 The Sample Data Tab The Sample Data tab Figure 4 4 is used to enter information about the sample which is stored with data file The three parameters presented on the right side of the dialog box must be included Information about these parameters is presented in Section 4 3 Measurement Setup X Measurement Intensity Sample data Sample record optional Concentra
62. idered undefined if they are set to zero If you want to specify zero concentration for the blank sample set the concentration to a very small but positive value Parameters can only be positive Radius is the average hydrodynamic radius of the selected fraction of the distribution If a different data output is used in the presentation e g molecular weight it will be reported instead Fraction is the percentage of the selected fraction in histogram that is used for the calculation Width is mean square width of the selected fraction in the distribution Intensity is the average intensity for the whole measurement Background is the percentage of the statistical baseline in the correlation function It is at least 50 and a value up to 80 90 is acceptable If the background is too high either the cuvette is dirty or it is misaligned 1 and 2 cumulants are the average decay time of the correlation function and the second momentum of the distribution of the decay times respectively a detailed discussion of these values is presented in Appendix A The numerical data for the distribution is listed at the bottom of the page The hydrodynamic radius diameter molecular weight or diffusion coefficient will be reported for each segment of the distribution depending on the mode of presentation 7 8 PrecisionDeconvolve3 Chapter 7 Appendix A General Principles A 1 WHAT IS LIGHT SCATTERING The propagation of light may be
63. in Section 3 5 Remove Active deletes the active file from the panel Delete Active deletes the active file from the diskette Insert Blank inserts a blank panel space Once you have generated a blank you can move a panelto it by right clicking on the panel and dragging the panel to the blank position Select All indicates that all files should be used in the task to be performed e g generating a plot When a file is selected the distribution has a gray background Inverse Selection changes the selection option for all files e g selected files are unselected and vice versa Remove duplicates deletes files that are present more than once from the screen one copy is left Reset presents the Reset Parameters dialog box Figure 6 11 The parameters that are included in this box are indicated via the Configure parameter lists dialog box Section 6 3 PrecisionDeconvolve3 Chapter 6 These parameters of the Active Data will be assigned to all selected data Concentration Cancel Save All to make the changes permane Figure 6 12 Reset Parameters Dialog Box The Reset Parameters dialog box allows you to select a set of parameters for a data file based on the selected parameter for the active file i e you can change the concentration parameter from that which is stored The changes are not saved to hard drive If you want to make then permanent use the Update all EB files command on the File menu
64. indow using up and down arrow keys and re center the line by pressing the space bar A typical Intensity window is shown in Figure 5 2 a detailed discussion of this window is presented in Section 3 3 2 intensity 9225 8565 Figure 5 2 Intensity Window The cutoff level red line indicates the maximum intensity that is permitted for a run It is selected by pointing the mouse to the desired level and clicking If the intensity during a run exceeds this value e g due to a dust particle entering the scattering volume the run is ignored The black line is a reference line pressing the space bar shifts the intensity curve so that the next run intensity will be on a reference line During a measurement you can change all parameters described in the previous chapter except the Sample Time Last Run Time and Path parameters in Measurement Settings dialog box These parameters can be changed only when the correlator is stopped If the Repeat field on the Measurements setting dialog box is non zero at the end of the measurement the data will be saved using the file name indicated in the Measurements setting dialog box After saving the data the data file name extension will be incremented by one the Repeat value will be reduced by one and data collection will resume When the Repeat parameter becomes zero the correlator stops and the Stop entry on the menu bar will revert to Start The last unsaved measurement will remain on d
65. ington 1977 5 D E Koppel J Chem Phys 57 4814 1972 65 Provencher Comput Phys Commun 27 213 1982 A 10 PrecisionDeconvolve3 Appendix A Appendix B Refractive Index and Viscosity Water deg C RIU P s pow 13568 pow 1357 0005 onm 1356 13335 001208 moo 33 00110 1339 w 1332 001056 1351 poros 13850 001002 PrecisionDeconvolve Appendix B B 1 Temperature Refractive Index Viscosity deg C RIU P 13323 0 00851 13322 0 00833 13321 0 00815 13319 0 00797 13318 0 00782 13u7 po 13 00750 1384 pos B2 o pow 1380 po 139 022 13306 0 00653 B 2 PrecisionDeconvolve 3 Appendix Index A About PrecisionDeconvolve Command 3 9 Accumulate 4 5 Active Data Command 3 5 6 12 Active Data Dialog Box 3 16 Conditions Tab 3 19 Fit Tab 3 17 Normalization Tab 3 20 Results Tab 3 19 Sample Tab 3 16 Additional Information 1 3 Angle 2 3 Average Error 3 18 Average Points 4 7 Background 3 17 Binary File 7 1 Brownian Motion A 5 Calibration Tab 2 5 Cascade Command 3 8 Channels 4 3 Coherence Area A 3 Collecting Data 5 1 Communication Diagnostic Tab 2 6 Configure Parameter Lists Dialog Box 6 11 Conditions Tab 3 19 Concentration Selecting 4 11 Correlation Function A 3 Correlation Tab 6 5 Correlation Window 3 13 Cumulants 3 17 A 8 Cutoff Intensity Level 4 7 Data Acquisition Windows 3 13
66. ion coefficients Note A sample usually consists of a collection of particles with different molecular weights and sizes thus the Dynamic Light Scattering experiment leads to a distribution for the diffusion coefficients The diffusion coefficient depends on particle size and shape and can be converted into related parameters such as e hydrodynamic radius Ry of the macromolecule e the molecular weight of the molecule when the concentration is known e the diameter of the macromolecule In this manual we will display data that refer to the hydrodynamic radius The reader should recognize that molecular weight distributions the diameter relaxation time and the rate of diffusion are also readily obtained A typical distribution for the hydrodynamic radius is shown in Figure 1 1 1 2 PrecisionDeconvolve3 Chapter 1 5 Figure 1 1 Hydrodynamic Radius Distribution 1 3 GENERAL CONVENTIONS USED IN THIS MANUAL PrecisionDeconvolve is a Windows application that follows the general Windows conventions All windows dialog boxes controls short cut keys scroll bars etc operate according to standard Windows procedures For the sake of brevity we use the following conventions e 15 understood that the OK button is to be clicked or the ENTER key on the keyboard is to be pressed to accept the settings and close the dialog box e It is understood that the CANCEL button is to be clicked or the ESC key on the ke
67. is box is checked the parameter s will be presented in the panel but the appropriate units will not be indicated Adjust height automatic manages the layout of the pane depending on the pane size and the number of parameters to show When you check this box the program will adjust the height of the pane to optimize the layout PrecisionDeconvolve2 Chapter 6 6 7 Number of columns indicates the number of columns that the panels should be arranged in Height of column in pixels indicates the height of each panel Show Last if this box is checked the Panels window automatically scrolls to the bottom when a new data pane is added this is to ensure that the last data is always visible The last data will have a thicker black frame in this case Allow duplicates of the same measurements indicates if two or more panels of the same data files are permitted 6 2 5 Intensity Record The Intensity record tab Figure 6 7 is used to manage the display of previously stored active data in the Intensity window use the Intensity tab on the Measurement Setup dialog box to setup Intensity parameters during new data accumulation Data presentation xj Distribution Correlation Panels Intensity record Data Printout Textfle content m Show active data intensity gt C Never Show Describtion C f not measuring Show Cutoff trace Always Center aligment m Time scale 4 r Intensity ran
68. isplay It can be saved manually via Save on the File menu 5 2 PrecisionDeconvolve3 Chapter 5 5 3 MONITORING DATA COLLECTION Note To monitor the accumulation process i e to determine the appropriate value for the various parameters set Run Time to 1 second minimum and set the Repeat parameter to 0 to avoid collecting saving preliminary data a b d e This section describes a number of items that the operator should visually monitor during the collection of data to ensure that useful data is being collected A detailed troubleshooting discussion is presented in Section 8 3 The Correlation Function with subtracted baseline should fall to 1 3 of its initial value at approximately 1 5 1 10 of the delay time indicated on the plot an acceptable plot is shown in Figure 5 3a while an unacceptable plot is shown in Figure 5 3b Correlation Function lol Correlation Function iof xi a b Figure 5 3 Correlation Function a Acceptable b Unacceptable Consecutive measurements on a sample should be reproducible The Correlation Function should not fall below the X axis In addition the baseline should not have any drift periodicity or extraneous spikes The Accumulate parameter should be large to provide sufficiently accurate correlation function and stable reconstruction of distribution The intensity should not exceed 1 000 000 counts The intensity on the Intensity window shoul
69. it Method The simplest approach is the direct fit method In this method the functional form of D is assumed a priori single modal bimodal Gaussian etc and the parameters of the assumed function that lead the best fitof G to Ge 9 A T then are determined This method is only as good as the original guess of the functional form of D Moreover using the method can be misleading because it may confirm nearly any a priori assumption made It is also important to note that the more parameters there are in the assumed functional form of D the better the experimental data can be fit but the less meaningful the values of the fitting parameters become In practice typical QLS data allow reliable determination of about three independent parameters of the size distribution of the scattering particles A 5 4 The Method of Cumulants The second approach is not to attempt to reconstruct the shape of the scattering particle distribution but instead to focus on so called stable characteristics of the distribution i e characteristics which are insensitive to possible fast oscillations In particular these stable characteristics are moments of the distribution or closely related quantities called cumulants 2 The first cumulant moment of the distribution D that gives the average diffusion coefficient D can be determined from the initial slope of the field correlation function Indeed using equation A 12 it is straigh
70. laced on the plot that indicates the fit of the correlation function h Overlay selected data Whenthe Overlay selected data command is selected the data for each run is presented in the correlation plot 6 2 4 Panels Tab The Panels tab Figure 6 6 is used to indicate the desired format of the panel s for data presentation The field in the left indicates the parameters that can be displayed in the panel the selection of the allowable parameters is made via the Configure Parameter Lists dialog box Section 6 3 To display a given parameter check the corresponding box and verify that the Show Parameters box is checked Data presentation x Intensity record Data Printout Text file content Distribution Correlation Panels Show parameters Tab 10 characters Parameter names no than characters ivi Operator Do not show units 3 Number of columns 100 Height of panels in pixels Show last Adjust height automatic Cancel Figure 6 6 The Panels Dialog Box Show Parameters if this box is checked the items in the field below will be presented in each panel if they are checked The parameters listed in the block are selected on the Configure Parameter Lists dialog box Section 6 4 Tab number of spaces between the title of the parameter and the value in each panel Parameter Names no than xxx characters number of spaces for the parameter value in the panel Do not show units if th
71. le preparation device that is integrated and computer controlled for maximum throughput It includes a Peltier cooled heated autosampler with provision for 96 well plates that injects into the DLS system The system is completely controlled through the serial port The temperature is displayed on the front panel but can be set and monitored in the software The automated sample preparation system has access to larger volume containers allowing for automated step titration serial dilutions and kinetic studies PrecisionDeconvolve 2 Chapter 1 1 1 e ALS4000 Automated Expert DLS System employs the Automated Sample preparative system used in the ALS3000 but directly injects into the Expert DLS system The system can automatically collect correlation functions from three detectors at angles from 5 to 355 degrees in 5 degree increments allowing for the monitoring of the nucleation of aggregates in the presence of larger particles In addition it fully characterizes nanoparticles and also is able to measure slow diffusion characteristics in and out of a sophisticated matrix The fluctuations in scattered light intensity caused by the sample are monitored their correlation function calculated and deconvoluted into a distribution of diffusion coefficients and the corresponding radii for the scattering particles 1 2 INTRODUCTION TO LIGHT SCATTERING Note This section provides the analyst with a qualitative description of the Dynamic Light Scatteri
72. lected data ag Note Plot is shown only if it contains at least two points and if both X and Y parameters are not identical for all data Each data point corresponds to a selected file and is represented by a square The square representing the active file is highlighted by red border The equation used for the plot is shown in the upper left corner in green and X and Y parameters of the active file are shown in the top right corner of the Plot window in red 6 14 PrecisionDeconvolve3 Chapter 6 ME Plot of 1st Cumulant vs Diameter nj x Y B K X X Diameter 16 12 B 2098e 004 K 3 16e 002 1st Cumula 16967 20 30 40 50 60 70 Figure 6 15 User Generated Plot The Plot window operates in the following manner e Clicking on a data point will designate the corresponding file as the active file The Correlation Function and the Distribution windows as well as any open dialogs will be updated e Pressing the Delete key will remove the active file from the Plot window but not from the Main window ag Note If a data file does not contain the selected parameter s the plot will not contain a data point for that file If a linear or quadratic fit is selected a blue line or parabola respectively will be drawn through the data points The mathematical formula for the curve and the values of the parameters will be shown in blue font above the plot If you remove a point from the plot by clicking it to make it Active Data
73. ll files Selected Data Update and Intensity options to save the present data A detailed discussion of these options is presented in Section 7 2 e Print presents the Printer Output Properties dialog box Figure 3 3 which is used to select the information to be printed and initiate printing A detailed discussion of the printing is presented in Section 7 3 Printer Output Properties x Print Data Printout Margins Choose what to Print Preview Printer setup Figure 3 3 The Printer Output Properties Dialog Box e Exit Closes the program 3 2 2 2 Measure The Measure menu Figure 3 4 includes the following commands Measure Setup Ctrl E Start Stop Make Active Motor Figure 3 4 The Measure Menu e Setup accesses the Measurement Setup dialog box Figure 3 5 which is used to establish data acquisition parameters A detailed discussion of data acquisition is presented in Chapter 4 PrecisionDeconvolve 2 Chapter 3 3 Measurement Setup x Measurement Intensity Sample data Sample record Sample Time 1 Run Time 256 Channels 150 Accumulate 1024 5 Repeat Don t save 10 Smoothness Keep in memory Cancel Apply Help Figure 3 5 The Measurement Setup Dialog Box Start initiates a run using the parameters indicated in the Measurement Setup dialog box Stop halts the present run The current data is not automatically saved when this co
74. log Box pag Note Correlator parameters Run Time parameters and the Path cannot be changed while the correlator is running other parameters can be changed at any time The Apply button is used to accept a change in the Smoothness parameter for data from a completed run 4 2 1 The Measurement Tab 4 2 1 1 The Sample Time The instantaneous intensity of the scattered light is represented by the number of photons detected by the photodetector during one Sample Time interval The sample time interval is the delay time for the first channel of the correlation function in microseconds other channels are delayed by integral numbers of sample times as described in the Allocation of channels topic The minimum sample time is 1 usec The set of intensities over the Sample Time interval is the time dependent intensity of the scattered light that is used to calculate the correlation function The range for the sample time is from 1 usec to 30 msec and the sample time should be short compared to the correlation time of the intensity fluctuations Short sample times are used for very small particles or molecules that move rapidly and therefore have a small correlation time On the other hand the maximum delay time in the correlation function corresponds to 1024 sample times When very large macromolecules which move slowly are studied long sample times must be used We suggest that you start with a relatively large value for the sample time so
75. lve3 Chapter 7 e Summary The Save Summary command presents the Summary dialog box Figure 7 2 which is used to select the parameters to be placed in a summary table The items that are indicated in the Summary dialog box are selected in the Parameters list dialog box Section 6 3 and the desired items should be checked on this dialog box Parameters of the selected data to be summarized in a print out or in a text file Retr index Sample time dn dc lt lt lt lt lt Figure 7 2 Summary Dialog Box The operator should highlight the parameters that should be included in the table Once a table has been generated it can be imported into a data processing and plotting program A typical report is presented in Figure 7 3 Table of parameters File name Radius Intensity Date Operator Aug01 001 36 15 1 00e 004 Aug 01 14 35 27 T Havard Aug01 002 25 96 8 35e 004 Aug 01 18 57 25 T Havard Aug03 001 25 96 8 35e 004 Aug 03 16 40 11 T Havard Aug03 002 25 96 8 35e 004 Aug 03 08 29 21 T Havard Aug03 003 19 16 8 35e 004 Aug 03 08 29 21 T Havard Aug03 004 30 34 4 18e 004 Aug 03 08 45 05 T Havard Figure 7 3 Summary Table PrecisionDeconvolve2 Chapter 7 7 3 7 3 PRINTING A REPORT The Print command presents the Printer Output Properties dialog box Figure 7 4 which is used to select the items to be sent to the printer Print options Print Data Printout Margins Choose what to
76. menu 3 8 Window Panels window Plot window Layouts 4 Cascade Tile Figure 3 12 Window Menu PrecisionDeconvolve3 Chapter e Panels Window presents a window in which the distribution for a number of files can be shown Chapter 6 e Plot Window presents the Plot dialog box which is used to indicate the format of the Plot Section 6 41 e Layouts accesses a sub menu which includes Measurement presents the windows normally used during data acquisition Correlation Function Distribution and Intensity Data Analysis presents the windows normally used for data processing Panel Correlation Function and Distribution 3 Restore Custom presents the Window format that was stored with Store As Custom Store as Custom saves the present Window format e Cascade standard Windows function whereby windows partially overlay others e Tile standard windows function where all open windows are positioned so that the contents are visible 3 2 2 6 Help The Help command Figure 3 13 presents a drop down menu which can be used to access the following Help Help Topics About PrecisionDeconvolve Figure 3 13 The Help Menu e Help presents a drop down menu which can be used to access information to assist the user e About PrecisionDeconvolve presents a dialog box with the version number of the program 95 Note Context sensitive help is available for most controls within
77. mmand is used If you want to save the data it will be necessary to use the Save data command Make Active makes the data that is being collected the active file in the data panel at the end of the data collection process Motor accesses the Positioning dialog box which is used to select the position of the stirrer in the cuvette This dialog box is described in Section 4 4 PrecisionDeconvolve3 Chapter 3 2 2 3 Data The Data menu Figure 3 6 includes the following Data Active Data Ctrl D Remove Active DEL Delete Active Insert blank Ctrl Space Select All Inverse selection Remove selected Ctrl DEL Remove duplicates Reset Sort Find Plot Smoothness Ctrl R Figure 3 6 Data Menu e Active Data presents the Properties dialog box for the active file Figure 3 7 and is used for viewing processing stored data This dialog box is described in detail in Section 3 5 Dec15 1 Properties xj Sample Fit Results Conditions Measurement Normalization File Dec15_1 Date Sun Dec 15 16 56 14 200 Path C PDIdata Dec15_1 pd Operator fa Smith Comment Samples from ED Cancel Apply Figure 3 7 Properties Dialog Box e Remove Active erases the present data from the display window but leaves the data in memory e Delete Active erases the present data from the window and from memory If you only want to remove active
78. mple should be obtained from the literature or measured in your laboratory The units for viscosity should be entered in Poise P For aqueous solutions the value 15 0 0089 cP at 25 A table presenting the viscosity of water as a function of temperature is presented in Appendix 4 and in the on line help file A table of viscosity as a function of temperature can also be obtained by pointing the cursor to the field and pressing F1 4 3 2 Temperature Temperature should be set to the ambient temperature in degrees Celsius C If the CoolBatch system is used set the temperature to the sample temperature If the Expert system is used see Section 2 2 PrecisionDeconvolve2 Chapter 4 4 9 4 3 3 Refraction The refractive index of the sample should be obtained from the literature measured in your laboratory For aqueous solutions the value 1 332 should be used at 25 A table presenting the refractive index of water as a function of temperature is presented in Appendix 2 This table can also be obtained by pointing the cursor to the field and pressing F1 4 3 4 Annotative Information The Concentration pH Molarity Other operator and info fields should be completed if these parameters should be stored with the data While they are not relevant for the collection of data they are used for various calculations and data presentation see Chapter 6 These parameters can be entered before the data 15 collected or after the data 1
79. n agreement signed by PDI and you Unpublished rights reserved under the copyright laws of the United States Precision Detectors Inc 34 Williams Way Bellingham MA 02019 Your acceptance or decline of the foregoing Agreement was or will be indicated during installation PrecisionDec onvolve License Agreement v This page intentionally left blank PrecisionDeconvolve3 License Agreement Table of Contents Precision Detectors Inc Electronic End User License iii Chapter 1 Introduction msna nanna E EE EIN EVE S VEEE tea 1 1 I E OE BAT AEAT A SANSE EIEE M ec S A 1 1 1 2 Introduction to Light 1 2 1 3 General Conventions used in this Manual cc cece cece ceccesccesceesceeceesceescesccesccesceusseusceuss 1 3 1 4 For Additional Information ceeseeeeeeeeeeeeenee e eene 1 4 1 5 Contents of this Manual iter ovine ee eoi Pete tette moe deer 1 4 Chapter 2 Installation ccscscssssscssssscscssscscssscscscssscscscscssscscssssssssssssssssssssssssssssssssssssssssssess 2 1 2p sais utet Lote co rame Ces e remeare 2 1 2 2 Loading the Software and Configuring the Communic ations Port on the Personal Computer T 2 1 2 35 A Test RUM Suo Deest t er n Eo ua c 2 8 Chapter
80. n sample times should be determined The number n is referred to as a channel number Up to K channels in principle can be measured simultaneously but usually a smaller subset of M equidistant or logarithmically spaced channels is used Clearly the shortest delay time at which the correlation function is measured by the procedure described above is Av channel 1 The longest delay time cannot exceed the duration of the digitized copy Thus it is important that the correlation time T fit into the interval At lt lt lt lt This condition determines the choice of the sample time for the particular measurement To increase the statistical accuracy with which the correlation function is determined it is essential to maximize the number of count pairs whose products are averaged within the measurement time If the correlation function is being measured in M channels simultaneously ideally M products should be processed for each new count i e during sample time Ar The instrument capable of doing this is said to be working in the real time regime The real time regime means that the information contained in the signal is processed without loss The PDI correlator works in real time with a minimal sample time Ar of 1 microsecond and the length of the digital copy 1024 The number of channels M processed in real time is determined by formula 19 5 At 4 5 and cannot exceed 256 A 4 PrecisionDeconvolve3 Appendix
81. n the Data menu Configure Appearance of the Data equivalent to the Data Presentation command on the Setup menu Hardware Setup equivalent to the Hardware command on the Setup menu Default Measurement Layout presents the windows normally used during data acquisition Correlation Function Distribution and Intensity Default Layout for Data Screening and Analysis presents the windows normally used for data processing Panel Correlation Function and Distribution Custom saves the present window format PrecisionDeconvolve3 Chapter 3 2 4 The Data Display Window The format of the data display window is dependent on the selections made on the Windows menu The default display when data is being acquired is presented in Figure 3 15 and is discussed in Chapter 5 The default display for post run data processing is presented in Figure 3 16 and is discussed in Chapter 6 Pr PrecisionDeconvolve File Measure Data Setup Window Help sHE IN NN Br EI HH iB xi TUTO All Ph A F j N Test Mode LE Intensity qm 9376 8705 For Help press F1 Figure 3 15 Display Data Collection PrecisionDeconvolve 2 Chapter 3 11 111 PrecisionDeconvolve File Measure Data Setup Window Help m Bs See 88 Panels window 100 Mw 9 66 101 KDa 100 Mw 8 97 10 1 st Cumula 554 1sec 1503sec 151 Cumula 14742sec Rn
82. nce q along the vector 4 As stated above Ax op thus for q pto 1 Dq e Rigorous mathematical analysis of the process of light scattering by Brownian particles leads to the following expression for the correlation function of the scattered light x x exp Dq 9 A 5 A 4 7 Determination of the Sizes of Particles in Solution According to Equations A 4 and A 5 measurement of the intensity correlation function allows evaluation of the diffusion coefficients of the scattering particles The diffusion coefficient in an infinitely dilute solution is determined by particle geometry For spherical particles the relation between the radius R and its diffusion coefficient D 15 given by the Stokes Einstein equation k T 6 D where k is the Boltzmann constant T is the absolute temperature 1 is the viscosity of the solution For non spherical particles it is customary to introduce the apparent hydrodynamic radius defined as R app kT nnp A 7 where D is the diffusion coefficient measured in the QLS experiment PrecisionDeconvolve Appendix A A 5 For non spherical particles it is important to note that the diffusion coefficient is actually tensor the rate of particle diffusion in a certain direction depends on the particle orientation relative to this direction As small particles diffuse over a distance q their orientation may be changed many times QLS measur
83. nd select the Delete Active command on the Data menu this action will not remove the file from the disk A file can be added to the display by selecting Open on the File menu and selecting it from the Open dialog box To select more than one file in a contiguous set depress the Shift key and click the first and last file to be selected To select more than one file in a non contiguous order depress the Ctrl key and click on the files of interest Panels can be moved by creating a blank panel Section 6 4 ands then dragging a panel via the right mouse button 6 2 SELECTING THE DESIRED DATA PRESENTATION FORMAT 6 2 1 Data Presentation Dialog Box The Data Presentation command on the Format menu accesses the Data Presentation dialog box Figure 6 2 which is used to indicate the general design of the data presentation Each of the six tabs addresses a specific aspect of data presentation The Apply button at the bottom of the dialog box can be used to see the effect of changing a field without having to close the dialog box As an example if you just want to see the effect of MW normalization on the distribution you can check the appropriate check box and press Apply If the result is not acceptable simply remove the checkmark and press Apply again After you make your selection s press OK 6 2 PrecisionDeconvolve3 Chapter 6 Data presentation x Distribution Correlation Panels Data Printout Text file content Distrib
84. ng method A detailed discussion of the technology is presented in Appendix A The term Light Scattering is used to describe the process in which light from an incident light beam is scattered in all directions upon interaction with particles in the beam Light is an electromagnetic wave and the light scattered by an ensemble of particles is the sum of light scattered by individual particles When the incident light is coherent the intensity variations or speckles are produced at the observation plane These speckles are due to the variation in phases of the waves scattered by different particles At one point waves arriving at different phases cancel each other more fully than at another As the scattering particles move over distances that are comparable to the wavelength of the incident beam the phases of the scattered waves and the speckle pattern are dramatically changed Monitoring the fluctuations of intensity of the scattered light passing through a small pinhole smaller than the size of the speckle make it possible to tell how fast the scattering particles diffuse over a distance equal to the wavelength of the scattered light In Precision Detectors systems this task is achieved by detecting the intensity of scattered light by an avalanche photodiode computing the correlation function of the photocurrent by a specialized correlator and deconvoluting this correlation function into contributions from particles with different diffus
85. nts per sample time the scattering intensity will be sufficient any further increase in concentration will not improve the accuracy If there are less then 0 2 counts per correlation time the scattering intensity will be too low the correlation function will be very noisy and it will be very difficult to obtain good result In this case increase the concentration e If the count rate exceeds 1 000 000 it is possible that there will be a significant contribution from multiple scattering Furthermore above this count rate photons often strike the photo detector in a too quick a sequence to be detected as separate events which can lead to undesirable distortions PrecisionDeconvolve Chapter 4 4 11 Chapter 5 Collecting Data 5 1 OVERVIEW This chapter describes the collection of data using PrecisionDeconvolve it is assumed that the user has selected appropriate parameters see Chapter 4 for details and the sample has been placed in the cuvette that has been inserted into the system or the flow has been initiated 5 2 STARTING THE MEASUREMENT A measurement is started by clicking the Start command on the Measure menu of PrecisionDeconvolve Each measurement consists of several short runs and the number of runs is specified by the Accumulate parameter and the duration of each run is specified by the Run Time parameter When the data collection is initiated run is completed the correlation function will be displayed in the
86. number P 0834 Settings appropriate for this sample are indicated below Once you have collected some data we recommend that you refer to Chapter 3 and determine the effect of changing each parameter in a controlled fashion Note Let the system warm up for at least 30 minutes before collecting data To set up the appropriate parameters a Start PrecisionDeconvolve b Select the Setup command on the Measure menu to display the Measurement tab of the Measurement Setup dialog box Figure 2 7 Measurement Setup x Measurement Intensity Sample data Sample record Sample Time 1 Run Time 256 Channels 150 Accumulate 1024 Last 5 Repeat Dontsave 10 Smoothness Keep in memory Cancel Apply Figure 2 7 The Measurement Setup Dialog Box 2 8 PrecisionDeconvolve3 Chapter 2 Setthe following parameters e Sample Time 3 e Last 220 e Run Time 1 e Accumulate 50 Repeat 1 e Smoothness 10 Verify that the Don t Save and Keep in Memory boxes are not checked d Open the Intensity tab Figure 2 8 and select the settings as indicated in Figure 2 4 the value in the absolute Counts sec is not relevant Measurement Setup B xj Measurement Intensity Sample data Sample record Cutoff intensity level C Absolute 4078 Counts sec Average 10 points v Trace average Reset on start Track average Auto scale Cancel Apply Figure 2
87. nvolution problem means that distributions differing by the presence or absence of a fast oscillating function produce very similar correlation functions The regularization requirement that the distribution should be sufficiently smooth eliminates this ambiguity allowing unique solutions to the minimization problem There are several methods that utilize this approach for reconstructing the scattering particle distribution function from QLS data of these methods impose the condition of smoothness on the distribution but differ in the specific mathematical approaches used for this purpose One popular program originally developed by Provencher is called CONTIN Precision Detectors use a proprietary algorithm of superior quality regularization algorithms produce similar results and incorporate the use of a parameter that determines how smooth the distribution has to be The choice of this parameter is one of the most difficult and important parts of the regularization method If the smoothing is too strong the distribution will be very stable but will lack details If the smoothing is too weak false spikes can appear in the distribution The rule of thumb is that the smoothing parameter should be just sufficient to provide stable reproducible results in repetitive measurements of the same correlation function Two facts are helpful for choosing the appropriate smoothing parameter First the lower are the statistical errors of the
88. ommand on the Save sub menu of the File menu The vertical scale in the intensity window can be changed using up and down arrow keys an expanded display is shown in Figure 3 18 Figure 3 18 Expanded Vertical Scale The black horizontal line is provided as a reference guide If the Intensity window is the active window pressing the space bar will shift the whole intensity curve so that the next point will be on this line Closing and reopening the ntensity window removes previous intensity data If intensity data becomes too long to fit into the Intensity window a horizontal scroll bar will automatically appear When the intensity window is active e Clicking the right button the mouse sets the cutoff level to the Y coordinate of the point where the mouse was clicked Pressing the space bar moves the whole graph up or down so that the current average intensity green line is on the black horizontal eye guide line e The left and right arrow keys scroll the display if scrolling is necessary 3 3 3 The Correlation Window The Correlation window shows the correlation function and is updated after each run A typical correlation window is presented in Figure 3 19 Correlation Function lal x Figure 3 19 The Correlation Window 3 14 PrecisionDeconvolve3 Chapter The correlation curve presents the following information e Black trace The correlation function of the active data e Red trace The fit of the co
89. on to be shown on the window A detailed discussion is presented in Section 6 2 3 6 PrecisionDeconvolve3 Chapter Data presentation ES Text file content Correlation Data Printout Distribution Distribution over Show Y axis Show Average Load with main peak selected Hydrodynamic radius Mol weight 1 R E MW narmalization LogScale Intensity record Cancel Apply Figure 3 9 Data Presentation Dialog Box e Load Settings Used to access the Load settings dialog box which allows you to select the Data Presentation Settings file configuration that you want to use This is a standard Windows Open file dialog box e Save Settings Presents the Save settings as dialog box which is used to save the present settings as a file This is a standard Windows Save as dialog box e Parameters List Presents the Configure parameter list dialog box Figure 3 10 which is used indicate the parameters and general display The Configure parameter list dialog box is described in detail in Section 6 3 Configure parameter lists E List of parameters available 11 Cumulant 2nd Cumulant Panel display Accepted runs C X axis in PLot Angle C Y axis in Plot JComment C 7 Concentration Selection reset Date C Printout Diameter Summary file n ac C Text file iscarded runs 5 Sort utility
90. opened will depend on the configuration that was present used when the program was previously used If a different configuration is presented select the Layout command on the Window menu and choose the Measurement option PrecisionDeconvolve j g x File Measure Data Setup Window Correlation Function Intensity For Help press F1 ej i Figure 2 11 The Measurement Window i Press the Go button After the data acquisition is complete the display should appear similar to that shown in Figure 2 12 PrecisionDeconvolve 2 Chapter 2 2 11 111 PrecisionDeconvolve File Measure Data Setup Window DEES Br Be eee Correlation Function M 100 R 10 7nm 75255 180074 Figure 2 12 The Measurement Window after Measurement PrecisionDeconvolve32 Chapter 2 Chapter 3 Introduction to PrecisionDeconvolve 3 1 OVERVIEW PrecisionDeconvolve is used to collect process store and print light scattering data The program can 3 be opened by clicking on the PrecisionDeconvolve icon or by selecting it on the Program list which is accessed from the Start menu This manual describes Version 4 4 of PrecisionDeconvolve This chapter discusses e The format of the Main window of PrecisionDeconvolve and the role of the various commands Section 3 2 e The format of the windows used during data acquisition Section 3 3 3 2 THE
91. printout is shown in Figure 7 9 7 6 PrecisionDeconvolve3 Chapter 7 File junQ6 024 printed on Oct 23 08 01 02 2000 Path A Date Tue Jun 06 15 24 50 2000 Operator T Havard Info 91 R 3 99nm 1 0 0 9 0 5 0 5 0 3 0 1 0 01 02 03 04 05 msec 1o 10 10 10 nm Sample time 3 mesec Temperature 29C Radius 40 nm Total channels 54 Viscosity 0 814 cp Width 0 014 Lastchannel 220 Refractive index 1 330 Fraction 90 995 Duration 52 sec Intensity 540697 cnt s Smoothness 1 Background 85 6 Fluctuations 2 Ist cumulant 1 3910 0 01 2st cumulant 1 5810 Distribution over hydrodynamic radius R nm R 54 3 453 377 314 262 5 08 423 152 047 2 17 2 78 304 0 63 8 08 42 3 404 Figure 7 9 A Typical Report from The plot shows the content of the Correlation Function and Distribution window and the five blocks of information The top of the report presents a description of the sample including data file name and location date of measurement name of operator and comments entered in the Sample dialog box see Section 3 2 1 5 The information on the bottom of the report includes a detailed description of the data acquisition and processing grouped into three columns The left column describes the measurement process the middle column describes the sample and the right column describes results Additional information about the various parameters is indic
92. r will shift the whole intensity curve so that the next point will be on this line Closing and reopening the Intensity window removes previous intensity data If intensity data becomes too long to fit into the ntensity window a horizontal scroll bar will automatically appear When the window is when the length of the history record requires scrolling points for the current run appear next to the right edge of the window and the whole graph is scrolled to the left e Red cutoff level If the run has the intensity above the cutoff level it is discarded The cutoff level is reported in counts per second in the left top corner of the window in red e Green intensity exponentially averaged over a number runs specified in the Intensity Management dialog box The photon count rate of the last run is reported in counts per second in the right top corner of the window PrecisionDeconvolve 2 Chapter 3 13 Each point in the intensity history plot represents one run The duration of the run is determined by Parameter Run time in the Measurement setup dialog box and may vary from one measurement to another In addition it should be noted that intervals between measurements are not shown so that the intensity history plot may not be a proper representation of the intensity time dependence and you cannot print the intensity history If you want to preserve and plot the intensity as a function of time during your measurements use the ntensity c
93. rage value will be shown in green 6 2 6 Data Printout The Data Printout tab Figure 6 7 is used to describe how the data should be printed out when the Print command is given Note You can control the content but not the layout of the printout which is designed to fit on a single page If too mach information is requested or printer resolution is low the printout may become crowded The Print preview command File menu can be used to develop a satisfactory printout format Data presentation x Distribution Correlation Panels Intensity record Data Printout Text file content Print parameters Diff coef 1st Cumulant Do not show units Date Comment Tab 10 characters Concentration Parameter names no Angle than characters Parameters in columns Print distribution plot 1st 2nd 3d Print corfunction plo Print distribution values Print intensity plot Apply Cancel Figure 6 8 The Data Printout Parameters The upper portion of the dialog box is similar to Panels tab Section 6 2 4 The lower portion includes the following selections PrecisionDeconvolve2 Chapter 6 6 9 Print in columns Click one of the radio buttons to indicate parameters to be printed in the header or in one of three columns of the printout Parameters listed here can be selected in Configure parameter lists dialog accessible through Parameter Lists command
94. ray on the menu for the sake of clarity all commands on the menus are presented in black in this manual e Open Presents a standard Windows Open dialog box which lists files in the indicated directory PrecisionDeconvolve data files have the pdi suffix e g 123456789 pdi Two or more files can be open at one time The last file that was opened is determined to be the active file e Save data Stores the current data as a binary file that can be opened via the Open command The data is automatically stored in the file and directory indicated on the Sample record tab of the Measurement Settings dialog box Section 4 3 unless the Don t Save check box on that tab is selected or the measurement process was terminated by the Stop command The Save Data command cannot be executed while the correlator is running e Save data As Presents the standard Windows Save Data as dialog box to allow the user to name the data file and store it in the desired folder or to save a duplicate copy of the data file in another directory The data will be stored as pdi file e g 123456789 pdi The correlation function the distribution in the same mode as it is presented in the Distribution window and the intensity record are saved as columns of ASCII numbers ready to be imported or cut and pasted into an external program e g Microsoft Excel 3 2 PrecisionDeconvolve3 Chapter e Save accesses a sub menu which includes the As a text file Update a
95. re 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 United Nations Convention on Contracts for the International
96. red for the correlation window x Intensity record Data Printout Text file content Distribution Correlation Panels v SubtractBase V Show Deviation Show Y axes Scale factor Log time Show Fit Log CF Overlay selected data Figure 6 4 The Correlation Tab PrecisionDeconvolve 2 Chapter 6 6 5 Subtract Base When the Subtract Base check box is selected the baseline is subtracted from the correlation curve To see the baseline of the correlation function deselect this box Only the part of the correlation function above the baseline is of interest ag Note In an ideal measurements the baseline is approximately one third to one half of the initial value of the correlation function Too large a baseline may result from dust in the sample a dirty or scratched cuvette instability of the laser or misalignment of the optics b Show Y axes When the Show Y axes box is checked the Y axis in the Correlation window and the Distribution window will be labeled Both the correlation function and the distribution are normalized so the Y axis in both plots varies from 0 to 1 c Log time When the Log Time box is checked the Time x axis is indicated in Log format d Log CF When the Log CF box is checked the Correlation Function y axis is indicated in Log format e Show deviation When the Show Deviation check box is selected the deviation of the experimentally determined correlation fun
97. rrelation function re computed from the distribution shown in the Distribution window shown if Show fit box is checked on the Correlation tab of Data Presentation dialog box Section 6 2 3 e Green trace The deviation difference between the correlation function and the fit magnified by Scale factor shown if Show deviation check box is checked on the Correlation tab of Data Presentation dialog box Section 6 2 3 e Grey trace The correlation functions of all selected data shown if Overlay selected data check box is checked on the Correlation tab of Data Presentation dialog box Section 6 2 3 When new data is being collected this correlation function window updates after every run Duration of a run is determined by the Run time parameter in the Measurement dialog box 587 Note You can change the scale of delay time axis via the left right arrows 3 4 THE DISTRIBUTION WINDOW The Distribution window Figure 3 20 shows the distribution of scattering particles computed from the correlation function of the active data The histogram represents the intensity of scattering from particles of a certain diffusion coefficient Hydrodynamic radius diameter or molecular weight depending on which option is chosen in the Distribution over list in the Distribution dialog box Section 6 2 2 A discussion on how the distribution is calculated is presented in Appendix A As an alternative you can view the relaxation time distribution
98. s are saved first one after another then the correlation function then the distribution over the same parameter and normalized in the same way as in the Distribution window with highlighted columns marked and finally the intensity record with the cutoff level and accepted runs marked 6 3 PARAMETER LISTS The Configure parameter lists dialog box Figure 6 10 is used to indicate the parameters that should be indicated in the various areas where the user can select the parameters that are to be used displayed printed sorted etc Configure parameter lists 4 List of parameters available 1st Cumulant 2nd Cumulant amp ccepted runs Angle jComment C Panel display C X axis in Y axis in Plot C Selection reset Concentration Date Printout Diameter C Summary file id w C Sort utility Discarded runs Ses 1dn dc C Find utility Figure 6 10 Configure Parameter Lists Dialog Box The field on the left side indicates all of the parameters which are available via the program and the radio buttons on the right are used to indicate the area for which you want to select parameters If for example you want to select various parameters for the Panel Display click that radio button then check the various boxes and finally click apply After you have programmed all of the items for a given item e g Panel display press Apply then select the parameters for some other item W
99. s or diameter e Polydispersity D relative dispersion of the selected part of the distribution over diffusion coefficient If all distribution is selected and smoothness parameter is close to zero this parameter is close to the ratio of square root of the 2nd cumulant to the 1st cumulant e Mw Mz ratio of weight concentration averaged molecular mass to molar concentration averaged molecular mass This is one of the standard indicators of polydispersity in polymer science This value is computed using a relationship between particle size and its molecular weight supplied in the Normalization tab The Conditions tab Figure 3 24 presents analytical conditions These values can be changed by the user as appropriate Deci5 33 Properties x Sample Ft Results Conditions Measurement Normalization Concentration 25 Temperature pH 7 5 0 0083 Viscosity Molarity 10 1 33 Refractive index dn dc 0 002 30 Angle Apply Figure 3 24 Active Data Dialog Box Conditions Tab PrecisionDeconvolve 2 Chapter 3 19 The Measurement tab Figure 3 25 presents information about the parameters used to obtain the data This tab cannot be edited by the operator Dec15 33 Properties Sample Fit Conditions Measurement Normalization Sample Time 1 Run duration Channels 256 0 Total runs Last channel 1024 0 Rejected runs Cancel Apply Figure 3 25 Acti
100. s the intensity of light scattered by all of these particles J N 1 where is the number of particles of i th kind in the scattering volume and 7 is the intensity of the light scattered by each such particle For a continuous distribution of scattering particle size equation A 10 is generalized as follows I D Dq T dD 1 10 where D dD N D I D dD is the intensity of light scattered by particles having their diffusion coefficient in the interval D D dD N D dD is the number of these particles in the scattering volume I D is the intensity of light scattered by each of them The goal of the mathematical analysis of QLS data is to reconstruct as precisely as possible the distribution function 7 D or N D from the experimentally measured function G Es It should be noted that polydispersity is not the only source of non single exponential correlation functions of scattered light Even in perfectly monodisperse solutions interparticle interactions orientation dynamics of asymmetric particles and conformational dynamics or deformations of flexible particles will lead to a much more complicated correlation function than described by equation A 6 These effects are usually insignificant for scattering by particles small compared to the length of the inverse scattering vector q but become important and often overwhelming for larger particles In those cases QLS probes not the p
101. save the data in the Intensity window as a text file Intensity txt The time basis for the intensity data is the time when the first run was initiated Each line in this file describes one run When the Save Intensity command is selected a dialog box is presented that asks if you want to Save with history If you answer Yes each line in the file will contain three fields 1 Time elapsed between the moment when the program was started and the moment when the run was completed 2 Average intensity during the run in counts per second Asterisk if the run was rejected because the average intensity exceeded the cutoff level Section 5 2 The letter 5 is used if the run was stopped and the file was not saved if data was saved the three digits extension of the data file name is indicated If you answer No only the time and intensity are saved A portion of a typical intensity file saved without history is shown in Figure 7 1 Thu Aug 03 09 11 42 2000 time sec counts sec 198 4 174e 004 1198 4 182e 004 198 4 172e 004 197 4 171e 004 197 4 177e 004 1197 4 183e 004 197 4 180e 004 197 4 170e 004 197 4 175e 004 197 4 177e 004 Figure 7 1 A Typical Intensity File The top line of the file shows the date and the time when the program was started Note The data is always saved to the Intensity txt file if you want to start a new file and save the existing data rename the existing file 7 2 PrecisionDeconvo
102. t 1 5 CONTENTS OF THIS MANUAL e Chapter 2 Installation describes how the software is installed e Chapter 3 Introduction to PrecisionDeconvolve describes the main window and all of the commands that are provided in the program In addition it describes the windows that that are presented during data acquisition e Chapter 4 Selecting Operating Parameters describes the various factors that should be considered in selecting data acquisition and display parameters e Chapter 5 Collecting Data describes the data acquisition process e Chapter 6 Viewing and Processing Stored Data discusses the various ways in which stored data can be processed reprocessed to meet the needs of the laboratory e Chapter 7 Saving and Printing Data describes how you can save data and generate reports In addition an appendix that describes the theory of light scattering measurements is included and an appendix with the Refractive Index and Viscosity of water as a function of temperature is included 1 4 PrecisionDeconvolve3 Chapter 1 Chapter 2 Installation 2 1 OVERVIEW This chapter describes the steps required to install PrecisionDeconvolve application software in your personal computer 2 2 LOADING THE SOFTWARE AND CONFIGURING THE COMMUNICATIONS PORT ON THE PERSONAL COMPUTER To load the software onto the personal computer a Place the distribution diskette in the CD ROM drive If your computer is configured for Autorun a Welcome s
103. t If the phase of a wave scattered at the origin is used as a reference the phase of a wave scattered at a point with radius vector r is q 9r as shown in Figure 1 The vector 4 is called the scattering vector which is a fundamental characteristic of any scattering process The length of the vector is indicated in equation 1 47 q ql sin 2 1 where n is the refractive index of medium X is the wavelength of light is the scattering angle Partial cancellation of waves scattered by different parts of the large aggregate reduces the intensity of light scattering by a factor of lot where is an averaged value of the phase factors exp i q r for all monomers The factor should be averaged over all possible orientations of the particle The result of this averaging yields the structure factor 5 Expressions for the structure factors for particles of various shapes can be found elsewhere LASER SAMPLE Ko Figure A 1 The Scattering Vector 4 The path traveled by a wave scattered at the point with radius vector r differs from the path passing through the reference point by two segments and 2 with lengths and respectively The phase difference 15 i 1 where k k k l 27tn X is the absolute value of the wave vector k or k The segment is a projection of r on the wave vector of the incident beam k i e 1 rek k Similarly 1 exfk and thus Ad r
104. t is defined in equation A 2 lt i t i t T gt A2 The notation G T is introduced to distinguish the correlation function of the photocurrent from the correlation function of the electromagnetic field Ge T which is the Fourier transform of the light spectrum G t E t E t t gt A 3 In the above formulae the angular brackets denote an average over time This time averaging an inherent feature of the QLS method is necessary to extract information from the random fluctuations in the intensity of the scattered light PrecisionDeconvolve Appendix A A 3 For very large delay times the photocurrents at moment and t t are completely uncorrelated and G co is simply the square of the mean current i At T 0 GC 0 is obviously the mean of the current squared i Since for any i t i gt i the initial value of the correlation function is always larger than the value at a sufficiently long delay time The characteristic time within which the correlation function approaches its final value is called correlation time For example in the most practically important case of a correlation function that decays according to an exponential law exp T correlation time is the parameter In the majority of practical applications of QLS the scattered light is a sum of waves scattered by many independent particles and therefore displays Gaussian statistics This being the case
105. tforward to show that d 1 n s x jiw Dq dT 0 12 The second cumulant moment of the distribution can be obtained from the curvature second derivative of the initial part of the correlation function As in the direct fit method the accuracy of the real QLS experiment allows determination of at most three moments of the distribution D The first moment D be determined with better than 1 accuracy The second moment the width of the distribution can be determined with an accuracy of 5 10 The third moment which characterizes the asymmetry of the distribution usually can be estimated with an accuracy of only about 100 A 8 PrecisionDeconvolve3 Appendix A A 5 5 Regularization The regularization approach combines the best features of both of the previous methods The advantage of the cumulant method is that it is completely free from bias introduced by a priori assumptions about the shape of J D assumptions that are at the heart of the direct fit method On the other hand reliable a priori information on the shape of the distribution function in addition to the experimental data improves significantly the quality of results obtained by the QLS method The regularization method assumes that the distribution 7 D is a smooth function and seeks a non negative distribution producing the best fit to the experimental data As discussed above the ill posed nature of the deco
106. that you can get an overview of the data As a rough rule of thumb the numerical value of the expected R in nanometers can be used if Ry is unknown 3 5 usec is a good starting point 4 2 PrecisionDeconvolve3 Chapter 4 To change the sample time use up down arrows adjacent to the field or highlight the field and enter the desired value After you have run a short preliminary measurement of your sample note the period of time required for the correlation function to decrease to about 0 4 e times Ideally this time should be approximately between 25 35 of the time scale e g see Figure 4 2 as this is the optimum for the accurate reconstruction of the distribution of the scattering particles If the curve falls too rapidly reduce the sample time and repeat the process until an acceptable time scale for the correlation function is obtained Note In the case of small particles lt 10 nm do not reduce the sample time in psec to less than the expected value of Ry dn nm As an example if the expected R is 5 nm the sample time should not be less than 5 psec m Correlation Function Figure 4 2 Correlation Curve 4 2 1 2 Channels The Channels field is the number of channels in which the correlation function is calculated during measurements channels are updated every sample time The number of channels that can be processed depends on the sample time as shown in Table 4 1 the operator cannot set this numb
107. there is a relation between the intensity correlation function T and the field correlation function Ge T 2 Ero 1 2 G x i 1 y g a 4 amp n Gy 0 is the normalized field correlation function is the average intensity of the detected light and Y is the efficiency factor For perfectly coherent incident light and for scattered light collected within one coherence area the efficiency factor is 1 If light is collected from an area J times larger than the coherence area fluctuations in light intensity are averaged out and the efficiency factor is of the order of J lt lt 1 Low efficiency makes the quality of measurements vulnerable to fluctuations in the average intensity caused by the presence of large dust particles in the sample or instability of the laser intensity A 4 4 Determination of the Correlation Function In PDI instruments the correlation function is determined digitally The number of photons registered by the photodetector within each of a number of short consecutive intervals is stored in the correlator memory Each count in a given interval termed the sample time and denoted Av represents the instantaneous value of the photocurrent i t The series of K counts held in the correlator memory is termed the digitized copy of the signal According to Equation 1 to obtain the correlation function T at t nN n 1 K the average product of counts separated by
108. tion 7 pH 10 Molarity 0 002 dn dc 0 Other required 0 0083 Viscosity 25 Temperature 1 33 Refraction Cancel Apply Help Figure 4 4 The Sample Data Tab 4 2 4 The Sample Record Tab The sample record tab is used to indicate the name of the file etc The default name will be today s date and the number will increment from one for each run These can be readily changed as desired 4 8 PrecisionDeconvolve3 Chapter 4 Measurement Setup E Measurement Intensity Sample data Sample record file name Dect uam Browse path EAPDIdata operator A Smith info B54 1 1Samples Cancel Figure 4 5 The Sample Record Tab 4 2 4 1 Path The Path field is used to indicate the directory into which the data should be stored The absolute path which starts with the drive letter CAdatasample1 should be provided If the path does not exist the error message Provide absolute path will appear If only the last directory in the path does not exist this directory is automatically created 4 2 4 2 Next File The Next File field shows the file name that will be used to save the results of the next measurement The extension which must include three digits 001 is used to consecutively label files with a common file name The date e g Jul20 is the default file name and can be changed as desired 4 3 THE SAMPLE PARAMETERS 4 3 1 Viscosity The viscosity of the sa
109. ts orn eerte e Deeds 6 4 The Role of the Data Menu with Stored Data Display eee 6 12 eM 6 14 Chapter 7 Storing and Printing eee eee eee ee eee ee eret eee Tel SOVELVIEW EE 7 2 SAVING E 7 2 1 Saving Data as a Binary File 1232 The save Command ire fre D o RR ECRIRE Ob EP e DERE sahasbs 733 Printing a R pott ener te Tre eere do Re eder e ec dee bere dore Appendix A General Principles eee e eee eee ee esee eene nee e tea a ana sana sese sa sese caso sapo sa na A 1 Appendix Refractive Index and Viscosity Water eee eee eee eee esee B 1 viii PrecisionDeconvolve32 Table of Contents Chapter 1 Introduction 1 1 OVERVIEW The Precision Detectors PrecisionDeconvolve software application package is designed to provide for Collection of dynamic light scattering data with the Precision Detectors Model PDDLS Batch Light Scattering System the Precision Detectors Expert Series with the Dynamic Light Scattering Module the Precision Detectors Model ALS3000 Automated DLS System and the Precision Detectors Model ALS4000 Automated Expert DLS System Display reporting and exporting of raw and processed data from the above detectors Processing of dynamic light scattering data from the above detectors and dat
110. ure diffusive Brownian motion of the scatterers but also other types of dynamic fluctuation in the solution PrecisionDec onvolve Appendix A A 7 A 5 2 Deconvolution of the Correlation Function 41 Problem The values of T contain statistical errors We have described previously the features of the QLS exp instrument that are essential for minimizing these errors It is equally important to minimize the distorting effect that experimental errors in G T have on the reconstructed distribution function I D exp The distribution D is a non negative function A priori then a non negative function D should be sought that produces via equations A 3 and A 10 the function G e which is the best fit to the experimental data Unfortunately this simplistic approach does not work The underlying reason is that the corresponding mathematical minimization problem is ill posed meaning that dramatically different distributions D lead to nearly identical correlation functions of the scattered light and therefore are equally acceptable fits to the experimental data For example addition of a fast oscillating component to the distribution function D does not change CN considerably since the contributions from closely spaced positive and negative spikes in the particle distribution cancel each other We discuss below three approaches for dealing with this ill posed problem A 5 3 The Direct F
111. ution over f Show Y axis Mol weight 1 R E Show Average Load with main M W normalization peak selected B Cancel Apply Figure 6 2 Data Presentation Dialog Box Distribution Tab 6 2 2 Distribution Tab The Distribution tab Figure 6 2 is used to select the general format of the Distribution window The Distribution field is used to select the type of distribution to be presented The drop down menu presents the Diameter Hydrodynamic Radius Diffusion Coefficient Molecular Weight and Relaxation Time options e Diffusion presents the distribution with respect to the diffusion coefficient cm sec e Diameter presents the distribution with respect to the diameter nm e Radius presents the distribution with respect to the hydrodynamic radius nm Molecular weight presents the distribution with respect to the MW kDa Relaxation time presents the distribution with respect to the relaxation time usec sg Note The directly measured quantity is the diffusion coefficient other parameters are calculated from the diffusion coefficient and are model dependent When the distribution over molecular weight is chosen the user supplied parameters and in Eq 6 1 are employed see next page The reader should note that the software provides a rough estimate of the molecular weight of the particle with given diffusion coefficient The relationship between these
112. ve Data Dialog Box Measurements Tab The Normalization tab Figure 3 26 presents information about the scaling of the data A detailed discussion about scaling is presented in Section 4 4 4 15 33 Properties Sample Fit Results Conditions Measurement Normalization Scaling Law Mol weight 1 R E Cancel Apply Figure 3 26 Active Data Dialog Box Normalization Tab 3 20 PrecisionDeconvolve3 Chapter 3 6 THE SMOOTHNESS DIALOG BOX The Reset Smoothness dialog box Figure 3 27 which is accessed by selecting the distribution function window and selecting Smoothness from the Data menu is used to change the smoothness of the correlation The Smoothness parameter is used to eliminate false spikes or transients in the distribution Reset smoothing i x 10 Smoothness parameter Relative increase in mean square difference between the data and the fit 1 02 due to smothing requirement Choose smothness by deviate increase May take some time Selected Figure 3 27 Reset Smoothness Dialog Box We recommend that a small value e g 10 should be used at first If you see bimodality spikes or transients raise the value until the bimodality disappears and then back off slightly If the smoothness factor is too large the resulting distribution will be broad and stable reproducible but will lack detail which may contain important information On the oth
113. yboard is to be pressed to close the dialog box and preserve the original settings e The APPLY button is to be clicked to change settings without closing the dialog box e Common dialog boxes and commands that are similar to other Windows programs are not described e g the Open dialog box is identical to that used in programs such as Word When we are describing a dialog box or window the name of the window will appear in italics Access the Correlation Function dialog box When a button or a command from a menu is to be chosen the button command is shown in italics To initiate data collection click Start on the menu bar On line help is available by pointing to the field of interest and pressing F1 PrecisionDeconvolve2 Chapter 1 1 3 1 4 FOR ADDITIONAL INFORMATION Precision Detectors maintains a number of facilities to provide additional assistance to the user Technical assistance and service assistance can be obtained from Precision Detectors Inc 34 Williams Way Bellingham MA 02019 Tel 508 966 3847 Tel 800 472 6934 US only Fax 508 966 3758 e mail pdi precisiondetectors com website www precisiondetectors com or your local distributor Precision Detectors publishes Biomolecular Characterization Notes and Polymer Characterization Notes which are newsletters that provide a discussion of light scattering issues of current interest Please contact Precision Detectors to be added to the mailing lis
114. you want to get eliminate duplicate files that are already loaded use the Remove duplicates command in the Data menu The Test mode check box indicates if the system should operate in Test Mode which generates test data without the need for a sample A series of runs in Test Mode is useful to become familiar with the general operation of the PrecisionDeconvolve software and will show you what you can expect in a live measurement situation If a check mark is present in this field it will not be possible to collect actual data b The Hardware settings tab The Hardware settings tab Figure 2 2 is used to set a variety of instrument and communication parameters Hardware configuration E Input Output Laser switch PD Expert Hardware settings Calibration Communication diagnostics Port Baud 38400 800 Wavelenath 90 Angle Laser switch IV Figure 2 2 The Hardware Settings Dialog Box Hardware Settings Tab Set the Port setting to match the communication port to which the correlator board is connected The Baud rate should be set to 38400 the Wavelength should be set to the laser wavelength and the Angle should be set to 90 and should not be edited PrecisionDeconvolve 2 Chapter 2 2 3 c Note In order to use a Baud rate other than 38 400 an internal change must be made to the detector This change requires the assistance of Precision Detectors or an authorized Precision Detectors distri

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