Data Acquisition and Analysis Software Quick Start Guide
Contents
1. Customise gt Extensions and check Calibrations Not all versions of DAx contain this option 9 This is true for cubic spline calibrations even if the Derive from data boxes had not been checked in the GPC calibration dialog This type of calibration does not support extrapolation beyond the data range Multi linear and polynomial calibrations do 31 Chapter 2 5 1 Step 1 Analysing the Calibration Sample Once again start DAx Load the file test3 DAx and analyse it the first exercise took you through the steps required to do this Zoom in on the part of the graph following the big peak You should get something like this but without the numbers on top of the peaks they are added in the next step DAx 5 0 PP 09 03 96 16 01 55 AU 0 005 time min Next make sure a peak list window is displayed We know the base pair counts for the peaks in this data set and should enter them in the annotations column in the peak list window NB we do not care about the earlier peaks in the peak list window Only the peaks with migration times over 4 minutes are of interest To locate a peak in the peak list window simply move the mouse cursor over the top marker for the peak in the graph the appropriate peak in the peak list window will be highlighted The peak list might end up containing something like this Peaks list STAND 1 Da1 not saved Measured 00 00 00 00 00 00 by PP RMS Noise AU 0 00023251 0 000116262. Peak Begin Top End Top AU Annotation 1 0 168 0 182 0 245 0 0009542 2 1 433 1 442 1 450 0 00028247 lt some peaks skipped gt 13 3 770 3 808 3 838 0 002147 14 4 155 4 170 4 182 0 00030184 32 15 4 360 4 423 4 443 0 003394 106 16 4 460 4 485 4 507 0 0029842 112 17 4 908 4 938 4 970 0 0028615 156 18 4 970 4 983 5 013 0 0037159 160 19 5 013 5 028 5 060 0 0059983 164 20 5 060 5 073 5 105 0 0035568 168 21 5 105 5 118 5 140 0 0029702 172 22 5 140 5 153 5 175 0 0019518 175 23 5 187 5 207 5 230 0 0016548 180 24 5 590 5 607 5 628 0 0017969 214 25 5 633 5 652 5 685 0 0020277 218 26 5 685 5 695 5 722 0 0015195 222 27 5 722 5 740 5 757 0 001 7602 226 28 5 763 5 783 5 810 0 0016279 230 29 5 810 5 828 5 847 0 0025198 234 30 5 850 5 872 5 895 0 0014169 238 We ve now made sure of the following crucial steps for creating a DNA calibration e we have a data set with peaks e base pair counts have been entered as annotations for the peaks Chapter 2 5 2 Step 3 Creating the Calibration Execute the Calibration Calibrate menu option The DNA Calibration dialog box is displayed For details refer to the chapter Calibration Dialog Box in the DAx manual The dialog box looks like this 33 Edit Main Calibration Set Calibration Type Derive Main Calibration from m Calibration Curve Type Copy Main Calibration from Te Store Main Calibration in ER C i li Delete Calibration from Multilinear c p DAXSI
3. 2 1 2 Step 2 Loading a Data Set 6 Chapter 2 1 2 1 Data Tags 8 Chapter 2 1 2 2 Data Tags Moving Data 9 Chapter 2 1 2 3 Which mouse button should use 10 Chapter 2 1 2 4 Data Tags Other Uses 10 Chapter 2 1 3 Step 3 Zooming In on the Graph 11 Chapter 2 1 4 Step 4 Printing the Graph or Exporting It 12 Chapter 2 1 5 Step 5 Analysing the Data Set 13 Chapter 2 1 5 1 Manually Adjusting Peaks 14 Chapter 2 1 6 Step 6 Displaying Peak Data 15 Chapter 2 1 7 Step 7 Saving Your Work End of first workout 16 Chapter 2 2 Second Exercise Shortcuts and More 17 Chapter 2 2 1 Step 1 Opening Several Data Files and Analysing Them 17 Chapter 2 2 2 Step 2 Adjusting a Baseline 19 Chapter 2 2 3 Step 3 Finding Peaks 22 Chapter 2 3 Third Exercise Capillary Electrophoresis 23 Chapter 2 3 1 Step 1 Calculating Apparent Mobilities 23 Chapter 2 3 2 Step 2 Calculating Effective Mobilities 25 Chapter 2 4 Fourth Exercise Gel Permeation Chromatography 27 Chapter 2 4 1 Step 1 Analysing the Calibration Sample 27 Chapter 2 4 2 Step 2 Setting the Flow Rate 28 Chapter 2 4 3 Step 3 Creating the Calibration 29 Chapter 2 5 Fifth Exercise DNA Base Pair Count Determinations _ 31 Chapter 2 5 1 Step 1 Analysing the Calibration Sample 32 Chapter 2 5 2 Step 3 Creating the Calibration 33 Chapter 2 5 3 Step 4 Using the Calibration 35 Chapter 2 6 Sixth Exercise Qualifying amp Quantifying Peaks 36 Chapter 2 6 1 Step 1 Analysing the Sample with Known Components__ 37
4. 299 000 ethanal 0 365 0 0036532 0 00012213 19 301 700 propanal 0 576 0 0057591 0 00019085 20 304 400 butanal 0 375 0 0037514 0 00012321 21 307 100 pentanal 0 261 0 0026055 8 4843E 05 22 309 200 hexanal 0 207 0 0020745 6 7091E 05 23 312 400 heptanal 0 162 0 0016157 5 1711E 05 24 336 400 methanol 5 12 0 0017238 5 1231E 05 25 339 100 ethanol 4 14 0 0020816 6 1378E 05 26 341 700 propanol 4 4 0 0015037 4 3998E 05 27 344 400 butanol 5 73 0 0019723 5 728E 05 28 347 000 pentanol 5 16 0 0017918 5 1635E 05 29 349 700 hexanol 8 21 0 002869 8 2068E 05 30 352 300 heptanol 4 48 0 0015776 4 4788E 05 As you may see the alkanals have been assigned concentrations equaling 100 times the peak area and the alkanols have a concentration equaling 100 000 times the migration time corrected peak area We ll now use these data to create an Identification Database Chapter 2 6 2 Step 3 Creating the Identification Database Execute the Analysis Edit Database menu option The Identification Database edit dialog box is displayed For details refer to the chapter Identification Database Edit Dialog Box in the DAx manual The dialog box looks like this 38 Peak Identification amp Quantification xl Fetch components from Quantification Concentration limits Remarks ESTanD Qualification Attributes STAND 1 Dai _ Qualification coordinate Peak Top Coordinate Moment Capacity factor x Tolerance poo A Absolute IV Intermediate q
5. 96 19 57 54 Peak Top s Component Conc Area AU s STAND 1 Da1 8 195 600 0 094012 S158 4 Dal 4 194 500 0 012663 Average 569 900 0 05943 St Dev 530 118 0 058164 lt some peaks skipped gt STAND 1 Da1 23 269 100 0 0027982 S158 4 Dal 19 269 100 0 0018149 11 The exact contents may vary especially which data columns are present 43 Average St Dev STAND 1 Da1 S158 4 Da1 Average St Dev STAND 1 Da1 158 4 Da1 Average St Dev STAND 1 Da1 158 4 Da1 Average St Dev STAND 1 Da1 158 4 Da1 Average St Dev 26 20 27 21 33 22 35 23 269 100 0 000 301 700 301 500 599 250 420 940 304 400 304 100 304 250 0 212 341 700 341 700 1143 799 1134 340 347 000 346 800 519 100 243 527 propanal propanal butanal butanal propanol propanol pentanol pentanol 0 58895 0 088674 0 66902 0 58585 0 38844 0 064189 0 22632 0 22928 4 4787 8 8621 11 693 7 7497 5 1775 11 001 11 006 5 8286 Chapter 2 7 Seventh Exercise Gradients 0 0023065 0 00069536 0 0058905 0 00088191 0 0066915 0 0058645 0 0038913 0 00064033 0 0022658 0 0022988 0 0015328 0 0030258 0 0075036 0 0074632 0 0017986 0 0038187 0 0038134 0 0020148 Some versions of DAx contain extensions that allow you to correct for signal drift in gradient HPLC and temperature programmed GC You can also include a plot of the gradient percentage or programme temperature in yo
6. Chapter 2 6 2 Step 3 Creating the Identification Database 38 Chapter 2 6 3 Step 4 Using the Identification Database 41 Chapter 2 6 4 Step 5 Setting up a Comparison Sheet 42 Chapter 2 7 Seventh Exercise Gradients 44 Chapter 2 7 1 Step 1 Defining the gradient 44 Chapter 2 7 2 Step 2 Adjusting the gradient 46 Chapter 2 7 3 Step 3 Adjusting gradient percentages 47 Chapter 3 Index 49 Chapter 1 Installation Chapter 1 1 System Requirements To install DAX you must have permission to make changes to the system registry and permission to create the DAx directory If you are unsure whether or not you have these permissions please ask your systems administrator Chapter 1 2 Installing the Software Insert the DAx distribution CD into the CD ROM drive We will assume drive R is being used From the Start menu execute the Run option In the Command Line box type R autorun Click the OK button The set up menu will now start Select DAx Master Setup and follow the instructions on screen Run the Install USB dongle program that has been installed in the DAx program group This should be run before the USB dongle is attached Follow the instructions on screen 7 Attach your USB dongle after installation completes This completes the installation Set up has created a program group in the Start Menu containing the DAx icon click this to start DAx OP Oo D Refer to the Measurement window users manual for det
7. Chromatography to enter a molecular weight for the peak s component To enter a Component name or Annotation simply click the mouse cursor in the peak list window Use the cursor keys and the Shift Tab key to navigate 4 The other columns that can be edited are the annotation and the concentration 15 The contents of the peak list window can be printed Use the print button They can also be saved as a text file Use the File Export menu option for this Finally the peak list can be sent to the clipboard as a table using the Edit Copy menu option Note that the copy command will copy highlighted lines in the peak list window Use Edit Select All to highlight the entire window contents or drag the mouse cursor over the lines you want to select or press the Shift key and use the cursor keys to select lines Chapter 2 1 7 Step 7 Saving Your Work End of first workout Use the File Save As menu option to save your work Better yet use the Save button Dle lale efi aldada se AM 2 The File Save dialog will appear It will look like this save as 21x Select Data ans m Save in Sy DAxSeq e l manuals f TEST1 DAX UI TEST2 DAx File name TESTI DAX Save as type Dax files dax da1 7 Cancel Enter a name in the File Name item then click the OK button to save the data sets By default DAx files will be written but several other file types may be selected in the Save
8. Time 5 Most of this window is used to display the data set Data tags representing the data sets are displayed at the top of the window Chapter 2 1 2 1 Data Tags At the top of each graph window one or more data tags represent the data sets in the window Data tags have the appearance of pushbuttons e data tags can be clicked to display a data tag menu e data tags can be dragged and dropped see the next section e data tags flash blue when a popup menu appears that pertains to the data set such as when the right mouse cursor is clicked on a peak marker e data tags flash blue when a data set is changed such as when a peak marker is dragged to a new location e data tags are depressed when the mouse cursor is placed over the data set s curve e data tags are depressed when the mouse cursor is placed over a peak tack or spline marker e data tags are depressed in stacked views created using the Data Stack data menu option when the mouse cursor is in the vertical range of one or more of the stacked data sets The last three of these are tracking behaviour Tracking behaviour is suppressed when the Shift key is held down By default up to three lines of tags are displayed at the top of graphic windows For windows containing many tags the number of lines of tags can be changed by placing the mouse cursor just below the data tags where it will change shape to an up down arrow Click and drag the mouse to change the amou
9. on red Click on the popup button at left of the status bar and keep the mouse button depressed to see up to 32 of the most recent important messages When more than one important message has been added recently the button will flash e A waste barrel bottom right Data sets can be dragged to the waste barrel to remove them However they can also be retrieved back from the waste barrel Refer to the chapter The Waste Barrel in the DAx manual for details on how to use the waste barrel Now that you know a little more about the various items on the screen enter your name in the Operator item and your password in the Password item If no one else has changed it the password is XYZZY Press enter to start the program proper Chapter 2 1 2 Step 2 Loading a Data Set Now that DAx is running we need to load a data set This is done using the File Open menu option However the quick way to open a file is to click the file open button Click here to open a file A dialog box appears listing the available data files Two or more example files have been installed along with DAx The dialog box should look something like this OPen le Files of type Dax files dax da1 Cancel I Add to window 7 Multiple Windows I AutoAnalyse Config Double click on test1 DAx to load it into memory A data set graphic window will be opened It will look like this EI E DAxSeq TEST1 DAX eis 100 200 300
10. Click here to print the contents of a window You may also put the contents of a data set graphic window on the clipboard To do that use the Edit Copy Graph menu option A so called metafile picture is put on the clipboard You can insert such a picture into a word processor document That s how the following picture was created 3 The Print button actually prints a report if a report definition has been set up This can be done using the Report Load Report menu option 12 DAx 5 0 PP van Mierlo 07 02 96 21 42 37 Volt time s Chapter 2 1 5 Step 5 Analysing the Data Set Now let s try to find some peaks The quickest way to do this is to click the Baselines amp Peaks button Die S fT of se as 8 El Elm 2 Click here to create baselines for and find peaks in the data in a window This is what we get provided the analysis parameters have not been changed since DAx was installed EI E DAxSeq TEST1 DAX 5 x S HA DAXSIM1 PRN B DAXSIM1 PRN U Time It Chapter 2 1 5 1 Manually Adjusting Peaks is possible to manually adjust peaks In the example above the triangular peak top markers are clearly visible There are similar markers for peak begin and end these markers are obscured by the signal in the example above but are clearly visible on screen To move any of these markers place the mouse cursor on them click the left mouse button and
11. DAX Data Acquisition and Analysis Software 2014 PP van Mierlo Quick Start Guide Edit View Data Peaks HPLC GC GPC CE Calibration Analysis Report Datalist Window Help Sleinie e _ 2l als Ll z A EM al 205 83 0 2369 V Mw lt na gt AppMob 338 8 CAL lt na gt lax test3 dax a i sTanD 1 0a1 _ B STAND 1 Da1 methanal 0 61 216 20 13 221 70 223 00 225 70 4 0000 0 5238 14 227 40 228 50 229 40 2 0000 0 3727 15 261 60 265 40 267 20 5 6000 1 0290 16 267 40 269 10 270 20 2 8000 Ds 4112 2 0 methanal 0 275 0 100 0 400 5002 297 80 ethanal 0 365 0 000 0 400 19 300 50 301 70 303 20 propanal 0 568 H 0 000 0 400 2 7000 0 4453 20 303 20 304 40 306 10 butanal 0 360 0 000 0 400 2 9000 0 4900 21 306 10 307 10 308 20 pentanal 0 246 0 000 0 400 2 1000 0 3624 22 308 20 309 20 310 30 hexanal 0 189 0 000 0 400 2 1000 0 3875 23 311 20 312 40 313 90 heptanal 0 177 2 7000 0 4123 24 335 40 336 40 337 70 methanole 5 289 H 0 300 0 510 2 3000 0 3968 25 337 90 339 10 340 80 ethanole 4512 2 9000 0 4878 26 340 80 341 70 342 90 propanole 5 028 2 1000 0 4187 27 342 90 344 40 345 60 butanole 6 764 2 7000 0 4989 345 60 bentanole 6 353 www dax nl Contents Chapter 1 Installation 3 Chapter 1 1 System Requirements 3 Chapter 1 2 Installing the Software 3 Chapter 2 Exercises 5 Chapter 2 1 First Exercise Basics 5 Chapter 2 1 1 Step 1 Starting DAx 5 Chapter
12. MI PRN ak DAXSIM1 PRN Boiynomial Degree 1 A m Peak Coordinate Offset Limits Low 0 Pd Hick fie 20 pA Derive from data m Base pairs Limits ID Iter poi o not alter point usage toe oo WA Hak E20 A Add to existing calibration Tl Derive from data Cancel Help pa Now check Derive main calibration from check the topmost data set in the list box at left do not select Logarithmic Base Pair counts select a Polynomial curve with degree 1 do not check the Derive from data boxes and click the OK button A DNA calibration has been created To see how it looks execute the Calibration Calibration Curve menu option To see a table execute the Calibration Calibration List option Here s the curve 34 DAx 8 1 18 03 2008 17 46 43 PP DNA Calibration not saved Linear order 1 Polynome BP 4 57757 0 0242333x Correlation 99 9965 Ave Diff 0 251134 Base pairs 250 200 150 100 l 4 5 5 0 5 5 Peak Top Coordinate The calibration does not seem to take the leftmost two points into account this is because we double clicked the mouse on those points in the calibration curve window to exclude them from the calibration Now all that remains is to use the calibration to analyse some real data Chapter 2 5 3 Step 4 Using the Calibration Load the file test4 DAx and analyse it Zoom in on the part of the graph following the big peak You should g
13. ails on hardware installation if applicable Chapter 2 Exercises This guide helps you make a quick start in using DAx Chapter 2 1 First Exercise Basics Chapter 2 1 1 Step 1 Starting DAx Start DAx using the DAx icon in the DAx program group in the Start Menu This is what you will see DAx Data Acquisition amp Analysis l8 x File Edit Sequence CE Calibration Analysis Report Help Dells tfs on el 3s w e Hz Sl 2 Operator Name xl PP Ser Hr PP 7 1 2 8 22 DAx Data Acquisition amp Analysis Version 7 1 2002 Yan Mierlo Software Consultancy fax 31 40 241 1945 email support dax nl Operator PP Password Cancel Change Password This screen contains the following items e A caption with the program name and buttons to minimise maximise restore or close the program e A menu bar Initially this has only a limited number of options depending on the version of DAx you re using Two options are always present File and Help e A toolbar containing buttons that let you quickly access the most common menu options The toolbar is also used to display tracking coordinates whenever the mouse cursor is over a graphics window e A status bar at the bottom The status bar is used to display messages of varying importance Normal messages are black on gray and more important messages are black on yellow or white on green or blue Messages that require immediate attention are a white
14. aks list DAXSIM1 PRN not saved Measured 00 00 00 00 00 00 by PP RMS Noise AU 0 189 0 189 Peak Begin s Top s End s Top AU Annotation 93 200 94 300 95 400 5 9427 50000 118 300 119 700 120 800 4 9746 45000 136 500 137 800 139 300 4 6067 40000 144 200 146 000 147 200 4 1538 35000 162 600 164 400 165 800 3 6891 30000 177 000 178 700 180 200 3 6716 25000 248 600 251 000 252 900 1 97 257 900 260 100 261 900 1 8359 271 700 274 000 276 300 1 7433 0 279 500 281 700 284 200 1 6588 OONDOBRWD We ll now use these data to create a GPC calibration Chapter 2 4 2 Step 2 Setting the Flow Rate It is important to realise GPC calibrations work with elution volumes not time coordinates So we must make sure the flow rate for our calibration measurement is correct Click the data tag and execute the Sizing Sizing Dialog menu option This is what you ll see amp Data sizing DAXSIM1 PRN 1 x Data Generic knone gt m Horizontal Axis Unit DE paxsm prn Freg Hz fio A Name Vot w Offset s 0 00 A Offset fo Z Inert s 0 00 A Factor 6 97384 WA Unit Time s gt Move first point to 0 0 Flow Rate ml min 3 WA D New Window I New Curve Total Concentration RB A Cancel Help Dilution jo A 28 Change the Flow Rate to 300 The flow rate setting for the baseline will also change All data sets that belong together will always have the same flow r
15. an remove the spline or more accurately hide it Just click the add spline button on the toolbar again or execute the Splines Add Spline button in the data tag menu again Now your baseline should look like this 25 20 15 10 sn an 5 700 Now you re ready to find some peaks What if peaks had already been detected DAx will notice the baseline has changed and will recalculate the peaks Chapter 2 2 3 Step 3 Finding Peaks To find peaks use the Peaks Find Peaks menu option to invoke the peak find dialog You may also use a toolbar button Dlelals 5 ls al ESIC 2 Click here to invoke the peak find dialog 22 Refer to chapter Peaks Find Peaks in the DAx manual for details on the peak find dialog box For now you might just click the OK button This ends the second exercise Chapter 2 3 Third Exercise Capillary Electrophoresis The third exercise takes you through some aspects of Capillary Electrophoresis If the menu option CE is not present you need to use File Customise gt Extensions and check CE Options Not all versions of DAx contain this option Chapter 2 3 1 Step 1 Calculating Apparent Mobilities In capillary electrophoresis CE the apparent mobility of a Liot dag app i tay component is calculated as m with Mapp apparent mobility 10 m7 Vs lot length of capillary m let length between injection point and detector m tmig migration time s V voltage drop over ca
16. and new points are added To see what the quantitative calibrations look like click the right mouse button on a line in the Identification Database list window Now execute the Draw Quantitative option and a graph depicting the calibration will be displayed It will look like this DAx 5 0 propanal calibration PP printed 10 04 96 14 57 14 Area AU s 0 006 0 004 0 002 0 000 0 0 0 2 0 4 0 6 concentration Remember that the concentrations for the alkanols were based on migration time corrected peak areas rather than peak areas For this reason the quantitative calibration for the alkanols should use not the peak area but the migration time corrected peak area as the quantifying parameter Go to the Identification Database list window Click the left mouse button on the line containing the first alkanol methanol then while keeping the mouse button depressed drag the mouse cursor to the last line containing an alkanol Now click the right mouse button Execute the Config Quantification menu option in the pop up menu This is what you will see 40 Quantitative Calibration xl Quantification coordinate Normalised Peak Area Peak Area Migration Normalised Peak Area Migration m Calibration Curve Type C Multilinear C Cubic Spline Degree Polynomial fi m Concentration Limits tow fot YZ Hin fos A Tl Derive from data Relative limits in Cancel Help Change th
17. as type item In the example there are two data sets listed in the list box at left If you try to deselect one of them they will both be deselected That s because these data sets belong together as indicated by the line connecting them which means they cannot be saved separately You must use a new file name If you try to use an existing file name DAx will refuse to save the files That s because it s not Good Laboratory Practice to overwrite any data This concludes the first exercise Chapter 2 2 Second Exercise Shortcuts and More The second exercise will show you some shortcuts but it will also dig a little deeper Chapter 2 2 1 Step 1 Opening Several Data Files and Analysing Them Let s begin the second exercise We ll assume you have opened DAx the same way as before chapter Chapter 2 1 1 Once again click the File Open button to start loading data This time we ll use two shortcuts You can select more than one file in the file open dialog box Click on one file name then press the Ctrl key and click on another filename You might get something like this 5 This behaviour can be changed using the File Customise gt GLP menu option 17 Open eee 2x Look in Sy DAxSeq JB ec l manuals Filename TEST2 DAX TEST1 DAX Files of type Dax files dax da1 Cancel I Add to Window I Multiple Windows I AutoAnalyse Config If you were to press the OK button now
18. ate We ve now made sure of the following crucial steps for creating a GPC calibration e we have a data set with peaks e molecular weights have been entered as annotations for the peaks e the flow rate has been set Chapter 2 4 3 Step 3 Creating the Calibration Execute the GPC Calibrate menu option The GPC Calibration dialog box is displayed For details refer to the chapter GPC Calibration Dialog Box in the DAx manual The dialog box looks like this m Calibration Curve Type DAXSIM1 PRN tame DAXSIM1 PRN IV Use Logarithmic Mw s C Multilinear Cubic Spline Polynomial Degree fi A m Elution Volumes ml Low fo A Hion 20 IV Derive from data Il Do not alter point usage m Molecular Weight Heeale Calibration Add to existing calibration Cancel Help T Detector measures weight Now e select the topmost data set in the list box at left e check the logarithmic Mw s box e select a cubic spline curve 8 As soon as you change the flow rate for the top data set the baseline will also become selected in the list at the left side of the dialog box 29 e check both Derive from data boxes e and click the OK button A GPC calibration has been created To see how it looks execute the GPC Calibration Curve menu option To see a table execute the GPC Calibration List option EN GPC Calibration Graph L Iolx lag Malecular Weight Valume
19. both files would be loaded e check the Multiple Windows box to load each data set into its own window Otherwise the data sets will be loaded into a single data set graphic window e check the Add to Window box to add the selected data sets to an existing data set graphic window Now the second short cut Check the AutoAnalyse box in the lower right corner of the File Open dialog the data files will automatically be analysed when they have been loaded Depending on how AutoAnalysis has been set up various operations may be performed e automatic baseline construction e automatic peak detection e automatic printing of the data as a graph e automatic printing of a peak list e automatically saving the peak list to a file Automatic analysis is set up using the Config button that is displayed next to the AutoAnalyse box in the File Open dialog Refer to the chapter Setting up Automatic Analysis in the DAx manual for details To clean up DAx in preparation for the next step use the Window Close All menu option to close all data windows If you were using automatic analysis two things can happen e the contents of a window have not yet been analysed DAx will see this and ask if the analysis should be performed before the window is closed e the contents have been analysed but not saved DAx will ask you to confirm the loss of the modified data Chapter 2 2 2 Step 2 Adjusting a Baseline The file test2 DAx contains a pretty d
20. drag the marker to its new position Press the Ctrl key then double click the left mouse button to add a new peak Remove a peak by moving its top past its begin or end marker Made a mistake Press Alt BackSpace to undo the operation Alt Shift BackSpace redoes it or use Edit Undo and Edit Redo Place the mouse cursor over the top marker then click the right mouse button to display a popup dialog box This dialog box let s you enter a peak name or concentration It also lets you remove the peak or mark the peak as the reference or normalisation peak cf chapters Marking Normalisation Peaks and Marking Reference Peaks in the DAx manual Chapter 2 1 6 Step 6 Displaying Peak Data To display a list of peaks click the Peak List window button on the left side of the data tag 5 DAXSIM1 PRN Click here to open a peak list window Peak list windows can contain a variety of columns of information Below is an example lolx A Peaks list DAXSIM1 PRN not saved Measured 00 00 2000 00 00 00 by Description Point to point noise Y 0 18635 3 07 RMS Noise Y 0 056352 0 93 potassium sodium lithium The Component column is one of the columns in the peak list window that can be edited In the example names for the peaks are being entered Values entered in the annotations column are used in Capillary Electrophoresis to set the apparent mobility of a reference peak and in Gel Permeation
21. e quantification parameter to peak area migration time then click the OK button You can also set lower and upper limits for the concentration Concentrations outside this interval will still be calculated but will get an L or H flag This is useful in quality control situations Now all that remains is to use the Identification Database to analyse some real data Chapter 2 6 3 Step 4 Using the Identification Database Load the file test4 DAx and analyse it Zoom in on the part of the graph following the big peak You should get something like this 41 DAx 5 0 PP 03 04 96 15 42 11 AU 0 15 0 10 0 05 propanal butanal propanol pentanol 0 00 LAA T T T T T I T T T T T 250 300 350 time s To make the names visible you can either inspect the peak list window or mark peaks with component names Use the File Customise gt Plotting Peaks menu option to display a dialog box in which you can select a peak label To see which concentrations DAx has derived again use the peak list window Notice that the concentrations found for the alkanals are roughly equal to 100 times the peak area The concentration found for the alkanols is roughly 100 000 times the migration time corrected peak area You may wonder if DAx will overwrite any component names you have entered manually for a peak It will not Chapter 2 6 4 Step 5 Setting up a Comparison Sheet DAx has the option of creating a
22. es To change the gradient percentages invoke the data set information dialog box by clicking on the data tag and selecting the Info menu item In the information dialog click the HPLC button This displays another dialog box in which you can now change the gradient percentage values 47 ters Parame HPLC Gradient GC Temperature Programme ELLLLLLLLL eLLLLLLLLL eLLLLCLLLL 1 2 3 i 3 ARNHNNNN SE 48 Chapter 3 Index adjusting baselines 19 adjusting gradients 46 adjusting peaks 14 analysing data 13 annotations 15 apparent mobilities 23 automatic analysis 17 baselines 13 19 Capillary Electrophoresis 23 changing peaks 14 copying data 9 data tags 9 10 displaying a peak list 15 DNA analyses 31 editing baselines 19 editing gradients 46 editing peaks 14 effective mobilities 25 finding peaks 13 22 flow rates 28 GC gradients 44 Gel Permeation Chromatography 27 gradients 44 46 HPLC gradients 44 installation 3 loading data 6 manually adjusting peaks 14 mouse buttons 10 moving data between windows 9 opening more than one data file 17 peaks 13 14 15 22 36 annotations 15 printing 12 qualifying peaks 36 quantifying peaks 36 recognising peaks 36 saving data 16 shortcuts 17 Size Exclusion Chromatography 27 starting DAx 5 status bar 6 system requirements 3 toolbar 6 waste barrel 6 zooming in 11 49
23. et something like this DAx 8 1 18 03 2008 17 51 04 PP Time min 35 As soon as you find peaks the base pair counts are automatically calculated But you don t see them yet To make them visible you can either inspect the peak list window or mark peaks with base pair counts Use the File Customise gt Plotting Peaks menu option to do this This will display a dialog box in which you can select Base pairs as one of the peak labels This is what you get DAx 8 1 18 03 2008 17 49 57 PP 111 4 Time min Finally try using the Calibration Calibrated Axis menu option to display test4 DAx with base pair counts as the horizontal axis Chapter 2 6 Sixth Exercise Qualifying amp Quantifying Peaks The sixth exercise takes you through the steps needed to set up an Identification Database and to use the database to qualify peaks in an unknown sample as well as determine the concentrations of the components If the menu option Analysis is not present you need to use File Customise gt Extensions and check Analysis Options Standard analysis should be unchecked 36 Chapter 2 6 1 Step 1 Analysing the Sample with Known Components Once again start DAx We ll use the same test file as for the DNA calibrations So load the file test3 DAx and analyse it the first exercise took you through the steps required to do this Zoom in on the part of the graph following the big peak You should get s
24. if no CE calibration parameters have been stored with a data set The default parameters will be used The default parameters are always equal to the last set of parameters stored with a data set When you now move the mouse across the graph in the data set graphic window for test1 DAx one of two things can happen e the apparent mobility is tracked in the toolbar e the toolbar displays EffMob lt na gt That s because no reference peak has been set yet so the effective mobility cannot yet be calculated To change between tracking apparent and effective mobilities use the CE Apparent Mobility and CE Effective Mobility menu options Use CE Track Mobility to turn mobility tracking on and off 24 Chapter 2 3 2 Step 2 Calculating Effective Mobilities Now let s mark a reference peak First analyse the data use the Peaks Baselines amp Peaks menu option or the Baselines amp Peaks button on the toolbar Move the mouse cursor over the peak top of the third peak and press the right mouse button You ll see something like this DAX ioj x EioaxsimieRN B oaxsim ern Name Poo Concentration WA Annotation 2 Peak Type cnotyper rend lt none gt x knone gt Reference J Normalisation V Active for areas Help Time 5 Cancel Delete Peak Check the Reference Peak box to make the fourth peak the reference peak The peak marker will change and the toolbar will start t
25. ifficult measurement to analyse It s quite conceivable you re not completely satisfied with the job DAx does of constructing a baseline Simply adjust it yourself Let s see how this is done First load the file test2 DAx Now you can use the analysis button to analyse the data or you may just want to create a baseline without searching any peaks To do that use the Peaks Construct Baselines menu option or click a button Delals fis aalala e El EM e Click here to start constructing baselines You will get a baseline construction dialog box For details refer to the chapter Peaks Construct Baselines in the DAx manual For now just click on the OK button A baseline will be constructed The resulting window may look something like this ElE DAxSeg TEST2 DAx loj x D Bienen Bas AT 0 500 1000 1500 Time It seems likely the baseline needs to be a straight line beneath the two major peaks To make this so we need to add splines to the baseline Click on the data tag for the baseline the tag with the yellow B at left Now either execute the Splines Add Spline menu option or click the add spline button on the toolbar Dlelals fis a a als SIO Click here to add a spline to a data set A spline is a collection of nodes connected by straight lines or by curves The nodes are drawn as squares in the same colour as the curve You can change a spline in a number of ways e
26. indow Invoke the File Customise menu option Select Plotting Peaks and select Effective Mobility as the first or second peak label Effective mobilities will now be displayed above the peak tops You can also select a variety of other labels Finally in this exercise close the peak list window and then use the CE Mobility Axis menu option in the data set graphic window for file test1 DAx to display a graph where the time axis has been replaced by a mobility axis 26 DAx 5 0 Mobility versus signal plot PP van Mierlo printed 09 02 96 14 06 33 AU 400 Effective Mobility Since mobility varies with the reciprocal of the migration time the order of the peaks has been mirrored Chapter 2 4 Fourth Exercise Gel Permeation Chromatography The fourth exercise takes you through a Gel Permeation Chromatography Calibration If the menu option GPC is not present you need to use File Customise gt Extensions and check GPC Options Not all versions of DAx contain this option Chapter 2 4 1 Step 1 Analysing the Calibration Sample Once again start DAx Load the file test1 DAx and analyse it the first exercise took you through the steps required to do this Make sure a peak list window is displayed Now we ll assume we know the molecular weights for the first six peaks in this data set Enter them into the annotations column in the peak list window The peak list might end up containing something like this 27 Pe
27. mf ft GPC Calibration Table _ O x GPC Calibration Parameters Polyn Par Used E ml Mw Check Data 0 5 893 375 25000 568789952 DAXSIM1 PRN 1 2 2247E 06 521 360 352526304 DAXSIM1 PRN 2 5705 YES 729 525 35000 164316640 DAXSIM1 PRN 3 4 7943 YES 659 068 40000 108906760 DAXSIM1 PRN 4 YES 598 546 45000 31950158 DAXSIM1 PRN 5 YES 471 571 50000 632817 DAXSIM1 PRN Let s verify this calibration Activate the data set graphic window for the file test1 DAx and execute the GPC Mw Axis menu option You ll get something like this 30 DAx 1 0 Molecular Weight versus signal plot PP van Mierlo printed 08 02 96 20 52 37 AU log Molecular Weight The curve starts out and ends in peak tops The reason is that valid elution volumes for molecular weight calculation were limited to the range of the calibration data points by checking the Derive from data box Elution volumes smaller than the smallest elution volume in the calibration list or larger than the largest volume cannot be converted to molecular weights You may also notice that the curve has been flipped over in horizontal direction This is because high molecular weights correspond to low elution volumes Chapter 2 5 Fifth Exercise DNA Base Pair Count Determinations The fifth exercise takes you through the steps needed to determine base pair counts in a DNA electropherogram If the menu option Calibration is not present you need to use File
28. nt of space allocated to data tags 1 The data tags can also be displayed at left using the View Tags left menu option 8 Chapter 2 1 2 2 Data Tags Moving Data Data tags can be moved around Drag them to the waste barrel to remove the data don t try that now Drag them to another data set window to move the data If you press the Ctrl key while dragging the data will not be moved but will be copied It s also possible to drag a data tag to an empty part of DAx where no window is being displayed If you try that a new data set graphic window will be created Let s try data copying Move the mouse cursor over the data tag click now press the Ctrl key While keeping both the mouse button and the ctrl key depressed move the mouse cursor over an empty part of the DAx window Now release the mouse button A new window containing a copy of the data is created You should get something like this s DAx 7 1 Data Acquisition amp Analysis PP 1 File Edit View Data Peaks Sequence HPLC GC GPC CE Calibration Analysis Report Window Help Ole ma S ES bret A As Bi amp E 12 84 2 28 V Mw lt na gt AppMob lt na gt CAL lt na gt TEST1 DAX as lbaxs m een PRN EI E DAxSeg TEST1 DAX i x g DAXSIM1 PRN Y rama Drag curves to the waste barrel to delete them Note that the Waste Barrel has been moved out of the way again by clicking the mouse button on i
29. omething like this but without the names on top of the peaks they are added in the next step DAx 5 0 PP 03 04 96 15 31 25 AU 0 02 methanal _ ethanal propanal butanal pentanal J exanal heptanal methanol ethanol propanol butanol pentanol hexanol heptanol 0 00 T 350 time s 250 wo fo Oo Next make sure a peak list window is displayed We know the components in the data set and should enter their names in the component name column in the peak list window19 We ll also enter concentrations in the concentration column NB we do not care about the earlier peaks in the peak list window Only the peaks with retention times over 4 minutes are of interest To locate a peak in the peak list window simply move the mouse cursor over the top marker for the peak in the graph the appropriate peak in the peak list window will be highlighted The peak list might end up containing something like this 10 Obviously the names of the components have been pretty randomly picked 37 Peaks list STAND 1 Da1 not saved Measured 00 00 00 00 00 00 by PP RMS Noise AU 0 00023251 0 00011626 Peak Top s Comp Conc Area AU s Area Mig 1 10 900 0 0020676 0 0017258 2 190 800 0 004007 0 00020978 lt some peaks skipped gt 13 226 800 0 0013924 6 1429E 05 14 228 500 0 003491 1 0 00015278 15 265 400 0 0039531 0 00014919 16 269 100 0 002658 9 8766E 05 17 296 300 methanal 0 266 0 0026634 8 9883E 05 18
30. pillary kV The effective mobility is Met Mapp EOF with Mer effective mobility 10 m Vs EOF electro osmotic flow 10 m Vs To be able to calculate apparent mobilities DAx needs to be told the length of the capillary being used the distance from injection to detector and the voltage drop To then be able to calculate effective mobilities DAx needs to be told the electro osmotic flow Generally instead of entering the electro osmotic flow you will mark a reference peak and enter its effective mobility Since the apparent mobility for the reference peak is also known the electro osmotic flow can then be calculated 23 Let s assume the file test1 DAx contains a CE measurement So once again start DAx Load the file text1 DAx refer to the first exercise for instructions Now invoke the CE Calibrate menu option The CE calibration dialog will be displayed CE Capillary Parameters i xl DAXSIM1 PRN a Length of capillary m 1 DAXSIMI PRN p n A Length till detector m 0 3 Voltage kv 10 000 A Default Reference Mobility 10 9 m s 0 00 A Parameters will be stored for selected data sets and as global defaults Cancel Help Enter the values shown into the dialog box Now click the OK button to store these calibration parameters You should realise that the parameters will only be stored in the data sets you select in the list displayed at left What happens
31. put the mouse cursor over a node then click the left mouse button and drag the node to a new position Keep the Ctrl key depressed to add a node e f you drag a node past other nodes those will be deleted e Add a node by depressing the Shift and Ctrl keys then double clicking the mouse at the position where you want to add a node 6 It s not enough to just click the add spline button if the data tag for the baseline is not the default data tag The default data tag is the one with the thicker edge Clicking the mouse button on a data tag makes it the default data tag 20 e Use Alt BackSpace to undo node modification operations Alt Shift BackSpace redoes them o Initially the spline for the example may have looked something like this the spline node markers have been enlarged for clarity Now the spline has been modified but the baseline hasn t yet been updated Click on the data tag for the baseline and use the Splines Replace Curve With Spline Changed Nodes Only menu option to replace the baseline with the spline Only the parts of the baseline where you adjusted the spline will be changed You can also use a toolbar button for this 7 In fact DAx offers to do this automatically as soon as the spline is first changed 21 Dlelals fis 2 alas t ce Em 2 Click here to replace a data set with a spline A menu appears to let you choose how much of the curve to replace with the spline Now you c
32. racking effective mobilities if menu option CE Effective Mobility has been checked otherwise you should do that now In the first step of this exercise it was explained that to calculate effective mobilities the effective mobility for the reference peak needs to be known But we didn t enter it However in the CE calibration dialog we did enter a default reference mobility and that s now being used If the effective mobility for the reference peak does not have the default value you should enter the correct value as an annotation in the peak top popup dialog or in the annotations column in the peak list window The peak list window might end up looking like this 25 15 x A Noise W 0 056352 0 93 End s Height y Annotation Rel Conc AppMob EffMob Areaiig V 121 10 5 0205 11 40 187 97 215 14 0 0433 139 40 4 6275 190 45 11 18 163 25 190 45 0 0368 147 30 4 1549 10 99 154 11 181 28 0 0342 166 10 3 7062 11 45 136 36 164 03 0 0316 180 30 3 6471 11 17 125 91 153 08 0 0284 253 00 1 9579 8 05 89 64 116 81 0 0145 262 60 1 8735 8 12 86 51 113 68 0 0142 276 40 1 8195 8 32 82 12 109 29 0 0138 284 30 1 7049 8 09 79 87 107 04 0 0130 ss n104 gt s 6 Notice the column with effective mobilities for the peaks Naturally the effective mobility listed there for the reference peak equals the value entered as the annotation Would you like to print the effective mobilities for each peak in the graphic w
33. sheet that compares the peaks found in various data sets Peaks with the same qualifying parameter are grouped and the averages of all of their parameters as well as standard deviations are calculated Let s try it now Load the files test3 dax and test4 dax into a single window and analyse them refer to chapter Chapter 2 2 1 for information on opening several data files at once Make sure the data files are in a single window If they are not use the mouse cursor to drag the data tags to a single window 42 Now execute the Analysis Comparison Sheet option You will get a dialog box that looks like this STAND 1 Dal IV Only show matching peaks aan E A A STAND 1 Da1 I Only show differences 5158 4 Dal 5158 4 Dal C Use peak names Qualification coordinate Peak Top Coordinate Moment Peak Coordinate Offset gt Peak Renin Conrdinate m Tolerance a A Absolute C Relative m Statistics Il Do not show statistics V Average M Stand Dev Help V Skew J Stand Dev Cancel Now e check the Only show matching peaks box This will make sure a comparison sheet is created that only lists the peaks that occur in both data sets e select the peak top time as the qualifying parameter e set an absolute tolerance of 2 seconds Now click the OK button A text window will be created with the following contents DAx 5 0 Comparison sheet 17 04
34. t then dragging it to a new position Chapter 2 1 2 3 Which mouse button should use When dragging data tags you can use either the left or right mouse button If you use the left button only the data tag you clicked on will be moved2 If you use the right mouse button the data set and any other data belonging to it will be moved Now that you have created a copy of the data try sending the copy to the waste barrel Simply click and drag the data tag to the waste barrel An empty window will be left Chapter 2 1 2 4 Data Tags Other Uses If while trying to move a data tag you inadvertently released the mouse button while the mouse cursor was still over the tag you will have seen the data tag menu This is a menu that contains a number of options that affect a single data set The menu options in the main menu displayed at the top of DAx affect all data sets in a data set graphic window If you execute the Info option in the data tag menu you will see the data set information dialog box Something like this 2 You can also select multiple data tags by pressing the Ctrl key when clicking additional tags then move all selected tags at once 10 Data Information xl Data Set EASE Measuring File E DAxSeq TEST1 DAX I AutoAnalysis File Date Time 2770371 996 22 46 28 Modified Operator Generic Measurement amp Measured 00 00 2000 00 00 00 Flow Rate Adjustment a Curve Vertical Moving C
35. ualification pass C Relative Change Parameters Add to existing calibration OK Cancel Now e select the topmost data set in the list box at left is selected by default click on the Peak Top Time qualification coordinate enter a value of 2 Seconds for the tolerance select the Quantification tab select peak area as the quantifying parameter select a polynomial calibration with degree 1 and click the OK button Help An Identification Database has been created To see a table of the database execute the Analysis Display Database menu option This will create an Identification Database list window Note that in the Quant Points column the number of quantitative calibration points for each component is 2 This is because DAx automatically adds the point 0 0 to each calibration assuming that peak area is 0 if the concentration is 0 If more than one quantitative calibration point is found for a component because several measurements with different component concentrations are being included in the Identification Database the 0 0 point is de activated NB The procedure for adding quantitative calibration points is exactly the same as the procedure used above to set up the initial calibration points with one exception before clicking the OK button in the Identification Database Edit dialog box you must check Add 39 to existing calibration That way the previous points are kept in memory
36. ur graphs If the menu option HPLC GC is not present you need to use File Customise gt Extensions and check HPLC Options or GC Options Not all versions of DAx contain this option Chapter 2 7 1 Step 1 Defining the gradient As always start DAx then load the file test5 dax There is no need to analyse the data yet The data look like this 44 DAx 5 1 uit dax 3 04 08 96 15 43 28 AU gradient time min There is clearly a gradient which runs from approximately 1 minute to approximately 2 5 minutes To tell DAx about this gradient use the HPLC GC Set Gradient menu option A dialog box appears El amp C win SETUP DAx test5 dax Number of steps fi V Derive signal values V0 Y1 from data TR F ro 1xT to min 1 A Al A A A nv ee A za f 54 Gradient Type t1 min 3 WA A WA WA WA C GC Temperature Programme Y1 Ge _z _ HPLC Solvent Gradient WA WA 2A VA a m Alera Enter the values shown above in the dialog box That is t0 1 minutes t1 3 minutes Derive signal values from data has been checked so no values for YO and Y1 can be entered In the example an HPLC Gradient has been selected The initial percentage has been entered as 0 and the final percentage as 30 Click the OK button This is what you will see 12 In most practical applications you would know the exact timing of the gradient In this example we assume the exact timing is unkno
37. urve Vertical Sizina 7 Description p ze Gradient BE Points sooo Freq Hz m Offset s oo Ordinate var Total Core Inert s 0 00 Unit Offset fo Dilution 0S Flow min 3 Unit Factor 6 97384 Dax 7 1 24 08 2002 14 56 50 PP Read data from disk file E DAxSeg TEST1 DAX Refer to the chapter Data Set Information Dialog in the DAx manual for details Chapter 2 1 3 Step 3 Zooming In on the Graph You may want to enlarge part of the graph being displayed for a data set To do that you must once again drag the mouse Place the mouse cursor in the bottom left corner of the part of the graph you want to enlarge Now click the left mouse button and while keeping the mouse button depressed move the mouse cursor to the top right corner of the part you want to enlarge Then release the mouse button 11 E E DAxSeq TEST1 DAX BE Ioj xj al 60 80 100 Time 5 v Some remarks e Click the right mouse button to zoom out e You do not have to go bottom left to top right drag in any direction you like e You can zoom in up to ten times However you may reach maximum zoom before then e Press the Shift key and double click anywhere in the graph to set a relative origin for mouse tracking coordinates Chapter 2 1 4 Step 4 Printing the Graph or Exporting It To print the graph use the File Print menu option More conveniently click the print buttons Dlelals fis el amp SIO
38. wn to be better able to demonstrate how gradients can be modified 45 TEST5 DAX Iof x feipors time min Two new lines have been added The lower one displays the gradient percentages that were entered running from O to 30 percent We will come back to this line later For now you may hide it using the View Gradient Percentages menu option Chapter 2 7 2 Step 2 Adjusting the gradient Clearly the time coordinates for the gradient are not yet correct To change them move the mouse cursor over either of the triangles used to denote the beginning and end of the gradient The mouse cursor will change to a four pointed arrow Click the left mouse button and simply drag the gradient node to a new location The exact time coordinates for test5 dax by the way are 50 and 150 seconds You can now subtract the gradient from the date set Invoke the HPLC GC Subtract Gradient menu option to do this Put the new data set in a new window You ll probably end up with something like this 46 DAx 5 1 uit dax 3 04 08 96 16 09 42 AU gradient 100 4 i 50 T T T l T T T I T 0 100 200 time s Initially the gradient signal value curve was still displayed in the new window You can use the data tag Gradient Show Gradient menu option to hide it This leaves only the data and the gradient percentage curve as shown above Chapter 2 7 3 Step 3 Adjusting gradient percentag
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