User Manual - Andreas Håkansson
Contents
1. 0 07380216 9 07152722 0 06925228 0 06697734 differential refraq r olar mass 7 0 0647024 7 0 06242746 7 0 06015252 0 05787758 7 0 05560264 0 0533277 7 _9 05105276 7 _0 04877782 7 0 04650288 7 0 04422794 0 041953 7 0 03967806 e61 time min Jtime min 99 d 99 r 99 d 99 r 99 r 99 r 99 r 99 r 99 r 99 r 99 r 99 d 99 r 99 r 99 r 99 r 99 r 99 r 99 d 99 r 99 r 99 r 99 r 99 rr 7 720142401 7 722417341 7 724692281 7 726967221 7 729242161 7 731517101 7 733792041 7 736066981 7 738341921 7 740616861 7 742891801 7 745166741 7 747441681 7 749716621 7 751991561 7 754266501 7 756541441 7 758816381 7 761091321 7 763366261 7 765641201 7 767916141 7 770191081 7 772466021 4 rms radius Figure 3 9 reducedTemplate xls 3 3 A Note on time As seen in Fig 3 2 the Excel file used as an input to the software allows the user to use different time vectors for different detectors Fig 3 10 displays the Rayleigh ratio in black and r ms radius in red from the input file particle80nm xls Here the concentration signal _13 FFF HydRad 2 0 User s Manual covers all of the experiment whereas the r ms signal was only captured where the material actually eluted Also note that the concentration detector was switched on before starting elution The time before elution tpe in Fig 3 10 must2. 0 User s Manual x WHI S gt particle80nm Kompatibilitetslage Microsoft Excel no o eia Start Infoga Sidlayout Formler Data Granska Visa amp d ogr amp j a mes Autot B a Calibri Jui An e B Biraddnyt text Alimant Fi a E pan 7 8 a AY BR J Hsmta format FZU B D A EER i H Centrera verkoume B gt e A eszek Paro Cellformat Infoga Pl Format 2 tenn ae a hoethsred Jrklipp Tecken Justering Tal gt Format Celler Redigering H8 fe 75 8772661812767 A c D E F G HA 1 time min differential refractive index time min rayleigh ratio time min molar mass time min rms radius 2 0 28665266 0 205898995 0 103316164 0 031895025 6 047212581 16743491 75 6 047212581 108 8621033 3 0 278462874 0 20620559 0 095126379 0 02725653 6 055402366 16313139 61 6 055402366 101 265788 4 0 270273089 0 206512185 0 086936593 0 023316038 6 063592151 14841434 45 6 063592151 93 07671129 5 0 262083304 0 21275721 0 078746808 0 019906949 6 071781937 12521215 78 6 071781937 84 25816992 6 0 253893519 0 210456954 0 070557023 0 017034674 6 079971722 11453799 4 6 079971722 77 74266456 7 0 245703733 0 200661343 0 062367238 0 014635608 6 088161507 11292729 84 6 088161507 75 25092709 is 0 237513948 0 196525903 0 054177452 0 012594147 6 096351292 11484373 63 6 096351292 9 0 229324163 0 194810948 0 045987667 0 010855086 6 104541078 13058203 5 6 104541078 86 6260547 10 0 221134377 0 193942838 0 037797882
3. 0 009361385 6 112730863 14681388 96 6 112730863 95 59590929 11 0 212944592 0 193074727 0 029608096 0 008074824 6 120920648 16374610 27 6 120920648 101 9918029 12 0 204754807 0 19168847 0 021418311 0 006974634 6 129110434 17270372 14 6 129110434 102 5100372 13 0 196565022 0 186451665 0 013228526 0 006032936 6 137300219 16839504 55 6 137300219 99 88933685 14 0 188375236 0 1846136 0 005038741 0 005222344 6 145490004 15623646 56 6 145490004 95 67573565 15 0 180185451 0 183420773 0 003151045 0 004560906 6 153679789 14281663 16 6 153679789 88 93757123 16 0 171995666 0 182227945 0 01134083 0 004003181 6 161869575 13499570 44 6 161869575 83 67135551 17 0 163805881 0 180513198 0 019530615 0 003530301 6 17005936 13926277 94 6 17005936 80 52304179 18 0 155616095 0 177366404 0 027720401 0 003109135 6 178249145 14956451 64 6 178249145 78 14322142 19 0 14742631 0 175770652 0 035910186 0 002731337 6 186438931 14915620 58 6 186438931 75 91659185 20 0 139236525 0 174523741 0 044099971 0 002420787 6 194628716 14406108 93 6 194628716 73 16258381 21 0 131046739 0 175925415 0 052289756 0 002206499 6 202818501 14314750 51 6 202818501 72 12253572 22 0 122856954 0 177327089 0 060479542 0 002029199 6 211008286 14296023 86 6 211008286 70 89021596 23 0 114667169 0 172405534 0 068669327 0 001876688 6 219198072 14075320 5 6 219198072 69 43273031 24 0 106477384 0 163285719 0 076859112 0 00174508 6 227387857 13722501 03 6 227387857 66 1985628
4. This opens the Geometry settings window see Fig 3 4 Use the edit boxes 1 in Fig 3 4 to specify the geometry of the channel A schematic image of the channel has been inserted 2 in Fig 3 4 in order to help the user FFF HydRad 2 0 User s Manual Geometry Settings Callibration UI gamma globulin Ferritin Thyroglobulin e 3 Hydrodynamic diameter nm 11 2 A Calibration Protein Geometrical parameters Measured retention time s 120 Channel width at injection point b0 m Channel width at outlet bL m Calculate channel thickness Tip to tip channel length L m Length of inlet triangle a0 m Calculated Channel thickness w m j Length of outlet triangle aL m Focusing point of sample z m Cross flow in callibration m3 s Miscallenous Flow from channel in calibration m3 s V Exclude tip in computing Rhyd V Use full expression for R Save Load setting Gamma 1 con e G 5 Close Figure 3 4 Geometry setting Next specify the hydrodynamic radius of the calibration particle used Values for a number of common calibration proteins has been inserted in the list 3 in Fig 3 4 and can be chosen by clicking However since these values has been calculated from tabulated diffusion coefficient see table 3 1 they depend on factors such as temperature solvent and accuracy in the tabulated values A recommendation is to always chose Manual
5. numeric settings compare with Fig 3 7 3 1 7 Resetting Closing FFFHydRad In order to reset the program clear results and loaded files press Reset in the upper left corner in the main window Note that settings are kept Close the program by pressing Exit in the lower right of the main window or use the menus File gt Exit 3 2 Tutorial 2 Using FFFhydRad with a reduced dataset FFFHydRad can be used if using other detectors than the ones described in Section 3 1 3 2 1 Using a template Excel file Start by opening the reducedTemplate xls file The fields that need to be altered by the user are marked in yellow First put whichever signal is used to calculate concentration and the corresponding time in the columns marked 1 in Fig 3 9 Then insert the times for which hydrodynamic radius needs to be obtained in the column marked 2 in Fig 3 9 note that multiple values are needed see Section 3 2 2 if data is needed for single time points Make ol s FFF HydRad 2 0 User s Manual sure the columns marked 2 and all columns to the right of it have the same number of rows with data and that the columns in 1 and the columns to the left of them all have the same number of rows with data Do not forget to save the file with a new name before closing Now run the analysis as described in Section 3 1 and save results to file Open the xls file created by the software to see the results 3 2 2 Manual estimation of single tim
6. 1 License and copying This software is free to download install use and modify for all applications if appropriately cited Users are encouraged to cite the papers Nilsson et al 2006 and Hakansson et al 2012 1 2 Version History MAPLE based version by Bj rn Bergenstahl and Lars Nilsson 2005 GUI Beta 1 for testing at Food Technology Lund University 2008 FFFHydRad 1 0 released 2009 FFFHydRad 1 3 released 2011 FFFHydRad 2 0 released 2013 1 3 New to version 2 0 Complete rework of documentation Improved speed for reading result files Support for reading data from xIsx files Users do not need MATLAB Statistic Toolbox Converted toa MATLAB app Output time is in the same scale as input time Results saved to both txt and xls format Rms and hyd based densities logically named in result files 1 4 Required Software FFFHydRad 2 0 is supplied as a MATLAB App and requires MathWorks MATLAB version 2012b or later Versions working with earlier versions can be supplied 1 5 Bugs questions or suggestions Reports of bugs questions about the software comments and enhancement suggestions could be sent to andreas a hakansson se Please include FFFhydRad 2 0 in the subject line 1 6 Developers FFFhydRad has been developed by Andreas Hakansson Lars Nilsson and Bjorn Bergenstahl The Food Colloids Group Lund University Sweden FFF HydRad 2 0 User s Manual 2 Installing and running FFFHydRad 2 0 2 1
7. 9 25 0 098287598 0 159921857 0 085048897 0 001607702 6 235577642 14001499 68 6 235577642 64 60783805 26 0 090097813 0 15842706 0 093238683 0 001481353 6 243767427 14533866 96 _6 243767427 63 86217235 _ 4 44 gt Hi 191130 3 ps 80nm a manm saaw F igure 3 2 A sample data file If a correctly formatted xls file has been chosen in the open dialogue 1 in Fig 3 1 after a few seconds the logarithm of molecular mass in g mol as a function of time position is shown in the main window 1 in Fig 3 3 Above the graph 2 in Fig 3 3 the name of the chosen file could be seen Also the status bar at the bottom of the window turns yellow 3 in Fig 3 3 and the complete path to the chosen file is displayed E FFFHydRad 2 0 S a File Help FFFHydRad 2 0 INPUT OUTPUT particle80nm xis 75 eu 7 45 1 Rms 74 Analyze 7 35 7 3 0 200 400 600 800 1000 First gt 100 Last K mf gt 600 9 iR g Settings Parameters Export Fig Time Rrms and MAL Export Fig ODE interpolat M lues eens Geometry settings ean values Po wera roe Normalize results Rhyd nm E Numerics Density rms kg m3 Density hyd kg m3 M based on Figure 3 3 Open data 3 1 2 Calculating channel height Before analysis could be performed the correct channel geometry must be specified and the channel height must be calculated from a calibration experiment To do so click Geometry settings 2 in Fig 3 1
8. FFFHydRad 2 0 User s Manual FFF HydRad 2 0 User s Manual What is FFFHydRad Flow Field Flow Fractionation is a versatile technique for separating and characterizing molecules and colloidal particles originally invented by J C Giddings During operation a mixture of molecules and or particles is injected and are allowed to elute through the channel Due to differences in hydrodynamic interactions different component will have different retention in the channel which gives rise to efficient separation Elution time depends mainly on the translational diffusion coefficient of the component Thus the translational diffusion coefficient henceforth the diffusion coefficient from a component can be estimated from the elution time For simple FIFFF configurations such as symmetrical channels and or constant cross flow the diffusion coefficient can be calculated using simple analytical expressions Wahlund amp Giddings 1987 Litz n 1993 Kirkland et al 1992 However most FIFFF experiments of today are run in more complex settings such as with asymmetrical channels and programmed cross flow since this has been shown to increase quality and decrease the time of analysis In these cases it is not as straight forward to estimate the diffusion coefficient or equivalently the hydrodynamic radius from a measured retention time FFFHydRad uses numerical methods to calculate diffusion coefficient from elution time The technique i
9. Installation The software is supplied as an installation file FF FHydRad mlappinstall Download the file and open MATLAB Click the APPS tab 1 and then click Install App 2 see Fig 2 1 4 MATLAB R2012b OME LOTS S Tis ap l earch c nentatior gt oe 5 Cr q A Get More Package Curve Fitting Optimization MuPAD PD Tuning System Signal Analysis Image Instrument SimBiology MATLAB Coder MATLAB Distributio Apps App tebook identification Acquistion Contro Compiier Figure 2 1 Installing a MATLAB app 2 2 Opening FFFHydRad To open FFFHydRad simply click on the APPS list 1 in Fig 2 1 and expand the list by clicking the down arrow 3 in Fig 2 1 Locate FFFHydRad in the list and click on the icon FFF HydRad 2 0 User s Manual 3 Using FFFHydRad 2 0 Tutorial This section is a detailed tutorial on how to use the software for reading and analyzing data A sample data file particleSOnm xls have is supplied with the installation and will be used in the tutorial The data is from a validation experiment described in Magnusson et al 2012 3 1 Tutorial 1 Using FFFhydRad with full dataset Running FFFHydRad will open the main window shown in Fig 3 1 BB FFFHydRad 2 0 Lo ja So File Help x FFFHydRad 2 0 INPUT 1 OUTPUT Read file Reset First af gt Last gt Mean values M fa mol Rrms nm Rhvd nmi Numerics Densitv rms ka m31 Densitv hvd f
10. be given in the Flow Settings 3 in Fig 3 5 for accurate determination Fig 3 10 also shows the void time t to clarify the difference the void time is calculated in the software from membrane geometry and channel height it does not need to be supplied by the user Concentartion signal i fims nm aT nm signal 0 5 10 15 20 time Figure 3 10 Zlustration of how to treat elution time in the evaluations _14 FFF HydRad 2 0 User s Manual References Hakansson A Magnusson E Bergenstahl B Nilsson L 2012 Hydrodynamic radius determination with asymmetrical flow field flow fractionation using decaying cross flow Part I A theoretical approach Journal of Chromatography A 1253 120 126 Kirkland J Dilks C Rementer S Yau W 1992 Asymmetric channel flow field flow fractionation with exponential force field programming Journal of Chromatography 593 339 355 Nilsson L Leeman M Wahlund K G Bergenstahl B 2006 Mechanical Degradation and Changes in Conformation of Hydrophobically Modified Starch Biomacromolecules 7 2671 2679 Li P Hansen M In Field Flow Fractionation Handbook Schimpf M E Caldwell K Giddings J C Eds John Wiley amp Sons Inc New York 2000 Litz n Anne 1993 Separation Speed Retention and Dispersion in Asymmetrical Flow Field Flow Fractionation as Functions of Channel Dimensions and Flow Rates Anal Ch
11. e points peak values When only hydrodynamic radius corresponding to a single time point is needed e g for peak value a MATLAB function manualRhyd m has been supplied First runt an analysis with FFFHydRad using a sample Excel file such as particle80nm xls The actual data in the file is not needed but make sure to fill in all geometrical and flow settings correctly in the FFFHydRad windows Hit Analyze and exit FFFHydRad Now in the MATLAB Command Window type load interm manualRhyd timeIwantRfor 1 utStruct while substituting timeIwantRfor with the time in seconds for which the hydrodynamic radius is needed Time should be given in the same time scale as the data in the Excel file see Fig 3 10 Example The peak in Fig 3 10 is located at 8 8 min 528 s load interm manualRhyd 528 1 utStruct will return At t 528 s Rhyd 42 039 nm r z men Fal a gt reducedTemplate Kompatibilitetslage Microsoft Excel 3 e Somme Infoga di t D nska Visa a a Fant z te oe Autosumma Ay oa Calibri in Aa Die say Radbryt text Allmant i F fs m a B gr a amp ri yi F Ez FS S a4 Cent 200 Villkorsstyrd Format ellformat Infoga Ta ormat Sortera o y KU Hi D A z zA Centr lum 4 formatering so bort ta iltrera Fl A B time min 0 092001681 _0 089726741 00874518 0 08517686 9 08290192 0 08062698 0 07835204 20 0760771
12. em 65 461 470 Magnusson E Hakansson A Janiak J Bergenstahl B Nilsson L 2012 Hydrodynamic radius determination with asymmetrical flow field flow fractionation using decaying cross flow Part II Experimental evaluation Journal of Chromatography A 1253 127 133 Wahlund K G Giddings J 1987 Properties of an asymmetrical flow field flow fractionation channel having one permeable wall Analytical Chemistry 59 1332 1339 Schimpf M E Cladwell K Giddings J C 2000 Field Flow Fractionation Handbook Wiley _15
13. er with a graph of apparent density over rms radius 3 in Fig 3 6 and radius quotient over Molar Mass 4 in Fig 3 6 However before looking at the results it is highly recommended to have a look at the interpolation underlying the estimations Press the Export ODE interpolation button 2 in Fig 3 6 This opens a plot showing the numerical solution to the ode and interpolation Make sure that the interpolations fits well to the ode solution see Fig 3 7 If there is a bad fit can be seen by a very jumpy saw tooth looking interpolation try decreasing the Imax setting under the Numeric settings until it looks smooth as Fig 3 7 E oaao ee a aM FFFHydRad 2 0 J INPUT OUTPUT Export figure a Export figure Y o 2 6 pA co Oo 3 3 x a oa 120 DA 7 45 E 0 9 7 za 115 m 4 E 0 85 Ssh i aS 7 35 S 3 4 T amp 105 0 at 0 200 400 600 800 First 100 0 75 4 s ae 31 35 32 325 3 35 M 7314 7 316 7 318 7 32 7 322 7 324 Last af ml gt 600 tyms nm Molar mass g mol y Export Fig Time M and Ri j Export Fig Time Ri and VC Settings Parameters Export Fig Time Rrms and MALS Export Fig ODE interpolation EE TEE Mean values Basic settings 7 M g mol 20720261 32 V Flow settings Ri ikesi 33 0996 Normalize results Time before elution s 150 Rhyd nm 40 5007 l Numerics initial cross flow m3 s 1 6667e 08 Density rms kg m3 105 4478 1 Half life of cross flow s se Den
14. fe of cross fow fs Oow Densty hyd kg m3 126 9552 particle8Onm M based on 74 1609 inear decrease rate m3 s2 3 476 12 Flow from channel m3 s 1 6667e 08 Save data to file Minimal cross flow m3 s 1 6667e 10 EXIT Figure 3 8 More settings 3 1 6 More settings Additional settings are available after clicking the Basic settings 1 in fig 3 8 or Numerics 2 in Fig 3 8 check boxes Basic settings include dynamic viscosity of the carrier liquid and experiment temperature FFFHydRad works by solving the retention equation for a large number of particle sizes 1 e the retention time for a large number of hydrodynamic radii are calculated An interpolation is needed in order to translate this to values for all the experimental times The first numeric option is the type of translation scheme The recommended setting is 20 which corresponds to a piecewise linear interpolation For historical reasons the software also allows for fitting the solution to a polynomial and evaluating the polynomial for intermediate values Different degree polynomials can be chosen by choosing degrees between 2 9 The setting DO is the ration between two succeeding simulated diffusion coefficient decreasing this number towards 1 increase the computational load and the resolution Imax is the total number of simulated sizes Always make sure that the interpolation fits the simulated data after analysing the date with new
15. ko m31 M based on Geometry settings Settinas Parameters Basic settings 2 Flow settings Status bar EXIT Figure 3 1 The main window 3 1 1 Reading data The first step is to open a file containing FFF data Click the Read file button 1 in Fig 3 1 This will open the standard file open dialogue of your operating system Select the appropriate Microsoft Excel file software supports both xls and xlsx files in the list and press OK For this tutorial pick particleSOnm xls The data in the xls file must be arranged in a manner recognized by the software See Section 3 2 for information of how to use the software if you do not have all these detectors but only need to calculate diffusion coefficient corresponding to a given retention time The data should be arranged in eight columns as displayed in Fig 3 2 The first two columns are the time and the corresponding RI readings The next two columns are time and Rayleigh ratio from MALS after that time and molar mass and then finally time and rms radius As could be seen in Fig 3 2 the time vectors can differ the program also allows for missing values in the rms Radius For more information on time scales see Section 3 3 In the sample file shown in Fig 3 2 the first row of the Excel sheet contains a text description this is recommended in order to keep track on the columns but optional for FFFHydRad the software will skip text rows in reading the data FFF HydRad 2
16. ls file was read The first file contains the calculated hydrodynamic radius together with the supplied data and some statistical information This file has the same name as the xls file but with _analyzed added at the end The second file contains all settings used in calculating the results and has the extension settings If the files already exist in your directory you will be asked if you want to overwrite them An xls file with results added to the original file name will also be created The names of the newly created files together with paths will be shown in the green status bar at the bottom of the screen when the files have been correctly saved a ik FFF HydRad 2 0 User s Manual Bi Razo i R TA E eon FFFHydRad 2 0 INPUT OUTPUT Read file Reset particle80nm xis Export figure Export figure N n N o Analyze ont k oO o a Apparent denisty kg m9 an ond Q O 7 Se a 0 200 400 600 800 1000 a A a 2 R 0 75 4 100 31 S15 232 325 ec ekon gl 7 314 7 316 7 318 7 32 7 322 7 324 600 Fms nm Molar mass g mol Export Fig Time M and RI Export Fig Time Ri and VC Export Fig Time Rrms and MALS Export Fig ODE interpolation a Mean values M g mol 20720261 32 v Flow settings Rrms nm sii Soon Normalize results Time before elution s 150 Rhyd nm 40 5007 Initial cross flow m s 1 6667e 08 Density rms kg m3 105 4478 Half i
17. ly specified and put the known hydrodynamic radius of the calibration particle in nm in the edit box Next put the measured retention time in seconds of the calibration particle in the box below When changes are made to the settings the w box 4 in Fig 3 4 turns red to indicate that the calculations have not been updated Press Calculate 4 in Fig 3 4 to update the value FFFHydRad allows for different degrees of completeness in the theoretical expression for the retention equations Many studies neglect the retention from the small tip of the channel tip retention can be excluded or included from the calculations by clicking a tick box 5 in Fig 3 4 Two different expressions for the retention parameter R are also available The full expression refers to the expression derived by Giddings et al 1987 including excluded volume effects Including the full expression is highly recommended see H kansson et al 2012 for a comparison the full expression corresponds to IV in table 1 of the reference It is possible to save and load channel settings for reuse 6 in Fig 3 4 Click the Close button when you are done with the calculation make sure that the w field is green indicating that the calculations have been updated after the last changes FFF HydRad 2 0 User s Manual Table 3 1 Some protein standards for channel calibrating Hydrodynamic diameter in PBS at 25 C nm BSA 06 Gamma globulin Thyroglob
18. ntific comments The main part of the user guide consists of the tutorials in Section 3 describing how to use the software for calculating channel height and analysing hydrodynamic radius Note that the software was developed for use with FIFFF coupled to both RI and MALS detectors A special tutorial is supplied in Section 3 2 for the user who only wants to calculate hydrodynamic radius from retention time without using these other detectors FFFHydRad 2 0 User s Manual Screenshot BB FFFHydRad 2 0 p File Help INPUT OUTPUT rticle80nm xls Export fiqure__ Export figure _ 75 130 0 95 A by 0 9 eu 120 2 Orms 74 Analyze E gt 0 85 a E 110 5 0 8 a a 1000 Fret IN o lt 100 0 76 31 32 a6 34 wale ashi mael poah ape maril lat ff ti CSCCC Y CCOC C NCC OS 6000 a EA Pra le Siar REER j Settinas Parameters 7 Sipain hG TO Geometry settings ean values C Basic settings M fa mon 20720261 32 J Flow settings 3 maaan PETOT Time before elution s 150 Rhvd Inmi 40 5007 E Numerics initial cross flow m3 s 1 68 6667e 08 Densitv rms ka m31 105 4478 Half life of cross flow fs 360 Density hvd Ika m31 126 9552 _ particle80nm Li j rate m3 s2 3 47e 12 M based on 74 1609 Flow from channel m3 s 1 6667e 08 Save data to file Minimal cross flow m3 s 1 6667e 10 FFF HydRad 2 0 User s Manual 1 General information 1
19. s based on the method originally proposed by Nilsson et al 2006 An accurate measurement of the channel height is needed in the calculations but cannot easily be obtained from direct measurements due to compression and solvent effects Channel height must therefore be obtained from a calibration experiments of a sample with known diffusion coefficient FFHydRad includes functions for performing these calculations according to the technique described by Hakansson et al 2012 The diffusion coefficient can be used to calculate the hydrodynamic radius of a particle or molecule Most molecules and particles are not spherical and it is often of interest to obtain some description of shape FIFFF is often coupled to a Multi Angle Light Scattering MALS detector The MALS detector will give information about a different length scale of the molecule most often the rms radius Combining a MALS detector with elution time analysis thus gives two different length scales of the molecule or particle under investigation The ratio of length scales has been used in order to obtain information about the geometrical shape A theoretical description of the algorithms used by FFFHydRad can be found in Hakansson et al 2012 A validation study can be found in Magnusson et al 2012 FFFHydRad is packed and distributed as a MATLAB app for ease of use and installation What ts in this User Guide 1 General information 2 Installation 3 Tutorials 4 Scie
20. sity hyd kg m3 126 9552 particles0nm Linear decrease rate m3 s2 3 47e 12 M based ont aiian Flow from channel m3 s 1 6667e 08 Save data to file 5 Minimal cross fiow m3 s 1 6667e 10 EXIT Figure 3 6 Displaying the results 10 FFF HydRad 2 0 User s Manual 44 Interpolation Numerical solution to ODE 42 hyd nm 38 36 330 340 350 360 370 380 390 time s Figure 3 7 A good ODE Interpolation From Fig 3 6 it can be seen that the mass weighted hydrodynamic radius of the sample is 40 50 nm The z weighted value can be seen in the command prompt of MATLAB after each evaluation together with some additional statistics DLS experiments indicate a diameter of 87 4 nm which fit rather well with the predicted result The rms hydrodynamic radius ratio can also be seen in the results For a spherical particle the ratio should be close to 0 77 The experimental results indicated a ratio in the interval 0 77 to 0 9 Variations seems unsystematic indicating a uniform shape across the investigated distribution Each graph has an export button in the top right corner Pushing this button will draw the graph in a new window here it could also be edited and exported to different image formats by MATLABs standard utilities Results can be saved to txt and xls files by pressing the save button 5 in Fig 3 6 Two new text files will be created in the same directory as where the x
21. tled Flow settings 3 in Fig 3 5 displays a number of new settings Chose time before elution see Section 3 3 initial cross flow flow rate from channel If using a decreasing cross flow it is also advised to enter the minimal cross flow allowed by the apparatus Note the SI units FFFHydRad allows for decreasing cross flows either from exponential decrease or linear decrease Exponential decaying cross flow Enter the half time of the cross flow flow rate in seconds in the box and press the enter key The box turns yellow to indicate that you are using exponential decay FFF HydRad 2 0 User s Manual Linear decaying cross flow Enter the decay rate in the appropriate box and press enter The yellow marking moves to the linear decrease box to display that the analysis 1s performed with linear decay Constant cross flow Put a very large number or inf MATLAB notation for infinity in the Exponential decay half time box and press enter Once satisfied with the time interval click Analyze 5 in Fig 3 5 to proceed Analysing the data will take a few seconds depending on your computer and operating system Once analysis is complete a summary of the results will be displayed in the MATLAB command window 3 1 5 Displaying and saving the results When completed the status bar at the bottom of the screen will turn green Resulting statistics of the selected data range will be shown in a list 1 in Fig 3 6 togeth
22. ulin Data from Schimpf et al 2000 3 1 3 Choosing interval If we now return to the main window Fig 3 5 A plot of the data showing either the logarithm of molar mass M or the rms radius in nm is displayed upon loading data Note that the x axis do not refer to time but to the numbering of time points in the experimental data i e 106 refers to the 106 th sampling time of the detector Sliders 1 in Fig 3 5 and edit boxes 2 in Fig 3 5 allows the user to choose what part of the data to include in the analysis The current position of the selected interval is displayed in the graph by the green rectangle The rectangle turns red if errors in the interval specifications are found i T O E o a eses FFFHydRad 2 0 INPUT OUTPUT Read file Reset particle80nm xis ba 78 5 Rms 7 4 Analyze 0 200 400 600 800 1000 First a Fj gt 100 2 Last Ki d 1 gt 600 Settings Parameters Geometry settings Mean values Basic settings 3 V Flow settings Rrms nm Time before elution s 150 Rhyd nm intial cross flow m3 s 1 6667e 08 Density rms kg m3 x Half life of cross flow s C Density hyd kg m3 z M based on Linear decrease rate m3 s2 3 47e 12 Flow from channel m3 s 1 6667e 08 Minimal cross flow m3 s 1 6667e 10 I m e a 2 e a C y y ll 2 a Figure 3 5 Choosing the analysis interval and altering flow settings i 3 1 4 Flow setting Ticking the box enti
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