Home

DSC Data Analysis in Origin® Tutorial Guide

Contents

1. Double click or press Return to set the markers 30 Lesson 3 Integration The area between the markers is the range that will be integrated To integrate the selected range Since you have created a baseline but have not subtracted it from the data integrate by choosing Integ from Baseline from the Peak Menu If you had subtracted the baseline from the data you would integrate by choosing Integ from 0 from the Peak menu The area between the two data markers is integrated The integration results copy to the Script Window 0 0014 8AMPLETDSC CP BASE 0 0012 0 0010 0 0008 o 0 0006 S n 0004 a 9 0002 Im Script Window x File Text Edit Hide Integ of Sampleidsc cp from SAMPLE1DSC_CP BASE i 86 212 47 80183 gt 73 080256 789 T1 2 8 080049 If you wish you can reset the markers and try again Each time you integrate the results are pasted to the Script Window To clear the integration shading from the plot window select Refresh from the Window menu The procedure that copies the integration results to the Script Window is a macro called EndInteg EndInteg is defined in the DSC CNF file and can be redefined to customize the output to suit your needs For more information see the LabTalk Reference Manual available separately from MicroCal Displaying the Integration Results The Script Window is a full featured text editor Use it to edit the integration text save th
2. Done 6 Now change the value of n from 2 0 to 1 0 Click on 10 Iter several times Note that the new fit curve is much worse than the original fit curve Chi 2 is ca Five times larger and that the experimental data curve is now substantially broader than the fit curve This means the data are most consistent with the idea that dissociation into subunits occurs simultaneously with unfolding for this polypeptide 7 Change the value for n from 1 0 to 4 0 and fit the data again It is again obvious that the fit is not as good as the original fit using an n value of 2 0 which suggests that the polypeptide is morre likely to contain two dissociable subunits rather than four 53 DSC Data Analysis Tutorial Guide Fitting Example 3 The Absolute Cp button will facilitate calculation of Absolute heat capacities for proteins this is discussed in more detail in Appedix IV starting on page 70 To illustrate the function of this button there are 5 raw data files of chymotrypsinogen solutions at five different concentrations covering a 10 fold range from 0 258 to 2 58 mg ml Step 1 Input the data The 5 raw data files used for this example are Vcht9bs dsc Vchtl5bs dsc Vcht12bs dsc Vcht13bs dsc and Vcht14 dsc The concentration of the sample in each scan is 258 516 1 03 1 55 and 2 58 mg ml respectively The samples were run on a VP DSC at 60 deg hr using the passive response mode for each scan A buffer buffer reference trace has
3. If you have not yet exited the baseline session do so now by clicking OK or Cancel in the baseline session menu bar Select Cursor Draw Baseline from the Peak menu in the standard menu display bar The cursor changes to a small cross hair Double click with the mouse to place each baseline point Each point connects to the next with a straight line The XY coordinates of the point appear in the Data Display Tool You can place any number of points you wish You can also use the arrow keys to place points Click once with the mouse to place a cross hair on the screen Use the UP DOWN LEFT and RIGHT arrow keys to move the cross hair Press RETURN to set the position of a point When you are satisfied click on the Pointer tool in the toolbox to set the baseline Once you have created a baseline you can use the Adjust Baseline option to fine tune the default baseline To adjust the baseline Select Adjust Baseline from the Peak menu The pointer becomes a cross hair To adjust a baseline point click and drag with the mouse Or use the arrow keys select a point with LEFT or RIGHT arrows move up and down with the UP or DOWN arrows move left and right with CONTROL LEFT or CONTROL RIGHT arrows When you are satisfied double click on a data point or press Return to set the baseline The baseline points in Cursor Draw Baseline and Move Segments by Cursor connect with a straight line by default Connect type and
4. Note that NormDATA contains two buttons which are used in the subtract baseline procedure Creating a Baseline The final step before deconvolution is to start the baseline session and plot a baseline for your data You will then decide whether or not to subtract the baseline and based on that decision choose from one of three curve fitting models To start the baseline session Choose the Start Baseline Session command from the Peak menu Based on the normalized data Origin calculates and plots in red the left and right linear line segments from which to determine the baseline A new menu bar appears containing the baseline session command menus iif Microcal Origin UNTITLED NormDATA BEE OK Adjust Baseline Cancel Window la xl Cessna cool cens Ran elal ms 4 pe alae ts Sis glo 15 10 E Sample 1dsc_cp Het 5 0 30 40 50 60 70 80 90 Temperature C Isles REC N00 For Help press F1 Astan BY Microsoft Word DSC T aJ Exploring Origin50 rrn n Microcal Origin U A MicroCal Origin C ORI zi Qv 330FM kcal m ole C Origin lets you adjust the left and right linear segments and provides several options for connecting the segments to create the baseline For this example we will simply accept the default segments as plotted and connect them using a progress baseline For greater detail see Lesson 4 Baseline Determinations 15 DSC Data Analysis Tutorial Guide To
5. Step Baseline There are two step baseline options Step at Peak is based on a linear connect baseline and places the step directly under the peak Step at Half Area places the step at the position of half the integrated area Move Baseline by Cursor This option lets you manually adjust the baseline by specifying the position of each of the baseline data points To use the Move Baseline by Cursor option first create a baseline by selecting one of the baseline creation commands described above Next select Move Baseline by Cursor from the Baseline menu Origin places twelve data points along the baseline you just created To move the data points either click and drag with the mouse or use the left and right arrow keys to select a point and the up and down arrow keys to move the selected point points can only move vertically When you are satisfied with the baseline double click or press Return A spline connection option is automatically applied to the new baseline You can remove the spline connection by changing the baseline s plot display options see page 42 below Lesson 4 Baseline Determinations Cursor Draw Baseline Cursor Draw Baseline is another method for creating a baseline The Cursor Draw Baseline method does not require you to enter the baseline session set linear segments or select a baseline option In this method you simply draw the baseline directly in the plot window To use the Cursor Draw Baseline option
6. To remove the data markers Select Data Markers from the Data menu to remove the check mark Origin hides the data markers Plotting the Integration Area Data 33 DSC Data Analysis Tutorial Guide The integration area data is saved as a function of X which is temperature for calorimetric data into a data set named integ area The leading underscore signifies that integ area is a temporary data set which will be deleted whenever memory is running low To access the worksheet of integ area data or use it for any other purpose you should first copy integ area to a permanent data set To copy the integration area data to a new data set Click on the Edit menu in the Script Window menu bar Note that the Script Execution command is checkmarked which means you can currently execute LabTalk scripts directly from the Script Window On a new line type the following script exactly as it appears Be sure to include the single space before integ area and also before my test Please note that the data set name must consist of two parts separated by the underscore character The first part will be the worksheet name in this case the worksheet will be named my the second part will be the column name of the data set copy integ area my test Press Return Origin should append a semicolon to the text you just entered This indicates that the script was executed For more information about the Lab Talk scripting language pl
7. 2 dete del pde reip ert e ipe tbe pedet teen 40 Cursor Draw Baseline iste e Deere snes UTC TORRES CEPR SEE OR ER ER A A ESAO EER 43 Lesson 45 General Comment PI pith cous coe ans cuaebuens a sie eaa Oa ESS 45 Fitting Example usos eve ouai ae e beri a Ve haa 46 Fitting Example p 51 Fittings Example 3 ins oce Deep pu eene ettet t entitas htt p eee 54 Lesson 8 Other Useful Detaills scsssssssssssessessssersessssessessssessesessessessssessessssessesessessessssessesessessesesseseesees 59 2C chi sqr Miiinizafioh conatus Aem otutegatuetA dumme utat etate etum A tu MI UE 59 Response Time VP DSQ eese epe reme pit de entree tnde ente ceste 59 Line Types for Fit Curves once EI REIR a rro E EE DR sunbed Seep be eb ERE EEES 62 Inserting an Origin graph into Microsoft Word sese nennen nennen rennes 63 Appendix Equations Used to Deconvolute DSC Data eerie eere eene entente tnn on statu stata tn sno 65 I Independent Trarisitions 4 nehme ete me Ree esed e it ee este iere dre inen 65 II Sequential Transitions up Beh ke eae RIP Rei ei aatis 67 III Single Two State Transition with Subunit Dissociation eseeeeeeeeeeeeeeeenen enne eene 69 TV Absolute Heat Capacity of Protel Sapes eine eae en pe o ee aee rite de eene 70 Introduction Introduction to DSC Data Analysis MicroCal Origin is a
8. Note the difference d nf p is usually referred to as the number of degrees of freedom The above equation states that the Chi squared value of the fit is equal to the sum of the squares of the deviations of the theoretical curve s from the experimental points divided by the number of degrees of freedom Since there is no weighting it can be seen that the calculated values are dependent on the magnitude of the scale and the number of data points Response Time VP DSC The VP DSC is the first calorimeter to have operator selectable response time It is made possible by passage of the amplified AT signal temperature difference signal between the two cells into the computer for digital processing before the feedback voltage is returned to provide feedback power to the sample cell This allows software control of the gain the ratio of power applied to the sample cell relative to the magnitude of the AT signal which in turn controls the response time of the instrument to heat effects occurring in the 59 DSC Data Analysis Tutorial Guide 60 cells The passive mode with zero gain is best to use for studying systems having very slow transients broad transition peaks slow scan rates or isothermal studies since the baseline is quieter and more stable due to the absence of feedback noise For systems with fast transients sharp thermal transitions and or fast scan rates shorter response times must be used to avoid distorting the shape
9. already been subtracted from each data scan 1 Click on the Read data button in the RawDSC window and open the Vcht9bs dsc Vcht12bs dsc Vcht13bs dsc Vcht14bs dsc and Vcht15bs dsc data files Import Multiple ASCII 21x Look in CN samples J e j 3 Vchtl3bs dsc 3 Vchtl 4bs dsc BEES B VchtSbs dsc 3 Vchti2bs dsc File name vehtt 5bs dsc Add File s OK Files of type osc Data dsc Delete File s Cancel C OriginS0 samples cht9bs dsc C OriginS0 samples chtl 2bs dsc C OriginS0 samples cht1 3bs dsc C OriginS0 samples cht1 4bs dsc C Origin50 samples cht1 Sbs dsc Your Origin graph will look like below 54 Lesson 5 Curve Fitting ii Microcal Origin UNTITLED RawDSC ile Edit View Graph Data Math Peak DSC Tools Format Window Help la xl IAA ce esas an aS AE all s Ges Blo Subtract Reference 0 0008 0 0006 4 0 0004 4 0 0002 4 0 0000 4 Cp cal C 0 0002 4 Response Time 0 0004 4 0 0006 4 T T 0 20 40 60 80 Temperature C Ralte r 7 s o ERFEZEEEEEEEENEEEENEEEEEEENI Not all points are plotted for Vch EPAR E sr xj Notice the square around the line type of the last file opened in this case data set Vcht15bs dsc in the legend of the graph This indicates it is the active data set When you click on the Absolute Cp button the routine will convert this data set Step 2 Converting raw
10. E ech vannintomnchbnashasnentsn thle batace deans E eben 7 subtracting Reference Data sz icc 6e Lame hide i oerte qu er ere eit iet ete ee ES 11 Viewing Worksheet Data ineeiieo in ni tra etr tpe eee dunes tee tpe eere epe cloves NER EN peau eror Epi e ech ee 12 Normalizing the Data oet REEL RARE UE CER E O E EA A OREO EF TERES 13 Creating Baseline reete n o dece oerte eee dedere eee oet e et dne ee gates 15 Fit ne the Datacenter ete p appe 17 The Fitting SesslOD siet re din ean iia E WERE E EUR I ee e EEG MIR oie 18 Calibration iore ire REPE EUER EE EE ATHE S RE EDT EK e SERRE VIP E ERE EQ REL I MEEA CH NERA 21 Lesson 2 Using the Data Selector To0l 4 eeeeeee eese eese eene eee ee seen ette seine tasto setas etas sense tas ease ease tosta 23 Lesson 32 Integration c ssis iSS 29 Setting Range and Integrating the Data eene nennen nennen trennen rennen ene 29 Displaying the Integration Results 1 itte terree te e te ge ete eges 31 Plotting the Integration Area Data sess nennen rene enr EE entente tenete 33 Lesson 4 Baseline Determinations eese esee eene tesa tn snnt tatus ots instat tata suse ss tasas essen sosta en sn sensn 37 Startin the Basehne SeSS10T e eee meret ot aequ deter beer cepe bite Erbe e Eee 37 User Adjustment of Linear Segments essere nnne trennen eene enne enne eret tenete 39 Choosing a Baseline Option
11. T have been indicated We can express AH T in terms of its temperature independent value at the midpoint T na and the heat capacity change for the transition AC A Le AH T AH AC T Ta 8 and then integrate eq 5 from Tma where K4 T is unity to an unspecified temperature T AH T Caf T K T ex a f E a ui a m 9 al P RT T RT mA a 9 mA Now eq 7 using substitutions from eq s 8 and 9 for each transition A B may be used to calculate the value of the system heat capacity C at any temperature T once values of the temperature independent fitting parameters Bo B1 T na AH AC A gt TB AHAB AC p gt are given To begin curve fitting with Origin the operator indicates the number of transitions needed to fit the experimental DSC heat capacity curve C Texp and then initializes all of the Tm values so that Origin can provide guesses for each of the other fitting parameters Knowing these it then calculates C T from eq s 7 9 and compares these values with C Texp Using Marquardt methods based on non linear least squares the guesses for each parameter are then improved the calculations carried out again and this iterative process continued until there is no further improvement in the fit of ze calculated CT to the experimental Cy T exp as indicated by a minimum value of chi Model for Independent Two State Transitions excluding DCp Effects When all heat capacity changes AC A ACB are assu
12. WYSIWYG in the graph window Type 12 into the center points text box Select Black Line from the Background drop down list box Note that if you select none for the Background the box may not be erased till you perform a screen refresh select Window Refresh 32 Lesson 3 Integration Click OK The Text Control dialog box closes and the integration results text updates to show your changes Click and drag on the integration results text to place the text in the upper right corner of the plot window To see the graph exactly as it will appear when printed switch to the Print View view mode View mode is chosen from the View menu To change view mode Click once on the View menu The View menu commands appear Notice that Page View is checkmarked showing it to be the selected view mode Page View uses the screen driver to display font information Select Print View from the menu Print View is now the selected view mode Print View gets font information directly from the printer driver and is a true WYSIWYG view mode The plot window text is now sized and positioned correctly rrr Iun unc cras so a Temperature PC Before proceeding with this lesson re select the View Page View command For more about view mode see Chapter 12 Plotting Customizing the Page Display in the Origin User s Manual or select the Index tab from the Help Origin menu option and enter View mode into the upper text box
13. also learn about the various baseline creation options that are available Baseline commands are located under the Peak menu Starting the Baseline Session When you are ready to create a baseline for your data you must start the baseline session In this lesson we will create a baseline for the SAMPLE DSC data file To open the SAMPLE data file If you are continuing from a previous lesson first select File New Project or click on the New Project icon to open a new Origin project Click on the Read Data button in the RawDSC plot window The Import Multiple ASCII dialog box opens Shortcut You can Double click on Samplel dsc in the Files list program Origin to SAMPLE1 DSC is located in the Samples sub folder of the Origin50 folder The always Look in the path and file name will be added to the lower list box Samples folder or any other folder by selecting Click OK File Set Default Folder and entering a new path SAMPLEI opens into the RawDSC plot window in the text box SAMPLES C cp x a so a 70 ao sa Temperature C 37 DSC Data Analysis Tutorial Guide Before creating the baseline you would normally subtract reference data and then normalize the data on concentration For the sake of brevity we will bypass those steps here They are discussed in detail in Lesson 1 Basic DSC Data Analysis To start the Baseline Session Choose Start Baseline Session from the Peak menu Origin ente
14. and Chi 2 is updated This option can be used for a quick simulation of curves 1 Iter One iteration of the Marquardt Levenberg routine At the end of the iteration the fit curve fitting parameters and Chi 2 are updated 10 Iter Same as above but carries out ten iterations The number of Iterations can be changed in the fitting sessions window by switching to the Advanced Mode of the fitting session click on the More button and selecting Options Control Then change the Max Number of Iterations from the drop down list box Select Function Opens the Select Function dialog box which allows the user to select a built in or previously saved function for fitting Select Dataset Opens the Select Dataset dialog box which allows the user to change the data file for fitting the temperature range and or Fitting Step Size used to fit the data More Switches from the Basic mode of the curve fitting dialog box to the advanced mode which contains its own menu bar for access to more options for fitting procedures Done Exits the Fitting Session dialog box returns to the NormData window the fit curve is plotted and the fitting results are plotted in the Results text window and to a text box in the Normdata window 19 DSC Data Analysis Tutorial Guide Use the 1 Iter amp 10 Iter buttons to control the iteration of the fitting cycles as described above Watch the fitting parameters and Chi 2 update in the Fitting Sessio
15. and click on the Add File s button Note When assigning filenames to raw data no hyphens dashes may be used in the filename the names should not begin with a number nor should the letter e be followed by a number Repeat the above procedure for the file Refl dsc note you may enter any number of files at the same time by this procedure Click OK to plot all files in the lower list box into the RAWDSC plot window ESsaMPLEIDSC cp REFIDSC_cp Temperature eC 10 Lesson 1 Basic Data Analysis You can read any number of data files into the same RawDSC window Note that when multiple data plots appear in the same window you can set the active data plot by clicking on the plot type icons next to the filename in the legend A black box around the line symbol type indicates the currently active data plot Editing fitting and other operations can only be carried out on the active plot Subtracting Reference Data In most experiments you will have obtained both sample reaction heat data and reference data and will need to subtract the reference data from the sample data To subtract the REFI reference data from the SAMPLEI sample data Click on the Subtract Reference button in the RawDSC window The Subtract Reference Data dialog box opens The first file opened in this case Samplel DSC will appear in both the Data and Reference drop down list box Note that the data set listed in the Reference box will be subtracted f
16. any order Lesson 2 Using the Data Selector Tool Lesson 2 Using the Data Selector Tool The Data Selector is a toolbar tool that is used to select a segment of plotted data Subsequent math editing or fitting operations will be carried out on the selected segment only In this lesson you will learn how to select a plot segment delete the segment set the data plot display range and reset the display range A sample DSC data file SAMPLE2 DSC has been included for your use with this lesson To open the SAMPLE2 DAT data file Select File New Project to open a new project Click on the Read Data button in the RawDSC window Shortcut Mp The Import Multiple ASCII dialog box opens Click the New Project button on the Standard toolbar Double click on Sample2 dsc in the Files list box Sample2 dsc is located in the Samples sub folder of the Origin50 folder The path and file name will be added to the lower list box Click OK The Sample2 dsc file opens into the RawDSC plot window Sample2dsc cp Temperature C 23 DSC Data Analysis Tutorial Guide To select a segment of plotted data Click on the Data Selector tool in the toolbox The Data Selector becomes highlighted The pointer turns into a cross hair Two data markers appear one at either end of the Sample2dsc_cp data plot in the RawDSC1 plot window Fe famplezdsc_cp m D oO a U Temperatur
17. happens simply click on the window Restore button d You will then be able to switch levels Simultaneously Running DSC and ITC Configurations If you purchased both the DSC and ITC software modules the installation program will have automatically created icons in the MicroCal Origin50 program group for the DSC and the ITC software This allows you to run two configurations simultaneously The most likely reason to do this would be if you have both the MicroCal ITC and the MicroCal VP DSC MCS DSC or MC 2 DSC calorimeters and you intend to run them both on the same computer Double click on either icon to run that configuration View Mode Each Origin plot window can be viewed in any of four different view modes Print View Page View Window View and Draft View Print View is a true WYSIWYG What You See is What You Get view mode This view mode displays a page that corresponds exactly to the page from your hard copy device Exact font placement and size is guaranteed at some sacrifice to screen appearance since the printer driver fonts must be scaled to fit their positions on the page this will not harm the appearance of true vector fonts This is a slow process and screen refresh speed suffers as a result Thus reserve the Print View mode for previewing your work prior to printing Origin automatically changes to Print View mode when graphics are exported to another application and when printing The view mode automatically
18. integrating 29 35 normalizing 13 15 opening worksheet for 12 13 44 plotting 34 35 selecting a plot segment 24 25 set as active 11 subtracting reference 11 12 Deconvolution see Curve fitting Dissociation w dCp 51 53 E EndInteg macro 31 F Fitting see Curve fitting Fitting parameters defined 45 46 for four models 17 Fitting Session 18 20 H H parameter heat change 45 46 Hv parameter van t Hoff heat change 45 46 I Import Multiple ASCII dialog box 9 10 Importing data 6 Installing Origin 3 4 Integration 29 35 copy integration area data 34 displaying integration results 32 EndInteg macro 31 formatting integration results 32 33 integrating selected range 31 integration results 29 31 plot integration area data 34 35 setting integration range 29 31 Isothermal scan 6 L LabTalk script language 34 Layer Control dialog box 34 35 Line Types for fit curves 62 63 M Math dialog box 21 Menu levels 4 5 71 DSC Data Analysis Tutorial Guide N Normalizing data 13 15 O Offsetting multiple DSC files 8 Opening and Analyzing Previous Versions of Origin ORG Documents 6 Opening DSC data files 9 10 Opening Worksheet window 12 13 P Page Preview mode 33 Page View mode 5 21 33 Plot window maximizing 38 Print View mode 5 21 R Reference data subtracting from sample data 11 12 Registering Origin 4 Response Time 59 62 Fee
19. of the thermogram even though this will result in lower sensitivity In the current version of VPViewer there are four response time selections in the experiment set up window No Gain Passive Low Gain Mid Gain and High Gain with respective relaxation time constants of 40 9 18 0 10 4 and 5 6 seconds The respective designations in the header of the data files are 0 Passive 5 Low Gain 75 Mid Gain and 1 High Gain Some protein transitions as well as those of polynucleotides are fairly sharp on the temperature axis so that detectable peak broadening may result when using the passive mode at usual scan rates In such a case faster response times or slower scan rates may be used to improve peak resolution in real time However Origin contains a Response Time button which when activated quickly removes small amounts of response time broadening for any of the four response times This post run capability sometimes avoids the necessity of introducing extraneous noise associated with faster response times as well as allowing the operator to quickly determine if peak resolution is satisfactory for any data set Whenever peak broadening is severe however this post run correction will not be completely accurate To illustrate the functionality of the Response Time button we have included the Origin project ResponseT opj in the C Origin50 Samples directory This Origin project contains the results of two successive experiments run on a sa
20. plot the baseline Select Progress Baseline from the Baseline menu Origin calculates and plots the progress baseline un a D E o E T x su E 70 Temperature CO The progress baseline is created such that each point of the baseline in the peak region reflects the extent of progress of the reaction For a single transition such as this the area between the progress baseline and the data approximates the total heat change due to the transition Click OK in the menu bar to exit the baseline session Origin asks if you want to subtract the baseline data Click YES Origin subtracts the baseline data from the normalized data then exits the baseline session kcal mole deg Temperature eC 16 Lesson 1 Basic Data Analysis Fitting the Data Origin provides four models with which to fit a curve to your data All four models use the Levenberg Marquardt LM non linear least square method In general you will want to get the best possible fit using the simplest model that fits the data Curve fitting is discussed in greater detail in Lesson 5 This section will serve to acquaint you with the basic procedure The four curve fitting models are located in the DSC menu They are Model Parameters Model 1 2 State T H Model 2 Non 2 State Tm H Hy Model 3 2 State w dCp Tm H Cp BLO BL1 Model 4 Dissoc w dCp Tm H Cp BLO BLI n All models except model 4 offer three menu selection options mo
21. returns to the selected view mode after the operation is complete Page View provides faster screen updating than Print View but does not guarantee exact text placement on the screen unless you are using typeface scaling software such as Adobe Type Manager Use Page View mode until your application is ready for printing or copying to another application Change to Print View mode to check object placement before exporting copying or printing Window View expands the page to fill up the entire graph window Labels buttons or other objects in a graph window that reside in the gray area of the page are not visible in Window View mode Draft View has the fastest screen update of the four view modes In Draft View the page automatically sizes to fill the graph window This is a convenient mode to use when you are primarily interested in looking at on screen data Note that view mode will not affect your print outs Only on screen display is affected Note About Data Import DSC Data Analysis Tutorial Guide MicroCal offers three versions of the DSC instrument the MC 2 the MCS and VP DSC All together there are four different data collection software packages available for use with these instruments 1 a DOS based and 2 a Windows based package for the MC 2 3 a Windows based package for the MCS and 4 a Windows based version for the VP DSC This version of Origin will accept data files from any MicroCal DSC instrument and fro
22. the graph as an object and it cannot be edited by Origin tools although it may be resized or reposition in the Word document When you share your graph Word displays the graph as an object which can be edited by Origin or linked to Origin and updated when the Origin graph changes Please refer to the Origin manual for more information about Creating a Graphic Presentation Importing your graph into Word e Create your graph in Origin and when you are satisfied with its appearance select Edit Copy page e Open your Word document and click at the location where you want the graph to be located e Select Edit Paste Special e Select Origin Graph Object from the As list box e Select the Paste radio button e Click OK Linking your graph into Word e You must first create your graph in Origin and then save it as part of an Origin project OPJ e Open the saved Origin project if it is not already opened that includes the desired graph window e Make the desired graph window active select Edit Copy Page e Open your Word document and and click at the location where you want to insert the graph e Select Edit Paste Special 63 DSC Data Analysis Tutorial Guide 64 e Select Origin Graph Object from the As list box e Select the Paste Link radio button e Click OK After your Origin graph is linked to Word you may return to the original Origin graph and make changes to the graph These changes can be reflected i
23. to plot the files from the lower list box SAMPLE1 DSC opens into the RawDSC plot window Sampletdse cp 60 Temperature C Before integrating you must plot a baseline for the data You will then choose to either integrate from the baseline or subtract the baseline and integrate from zero see Lesson 4 for more about baseline determinations Plotting a baseline 29 DSC Data Analysis Tutorial Guide Select Start Baseline Session from the Peak menu Origin starts the baseline session and plots two red linear baseline segments Select Progress Baseline from the Baseline menu Origin connects the linear segments with a progress baseline Select OK from the menu bar Origin asks if you want to subtract the baseline Click No Origin exits the baseline session Sampleidsc cp SAMPLE1DSC_CP BASE 2 sS amp a o Temperature C To set the integration range Click on the Data Selector tool in the toolbox Two data markers appear on the SAMPLE curve note that these appear on SAMPLE and not on the baseline curve because SAMPLE1 is currently set as the active data plot in the Data List under the Data menu Using the mouse or the arrow Keys position the data markers approximately as shown below If you are not sure how to position the markers refer to Lesson 2 in this tutorial s4MPLEIDSC_cp SAMPLEIDSC_CP BASE so a 70 a sa Temperature C
24. water then key in heat pulses to produce a calibration file as described in your DSC instrument manual You then open the calibration file into Origin and check that the heat pulses match Origin s Y axis scale readings 1 Note that the measured peak heights of 9 008 6 007 and 3 006 indicated on the figure below agree very closely with experimentally entered pulses of 9 6 and 3 mcal minute Pulse heights can be readily measured using the data reader tool from the Toolbox One way to do this is to take a reading immediately before the pulse and immediately after the pulse average these two readings and then subtract the average from a third reading taken in the middle of the pulse 9 008 mcal min 6 007 mcal min X axis title When you open a calibration file you must remember not to normalize on scan rate This means you should remove the checkmark from the Scan Rate Normalization checkbox in the RawDSC plot window before you read in the data file If you neglect to do this and leave the scan rate normalization on your calibration results will be wrong Also note that when unnormalized DSC data are read into Origin the Y axis units are mcal min rather than cal deg which is obtained when data are normalized You now know how to perform basic data analysis of DSC files using Origin The following lessons in this tutorial each look at a specific aspect of DSC data analysis in detail You may work through these lessons in
25. DSC Data Analysis in Origin Tutorial Guide Version 5 0 October 1998 e E MicroCal The Calorimetry Experts Using Gus scientific plotting software to analyze calorimetric data from the MicroCal MC 2 MCS or VP DSC differential scanning calorimeters Table of Contents Table of Contents Introduction to DSC Data Analysis ssiicsscsisessscsctesocsssscccsesscesscssencssnsassnssesscsssnscsseetdesnsssiecsesantenvnesSacsseuesssedessees 1 Getting Started assener anisa M 3 System Requirements eee eire d EEEE EEEE REA EE EE EEE EE EE E iS 3 Installing Oren sic n eee REDE Ina an Vere aue 3 Registering with MicroCal Software sees neen eren eene enne tnnt tenete 4 Starting OTIPID ctetu cete too UR nb mettait ice RE Ote foaeitest eee neret 4 Menu Levels eet be et ere itr ed itt d deed id te eR eR Pe EIS 4 Simultaneously Running DSC and ITC Configurations esee neret 3 MaewiMode estet ir HERR HIFU POENI EM ERE NUUAM ENREN E LEEREN REDE EF TE Re is 5 Note About D taImpott hee tree ter e eti e e e Ro eet esee 5 Opening and Analyzing Previous Versions of Origin ORG Documents eene 6 Lesson 1 Basic DSC Data Analysis c iscsccsersssssssssscsecssssesassssecsesseseecssnnssetstssssecersassndeseesesseasessedssnessosesssoacsecness 7 Star ng OfIglrz ee voices cr etr E ERR ERE REG INO Reset WI P Ee reste ree ERR 7 Reading DSC Data T nein aa a
26. Inc DSC program icon from the submenu Menu Levels This DSC version of Origin comes with three distinct menu configuration options or menu levels four if you also purchased the optional ITC software module Each menu level has its own distinct menu commands After Origin has opened you may change a menus level option under the Format Menu command option The four menu levels are General Full Menus Select this option to run Origin in the generic non instrument mode This menu level contains no instrument specific routines but does contain many general data analysis and graphics routines that you may find useful for other applications You will find these general routines described in your Origin User s Manual DSC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific DSC data analysis routines ITC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific ITC data analysis routines Note that this menu level is available only if you purchased the optional ITC software module Short Menus Select this option to run Origin with menus that are an abbreviated version of the General Full Menus configuration Getting Started Note that you cannot switch to a new menu level if there is a maximized plot window or worksheet in the current project A warning prompt will appear if you try to switch levels while a window is maximized If this
27. It is usually only when a given model is unable to fit the data that complexity should be increased by adding more fitting parameters Fitting Example 1 To better understand the flexibility of Origin s curve fitting process work through the following example The example uses data from a protein of structure ABA i e two identical and non interacting A domains and a single B domain Head to head covalently linked dimers can readily form ABA type of structures By comparison of results from different fitting models structures of this type can be recognized from DSC data If you are continuing from the previous lesson first select File New Project or click on the New Project button to open a new project Step 1 Prepare the data The first step in any fitting procedure is to open the DSC data file and reference data file then prepare the data by subtracting reference data and normalizing the result on concentration We have dealt with these data preparation procedures in Lesson 1 of this tutorial Here we will briefly review the steps involved 1 Click on the Read Data button in the RawDSC window and open the SAMPLES DSC and REFS DSC data files located in the Origin50 Samples folder 3 Click on the Subtract Reference button The Subtract Reference Data dialog box opens 4 Select SampleSdsc_cp for the Data drop down list box entry 5 Select Ref5dsc cp for the Reference drop down list box entry 6 Click OK to subtract Ref5
28. ains buttons that are used to import DSC data Two DSC data files SAMPLEI DSC and REF1 DSC are included for your use with this lesson SAMPLE1 contains sample DSC data REF1 contains the associated reference data Both are located in the Samples sub folder of the Origin50 folder These files were obtained on the MC 2 DSC instrument using the Windows data collection software Files from MCS or VP DSC data collection software packages are similar but have different header information To open the SAMPLEI DSC sample data file and REF1 DSC reference data file Click on the Read Data button in the RawDSC plot window The Import Multiple ASCII dialog box appears DSC Data Analysis Tutorial Guide Import Multiple ASCII 24 x Look in E samples J e B 3 Vchtl3bs dsc 3 Vchtl 4bs dsc 3 Vchtl5bs dsc 3 Vcht8bs dsc 3 Sample5 dsc 3 Vchtl 2bs dsc File name JRet1 dsc Add File s Files of type Dsc Data dsc Delete File s Cancel C S riginB samples S ample1 dse C A riginBOsamplesNRef1 dsc Navigate to the Samples folder from the Look in drop down list box Note You may configure Origin to always look in the Samples folder or your own folder by selecting Files Set Default Folder and entering the path in the text box To add the file Samplel dsc to the lower list box double click on the file Sample 1 dsc in the File Name list box alternatively you may click on the file
29. al data and any plots you have made from the data To save a project select the File Save Project or File SaveProject As command The project saves as a OPJ file in Origin 4 0 or higher To open a project use the File Open command To import a data file you will use the buttons that are attached to the RawDSC plot window The RawDSC plot window contains six buttons four of which are used for data import These buttons are described below DSC Data Analysis Tutorial Guide BUTTON NAME Read Data Offset Scan Rate Normalization Delete Time Column Response Time VP DSC Absolute Cp VP DSC FUNCTION Reads a data file into the project Use this button if the data were generated by MicroCal s data collection software This is useful for offsetting repetitive scans so they do not overlap on the Y axis Click once on the number 0 000 in the offset text box to open the offset dialog box Enter the offset you wish in the Step Size box enter the offset in mcal even though the data are plotted in cal and click OK Click on either scroll arrow so the proper positive or negative Y offset for the next data set appears in the offset field For each successive data set the offset will advance automatically by the same increment If this option is selected when you open a data file Origin converts from the experimental data units of mcal minute to units of cal degree You should always leave this box checked except when yo
30. anges for each step Using the K values all of the f values can be related to fo so that l Jed 4b vds 17 f l K K K K K K 1 K K K hehe Mae 9 ee 16 where Q 1 K K K K K K t 17 Eq s 16 may be substituted into eq 15 which may then be differentiated with respect to temperature to obtain the system heat capacity Since AH values are treated as temperature independent in Origin s sequential model most of the derivatives involve using the relation se AH AH pat T 2 KKK ae The final expression for the heat capacity of the system then becomes c m in ae enin MEY crm ripa eate ry e rye n NED 19 RT Q RT Q where the temperature dependent quantities have been indicated in the usual way The equilibrium constants can be evaluated in terms of the temperature independent AH parameters from 68 Appendix Equations Used in Deconvolution AH 1 1 n exl Fr a ml AH 1 1 K T ex 2 20 a T A E 20 Using eq s 19 20 and fitting parameters Kj AH K AH the fitting procedure is carried out as described above for the models involving independent transitions III Single Two State Transition with Subunit Dissociation It sometimes happens with biological macromolecules having subunits that subunit dissociation occurs simultaneously with thermal unfolding i e A nA K lay 21 4 where n is the number of dissociable subunits a
31. ased data collection software and so we will be using only the DSC data file opening procedure If your own data files are generated with DOS data collection programs you must open them via the MC 2 procedure Refer to Lesson 1 for more information about data import Opening and Analyzing Previous Versions of Origin ORG Documents To open a previous version of an Origin Document project select File Open This menu command opens the Open dialog box Select Old version ORG from the List Files of type drop down list Select the desired file from the list box and click Open to close the dialog box and open the document You may then make formatting changes and print the graph If you wish to analyze the previous version of the document you must update the document to version 5 0 To update a previous version of Origin Document project select File Update to Origin 5 0 Interface All templates will be updated to new templates compatible with version 5 0 You may then analyze the data with version 5 0 Please note when you update the Origin templates to Origin 5 0 most of the text labeling on the graphs will be lost including the fitting parameters If you want to save the old fitting parameters text you must copy the text before you update to Origin 5 0 To copy the fitting parameter text or any other text right click anywhere in the text box and select copy After you update to Origin 5 0 you may right click in any of the 5 0 templat
32. capacity for unfolding the A domain and where the term in brackets will be repeated for each domain involved in unfolding Remembering that fa 1 fa then the fractional concentration of the primed species can be readily expressed in terms of the corresponding equilibrium constant K 3 1 K Fis which can then be differentiated to give 4 j p i The derivative on the right of eq 4 is known from elementary thermodynamics 2554 AH 5 or RT 2 where AH N is the so called van t Hoff heat change for the reaction which corresponds to the heat change for the cooperative unit which actually participates in the reaction Substituting from eq s 3 5 into eq 2 then gives 65 DSC Data Analysis Tutorial Guide 66 K AC K AH AH C Cpu _ 4 e 6 I K 14 K RT Eq 6 is perfectly general at this point and can be applied to either two state or non two state transitions so long as all parameters are evaluated at the same temperature T Model for Independent Two State Transitions including ACp Effects If we assume each transition is two state then all of the above van t Hoff AH values become equal to the calorimetric AH values If we further assume that Con can be expressed as a linear function of temperature Con Bo B1 T then eq 6 becomes K T AC K T AH T 1 K T 1e k T RT C T B B T 7 where the temperature dependent parameters C T KA T and AH
33. cted plot segment is deleted from the plot window The selected values are also deleted from the SAMPLE2 worksheet The data markers are removed You may select Edit Undo to replace the data IE seMPLE2DSC cp su Temperature 9C 25 DSC Data Analysis Tutorial Guide Data markers are only meant to be displayed temporarily They will disappear as soon as another data set or plot window becomes active To select a segment of data for a longer period of time use the Set Display Range command Say that you would like to display or manipulate a central range of data in the SAMPLE2 data plot but you do not want to delete any of the data This is an ideal use for the Set Display Range command To set data plot display range Click on the Data Selector tool click on the right data marker and position it as shown below Then click on the left data marker and position it as shown IL SAMPLE2DSC cp eee Ren su Temperature 9C Double click or press Return to set the markers Choose Set Display Range from the Data menu The data outside the selected range are hidden from view seMPLE2DSC cp sa Temperature 9C 26 Lesson 2 Using the Data Selector Tool You may expand your data to fill the plot by selecting Graph Rescale to Show All if you wish All math editing and fitting operations will be done on the display range only The data outside the display range are not deleted they are merely hidden Use
34. dBack Modes 60 relaxation time constant 60 Response time button 60 S Scan rate normalization 8 and instrument calibration 22 Screen View mode 33 Scripting 34 Set Default Directory 10 Starting Origin 7 Subtracting reference data 11 12 System Requirements 3 T Text formatting 21 32 33 Tolerance 50 V View mode and screen refresh rates 5 available types 5 View Mode 33 W Window View mode 5 72
35. data to absolute heat capacity values 1 Click on the Absolute Cp button Absolute Cp The Absolute Cp dialog box will open with the following default parameters DSSS Cancel Specific volume ml gm Thermal expans 1 deg E41 Concentration mgm ml sis Cell Volume ml 0 512 File name vchtisbsds 2 For this example all the default parameters are correct Click OK Literature value for the partial specific volume of chymotrypsinogen is 717 ml gm and the coefficient of volume expansion is 7x10 deg 55 DSC Data Analysis Tutorial Guide A new window named AbsCp will be created With the converted data plotted as shown below if Microcal Origin UNTITLED AbsCp MEE EE Math Peak DSC Tools Fomat Window Help dei xl saaal 24 2E 2 gale t Sle Esc Subtract Reference wd Normalize Concentration Absolute Cp cal gm deg Temperature C fr a a 1 7 n o t For Heb press F1 DIM o ix 3 Now hold down the Ctrl key and press the tab key once to switch back to the RawDSC window 4 Click on the next data set in the legend to make it the active data set e chr bzusc cp Ne chri zbzdz cp Ne chri 3bzdz cp En chd aesas sp chri ebzaz cp 5 Click on the Absolute Cp button again Repeat steps 1 4 for each data set till you have completed the data conversion for all 5 data sets Your gra
36. del 4 offers only a Cursor Init option 1 Cursor Init allows cursor initialization of each transition T m s 2 No Init accepts the previously defined parameters if any 3 Simulate allows you to create a transition by entering user defined parameters Tools Format Window Help Set Indep Sequ Model Indep 2 State Cursor Init 2 State Mo lnit 2 State Simulate Non 2 State Cursor Init Non 2 State No Init Non 2 State from 2 State Non 2 State Simulate 2 State w dCp Cursor Init 2 State w dCp No Init 2 State w dCp Simulate Dissociation w dCp Cursor Init We will be using only the Cursor Init options in this lesson In the previous section you subtracted a baseline from your normalized data Models 1 and 2 require you to subtract the baseline before fitting while model 3 requires that you NOT subtract the baseline Model 1 is the simplest model so let s try fitting to this model first To fit a curve with the 2 state model model 1 17 DSC Data Analysis Tutorial Guide Select 2 State Cursor Init from the DSC menu Origin integrates the Samplel data then opens a dialog box asking you to enter the number of transitions You will fit this data with a single Tm so enter a 1 and click OK wT Ista p Number of peaks i Cancel Double click to set the T for this peak Or click once use the arrow keys to position the cursor then press RETURN Set Tm equal to abou
37. dsc by concentration To quickly switch between Origin windows press and hold down the Ctrl key while pressing the Tab key 14 Click on the RawDSC window to make it active or select RawDSC from the Window menu Click on the Normalize Concentration button A dialog box opens Enter the sample concentration in mM L and the cell volume in ml For this example enter 0 08 into the Concentration mM L text box Samplel dsc was at a concentration of 80 UM L and 1 12 in the Cell Vol ml text box The concentration will automatically appear if you originally entered it into the header of the raw data file Cell volume is set in the CELL1 CON file in the folder containing the data acquisition program and is stored in the header information of each data file cal deg gt cal deg M for Sampleldsc cp Cancel Concentration mM L 0 08 Cell Vol ml L 12 Click OK The Sampleldsc cp data plot is removed from the RawDSC plot window RawDSC closes and a new plot window NormDATA opens The normalized data are plotted as Sampleldsc cp in the NormDATA plot window iii Microcal Origin UNTITLED NormDATA 5 x E Eile Edit View Graph Data Math Peak DSC Tools Format Window Help B x seame B S nes es SIE Blo E Sample 1dsc_cp Cp kcal mole C Temperature C Talele r 7 n o aa For Help press F1 rrTITOuImNmm SRE 1 Lesson 1 Basic Data Analysis
38. dsc cp from SampleSdsc cp 7 Click on the Concentration Normalization button 8 Enter 0 074 mM L and 1 22 ml in the dialog box and click OK Lesson 5 Curve Fitting After completing these eight steps your screen will show the normalized SAMPLES data plotted in the NormDATA plot window cm a a E E so a Temperature C It is important to remember that data must always be normalized on concentration before doing curve fitting to any of the fitting models Step 2 Plot a baseline As we have discussed elsewhere in this tutorial the next step is to plot a baseline for the data Note that the SAMPLES plot shows flat baseline segments on either side of the transition with a AC in between The transition itself actually starts and ends at about the positions shown by the arrows below kcal moale deg so a Temperature C To create a good progress baseline for this data you may wish to manually adjust the linear baseline segments so that they are parallel to each other and coincident with the baseline data Origin does not use the position of the endpoints of the linear segments to calculate the progress baseline only the slope and offset You are thus free to create linear segments of any length which makes it much easier to align the segments parallel to each other 47 DSC Data Analysis Tutorial Guide 48 To create the baseline 1 Select Peak Start Baseline Session Origin calculat
39. e C Position the cross hair on the left data marker hold down the mouse button and drag the marker to the right approximately to the position shown below Or use the arrow keys to move the marker use the Left or Right arrow keys to select a marker use Control Left or Right to move the selected marker hold down the Control Shift key to increase the speed PLEASE NOTE If you are using the mouse to move the left cursor you must be directly on the marker before you click and drag the marker if the mouse cursor is away from the left cursor and you click and drag the mouse the RIGHT marker will be positioned at the mouse cursor To return the right marker to the desired position click and drag the mouse at the old position 24 Lesson 2 Using the Data Selector Tool EJ ample2dsc cp Oo e pa o 2 e Oo 4c Temperature Co To set the markers double click in the plot window press Return pressing the escape key with your left index finger is equivalent to pressing Return and this allows you to keep your right hand on the mouse or click on any toolbar tool The markers are set and the Pointer tool is re selected unless you selected a different tool Any math editing or fitting operation now performed will affect only the selected plot segment For example you could delete the segment To delete the selected plot segment Choose Cut or Clear from the Edit menu or press the Delete key The sele
40. e text to a file or copy and paste the text with the clipboard To copy the integration results to the plot window 31 DSC Data Analysis Tutorial Guide Click and drag with the mouse to highlight the integration results in the Script Window Choose Copy from the Script Window Edit menu The text is copied to the clipboard Script Window Alernatively to copy and paste you Eu may right click on the highlighted IUE AMILEiDSC CP BRSE text select copy from the menu then 8 t Ctrl4X right click where you want to C Ctrl C position the text label and select P Ctrl paste I Del v Script Execution Choose Edit Paste from the menu bar along the top of the Origin project window The integration text is pasted to the RawDSC1 plot window the active window 1s 4MPLEIDSC_cp SAMPLEIDSC_CP BASE anr LE1DSC p tom SAMP LEIDS Tm 61ID1z3 Ti 000 Temperature C The text as pasted is too large and placed poorly Let s reformat and reposition the text and add a text border To change the text style Double click on the integration results text in the plot window The Text Control dialog box opens From here you can edit the text set font style color and point size rotate the text and add a border Note that the Text Control dialog box is divided into two display boxes all text entries and formatting is done in the upper text box the lower text box displays the text as it will actually appear
41. e then approximates the molar heat capacity for the mixture of the states as they exist at any temperature in the transition region The area between this baseline and the data then approximates the total heat change due to the transition Note that only the slope and intercept of the linear segments are used in the calculation for the progress baseline The actual end points of the linear segments are not used During the calculation of a progress baseline Origin integrates over the entire display range of the active data set If you prefer you can restrict the region of integration to just the peak area After adjusting the low and high temperature linear baseline segments use the Data Selector tool described in Lesson 2 to set the region where integration should be carried out If you now select the Baseline Progress Baseline command integration will be restricted to the region that lies between the two data markers Linear Connect This baseline is formed by connecting the two linear segments with a straight line as shown below Whereas the progress baseline uses only the linear segment slope and intercept to form the baseline linear connect uses only the position of the two end points 41 DSC Data Analysis Tutorial Guide 42 Cubic Connect Similar to Linear Connect except the connection is a cubic polynomial The end points are used to create the baseline as with Linear Connect but with Cubic Connect the slope is used also
42. ease refer to the LabTalk manual or select the online Help LabTalk menu option My test is now an Origin data set that can be plotted and manipulated like any other data set To plot the my test integration area data Select File New Graph to open a new plot window The Graph1 plot window opens ortcut Double click on the layer icon in the Graphl plot window Click on the The Layer Control dialog box opens New Graph button Click on my test in the Available Data list then click on the gt button My test appears in the Layer Contents list All data sets in the Layer Contents list will plot in the active layer 34 Lesson 3 Integration Available Data eiste Layer Contents sampleldsc cp sampleldsc cp base integ area Layer Properties my_test Plot Associations fingani L Show Range C Rescale on OK Click OK The Layer Control dialog box closes and my test plots as a line graph in the Graph1 plot window d d 50 52 54 56 5 60 62 d 66 6 TO T2 74 35 Lesson 4 Baseline Determinations Lesson 4 Baseline Determinations Origin creates a baseline for the reaction heat data by establishing a left linear segment and a right linear segment that are considered to be the baseline region Several methods can be used to connect these two segments to form the baseline In this lesson you will learn how to create format subtract and open the related worksheet for a baseline You will
43. ein in question at a single temperature at the least The coefficient of thermal expansion is typically small 10 10 and exerts only a small effect on the Cp value over a moderate temperature range Index Index A Absolute heat capacity equations 70 fitting example 54 57 Active data set 11 B Baseline 15 16 37 44 adjust linear segments 39 40 adjust the default baseline 43 baseline menu commands 38 baseline session 38 42 change line connect type 43 44 cubic connect baseline 41 42 default name 44 linear connect baseline 41 move baseline by cursor 42 progress baseline 40 41 restoring 20 21 select baseline type 40 step baseline 42 subtract from DSC data 44 viewing worksheet data for 44 C Calibration 21 22 Chi Sqr button 19 Chi Sqr minimization 59 Curve fitting 17 21 choosing a model 46 fitting iterations 50 fitting models described 17 45 46 fitting session 18 20 formatting parameters text 21 Line types 62 63 to dissociation w dCp 51 53 to the 2 state model 18 20 to the 2 state w dCp model 20 21 D Data files automatic scan rate normalization 8 keeping time data 8 offsetting data 8 opening 9 11 Data Selector tool 23 27 hiding data markers 33 setting integration range 29 31 Data set change line connect type 43 44 create baseline for 15 16 create new data set 34 deleting selected segment 25 fitting a curve to 17 21 importing 6
44. es and draws the two linear baseline segments faintly visible in the figure below D 3 a E E 50 60 70 Temperature C 2 Select Adjust Move Segments by Cursor 3 Beginning with the left linear segment click and drag on first the left and then the right endpoint until the graph looks approximately like the figure below you can also use the arrow keys to move an endpoint Use the Up and Down arrows to move up or down Control Left and Control Right arrows to move left and right and Right and Left arrows to select a new end point n kcal mole deg Temperature C 4 Now use the cursor to adjust the right linear segment as follows Lesson 5 Curve Fitting n c r E 3 50 60 70 Temperature C 5 Select Baseline Progress Baseline Origin plots a progress baseline 6 Select OK from the menu bar 7 Click Yes in the dialog box to subtract the progress baseline Your graph should now look like this e E m Ei E cm n a P n e T 100 11D Temperature cy Step 3 Fit to the least complex model Generally you will want to use the simplest model e g with the fewest floating parameters that provides a good fit to the data You can then add complexity if necessary Begin the fitting session by using model 2 state with zero ACp to fit the data To do so 1 Select 2 State Cursor Init from the DSC menu This starts the fitting sess
45. es and select paste Lesson 1 Basic Data Analysis Lesson 1 Basic DSC Data Analysis This first lesson presents an overview of basic DSC data analysis procedures You will learn how to start Origin read a DSC data file into Origin open multiple DSC data files subtract reference data from sample data normalize data by concentration create and subtract baseline data and use Origin s curve fitting models to fit a curve to your data We will also briefly discuss instrument calibration After working through this lesson you will know all of the basic procedures involved in analyzing DSC data Subsequent chapters look at specific procedural elements in more detail Starting Origin Before starting this lesson you should have Origin up and running If you are in Windows From the Desk Top double click on the MicrCal Inc DSC icon Or alternatively select Start Programs MicroCal Origin50 MicroCal Inc DSC When you start Origin the program automatically opens the RawDSC plot window Note that this window contains several buttons These buttons let you execute certain DSC routines Reading DSC Data Before we proceed you should understand the difference in Origin between a data file and a project or document for versions of Origin previous to 4 0 A data file contains your experimental data In order to plot the data you must read the data file into an Origin project A project or document will contain both the experiment
46. general purpose scientific and technical data analysis and plotting tool In addition Origin can carry add on routines to solve specific problems Analyzing Differential Scanning Calorimetric data from the MicroCal MC 2 MCS or VP DSC instruments is one such specific application This version of Origin includes routines designed to analyze DSC data Most of the DSC routines are located in the Peak and DSC Differential Scanning Calorimeter menus in the Origin menu display bar A number of routines are implemented as buttons in plot windows This tutorial will show you how to use all of the DSC routines Lesson 1 provides an overview of the entire DSC data analysis and fitting process Please work through this lesson first The subsequent lessons each look in more detail at particular aspects of DSC data analysis and may be read in whatever order you see fit If you are unfamiliar with the basic operation of Origin you may find it helpful to read through the Origin User s Manual particularly the introductory chapters and first several chapters before beginning this tutorial Note that this DSC tutorial contains information about Origin only in so far as it applies to DSC data analysis For a complete discussion of Origin s capabilities please refer to the Origin User s Manual If you have questions or comments we would like to hear from you Technical support and customer service can be reached at the following numbers Toll Free in North Amer
47. ica 800 633 3115 Telephone 413 586 7720 Fax 413 586 0149 Email info microcalorimetry com Web Page www microcalorimetry com Getting Started Getting Started In this chapter we describe how to install Origin on your hard drive how to configure Origin to include the DSC add on routines and how to start Origin We recommend that you read your Origin User s Manual for a complete guide to all of Origin s features System Requirements Origin version 5 0 requires the following minimum system configuration e Microsoft Windows 95 or later or Windows NT version 4 0 or later e 486 DX or higher processor e 8megabytes MB of RAM 16 MB recommended e One3 5 inch high density disk drive e 12 MB of available hard drive spaces Installing Origin To install a new copy of Origin or to upgrade an existing copy run the program SETUP EXE located on Disk 1 the Setup disk The Setup program guides you through the installation process Installation requires 12 MB of free disk space on the drive where you intend to install Origin Additionally installation requires 8 MB of free disk space on the Windows drive where your Windows operating system is installed for temporary files Thus if you are installing Origin onto your Windows disk drive 20 MB of free disk space is required during installation only 12 MB of free space is required after installation is completed The Setup program prompts you to type in your Origin serial n
48. in50 folder then double click on the file name in the Files list Dissoc OPJ opens to show the Iv2p0 cp data set with its associated baseline plotted in the NormDATA window 51 DSC Data Analysis Tutorial Guide 52 S NormDATA OF x Tem perature c 2 Select fitting model 4 Dissociation w dCp Cursor init from the DSC menu 3 Step 2 may have caused an error message to appear warning you that no baseline is selected for this data If this happened you need to let Origin know that you want to use the baseline supplied with this data which is named lv2p0_cp base To do this select the Peak Cursor Pick Baseline command then double click on the plotted baseline data When you have finished again select Dissociation w dCp Cursor init from the DSC menu 4 Select 2 as the Number of Subunits Note that the number of subunits n and the concentration Ct appear in the Fitting Function dialog box as non floating parameters 5 Assign value of 0 to the BLO and BL1 parameters and remove the checks from each Vary Box so the low temperature baseline is not allowed to float Click on 10 Iter a couple of times You will note that the overall fit curve is fairly good Lesson 5 Curve Fitting i NonLinear Curve Fitting Fitting Session el E3 Parameter Value Var Eror Dependency po L m o FA 2 2 00 E 0 fo 90 37 Iv 02671 0 3308 xd eneo 377027 ChiSar titer E Select Function Select Dataset More
49. ion with model 1 selected The Cursor Init option allows you to initialize the parameters rather than accept any existing parameters 49 DSC Data Analysis Tutorial Guide 50 2 Origin prompts you to enter the number of transitions The simplest fit would be to two transitions so enter a 2 and click OK 3 Double click on or near each peak to set a T thermal midpoint for each transition As soon as you set the Tm s Origin initializes the fitting parameters in this case Ty Hj Ti H5 displays the parameters in the Fitting Session dialog box and draws an initial fit curve An Attention dialog box notifies you when initialization is complete Click OK to proceed with the fitting NormDATA olx ri NonLinear Curve Fitting Fitting Session Mi E3 Parameter Value Vary Eror Dependenc Tmi gas v s pis36 03622 H1 paes m afaa oao Tm2 oss m ajomas oaee H2 ias M s rszE3 0422 Fr Chi 2 1482618 ChiSar 1 Iter Lior CAU DEM e a e e a a Uc Mae aie 1 1 1 narah Select Function Select Dataset More Done Cp kcalimole tt z 4 The 1 Iter and 10 Iter commands let you control the iteration of the fitting cycles Click on the 10 Iter button a couple times Origin iterates the fitting cycle until the change in Chi 2 between two successive iterations is less than a pre set value this value is determined by the value set in the Tolerance Text box of the Control Parameters dial
50. itial fit curve 5 As before select 10 Iter from the menu bar to fit the curve to the data After several fitting cycles you should get a pretty good fit implying that the three transition model describes this data better than the two transition model Fitting Example 2 For multisubunit proteins it often happens that the subunits dissociate simultaneously with the unfolding of the protein i e A nA where n is the number of subunits When this happens the shape of the DSC peak is broadened relative to unfolding of the same protein where no dissociation occurs e g covalently linked subunits The mathematics for curve fitting to systems which undergo simultaneous unfolding and dissociation are included in the Appendix You may wish to try fitting the data set in the Origin project Dissoc OPJ contained in the Origin50 Samples folder The data was obtained on a helical polypeptide thought to contain two identical chains linked together in a coiled coil state These data have already been normalized on concentrationand a progress baseline has been created so curve fitting may begin immediately Note that whenever using the dissociation model the concentration used for data normalization should be that for the associated low temperature state not that for the dissociated high temperature state 1 To open Dissoc OPJ click Done to exit the fitting session then select File Open navigate to the Samples sub folder of the Orig
51. k at the curve fitting process in greater detail In the first section below we will make some general observations about the curve fitting models used in Origin Following that we will work through two fitting examples to illustrate some of the flexibility of the curve fitting process General Comment Origin provides four models with which to fit a curve to your data All four models use the Levenberg Marquardt non linear least square method but differ in the number of parameters involved as shown below Model Parameters Model 1 2 State with zero DCp Tm H Model 2 Non 2 State with zero DCp Tm H H Model 3 2 State with non zero DCp Ta H C BLO BL1 Model 4 Dissoc with non zero DCp Tmn H BLO BL1 n m C m C Tm is the thermal midpoint of a transition H is the calorimetric heat change AH H is the van t Hoff heat change AH C is the AC for each transition BLO and BL1 define the slope and intercept of the low temperature baseline segment and n is the number of sub units All of the models except model 4 can be used to fit to one or more transitions In the case of multiple transitions each transition has its own complete parameter set e g if model 1 is used to fit two overlapping transitions there will be two independent parameter sets Tm H and Tm2 H5 These specify the thermal midpoint Tm and the heat change AH at the T for each transition The BLO and BL1 parameters are an exception to this r
52. ludes both calorimetric and van t Hoff heat changes Indicating the temperature dependent parameters this can be rewritten as Cp T K TAH a AH y T 12 2 1 K T RT In this case the equilibrium constants will be calculated as in eq 11 with the important exception that the van t Hoff heat rather than the calorimetric must be used since now the two will be different i e AH T K T ex ma dq 13 A T exp T 13 Eq s 12 13 are then used for curve fitting in the usual way and the parameter set will be Tra AH AH A Tmp Aap AB an s mB mB II Sequential Transitions Sequential transitions may occur in proteins or other macromolecules when the stability of each domain A B C is dependent on whether other domains are folded or unfolded The sequential model imposes a precise order in which domains must unfold for every molecule in solution i e 67 DSC Data Analysis Tutorial Guide Kj K K3 K n P P P P AH AH AH AH 14 where each step in the reaction scheme involves the unfolding of one of the domains in the appropriate sequence i e step 1 is always the A domain step 2 always the B domain etc The total molar heat content of the system relative to a value of zero being assigned to the Po state will be H fy 0 f AH f AH AH fF AH AH AH 05 where the f s indicate the fractional concentration of each state and the AH s are the molar enthalpy ch
53. m any of the four versions of the data collection software To import a data file generated by the MC 2 DOS based data collection software you click on the Read Data button in the RawDSC plot window and select MC 2 data dat from the List Files of type box To import a data file generated by the Windows based data collection software either from the MC 2 DSC Data dsc the MCS or the VP DSC you click on the Read Data button iii PEL i in the RawDSC plot window and select DSC Data dsc from MC 2 Data dat the List Files of type box PLEASE NOTE Data file names Isoscan dsc should not begin with a number nor should they contain any Any File hyphens periods or spaces A new isothermal mode of collecting experimental data is available for the VP DSC This mode may be used when it is desirable to use the DSC to monitor a process at a constant temperature for varying periods of time such as when determining kinetics of a chemical reaction at several different temperatures e g estimates of shelf life For this mode data is plotted Vs time rather than temperature Selecting Isoscan dsc will read in data that has been collected in the VP DSC isothermal mode and will plot the data Vs time minutes Once a data file is called into Origin all further operations on the data are identical regardless of the original source of the data Note that in this tutorial all data were generated by a Windows b
54. med to be zero certain simplifications can be made in eq s 7 9 Before curve fitting begins the operator subtracts a baseline from the data which effectively sets Con equal to zero at all Appendix Equations Used in Deconvolution temperatures so that Bo and B1 are no longer used as fitting parameters Incorporating this setting all heat capacity changes to zero and recognizing that all AH A T for each transition may be replaced by the temperature independent heat AH na i e heat changes must be temperature independent if there is no change in heat capacity then eq s 7 9 simplify to K T AH T T 7 4C 10 1 K T RT AH T K T m j 11 a T exp 7 11 where again the right hand term in eq 10 must be repeated for each transition and where eq 11 must be evaluated for each transition In this case the fitting parameters are T na gt AHA Tmp AH mp but otherwise the curve fitting procedure is the same as described above Model for Independent Non two state Transitions Because of the extra fitting parameters required for the non two state vs two state model this model is only applied to transitions with no AC Before curve fitting with this model a progress baseline must be subtracted from the experimental data to remove the AC effects if they are present this also sets CpN to zero at all temperatures To treat non two state transitions the appropriate place to begin is eq 6 which still inc
55. mple at 60 deg hr The first experiment was scanned using the Passive FeedBack mode the scan was repeated using the High Gain Mode The data was scanrate normalized and a baseline has been subtracted from each trace Select File Open Navigate to the Origin50 Samples directory double click the project name ResponseT opj The project will open as shown below Lesson 8 Other Useful Details i Microcal Origin C OriginData ResponseTime ResponseT RawDSC ile Edit View Graph Data Analysis Tools Format Window Help 5 esas ao 86 BIS 2 dsl xi ms amp a Blo Subtract Reference 1 Normalize Concentration 0 00004 _ Passive 0 00002 4 0 00000 4 Response Time 0 00002 4 ae Temperature C I a s t rl o 7 n o ze Y imi Script Window xil Astan Dsc ogs WordPad Microcat Origin C KY Microsoft Word DSC Tut BOSSE 402PM You may see from the that the data collected in the passive mode dashed line has a slightly higher Tm and is slightly broadened from the data collected at the High Gain FeedBack mode fastest response time Make sure the data set Vcht20b2ds cp is the active data set box in legend is around the dashed line type of the Vcht20b2ds cp data set Vcni2 ndds cn EJ Vcni2 n2dz cn Click on the Response Time button The Response Time dialog box will open Enter 0 for the FeedBack Mode No Gain and r 40 9 f
56. mpleldsc tempi 12 Lesson 1 Basic Data Analysis The Plot Details dialog box opens Note that you can also open this dialog box by double clicking on the data plotted in the RawDSC plot window Plot Details Dataset s sampleldsc cp Plat Type TERETE NN Lowe SS Worksheet Line Symbol Color Black z V Line Symbol Gap Remove r Line 34 r Symbol aot Grey a Connect Straight wj Shape No Symbo v G anipende Type ssa Sue soid Inremental Width 05 Sizefptsy9 a co FillArea Drop Line Line Type EilCoer Black SkePts I Frea TF Symbol Click on the Worksheet button The Sampleldsc worksheet opens iii Microcal Origin UNTITLED Sa E File Edit View Plot Column Ma 30 79999 31 2 891 46E 5 31 20001 6 99336E 5 31 40002 3 04161E 5 31 60004 4 9134E 5 31 80005 5 97788E 5 32 00006 7 35419E 5 Refer to Chapter 3 Worksheets in the Origin User s Manual for a complete discussion on working with Origin data sets Normalizing the Data Before you proceed to fit deconvolute the data you will need to normalize the result by concentration Normalizing the data divides by the number of moles of the sample substance in the cell to convert from cal degree to cal mole degree Concentration normalization is always carried out after subtracting the reference data 13 DSC Data Analysis Tutorial Guide To normalize Samplel
57. n dialog box Sem pleidsc_qp Model MZSb e Chi 2 T005 6 To LU 2002649 aH B51E 2272 Aj Results BEE Data Sampleidsc cp Model M2State Chi 2 708039 6 Tn 68 77 8 025449 H 9 51E h 272 e a o E ur G o x a o Temperature C To fit a curve with the non 2 state model model 2 Select Non 2 State Cursor Init from the DSC menu Repeat the steps you followed above when fitting with the 2 state model The fitting procedure is identical for the two models Click Done when finished A fit curve is generated and the fitting results copy to the Results Window and to a text box in the Normdata Window Note the difference in parameter values between the two models Results OL x Model MN2State Chi 2 58971 6 Tn 68 79 8 82178 H 9 18bE 583 Hu 9 95E4 675 To fit a curve with the 2 state w dCp model model 3 Model 3 is designed for use with data having a non zero ACp Model 3 requires that a baseline be associated with the data so first we need to retrieve the baseline we subtracted previously To do so Select Simple Math from the Math menu The Math dialog box opens Select Sampleldsc cp then click on the upper gt button Sampleldsc_cp copies to the Y1 text box Select SAMPLEIDSC CP BASE this is the original baseline data set then click on the lower gt button 20 Lesson 1 Basic Data Analysis SAMPLEIDSC CP BASE copies to the Y2
58. n methods of connecting the fit curve data points are straight spline or B spline Straight A straight line is displayed between data points This type of line connection will not give a smooth representation of the fit curve if there are few data points Spline This option generates a cubic spline connection To use the connection the X values must be discrete and increasing Since curvature information is held in memory the spline resolution remains the same regardless of page magnification The SplineStep variable in the ORIGIN INI file controls the spline calculation increment Itis expressed in units of 1 point This is usually the most satisfactory representation of the fit curve but may exhibit an excursion from the actual fit curve if there is a sharp corner in the data Lesson 8 Other Useful Details B Spline The B spline curve can be described by parametric equations Unlike spline curves which pass through the original data points the B spline curve winds around the original data points without passing through them Thus this curve may not produce a satisfactory representation of the fit curve For a complete discussion of the B spline connection see the Origin User s Manual Inserting an Origin graph into Microsoft amp Word There are two ways to include your Origin graph into Word or other applications you may import your graph into Word or you may share your graph with Word When you import your graph Word will display
59. n the Word document by e Select Edit Update Client from the Origin menu to make immediate changes to the Word document graph Shortcut You may quickly start Origin and load the linked graph by simply double clicking on the graph while in Word Origin will be started with the original document loaded the changes can be made and by selecting Edit Update Client the changes will be transferred to the Word document Appendix Equations Used in Deconvolution Appendix Equations Used to Deconvolute DSC Data I Independent Transitions It will be assumed here that a protein or other macromolecule is composed of a number of structural domains A B C each of which is involved independently in a transition between the folded and unfolded forms A A B B Equilibrium constants will be expressed as fractions K4 fa fa Kp fg fg with the usual designation for the actual i e calorimetric molar enthalpy changes AH 4 AHg The total molar enthalpy of the system H will be H H f AH fp AH 1 The first term for the totally native form where all domains are in their folded state Hy must be included since all AH values are measured relative to the folded form The total molar heat capacity of the system Cp is the temperature derivative of the enthalpy in eq 1 so f C C f aC vani Sa Q where C y is the molar heat capacity of the totally folded state AC 4 is the change in heat
60. nd where the brackets signify molar concentrations of species If f is the fraction of macromolecule in the A state and 1 f the fraction in the A state then NEM 4l I f G bu 22 t where C is the total bulk molar concentration expressed as n mer equivalents The equilibrium constant using eq s 21 22 then becomes K f n C 23 Q3 Using eq 23 and the expressions lnK T AH T 1 R T AH T AH AC T T Q4 and integrating from Tma where f 0 5 to T then gives AH T C T K T 05 n C exp mA li a r Ta rm Q5 RT Tra RT T Once K T is known then f T may be solved from eq 23 using numerical methods and the excess heat capacity calculated from the equation 69 DSC Data Analysis Tutorial Guide 70 C T B B T f T AC pO 400 Q6 RT n penal tt etary T T The above equations are general for any value of n so long as the bulk concentration C is expressed as n mer equivalents For systems which associate when unfolding occurs i e A A Q7 the same equations are valid providing the bulk concentration C is expressed as monomer equivalents and that n in the above equations is equated to J IV Absolute Heat Capacity of Proteins The equation used to calculate Absolute Heat Capacity of Proteins is as follows AC g p V 1 00002 C e va ar C Where AC cal deg is the sample buffer baseline minus the buffer buffer ba
61. ntration normalization is carried out on a per mole of monomer basis while it will be 1 0 if concentration is entered in terms of moles of dimer present In model 1 it is possible to prescribe that overlapping transitions are either independent or sequential in nature For example if two structural domains are interacting strongly it is possible that their transitions will be coupled in a sequential manner whereas the independent model might better describe two transitions which are completely uncoupled from one another In practice this choice is often not critical since the sequential and independent models lead to virtually identical results whenever the Tm s of two transitions are separated by a couple of degrees or more To switch between select the DSC Set Indep Sequ Model command and answer the prompt that appears Origin provides the option to float all parameters during the fitting procedure or to assign some parameters as fixed and float others In fixing a parameter it may be given an invariant numerical value The mathematical derivations for each model are included in the appendix In general the objective of most investigators is to use the simplest model e g fewest floating parameters which provides a good fit of the data Thus if data are satisfactorily described by the two state model using two transitions this is to be preferred over a two state model with three transitions or a non two state model with two transitions
62. og box MicroCal has set the default value to be 10 Select 10 Iter once or twice more At the end of each fitting cycle Origin updates the Chi 2 and fitting parameters values and draws a new fit curve By now you should have a pretty good fit to the 70 degree peak but the fit to the 60 degree peak is not so satisfying At this point you can begin to think about improving the fit by adding complexity to the model Step 4 Add complexity You did not get a good fit using model 1 with two 2 state transitions While the high peak fit pretty well the low peak did not This could mean that the low peak is not really a single peak but two smaller overlapping peaks that combine and obscure each other To test this hypothesis see if you can get a better fit using model and three transitions 1 Click on the Close button x located in the upper right corner of the Fitting Session dialog box to abort the current fit 2 Select 2 State Cursor Init from the DSC menu 3 Type 3 in the Number of peaks dialog box and click OK 4 Double click to set each of the three Tm s one for each transition Place the first Tm just to the left of the low peak the second just to the right and center the third on the high peak Lesson 5 Curve Fitting a 3 E i zu x a so a 70 Temperature C Upon setting the third Tm Origin calculates all three sets of fitting parameters six parameters in all two for each transition and displays the in
63. or the Relaxation Response Time The scanrate will be automatically entered CE Please Note Normally the FeedBack Mode and Relaxation Response Time will be read VP FEEDBACK MODE 0 in from your experimental data file Response Time sec 40 9 SCANRATE deg hr 60 02849 Click OK A new curve will be generated and plotted as a blue line shown below as a wider line This is the modified curve for the passive mode that removed the small amount of response time broadening The smaller peak of the repeated scan using High Gain FeedBack is due to some non reversibility of the sample 61 DSC Data Analysis Tutorial Guide 62 Passive with Response AN Vcht20b2ds cp _ ff M Time broadening 6M Vcht20b3ds cp VCHT20B2DS_CP 0 00004 4 removed oN High FB 0 00002 Passive 0 00000 0 00002 50 Temperature C Line Types for Fit Curves You may select a line type to plot your data or fit curves from the Plot Details dialog box The Plot Details dialog box is available by double clicking on the data plot right clicking on the data plot and selecting Plot Details from the shortcut menu or selecting the desired data plot from the Data menu data list and selecting Format Plot The Line Group Select the desired line connection from the associated drop down list The line connection type affects interpolation results The default line type for fit curves is Straight line The most commo
64. other display options can be changed by double clicking on a point in the baseline and selecting new options in the Plot Details dialog box 43 DSC Data Analysis Tutorial Guide To change connect type for a baseline Double click on the baseline The Plot Details dialog box opens Select a connection option e g spline straight step from the Connect drop down list box Click OK To subtract the baseline from the DSC data Select Subtract Baseline from the Peak menu The baseline is subtracted Each time you create a baseline Origin names the baseline data set and appends a column to the active dataset worksheet for the baseline data The baseline data set is given the name NAME BASE NAME DRBASE in the case of Cursor Draw Baseline where NAME is the name of the data set for which you created the baseline To see the baseline data open the associated worksheet To open the worksheet containing baseline data Double click on the baseline to open the Plot Details dialog box or select the baseline name from the Data menu then select Plot from the Format menu to open the Plot Shortcut to the Worksheet Right click anywhere on the data trace and select Open Type dialog box Worksheet from the drop down menu Click on the Worksheet button The baseline worksheet opens 44 Lesson 5 Curve Fitting Lesson 5 Curve Fitting In Lesson 1 we discussed Origin s basic curve fitting procedures Here we will loo
65. ph should now look like below 56 Lesson 5 Curve Fitting Vcht15bsds_AbsCp Vcht14bsds_AbsCp Vchti3bsds AbsCp Vcht12bsds_AbsCp Vcht9bsdsc_AbsCp A Oo o o 2 E o ES Ri 2 o O o 2 3 o eo Q lt Temperature C You may wish to expand the view around 25 deg and add some grid lines a Vchti5bsds_AbsCp Vchti4bsds_AbsCp Vcht13bsds_AbsCp Vcht12bsds_AbsCp Vcht9bsdsc_AbsCp Absolute Cp cal gm deg 245 25 0 25 5 26 0 26 5 Temperature C 57 Lesson 8 Other Useful Details Lesson 8 Other Useful Details X chi sqr Minimization The aim of the fitting procedure is to find those values of the parameters which best describe the data The standard way of defining the best fit is to choose the parameters so that the sum of the squares of the deviations of the theoretical curve s from the experimental points for a range of independent variables is at a minimum For the ITC models where there is no weighting the theoretical models can be represented by y f x Dis Pas Poo where pi the fitting parameters the expression for x simplifies to 1 t a bi flesp Pal where n the total number of experimental points used in the fitting p total number of adjustable parameters y experimental data points J GDup2ps fitting function
66. re satisfied with the placement of the left linear segment double click in the plot window or press the Return key 39 DSC Data Analysis Tutorial Guide 40 O E nf o o E o o ee a Oo Temperature C Choosing a Baseline Option Once you have set the linear segments you are ready to select one of the six options for creating a baseline You will find the baseline options in the Baseline menu To select an option for creating the baseline Click on the Baseline menu option Choose a baseline option from the menu When you are satisfied with the baseline click on the OK command in the menu bar to exit the baseline session Origin gives you the opportunity to subtract the baseline then returns you to the RawDSC window or if you had normalized the data the NormDATA plot window The six baseline options are described in detail below Progress Baseline In this option the baseline in the peak region is created such that each point reflects the extent of progress of the reaction That is the placement of a baseline point at any temperature in the transition region 1 e relative to the left and right baseline extrapolated to the same temperature is determined by the fraction of the total area that has been completed at that temperature The progress baseline for the SAMPLE data is shown below Lesson 4 Baseline Determinations For a single transition such as this the calculated baselin
67. rom the data set listed in the Data box Select Sampleldsc cp from the Data drop down list Sampleldsc cp becomes highlighted and will be entered as the Data Select Refldsc_cp from the Reference drop down list Refldsc cp becomes highlighted and will be entered as the Reference Subtract Reference Data Data Reference Cancel Data Sampte1 dsc c Reference Refldsc cp Click OK Every point in Refldsc cp is subtracted from the corresponding point in Sampleldsc cp The result is plotted as Sampleldsc cp in the RawDSC plot window 11 DSC Data Analysis Tutorial Guide Sampletdsc cp Oo g oO o Q Oo 60 Temperature C Viewing Worksheet Data The series of values from which Origin creates a data plot is called a data set Each data set is contained in a unique worksheet column The data set is named after its worksheet and worksheet column separated by an underscore When you opened the Sample1 DSC data file the data were placed in a worksheet named Sampleldsc with the Y data in a column named cp Thus the Sampleldsc data set is called Sampleldsc cp To open the Sampleldsc worksheet Select Sampleldsc from the data list in the Data menu BAES Math Peak DSC Tools Fa E Get Tem Range Reset ta Full Range Data Markers Shortcut to worksheet Right z click on the data plot and Move Data Points select Open Worksheet from the menu Remove Bad Data Points w Sa
68. rs the baseline session and automatically analyzes the data to determine the linear segments to be drawn on either side of the peak ii Microcal Origin UNTITLED NormDATA Of x E OK Adjust Baseline Cancel Window l xl ne ze e wS 2E amp e alg 1e E sampletdse_cp Cp kealmolefC EE 3 46 so LE TO ac 20 Tem pe ratu ve C Rakelli E For Help press F1 Sampeidsc cp NomDATA 7 Note that a new command menu bar now appears at the top of the project These command menus provide complete control over the baseline determination process as follows OK Accepts the current baseline provides an opportunity to subtract this baseline from the data curve and then exits the baseline session Adjust Allows the user to adjust the two linear segments Baseline Choose one of five options for creating a baseline or draw a baseline manually Cancel Exits the baseline session unconditionally The current baseline if any is deleted If you are having difficulty seeing the linear segments you can maximize the plot window for a better view To maximize the RawDSC plot window Click on the Maximize button O at the upper right corner of the window The window zooms to full size 38 Lesson 4 Baseline Determinations User Adjustment of Linear Segments If you are not satisfied with the two automatically determined linear segments u
69. se the Adjust menu to change the segments as you see fit The Adjust menu provides two options refit the segments or move the segments by cursor To refit the linear segments Choose Refit Right Segment from the Adjust menu Baseline Cancel Wind Refit Left Segment Refit Right Segment Move Segments by Cursor Click the cross hair on or near the data you want as the first point of the linear segment A red cross appears on that point Use the left and right arrow keys to move the red cross until you have selected the point you want Press Return This sets the selected point as the first point of the linear segment Repeat the previous three steps to select the last point of the linear segment Origin now generates a new linear segment by fitting the data between the two points to a straight line Now for the left linear segment Instead of using the refit command lets adjust the left segment by cursor To move linear segments by cursor Select Move Segments by Cursor from the Adjust menu Select and move one of the left segment end points by either Clicking on one of the terminal black squares and dragging the mouse or Selecting an end point with the LEFT or RIGHT arrow keys then using the UP and DOWN arrow keys to move the point up or down Use the CONTROL LEFT or CONTROL RIGHT arrow keys to move the point left or right Repeat the previous step for the other segment end point When you a
70. seline g is the concentration of protein gm ml in the solution P t is the relative density of water stored in Origin as a polynomial in t V is the nominal volume ml of the sample cell t is the temperature in C C t is the absolute heat capacity cal deg gm of the protein in solution V is the partial specific volume of the protein ml gm Qt is the coefficient of thermal expansion of the protein and Cp t is the unit volume heat capacity of water cal deg ml CP t is calculated from literature data on the specific heat and density of water as a function of temperature by fitting to a polynomial in temperature The thermal coefficient of cubical expansion of tantalum is 00002 The above equation is solved for Cp t from the experimental data file AC using the stored polynomial expressions for Cp t and P t as well as the operator input parameters Zo V O and V This procedure assumes the relative density and the unit volume heat capacity Cp of the buffer solution can be replaced by the corresponding properties of pure water This should be a very good approximation at electrolyte concentrations commonly used in buffer solutions but would be less accurate at very high electrolyte concentrations gt 1M or in the presence of high concentrations of organic additives Also the value obtained for Cp depends strongly on the partial specific volume of protein so it is desirable to have an experimental value of V for the prot
71. t X 61 Origin initializes the fitting parameters displays the parameters and Chi 2 in the Fitting Session dialog box and draws an initial fit curve When initialization is complete the Attention dialog box appears with the message Done init with fixed Tm s Click OK Attention Dione fk oath Een The Fitting Session You are now in the Basic Mode of the Fitting Session dialog box Note that a new window displays the fitting button commands Please refer to the Origin User s Manual or press the F1 key for Online help for more information about the fitting procedures 18 Lesson 1 Basic Data Analysis az Microcal Origin UNTITLED NormDATA E Eile Edi View Graph Data Math Peak DSC Tools Format Window Help l x RRB ams Eel Ome saalam eal wsl al see E Sample 1dsc_cp dr NonLinear Curve Fitting Fitting Session Bie EG Value Vary Error Dependence 60 72 iv 0 0430 o 2312 H 9 53E4 M s a38 0 231 Cp kcal mole C sA fez i 2 v2400 38 CRS 11 aif rr ESEZ Chi 2 72400 38 qr ter i1 x dr 2 9p BS au Select Function Select Dataset More Done Temperature C Fee EIRP o MSPTETEETEEREERTETIT ET NN T Im Script Window x AMstar BY Microsoft Word Microcal Origin UNT BIO 1257PM Chi Sqr Allows the user to change the values of the parameters i e Tm AH to see an update of the fit using the new parameters
72. text box Click in the Operator text box and type a in the operator text box Click OK The original Sample data is restored since the baseline which was subtracted earlier has now been added back Now that you have retrieved the baseline data go ahead and fit a curve using model 3 Select 2 State w dCp Cursor Init from the DSC menu Then follow the same steps as outlined for the previous two models Click on Done to exit the fitting session To format the fitting parameters text Double click on the text in the plot window The Text Control dialog box opens Change the text style for example change font to Roman and point size to 10 Note that to ensure a true What You See Is What You Get text display on screen you should select the Print View mode from the View menu View mode does not affect output so for the sake of screen refresh speed it is usually adequate to use Page View Window View or Draft View all of which are faster Click OK bats Sample idsc cn Model dC pM ZT Chia 413510 Bia 2111 Z 1 256 ees 20006 SSSE 265 14163 21 e E 2 o E o x a o so 60 70 Temperature C Calibration 21 DSC Data Analysis Tutorial Guide 22 We will close this lesson with a brief note about instrument calibration Every two or three months you will want to calibrate the DSC instrument against the Y axis scale in Origin To do this you will fill both cells with
73. the Reset to Full Range command to bring the hidden data back into view To reset display to full range Choose Reset to Full Range from the Data menu Origin resets the data markers so as to display the entire curve 27 Lesson 3 Integration Lesson 3 Integration If you have created a baseline for a DSC data plot you can select a range of data to integrate Integration yields the following information about the data integration range temperature range area thermal midpoint and width of curve in degrees C at half height In this lesson you will use the Data Selector tool to set the integration range integrate the data copy the integration results to the plot window and plot the integration data Setting Range and Integrating the Data As you saw in Lesson 2 all math editing and fitting operations are performed on the currently selected plot segment or on the current display range This is true for integration as well The first step in integration is to use the Data Selector to set the integration range If you do not set the integration range the entire display range will be integrated Before beginning this lesson open a new project Select the File New Project or click on the New Project button command Click on the Read Data button in the RawDSC1 plot window The Import Multiple ASCII dialog box opens Double click on Samplel dsc located in the Samples sub folder of the Origin50 folder Click OK
74. this disk contains the DSC specific routines and click Next DSC Data Analysis Tutorial Guide The installation program automatically creates an Origin50 folder and window containing the program icons After installation is complete you may right click on the MicroCal Inc DSC icon and select copy from the drop down menu then right click anywhere on the desktop and select paste to install a desktop icon for Origin DSC Registering with MicroCal Software MicroCal Software Inc a separate company from MicroCal Inc produces and supports the Origin software package MicroCal Inc produces and supports the calorimetric fitting routines imbedded in the Origin for DSC and Origin for ITC packages MicroCal Inc will provide technical support for all aspects of the software without registration MicroCal Software will not provide technical support for the calorimetric fitting routines but if the copy is registered will provide standard technical support for the general purpose routines of the program Upon receipt of Origin please fill out and return the registration form included with your package to MicroCal Software You may also register at any time by contacting the Customer Support Department at MicroCal Software Starting Origin To start Origin double click on the Origin 5 0 program icon on the Desk Top Alternatively click Start then select Programs Point to the MicroCal Origin50 folder and select the MicroCal
75. u are checking calibration of the instrument Click on this checkbox to select or de select this option If the box is not checked then unnormalized raw data will read in as mcal minute In raw data files separate columns are included for Y axis temperature and time for each data point When data are being Scan Rate Normalized the time and temperature data are used to determine scan rate whereupon the time data is discarded and only the normalized Y axis readings and temperature data are imported into the project The operator has the option of importing or not importing the time data time extension into the project as active data though the time data is not plotted automatically This is used for removing small amounts of peak brodening due to using a feed back mode which has a response time too slow to resolve a peak adequately Refer to pages 59 62 for more information This is used to calculate Absolute heat capacity from raw DSC data Refer to pages 54 57 for more information Lesson Basic Data Analysis ii Microcal Origin UNTITLED RawDSC Fie Edt View Graph Data Math Peak DSC Tools Format Window Help lJel xl R j salasan ela as All ele eRe ts dls Eel iid Subtract Reference Normalize Concentration 0 001 O Io o O 0 000 p T T T T T T T T T DEn HE 30 40 50 60 70 80 90 Temperature C a s 1 T v 7 m o E For Heb pes Sit Wind ll The RawDSC window which cont
76. ule These parameters appear only once in the model they are not repetitive for each transition While all four models use a calorimetric heat change AH only the non 2 state model model 2 has a van t Hoff heat change AH The calorimetric heat H is determined only by the area under a transition peak while the van t Hoff heat H is determined only by the shape of the transition peak The sharper the transition the larger is H and vice versa The relationship between H and H can sometimes provide insights not accessible from H alone For example if a protein is composed of two identical domains which unfold independently with the same Tm and H then the ratio of H H will be 2 0 while it would be 1 0 if the protein had only a single domain If on the other hand the protein dimerized and the dimer underwent only a single coupled transition then the H H ratio would be 0 5 It is clear from this that the calorimetric heat H refers to heat change per mole while H is heat change per unfolding unit called the cooperative unit Thus the ratio H H can in simple cases be thought of as the number of cooperative units per mole Note It is important to realize that the ratio of calorimetric to van t Hoff heat depends on concentration normalization since calorimetric heat is always expressed on a per mole basis Thus for the protein dimer example considered above the ratio will be 0 5 if 45 DSC Data Analysis Tutorial Guide 46 conce
77. umber If you are upgrading your version of Origin your new serial number is located on the serial number label affixed to the Origin package or on the packing list If you are a new Origin user your serial number is located on your registration card or on the serial number label affixed to the bottom of the Origin box To start the Setup program perform the following Please refer to the Origin Getting Started Booklet for further information When choosing a Destination Directory or folder name to place Origin make sure this name or any other name in the path does not include a space otherwise Origin will not operate properly oo Start Windows 95 Windows 98 or Windows NT version 4 0 or later Close all Windows programs if any are open Insert the Origin Setup disk in the available floppy drive A or B Click Start the select Run Type A Setup or B Setup in the Open text box Click OK The dialog box closes and the Origin Setup program begins If you have a previous version of Origin you may want to install Origin 5 0 in a separate program folder Please change the program folder to be MicroCal Origin50 from the default MicroCal Origin name when prompted see Origin s Getting Started Booklet for more information Follow the instructions presented by the Setup program to complete the installation After disk 5 is completed make sure the X is in the box to select the Custom disk installation

Download Pdf Manuals

Related Search

Related Contents

IKEA LOVIKEN AA-291347-1 User's Manual  UHFSPLITAL - Proel Spa  csvsimple.    Herunterladen  "user manual"  

Copyright © All rights reserved. Failed to retrieve file