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square 1.5 User Manual

1. Method Assign compound for Blob 4194329 a Select the compund to be assigned to the blob p brass Hash Compound Name Sim Index LRI1 LRI2 LRI3 LRI4 LRIS Delta 1 monene 98 1030 o 0 0 o 0 a lt 0 0 0 0 0 p 3 Pseudolimonene 84 1004 0 0 0 o o ptas 4 Perillaldehyde 83 1278 o 0 o 0 o 5 a 83 953 o lo 0 0 y gt 6 arene lt delta 3 gt 81 1009 Jo 0 o o 7 enchene lt alpha gt 81 394 J o lo 0 0 _ F 8 Phellandrene lt beta gt 80 1031 o jo o o 0 3 9 Ocimene lt E beta gt 80 10 0 lo lo 0 0 10 Santolinatriene 79 902 o 0 o o o 11 erpinyl formate lt alpha gt 79 1306 0 lo lo 0 0 m 12 ricydene 78 923 o 0 o o 0 13 inene lt alpha gt 78 933 o o lo 0 0 14 inene lt beta gt 78 978 o o o 0 0 H 15 Sabinene 78 le72 Jo 0 o o o 16 erpinolene 78 1086 l0 0 o o o 17 eranyl benzoate 77 1965 0 0 10 0 0 18 erpinene lt gamma gt 77 1058 0 0 o 0 gt TI 1375 0 n in in lat Cancel Figure 7 23 The Assign Compound dialog box In fact the identified Blobs are not automatically added to the Method in order to avoid duplications The user should explicitly define which Blobs are to be added to the method using the following procedure e Once the automated search for one or more Blobs has been performed if the Blob is not already present in the Blob Table of the Method the entire row is redrawn in red in the Data Analysis table see Section 7 6 1 e The user can dec
2. Spine ag A RARE Reo 118 9 1 2 Set up the Method sea dia ia 123 9 1 3 Load a chromatogram Li a 126 9 1 4 Integrate the chromatogram aa 127 9 1 5 Identify the Blobs a crs o a a 129 9 1 6 Integrate again edo Bis dow A A o ee 130 9 1 7 Integrate All Chromatograms 131 9 1 8 Assign the concentrations 0 132 9 1 9 Save the Schedule p42 2 Gerd eg tak ee ge ee 134 9 1 10 Create the Calibration as op gee ge ee A 134 9 1 11 View Calibration results econ a ee ES 136 9 1 12 Save the Schedule again lt 136 9 2 Internal Standard Calibration 138 9 2 1 Set up the Schedule ine es egg RO See ee Da a 138 9 2 2 Set up the Method a ea a e AA Ga es 138 9 2 3 Load a chromatogram x6 ow dea A ee KE YK 138 9 2 4 Integrate the chromatogram 0 4 138 9 2 5 Identify the Blobs os fences Sap ag Exedy Ue ghee BR es 138 9 2 6 Integrat acallar Oly eae is YS 139 9 2 7 Integrate All Chromatograms 139 9 2 8 Assign the concentrations 20 139 9 2 9 Save the Schedule a 139 9 2 10 Create the Calibration 4 62 ts eg Be Sem eee 139 9 2 11 View Calibration results 139 9 2 12 Save the Schedule again 4 322 4 4 44 asd be ae 140 9 3 Quantitation of an Unknown Sample Chromatogram 140 9 3 1 Add unknown sample files to the schedule 140 9 3 2 Load a chromatogram ose dd Bek
3. data formats 168 3 In the the upper right side of the Dialog Box a combo box named Profile will be shown together with the two buttons New and Remove see Figure 10 8 Select a file of type GCxGC MS Look in o samples x 5 py a samples Profile v ES Data027 4sec Documenti Ef Data027 4secMIC1 recenti Mod Time 2nd El time o 1st Mod Time 2 Documenti GCMSsolution file Further info required Use a profile Risorse del computer e File name Data027 4sec qgd Risorse di rete p Files of type Files QGD GCD CDF AIA ANDI XRS Figure 10 8 Open Dialog Box with Profile Combo Box 4 Press the New button a new Dialog Box named New Profile will open See Figure 5 Assign a name for the new profile fill all other information requests displayed on the New Profile Window 6 Press Save this will close the window returning to File Open Dialog Box 7 5 In the Profile section of the File Open Dialog Box use the combo box to select the profile just generated Note that the combo box only shows the profile types that are compatible with the selected data file and software license The new profile is permanently stored in the ChromaConfig xml file and can be used when a similar data file should be analyzed The button Remove of the profile section allows the user to delete from ChromaConfig xml file the profiles that are no longer used 169 New P
4. The context menu is shown in Figure 5 5 69 ChromSquare License GCxGC MS Current file HC3 6sec xrs File Method Schedule View Options Action Tools 7 Filename Std Smp Peak PeakB Peake HC1 6sec ws Standard 0 0793 0 0792 0793 I HC2 6sec xrs Standard 0 3966 gt A HC3 6sec 15 Standard 1 983 faite cal 983 Schedule A pample wd Fil Row Only Null Values Fil Row All Values Chromatogram e Method Figure 5 5 The context menu of the Schedule Table 70 Chapter 6 Method Panel 6 1 Panel contents X ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS 8sec xrs Cal File Method Schedule View Options Action Tools Integration Shimadzu spectra search DA SCTE C Documents and settingsyaqualis Documenti ChromSquare My Samples 2010 Test Cases GCMS quantitate xcm Ej min area 1 0 8 A noise 10000 0 Schedule peak shift of Mod Time 6 0 blob recognition D name Ton Selection Background Subtraction i Laurate v gt Chromatogram delete Bg On All Bg Off All p 2 Method LL 27 262890 34 901460 Figure 6 1 The Method Panel Integration Page The Method Panel is shown when the user clicks on the corresponding large icon Method of the Side Bar Panel see Figure The Method Panel window consists of one or more pages tabs the main page 71 Integration which is al
5. e At the end of the identification process the Blobs Grouping becomes possi ble all Blobs which share the same Blob ID can be treated as single entities named Groups A series of new values sum of the areas other percentages with various normalizations can now be computed e After the Quantitative Analysis concentrations can be computed and stored in the Blob data structure e In the case of Spectral Search with LRI computation the relative data are stored in the Blob data structure as well See the section 7 6 1 for other details and samples of the Blob data structure 3 1 6 Blob IDs A Blob ID is the identification of a Blob or of a group of adjacent Blobs assigning a Blob ID means to assign a name to a region of the chromatogram map Blob ID is a data structure containing 3 elements 30 e the Name that is name of the chemical compound s corresponding to the region note that names may be very long character string e the JD that is an integer number a sort of abbreviation used for easier reference when more chromatograms are compared among them like in the calibration process e the Region that is the geometrical description of the polygonal area The user can directly set and modify the first two items IDs and names through the Method Panel see chapter xxx Regions are set through a graphical proce dure described in chapter xxx 3 1 7 Blobs vs Blob IDs It is important to highlight the difference b
6. e a Reset button this allow to restore the previously saved values of all scales 86 7 3 1 1 The Color Map The term Color Map refers to a set of instructions internally defined in the software which implements the transformation between intensity values and color values that is between an intensity scale and a color scale Only one Color Map is presently defined in the software this is a classical Color Map frequently used in this types of representations it scans from White to Black passing through a series of colors Magenta Cyan Blue Green Yellow Orange Red Brown 7 3 1 2 The intensity scale The minimum and the maximum values of the intensity scale are initially calculated by the software and can then be modified by the user in order to highlight some details instead of others The software calculates the initial default values of the intensity scale as the mathematical minimum and the maximum of the intensity values of the whole chromatogram Minimum and maximum of the intensity scale can be modified through the two slider controls upper and lower of the Graphical Parameters Window The upper control defines the maximum the lower control defines the minimum The user can manually change these values acting in two ways e directly writing the numerical values in the text fields e scrolling the corresponding sliders 7 3 1 3 The color scale In the middle between the two slider controls there
7. 8 1 2 1 Correct Modulation Time A wrong modulation time is generally the main responsible of an anomalous Map This is the most common case since this information cannot be supplied automat ically The user can easily change it and reload the chromatogram to view the changes in the Map Plot See Section 4 1 1 3 Note after the first loading of a Data File the software generates the correspond ing Result File see 3 3 2 If the user tries to load again the Data File using a different Modulation Time the software warns the user that the Result File is already present and asks whether he wants to overwrite it or not In this case the answer should be Yes 8 1 2 2 Correct Wrap Around The Wrap Around correction does not change the Blob patterns but moves the whole Map Plot towards the top or the bottom See Section 4 6 13 8 2 Integrate the Chromatogram The integration of a new chromatogram includes various steps 1 set up the Method 2 integrate the whole chromatogram 3 optionally define a smaller area Region of Interest or ROI and integrate only this one 4 check the integration results 5 optionally modify some integration parameters For new types of chromatograms the various steps may be repeated more times Once satisfactory results are obtained the Method can be saved and used for the integration of similar chromatograms this is normally the case in Quantitative Analysis The integration
8. e The third part contains switches which set the details of the linear map plot lower frame of the chromatogram panel 4 5 1 Interpolation switches 4 5 1 1 Enhance Small Peaks The switch enables an algorithm which enhances the visualization of the small peaks 4 5 1 2 Graphical Data Interpolation The switch enables the interpolation of data on the 2D map if ON the ab sorbance value at any geometrical point of the map is calculated as the result of a linear interpolation of the values of the nearest chromatographic data points generating an effect of a continuous shade of colors if OFF only the values of actual chromatographic data points are taken into account generating an effect of a pixelated map 4 5 1 3 Smooth Blobs The switch enables the smoothing of the Blob contours if ON the contours are bounded by smoothed curves if OFF by straight lines 4 5 2 2D Chromatogram Plot switches 4 5 2 1 Draw Names onto Map Plot The switch enables the writing of the names of the Blobs in the bi dimensional map plot 4 5 2 2 Draw TtR onto Map Plot The switch enables the writing of the Total retention time First Dimension reten tion time Second Dimension retention time of the Blobs in the bi dimensional map plot 52 4 5 2 3 Draw 2tR onto Map Plot The switch enables the writing of Second Dimension retention time of the Blobs in the bi dimensional map plot 4 5 2 4 Draw Area onto Map Plot The switch enables the
9. Data analysis Grouping aa A E M Polygon mode iia Da S Figure 10 5 Defining a Polygonal Region Figure 10 6 shows the assignment of the name to the defined region Figure 10 7 shows the integration results with the identification of the previously assigned polygonal Blob ID 166 hromSquare License LCxLC MS Current file Unknown 60sec xrs Method Schedule View Options Action Tools Insert name od 60 DP 445 TtR 60 593 2tR 0 593 1tR 60 000 Int 3225 69 Figure 10 6 Entering the polygon region name 4 ChromSquare License LCxLC MS Current file Unknown 60sec xrs File Method Schedule View Options Action Tools Chromal Graphical param Chromatogram Data analysis lt 2 4a 2 E O Polygon mode Index 1D Name en Srta 2621727 unknown 2818468 unknown 3015227 1 MY REGION 3211375 unknown il lod 40 DP 654 TtR 40 872 2tR 0 872 1tR 40 000 Int 2642 09 Method Figure 10 7 The polygonal Blob after integration 167 10 4 Profiles 10 4 1 Description Profiles are sets of information concerning the data files profiles have been intro duced in Chrom 1 software to simplify user input in fact e data files are not always self explaining and in many cases further informa tion must be supplied by the user e in many cases in laboratory routine the characteristics of data files being analyzed do not
10. cdf ex tension a suffix export is also added to the file name before the extension in order to avoid the presence of two files with same name and different extensions in the same folder which is not allowed by the software 4 1 6 Export as XML for Excel This command allows to save various information about the chromatogram being analyzed into a special xm1 file similar to the xrs file see 3 3 2 The exported file has xm1 extension these files can be easily read and managed by the latest versions of Microsoft Excel which allows a deep investigation of the file contents XML files can also be viewed with less functionality by Internet Explorer or other Internet browsers The software always asks for the filename of the exported file and the folder where it should be placed the folder path is remembered for future savings It is a good practice to use filename and folders different from those of the original chro matogram to avoid conflicts 4 1 7 Save Map as Image Any image displayed by the Chrom software can be saved as a graphic png file The command opens a File Selection Dialog Box for the selection of the folder and of the file name of the graphic file The Dialog Box represented in Figure 4 3 also contains some customization con trols INetCDF Network Common Data Form is a set of software libraries and self describing machine independent data formats that suppo
11. dimethyl gt 0 008 0 132 0 021 0 057 0 054 0 059 0 088 0 092 0 082 0 035 0 068 0 028 0 015 0 115 0 003 0 001 0 116 0 073 0 105 0 089 0 009 0 126 81281 101 0 Area 11138683 4 7424521 8 12326288 6 13236115 0 17064640 6 15110322 9 14006767479 241687437 5 167199420 14300792 2 42486605 1 18147053 4 7086734 8 12481074 1 102273925 5 11372425 0 27326860 5 56724782 9 94532457 7 65342969 9 9432852 0 26483227 7 Figure 4 12 The Chromatogram Report Preview 99 4 6 11 Search selected Blob The command calls the instrumentation software in order to search the selected Blob use the Data Analysis context menu described at Section 7 6 3 to select the Blob This command is presently implemented only for GCMSsolution LCsolution and LCMSsolution versions of the software 4 6 12 Search all Blobs The command calls the instrumentation software in order to search all Blobs This command is presently implemented only for GCMSsolution LCsolution and LCMSsolution versions of the software 4 6 13 Correct Wrap Around This command allows to add a time interval to the start modulation time in this way all modulations are shifted by the same time amount the visual result is that the map will appear shifted towards the top or towards the bottom with a wrap effect 4 7 Menu Tools The Menu Tools contains commands which calls some auxiliary modules which perform some independent operations that is oper
12. has to be used during loading procedure The operator can choose what are the ion s that have to be summed to calculate the Blob area using Jon Selection information stored in the Method Ion Selection information is specified through the last column the 4th column of the Blob recognition table of the Method Panel Once a Blob has been defined in the Method Panel in the same row of name and topology definition a text describing Ion Selection can be entered in the last column as shown in Figure 10 10 Moving the mouse around the Ion Selection column a small yellow window a tooltip is shown which specifies the syntax to be used to describe Ion Selection information There are 3 possibilities 1 If the cell is left blank the whole intensity is taken and all components are summed 2This folder varies according to the MS Windows version In Windows XP this is C Documents and Settings user Local Temp In Windows 7 it is C Users user Local Temp Where user stands for the current user login 171 method filename min area noise MI peck shift of Mod time 1 0 blob recognition 1D Int Std ID 1 H 60 833333333333336 Figure 10 10 Method Panel with Ion Selection 2 The text MAX means that the system should take into account the most abundant fragment 3 The user can also provide a list of ions by means of a set of items separated
13. Ok button the report template is automatically generated and saved 4 7 2 Compare Subtract Chromatograms The command opens a special window designed to perform the comparison and subtraction of chromatograms The tool is described in detail in Section 10 2 4 8 Menu Help The Menu Help is identified by a question mark and as usual is the last menu of the Menu Bar The menu contains items relative to various kinds of information tobe displayed The menu is shown in 4 15 File Method Schedule View Options Action Tools O H te Filename License Java3D Info Java Console A About Schedule Figure 4 15 The Menu Help 4 8 1 Help The command shows the ChromSquare User Manual in an Acrobat Reader window 4 8 2 License The command displays information about the license currently installed 62 4 8 3 Java 3D Info This command opens a window that displays information about the Java 3D ver sion installed if any This information is useful when the 3D visualization com mand see 10 1 does not generate an image or the image is not satisfactory in these cases the information displayed should be sent to the Technical Support in order to detect the origin of the problem A sample window is shown in Figure 4 16 Java3D Info i Java3D version 1 5 2 fcs build4 Java3D vendor Java3D renderer OpenGL Figure 4 16 The The Java 3D information window 4 8 4 Java Console T
14. The command opens a File Selection Dialog Box for the choice of an existing chromatogram which will become the Base Chromatogram e Open Reference Chromatogram The command opens a File Selection Di alog Box for the choice of an existing chromatogram which will become the Reference Chromatogram e Save Result Chromatogram As The command opens a File Selection Di alog Box for the definition of a new file name where will be possibly saved the Chromatogram arising from the subtraction operation in progress e Exit The command closes the tool window 163 10 2 3 3 The Tool Bar of the Compare Subtract window The Tool Bar is made up of 4 parts divided by a thin vertical lines e The first part contains just one item l The command resets the Zoom on both panels restoring the original chromatogram view at the maximum extent e The second part regards the Time Shift that is the shifting of one chro matogram with respect to the other one along the horizontal axis it includes two horizontal arrows a text field and a mouse icon du The command shifts the reference chromatogram left along the time axis by a number of data points indicated in the text field The command shifts the reference chromatogram right along the time axis by a number of data points indicated in the text fiels D oe The command allows the user to shift the reference chromatogram along the time axis by selecting the start and the end positions by
15. Zoom a Se det AS hah eae eee ate a 50 AAAS OO OU ED Sh DAA CRA E AE Fede 50 ATA Ae IN e TA ae ee Wee so 50 Meu Options o s becar Soh ene shih ch Be Be ae ae 51 4 5 1 Interpolation switches a 52 4 5 2 2D Chromatogram Plot switches 52 4 5 3 Linear Chromatogram Plot switches 54 M nu Actin IS a o Be ae Oe Be 54 4 6 1 Integrate ROL ito de a 55 4 6 2 Integrate Whole Chromatogram 55 46 3 QuantitatEn ote 2 a Ee a ii aa a EE a A a 56 4 6 4 Remove Integrations bic a A A a 56 4 6 5 Integrate all chromatograms 56 4 6 6 Create Calibration e ic Be ERS 56 4 6 7 Show Calibration hoi dk Sad ee Be A 57 4 6 8 Create Calibration Group saosaoa aa 57 4 6 9 Show Calibration Groupe soaa a ee a eG 57 4 6 10 Create Chromatogram Report 57 4 6 11 Search selected Blob vi AA A es 60 4 6 12 Search all Blobs 60 4 6 13 Correct Wrap Around aoa aa eae ee ee ee YS 60 Af Ment TOOLS 2 AA eae le ee we A Gee A 60 4 7 1 Report Template Editor 2 004 60 4 7 2 Compare Subtract Chromatograms 62 4 8 Menu Hel pr ssh p 4 bak ke ew ee ee ae a ae E 62 AGG Ae E O gee lo te race dco aad th eel den eben Ds ee E peta 2 62 48 2 o haana Ye cea Bess de HU eh ASM eh ee a 62 Arse Java SD fomi oe amp a A Be Se ee a a 63 4 84 Jaya OUSOLE scs A A ap A BO ek fd 63 48 9 ANOU 20h A one Btn SAG hag eed i tee a
16. e Abscissa axis is retention time ranging within the limits of modulation time set by the current zoom area that is between the lower and upper limits of the zoom rectangle e Ordinate axis is intensity ranging from 0 to an upper limit corresponding to the maximum intensity The user can change the limit through the control Max Int either directly writing a new value in the text field or using the two small arrows to quickly change it See Figure 7 8 7 4 2 Single modulation in whole modulation interval This representation shows the plot of the chromatogram concerning the current modulation that is the modulation corresponding to the current mouse pointer 90 HA Graphical param Chromatogram Data analysis ma Ca max int 330 930 0 000 0 400 0 800 1 200 1 600 lod 25 DP 601 TER 50 801 2tR 0 801 1tR 50 000 Int 18807 96 Figure 7 8 Single modulation in zoom interval position in the 2D Map Plot on a time domain corresponding to the whole mod ulation time More precisely e Abscissa axis is retention time ranging from o to the modulation time e Ordinate axis is intensity ranging from 0 to an upper limit corresponding to the maximum intensity The limits can be changed as described in Section 7 4 1 See Figure 7 9 HA Graphical param Chromatogram Data analysis GO max int E 330 930 300 100000000 0 000 0 400 0 800 1 200 1 600 Figure 7
17. la beled Debug Mode in the Program Group this second icon is not displayed on the desktop the icon allows to run the application displaying an infor mative window called Java Console see See Section 3 4 The Java Console may be useful to detect errors it does not affect the software performance only in some cases it can run a little slowly Normally the user should not use this second icon except when requested from the Technical Assistance anyway running in Debug Mode does not do any harm and the Java Console can be closed at any moment The next windows is Ready to Install This is a final review of the previ ous steps before proceeding with the very installation Select Install to continue The Installing window is then displayed showing a progress bar which lasts for some instants at the end the window will close automatically The installation procedure terminates asking the user for some optional in stallations e The first one concerns the installation of a set of sample files sample files are specific for the license purchased Answer Yes if this is the first installation or an upgrade of a previous installation sample files are very useful for testing the software before real use and they do not 20 require too much disk space answer No if you are just re installing the software starting from the same media in case of doubt answer Yes The installation of sample files is accomplished through a Co
18. 9 Single modulation in whole modulation interval 7 4 3 Multiple modulation in zoom interval This representation shows the plot of all chromatograms concerning the modula tions included in the current horizontal zoom interval on a time domain that is a fraction of the modulation time corresponding to the vertical zoom interval in the 2D Map Plot More precisely e Abscissa axis is retention time ranging within the limits of modulation time set by the current zoom area that is between the lower and upper limits of the zoom rectangle 91 e Ordinate axis is intensity ranging from 0 to an upper limit corresponding to the maximum intensity The limits can be changed as described in Section 7 4 1 See Figure 7 10 Graphical param Chromatogram Data analysis n m max int 5 330 930 300 10000000 o o o 17 20 34 40 51 60 68 80 86 0 Figure 7 10 Multiple modulation in zoom interval 7 4 4 Multiple modulation in whole modulation interval This representation shows the plot of all chromatograms concerning the modula tions included in the current horizontal zoom interval on a time domain corre sponding to the whole modulation time See Figure 7 11 EEE Graphical param Chromatogram Data analysis 170 moc ax int 330 930 5 t 300 10002001 o a t 17 20 34 40 51 60 Figure 7 11 Multiple modulation in whole modulation interval More precise
19. Blob ID 1 ID a counter of the Group ID 2 name the name assigned by the user to the Group ID 3 Blobs the list of the Blob IDs which belong to the Group ID comma separated Note that the table is read only and all modifications should be accomplished through the buttons 6 1 7 Buttons for Group Table management Below the Group Table there are 2 buttons to be used for table editing e Button add is used to add a new row that is a new Group ID to the table pressing this button will open a window containing a list of all currently defined Blob IDs the user can then select through the mouse one or more Blob IDs that will become part of the Group ID Pressing the CTRL key during the selection will allow to select more than one Blob ID e Button delete it is used to delete from the table the Group ID currently selected 74 6 1 8 ROI ROI means Region Of Interest this defines a smaller region of rectangular shape inside the chromatogram Map The ROI is defined by the user through mouse commands and Context menu in the Chromatogram Panel see 7 2 4 3 The ROI is unambiguously defined by the Total Retention Times of two opposite points of the rectangle remember that Total Retention Times actually identifies a point in the 2D Map Plot 6 1 9 Ion Selection for Map Display In this field the user can specify a list of lons which will be used for the graphical representation on the chromatogram Map Ions are repres
20. Cat Group CA Rut Id Stc ld Mee Group GA EG A 8573995 652176 50 000000 Group GA EG 2 12717590 572607 Group GA EG SoSo y Ol 3954704 030035 Linear Regression calculated with 3 points Regression Line Area m Conc b Coeff m Slope 111986 117 Coeff b Intercept 1882906 570 RF 8 929678278e 06 Linear Correlation Coeff R 0 975578 R squared 0 951753 Figure 9 31 Group Calibration Graph 153 9 4 5 Group Quantitation The last step of Group Calibration is the Group Quantitation of the unknown samples There is no specific command to achieve Group Quantitation for a given sample this is automatically performed together with normal Quantitation if a Group Calibration has been previously executed on the current schedule The results of Group Quantitation are shown in the last column of Grouping panel The next two figures show the Data Analysis page and the Grouping page with the Quantitation results of a given sample ChromSquare License GCxGC MS File Method Schedule View Options Action Tools 12648 25206 Chromatogram one Graphical param Chromatogram Deta analysis Grouping lt 48 2 BE C Potygon mode Index ID Name TR 2tR Area Area D Area Area ID Norm Norm Ar Conc 89128994 unknown A aE Sarto meee aa nee si m gt a unknown 40 768 0 370 302 5 ao 0 002 omoj ee 0 000 a 87294252 13 Palmitoleico 41 095 1 500 65
21. Figure 9 7 The Method Panel showing a new empty Method gt ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS Bsec xrs File Method Schedule View Options Action Tools method filename 3 154 Data ChromSquare 32 Quantitative quantitate xcm min area 100000 0 noise 10000 0 Ej peak shift of Mod Time 6 0 blob recognition Schedule ID name Int Std ID Ton Selection 1 O Chromatogram Ga ROI E ya 3 6686548331108146 3 9745293324432582 Method Figure 9 8 The Method Panel with some parameters entered 125 9 1 3 Load a chromatogram Once the Method has been set up switch back to the Schedule Panel select the first chromatogram and load it using the con with the Magnifying Lens third icon of the top tool bar See Section 5 1 The icon is pointed out in Figure 9 9 ChromSquare License GCxGC MS DEIR Fie Method Schedule View Options Action Tools a t 4 a H current method none Filename Std Smp Chromatogram gt lt Method Figure 9 9 The icon for loading the selected chromatogram Alternatively you can load the chromatogram by double clicking on the selected row Loading chromatogram may require some time during which a small window show ing the progress of the operation is displayed At the end of the loading process the software will automatically switch to the Chromatogram Panel where the loade
22. Selection Background Subtraction 1 The first column contains the ID of the Blobs normally the ID is generated automatically 2 The second column contains the names of the Blobs the names are normally assigned by the user in the Blob Definition procedure see 7 2 4 2 Blob names can be automatically generated using the Search Selected Blob or Search all Blobs commands The table allows the user to modify the Blob names after their original definition 3 The third column is only used for Calibrations with Internal Standards For each row the cell corresponding to the third column indicates an other Blob ID among those defined in the method that will be assumed as internal standard In order to simplify this assignment the cells of the third columns are in effect combo bores each combo box reports a list of all other Blob IDs the combo box allows the user to establish the mapping between the Blob ID and the Blob ID used as Internal Standard Internal Standard IDs can be different for each row 4 The fourth column is used to specify Ion Selection in the case of chro matograms generated by mass spectrometers see Chapter 10 6 for de tails 5 The fifth column is used to switch On Off the subtraction of the back ground of the Blob ID When selected the background is measured before each peak start and after each peak end The average spectrum of the background is subtracted when the total area of the Blob is cal culated This
23. The identification of the integrated Blobs has been already described in details for Qualitative Analysis Two kinds of identification are provided manual and automatic e Manual identification is always possible it must be done separately for each Blob that should be included in the Calibration e Automatic identification is provided for some types of license and it is per formed on all integrated Blobs in this case it is possible to use ROI see 8 2 3 to limit the number of Blobs Skip to Section 8 3 for a full description of both procedures The Figure 9 13 shows a the context menu and the dialog box for the manual identification of a Blob ChromSquare License GCxGC MS Current file HC2 6sec xrs File Method Schedule View Options Action Tools Ej Schedule Insert blob name Start TER 9 347 end TER 11 176 Hydrot Chromatogram 0 000 10 500 j Graphical param Chromatogram Data analysis 245 000 7 417245 od 101 DP 317 TER 10 142 2tR 2 539 1tR 10 100 Int 48 61 Method Figure 9 13 Manual identification of a Blob At the end of the identification switch again to the Method Panel you will find that the software has automatically filled the Blob Table in the Method adding the names and the IDs of each identified Blob 129 Once the identification has been completed save the Method with the main menu command Method Save As choosing a fil
24. a ea 140 9 3 3 Integrate the chromatogram 140 9 3 4 Execute the Quantitative Analysis 141 9 4 Group Calibration nia Bed a6 yl ae e eS 142 9 4 1 Description ni A a ie ea ee 142 9 4 2 Define Groups in the Method Panel 143 9 43 Integrate All Chromatograms 147 9 4 4 Group Calibration 109 Tica BE ERS AA 149 9 4 5 Group Quantitation a Bee ee a 154 10 Advanced Topics 156 OTID MEW ani 2 Gece Ae O E 156 101 1 Description us E Se Fd a ce ES 156 10 1 2 Changing the scene poca A E OR ES 158 10 1 3 The 3D View Tool Bar sae Ge E A EE ee 158 10 2 Compare subtract tool spa aa 161 10 2 1 Introduction 161 EZ Dennition gs tt a ANA E BR 161 10 2 3 Description of the Compare Subtract window 162 10 24 O ob soos ic OB Gotan aXe ae Sow Re Be Ge G 165 10 3 Define Polyaonal Blobs lt p RHE Oe RA 165 TOA Promlesy AI 168 10 4 1 Description aces ds Pe be hd E 168 10 4 2 Defining a new Profile lt 168 10 5 Configuratio EIA 170 T05 Des ripti n aae aen ea A A T a E 170 10 5 2 The file Options mb st a he at 170 10 6 TonrselectionZ E S RE Po 171 T061 Description ess ame ioc eR E ee TE Goa at ase ae 2 171 10 7 Assembling Data Files domed ed ee ee a ed 172 MUSE Description VERA AA A IE Fos 172 1072 Procedure rt a ee A AD E A A es 173 11 Technical Notes 177 11 1 Prerequisites to install ChromSquare with GCMSsolution spectra o Oy tha
25. a new row is added to the Schedule list Otherwise an error message is displayed and the Schedule list remains unchanged Read carefully the notes at Section 3 3 5 to fully understand the file mechanism 4 1 1 1 File Types The File Selection Dialog Box filters the directory contents and only shows the files which are compatible with the purchased license type The present manual applies to license GCxGC MS amp LCxGC MS version for Shimadzu GCMSsolution and GCsolution This license is compatible with the following Data files e Shimadzu file types gcd and qgd Shimadzu exported file types aia andi cdf csv txt e ChromSquare Result Data Files file type xrs 4 1 1 2 The Accessory Panel The command Open Datafile shows an Open File dialog box which is slightly dif ferent from the traditional ones in fact it contains on the right side an Accessory panel see the sample in the Figure 4 2 This panel is made up by three parts 41 Select a file of type GCxGC MS Look in samples E 5 gt d aa E Hc1 sec Profile v Fe m G E HC1 6sec xrs Ri aen E HC2 6sec xrs j E HC3 6sec E HC3 6sec xrs Mod Time 8 0 Es r Desktop HCX 6sec 2nd El time E HCX 6sec xrs j oe Sq Shimadzu GCxGC MS Bsec 1st Mod Time I E Shimadzu GCxGC MS 8sec xrs Documenti GCM3solution file Further in
26. according to the common rules of most software applications e Click on a point of the plot with a mouse button to start selection that is the point becomes first corner of a moving rectangle e Move the mouse holding the button pressed to change the size of the rectan gle the mouse pointer identifies the opposite corner of the moving rectangle the moving rectangle is shown in reverse colors e Release the mouse button to close the operation In Chrom software the following rule will also apply e when the selection is made by dragging the mouse with the left button clicked the result is directly the Zoom In of the selected region e when the selection is made by dragging the mouse with the right button clicked the result is the displaying of a pop up context menu which allows the choice among different operations to be made on the selected region Zoom In is however provided even in this case see Section 7 2 4 for a full description of this menu The Figure 7 1 shows a Map Plot selected region and the Plot Context Menu 7 2 3 Zoom The Zoom In operations are generally performed by selecting a region through the mouse selection as mentioned above However there are other Zoom operations like Zoom Out Reset Zoom or Move Zoom that can not be easily accomplished in this way For this reason the software provides two other ways to perform Zoom operation which complement the mouse ones e Zoom operations through the
27. be shown A sample of the Grouping Results table is shown in Figure 9 27 This table reports a row for each Group defined in the Method and 5 columns 2Note that if the schedule had been previously integrated with a different method for example with a similar method not having Named Groups the software will warn that the current Method is different from the Method stored in the Results files and will ask if you which Method should be used for the integration the correct answer in this case is to preserve the Method file since we are applying a new Method 147 i ChromSquare License GCxGC MS File Method Schedule View Options Action Tools Chromatogram 12608 _ O Grephical param Chromatogram Data analysis Grouping lt 2 Gt 26 BEC Potygon mode 7 ID O unknown 06 631 DP 0 TtR 19 456 21R 0 000 1tR 19 456 Int 15 56 Figure 9 26 Integration Results with Named Groups 1 in the first column the JD of the Group as defined in the Method 2 in the second column the Name of the Group as defined in the Method 3 in the third column the Area of the Group calculated as the sum of the areas of the Blob IDs which constitute the Group 4 in the fourth column the Area Percent of the Group calculated as the sum of the areas percent of the Blob IDs which constitute the Group 5 in the fifth column the Concentration of the Group empty at the moment will be calculated after that
28. by commas each item can represent a single ion number M Z or a range of ion numbers the range is described by the starting and ending M Z numbers separated by dashes All numbers should be in ascending order For example the following text 60 80 96 99 105 107 means that all ions between 60 and 80 those between 105 107 plus ion 96 and 99 concur in determining the Blob area The results are computed upon those values 10 7 Assembling Data Files 10 7 1 Description This feature applies mainly to LCxGC MS The feature becomes necessary when the chromatographic instruments that op erate the first and second eluition are managed separately and a new injection 172 is performed for each modulation It is then possible to assemble more chro matograms into a unique data file this feature is used when the modulations had been saved onto different files a single file for each modulation with files numbered as a sequence 10 7 2 Procedure To assemble more chromatograms please use the following procedure 1 Select the menu item File Assemble Datafile a File Selection Dialog Box with the title Select one of the files to assemble will appear See Figure 10 11 El Select one of the files to assemble iS LOGC CANA li LCXGC berg 008 0014 gcd Documenti 5 LOXGC berg 008 0002 gcd LCxGC berg 008 0015 gcd 2nd El time recenti LCxGC berg 008 0003 gcd LCxGC berg 008 0016 gcd sj LCxGC berg 008 0004 gcd r LC
29. chromatogram using menu File Open Reference Chromatogram Adjust the horizontal and or vertical shifting using the Tool Bar commands 10 2 3 3 or mouse commands 10 2 3 4 Press button Execute Subtractions Where necessary perform some zoom operations to emphasize the details Finally save the result chromatogram using the menu File Save result chromatogram as 10 3 Define Polygonal Blobs It is possible to identify one or more Blobs creating a polygonal region Every Blob whose top is included in that region gets the name and the ID defined together with the region itself The region is saved into the method so that every chromatogram processed with that method inherits the region settings To define such region l Select the page Data Analysis in the lower panel of Chromatogram view then check the check box Polygon mode Left click repeatedly the mouse onto the Map Plot to select the vertices of the polygon The area is automatically closed so there is no need to re select the first point Right click of the mouse deletes the last point inserted so undo is possible 165 3 When finished uncheck the check box Polygon mode a prompt for the name of the Blob is displayed Figure 10 5 shows a sample of a polygonal region being defined L ChromSquare License LCxLC MS Current file Unknown 60sec xrs File Method Schedule View Options Action Tools Graphical param Chromatogram
30. identified The Figure 9 15 shows the Chromatogram Panel with the Blob names both in the Map Plot and in the Data Analysis window pd ChromSquare License GCxGC MS Current file HC2 6sec xrs File Method Schedule view Options Action Tools E Schedule Chromatogram 14 856 Graphical param Chromatogram Data analysis Groupin 3 A ME O Polygon mode b LY ID Name TER 2R Area Area ID Norm Area Norm Area ID Conc Hydro2 3 34 457 Hydro3 23 460 3 597 3166637 2 3166637 2 35 196 Method lod 274 DP 696 TtR 27 493 2tR 5 575 1tR 27 400 Int 107 00 Figure 9 15 Integrated chromatogram with names of the recognized Blobs 9 1 7 Integrate All Chromatograms Analyzing the results of the second integration described in the previous Section you had been able to verify whether the Method which has been previously set up and saved entirely fulfils the needs of the current calibration in other words if all Blobs necessary to the calibration have been correctly integrated and identified If this is the case it is possible to proceed with the integration of all remaining chromatograms of the current Schedule using the current Method On the con trary you should modify the Method coming back to Section Set Up the Method 9 1 2 and repeat the various steps The integration of the remaining chromatograms may be done in two ways 131 e manually one chrom
31. in Figure 10 3 contains e a File menu e a Tool Bar with various icons and controls shown in Figure 10 4 e an Upper Panel where will be plotted the Base Chromatogram in red color and the Reference chromatogram in green color e a Lower Panel where will be plotted the Result Chromatogram in red color e a Status Bar which reports continuously the coordinates of the mouse pointer in the Upper Panel 162 10 2 3 1 Zoom Both upper and lower Plot Panels initially show chromatograms at the maximum extent the user may however perform zoom operations on the panels in order to view more details Zoom operations are managed in the traditional way dragging the mouse with the left button pressed and releasing it when the desired region has been selected See 7 2 3 for a general description of the zoom operations Zoom may be applied repeatedly so that any desired enlargement may be achieved on both Panels Use the first icon of the Tool Bar 10 2 3 3 to reset the Zoom in both Panels and go back to the original representation of the chromatograms Zoom is managed separately for each panel the reset command however applies to both panel at the same time File 1 Time shift 4m 1100 3 Intensity shift J 10000 A l Execute subtraction Figure 10 4 The Compare Subtract Window Tool Bar 10 2 3 2 The Menu File of the Compare Subtract window The menu File contains the following items e Open Base Chromatogram
32. samples and save the corresponding xrs files with the area data 9 3 4 Execute the Quantitative Analysis To execute the Quantitative Analysis of all Unknown Samples included in the Schedule give the main menu command Action Quantitate The command will use the Calibration defined within the current Schedule Error messages will appear if the Schedule does not contain a correct Calibration The Quantitation results will be shown in various places Perform the following actions to see all results 1 Switch to the Schedule Panel the concentration results are shown for each column corresponding to a Blob on the yellow lines corresponding to the unknown Samples these concentration were all zero before executing the Quantitation command 2 From the Schedule Panel load a Chromatogram corresponding to an un known Sample double click on the row or use the Magnifying Lens icon The current panel automatically switches to the Chromatogram Panel 3 Look at the Map Plot the concentrations will be shown near to each Blob 4 Look at the Data Analysis Table 7 6 1 in the Data Analysis window of the lower panel the Concentration column will now be filled with the calculated concentration values The Figure 9 20 is an example of the Schedule Panel which shows the Quantitation Results in the yellow row corresponding to the sample 141 Xx ChromSquare License GCxGC MS Current file Unknown 6sec xrs File Method Schedul
33. selection has no effect if a Jon Selection is defined for the given Blob When background subtraction is selected the baseline calculated upon the TIC is substituted by the average background total count When the user defines a new method rows are generated dynamically as soon as the user defines a new Blob a corresponding new row is added to the table using the name assigned in the Blob Definition procedure see 7 2 4 2 See Sections 3 1 5 3 1 6 3 1 7 for more details about Blobs and Blob IDs 6 1 5 Buttons for Blob ID Table management Below the Blob ID Table there are 3 buttons to be used for table editing 73 e Button delete it is used to delete the currently selected Blob ID row of the table take note there is no corresponding add button since Blob Ids rows may be added to the table only through the aforementioned procedure see 9 2 3 e Button Bg On All Background On All pressing this button all Back ground Subtraction check boxes are set to On that is the background sub traction process is scheduled for all Blob IDs e Button Bg Off A11 Background Off All pressing this button all Back ground Subtraction check boxes are set to Off that is no background sub traction process is scheduled 6 1 6 Groups ID Table Groups ID Table contains a set of Group IDs that will be used by the software for the recognition of the Group IDs The Table contains e a row for each Group ID e three columns ID name
34. the written text and to avoid misunderstandings about the use of the graphical objects They do not always represent an exact reproduction of software behaviour since this is affected by to many factors like license types and choice of sample data files In same cases where the type of license makes the difference the Figures refers to that very license in other cases when the Figure is just used to explain an operation not depending on any particular license the Figure may contain references to a license different from that to which the manual refers 18 Chapter 2 Installation and Startup 2 1 Installing the software The Chrome is a Java application this means that it may run under any operating system provided that a suitable Java Virtual Machine is installed on ibe Before installing the Chrom 2 software verify that a recent Java Runtime Environment version 1 6 or later is installed For free download visit the site http www java com The install disk is provided as a CD ROM To install the Chrom software insert the CD ROM into the driver The disk contains a bootstrap installation The installation procedure is a very straightforward very similar to the most common installation procedures The user should navigate among various dialog boxes answering to very simple questions In the following steps an outline of the installation procedure is reported The various windows make a sequence that may b
35. undefined In order to let the Schedule know that the Method has changed it is necessary to give the menu command Method Open Method and reload the the Method that have been just saved You will see that new columns one for each Blob will be automatically placed in the table and temporarily filled with zero values of concentration The Figure 9 16 shows the Schedule Panel with the new columns corresponding to the Blobs 132 E ChromSquare License GCxGC MS Current file HC2 6sec xrs File Method Schedule view Options Action Tools a t y C fed current method Hydro xcm Filename Std Smp Hydrol Hydro2 Hydro3 HC1 6sec xrs HC2 6sec xrs A HC3 6sec xrs Schedule Chromatogram gt Method Figure 9 16 Schedule panel ready to insert concentration values Now you can write the concentrations in the cells of the Schedule table Rows are the Standards columns are the Blobs To facilitate the input select a cell and give a double click the cell will be shown in reverse and you can directly write on it You can also navigate through the cells using the Tab key The Figure 9 17 shows the Schedule Panel while a concentration value is being inserted For each row of the Table you must also specify whether it corresponds to a Standard or an Unknown Sample Use the combo box in the second columns to make this choice Note that rows corresponding to the Standards will be coloured in green rows corresp
36. versions of GCMSsolution 177
37. writing of the Areas of the Blobs in the bi dimensional map plot 4 5 2 5 Draw Area Perc onto Map Plot The switch enables the writing of the Area Percents of the Blobs in the bi dimensional map plot The Area Percent of a Blob is computed as the percent ratio of the area of the Blob with respect to the sum of the areas of all Blobs both identified and unidentified 4 5 2 6 Draw Normalized Area Perc onto Map Plot The switch enables the writing of the Normalized Area Percents of the Blobs in the bi dimensional map plot The Normalized Area Percent of a Blob is computed as the percent ratio of the area of the Blob with respect to the sum of the areas of all identified Blobs 4 5 2 7 Draw Concentration onto Map Plot The switch enables the writing of the Concentrations of the Blobs in the bi dimensional map plot 4 5 2 8 Tick Blob Contour IDed Blobs The switch enables the drawing of the Blob contour of the dentified Blobs in the bi dimensional map plot 4 5 2 9 Tick Blob Contour Unknown The switch enables the drawing of the Blob contour of the unidentified Blobs in the bi dimensional map plot 53 4 5 2 10 Draw Only selected Ions The switch enables the drawing of a bi dimensional map plot based upon a selection of ions instead of the normal map plot based on all ions The selection of ions is defined in the Method see Section 6 1 9 4 5 3 Linear Chromatogram Plot switches 4 5 3 1 Enhance Blob Line in Chromatogram Th
38. 2 1116659119 Neral 29 547 4 830 169894187 8 169894187 8 34 294 12779629 1665957737 30 091 5 484 102736439 8 102736439 8 20 738 13328201 648969502 Geranial 32 221 5 283 222768001 3 222768001 3 4 967 Figure 7 14 Data Analysis page Left view 7 6 1 The Data Analysis table The table has a vertical scrolling bar since the number of Blobs can exceed the available space 94 60 961 81 Area ID Norm Area Norm Area ID Conc LRI LRI lib Sim Idx 34 294 34 294 34 294 1245 1238 97 20 738 20 738 20 738 1256 1246 97 44 967 44 967 44 967 1301 1268 96 Figure 7 15 Data Analysis page Right view The different columns have the following meaning 1 Index The Index is an integer number that unambiguously identify the Blob intended as a geometrical entity see Section 3 1 5 for a description of this and other quantities ID The Blob ID see 3 1 6 Name Name of the Blob TtR Total retention time of the Blob 2tR Retention time in 2nd dimension of the Blob Area Area of the individual Blob Area ID Sum of the areas of all Blobs having the same ID In the case of Blobs Grouping this is the overall area of the Group of Blobs it is the same for all the Blobs belonging to the Group Area Area percent of the individual Blob calculated with respect to the sum of the areas of all Blobs 3Accord
39. 3 Schedule Library File 4 Library File 5 C Delete duplicates Reverse search minimum similarity index 70 00 max num of hits Chromatogram LRI upper tolerance LRI lower tolerance LRI index in library gt Use current Chromatogram to define LRIs Figure 6 2 The Method Panel Shimadzu Spectra Search Page 6 2 2 Delete duplicates If this box is checked the duplicate hits found during the search are deleted and only the one with the higher similarity index is displayed 6 2 3 Reverse search If this box is checked the reverse search is performed instead of the direct one 6 2 4 LRI upper tolerance lower tolerance When using Linear Retention Indexes LRI the result set is filtered to the only hits that match the LRI within a range defines by these two numbers 6 2 5 LRI index in library Shimadzu libraries allow to store up to 5 Linear Retention Indexes for each refer ence compound This parameter lets the user to choose which one has to be used 76 for the library search If no LRI is selected no filtering is performed against the Linear Retention Index 6 2 6 LRI Table This table is made by three columns 1 Name is the name of the Blob identifying the LRI milestone e g the ap propriate alkane This is only for a documentation purpose and to remind the user in fact it is equivalent to wri
40. 3D View The command generates a 3D View of the region currently selected in the map plot See Section 10 1 for details about the 3D representation 50 4 5 Menu Options The Menu Options contains the commands which allow a fine tuning of the bi dimensional plot both the plot details and the drawing of various labels All these commands work as switches each time that an item is selected the switch changes from ON to OFF and back when the switch is ON a small check sign is shown to the left of the menu item The menu is shown in Figure 4 9 File Method Schedule View Options Action Tools le QQ Y Enhance small peaks An Y Graphical data interpolation Smooth blobs Y Draw names onto map plot f Draw TtR onto map plot Draw 2tR onto map plot Schedule vV Draw area onto map plot Draw area onto map plot Draw normalized area onto map plot Draw concentration onto map plot Tick blob contour IDed blobs Tick blob contour Unknown Draw only selected ions Chromatogram Y Enhance blob line in chromatogram Draw names onto chromatogram Draw TtR onto chromatogram Draw 2tR onto chromatogram Figure 4 9 The Menu Options The menu Options is divided in three parts separated by horizontal lines e The first division contains switches regarding interpolation e The second part contains switches which set the details of the 2D map plot upper frame of the chromatogram panel 51
41. 6 7 17 7 18 7 19 The The Java 3D information window 63 The Tool Bar of the Schedule Panel 64 The Schedule Panel before loading a Method 68 The Schedule Panel after loading a Method 68 The Schedule Panel after the assignment of the Concentrations 69 The context menu of the Schedule Table 70 The Method Panel Integration Page o 71 The Method Panel Shimadzu Spectra Search Page 76 Map Plot Context Menu iaa y at SE Ge a 81 Map Plot Zoomed Region imac a a e ad 82 Insert Blob Name window o 84 The Method Panel with the new BlobID 84 Graphical Parameters Page ees 86 Navigator Panel with Zoomed Area 88 The Toolbar of the Chromatogram page 90 Single modulation in zoom interval 91 Single modulation in whole modulation interval 91 Multiple modulation in zoom interval 0 92 Multiple modulation in whole modulation interval 92 The Spectrum page Eds Sa re oe a he a ey 93 Data Analysis page Overall view o 94 Data Analysis page Left view o 94 Data Analysis page Right view o 95 Data Analysis page Example showing ID Grouping case 98 Data Analysis page The Data Analysis Toolbar 99 Enhanced representation
42. 7 Ac Tridecanolco 21 groups D Name Blobs ROI 6 4135 62 9 Figure 9 21 Method Panel with some Blob ID defined number the name could be later changed by the user A sample of the list is shown in Figure 9 22 3 Select two or more Blob IDs holding the CTRL key the selection should be done according to analytical criteria in the example we just selected compounds having similar names A sample of the list with selection is shown in Figure 9 23 144 Select the one or more Blob ID hold CTRL key for multiple selction 1 Gallic acid 2 Tyrosol 3 Catechin 4 Caffeic acid 5 Ethylgallate 6 Rutin Figure 9 22 List of all Blob IDs Select the one or more Blob ID hold CTRL key for multiple selction 2 Tyrosol 3 Catechin 4 Caffeic acid 6 Rutin Figure 9 23 Selection of the Blob IDs Press Ok a new row is inserted in the Group table showing the new Group the row displays the following information e a Group ID that is a sequential identifier assigned by the system e the Group Name which is initially assigned by default as the word Group followed by the ID number e the list of the Blob IDs belonging to the Group See Figure 9 24 for a sample of the Method Panel with just one Group added When one or more groups have been defined it is possible to verify whether a given Blob ID is included in one or more Groups by simply moving the mouse on the upper table whe
43. 9 8 8351 6 0 0 064 0 773 9 785 9563 363 87621330 13 Ac Palmitoleico l 41 249 1 490 7603 1 E 0 0 ow aote 9 785 9563 362 87621998 unknown 41 255 _ 1 830 27 7 0 0 0 000 0 000 0 000 0 000 Pot 13 Ac Palmitoleico 41 465 1 515 68 8 A 0 001 0 064 0 104 9 785 9563 363 88801532 12 Ac Palmitico 41 800 1 260 219 2 25415 0 0 0 194 _0 29 776 25872 37 88867064 1 Palmitico 41 831 1 240 1544 5 25415 0 0 0 HITA 1 810 29 776 25872 37 89063668 _12 Ac Palmitico 41 923 1 220 23651 4 25415 0 0 181 0 194 27 710 29 776 25872 378 41 935 0 120 12 0 0 0 0 0 0 000 0 000 0 000 a Sava nA AAA nena nana AAA od 883 DP 205 TtR 27 243 2R 1 025 11R 27 226 Int 15 81 Figure 9 32 Group Calibration Quantitation Results Data Analysis Page 154 E ChromSquare License GCxGC MS File Method Schedule View Options Action Tools Graphical param Chromatogram Data analysis Grouping FER Index Name 1 Group Capr 2 Group Paim 3 Group Lino od 1667 DP 169 TIR 51 413 2tR 0 845 1tR 51 399 Int 16 30 Figure 9 33 Group Calibration Quantitation Results Grouping Page 155 Chapter 10 Advanced Topics 10 1 3D View 10 1 1 Description Chrom 1 ere includes a 3D facility which allows a full 3D representation of the chromatograms which is displayed in a separate pop up window To generate this representation the user must e select a rectangular area on the 2D Map Plot see Section 7 2 2 for how to selec
44. Blob including a list of all peaks corresponding to the various modulations with a very detailed description for each of them A sample is shown in Figure 7 22 This window may be useful in special cases when the user wish to investigate the inner functioning of the software It is not generally used in the daily practice 103 Blob Info E 0 Index 13828201 ID 648969502 Name Geranial Top Mod 211 Top Time 105 Top value 23899538 000 Start Mod 210 End Mod 219 TER 32 221384 2tR 0 088050 Total Area 222768001 334 Block Point Start 210 88 Dimension 9 32 of peaks 10 Peak 0 modulation 210 start 94 top 103 end 120 area 50045890 308 height 10171567 731 Peak 1 modulation 211 start 88 top 105 end 119 area 158256388 000 height 23880112 903 Peak 2 modulation 212 start 90 top 100 end 107 area 10704239 882 height 2532757 176 Peak 3 modulation 213 start 90 top 100 end 111 area 1238540 000 height 289731 810 Peak 4 modulation 214 start 90 top 99 end 108 area 636315 944 height 148369 500 Peak 5 modulation 215 start 93 top 98 end 104 area 443504 000 height 104797 455 Peak 6 modulation 216 start 92 top 98 end 103 area 425695 000 height 71512 000 Peak 7 modulation 217 start 91 top 97 end 101 area 348390 700 height 54655 800 Peak 8 modulation 218 start 90 top 97 end 102 area 336283 750 height 45083 750 Peak 9 modulation 219 start 90 t
45. CxGC berg 008 0011 gcd LCxGC berg 008 0012 gcd es Figure 10 12 List of the files to assemble E Select files to assemble LCxGC berg 008 0001 gcd LCxGC berg 008 0002 gcd LCxGC berg 008 0003 gcd LCxGC berg 008 0004 gcd LCxGC berg 008 0005 gcd LCxGC berg 008 0006 gcd LCxGC berg 008 0007 gcd LCxGC berg 008 0008 gcd LCxGC berg 008 0009 gcd LCxGC berg 008 0010 gcd LCxGC berg 008 0011 gcd LCxGC berg 008 0012 gcd a ms Figure 10 13 Files selected for assembling 174 5 Press Ok when the desired configuration is achieved A new dialog box is finally shown asking for confirmation see Figure 10 14 2 The following files have been selected for the assembly LCxGC berg 008 0001 gcd LCxGC berg 008 0002 gcd LCxGC berg 008 0003 gcd LCxGC berg 008 0004 gcd LCxGC berg 008 0005 gcd LCxGC berg 008 0006 gcd LCxGC berg 008 0007 gcd LCxGC berg 008 0008 gcd LCxGC berg 008 0009 gcd LCxGC berg 008 0010 gcd LCxGC berg 008 0011 gcd LCxGC berg 008 0012 gcd LCxGC berg 008 0013 gcd LCxGC berg 008 0014 gcd LCxGC berg 008 0015 gcd LCxGC berg 008 0016 gcd LCxGC berg 008 0017 gcd LCxGC berg 008 0018 gcd Continue Figure 10 14 Asking for confirmation before assembling 6 The software will load all single datafiles assembling them into an unique Chrome datafile xrs that will be saved in the same directory In the assembly case the modulation time is given by the final ret
46. Group Calibration will be done By comparing the former two figures it is easy to verify for example that the area of the Group Tyr Cat 5672969 2 is the sum of the areas of the three Blob Ids which constitute the Group 3592113 7 592520 2 136974 5 461300 2 890060 6 148 ChromSquare License GCxGC MS Mi ib File Method Schedule View Options Action Tools 1 1 410 07 0 000 1265 B26 57 M4 50591 Graphical param Chromatogram Data analysis Grouping e 5 Index Name Area Area Conc 1 Group Capr 576 6 0 004 2 Group Palm 796 4 0 005 3 Group Uno 37 0 0 000 00 0 DP 0 TtR 0 000 2tR 0 000 1tR 0 000 Int 15 63 Figure 9 27 Integration Results Grouping Results Table 9 4 4 Group Calibration If the current Method defines Named Groups two new menu items appear in the Action Menu Action Create Calibration Group and Action Show Calibration Group These commands perform actions similar to Action Create Calibration and Action Show Calibration commands with reference to Groups rather than to Blobs To perform a Calibration of the schedule using Groups rather than Blobs execute the following steps 1 Execute the menu command Action Create Calibration Group A sample is shown in Figure 9 28 149 i ChromSquare License GCxGC MS Current file wax w File Method Schedule View Options Nei Tools Integrate ROI Integrate whole chromatogram Quantitate Remove integrati
47. If a Blob has to be calculated using an Internal Standard the cell corresponding to the Int Std ID column the third of the table must contain the Blob ID of the other Blob that should be used as Internal Standard If the cells contains 0 or 1 this means that the Blob does not use the Internal Standard Clicking an any cell of the third column a small Combo Box will be displayed enumerating all available IDs Select one of them different from 0 to assign the 138 Internal Standard Select 0 to delete the Internal Standard assignment 9 2 6 Integrate again See Section 9 1 6 9 2 7 Integrate All Chromatograms See Section 9 1 7 9 2 8 Assign the concentrations See also Section 9 1 8 You will see that in the Schedule Table the columns relative to the Blob defined as Internal Standards are coloured in green like the rows corresponding to the Standards You must fill in the concentration of the Internal Standard in the sample chro matogram Remember that green cells are those where the user can write the concentrations whereas yellow cells are those where concentrations will be calculated 9 2 9 Save the Schedule See Section 9 1 9 9 2 10 Create the Calibration See also Section 9 1 10 Note in the regression line plot the concentrations and the areas are divided by the concentration and the area of the internal standard so they assume very low values indicatively the magnitude of the unity 9 2 11
48. Interface consists of a Main Window and many secondary windows It is important to know the exact names of these windows in order to easily understand the description reported in the manual In this manual the windows names and other objects of the Graphical User Interface menu items toolbar icons etc are represented with a monospaced font that is the font used for the present paragraph The Chrome Main Window is represented in Fig 3 1 It includes together with the usual Windows tool bars like the Menu Bar and the upper Tool Bar an important feature called ChromSquare Side Bar placed in the left side of the main window This bar contains some large icons presently 3 but others could be added in future releases each icon behaves as a button which lets the user to switch among different panels the Schedule Panel the Chromatogram Panel the Method Panel In other words the contents of the main window varies according to the Panel selected in each moment the user can easily shift from one panel to another without opening and closing specific windows The following table summarizes the list of items reported in Fig 3 1 32 Add a File to the Schedule Remove a File from the Schedule Display selected File Move Up and Down selected File Menu Bar Ei ChromSquare License UGxL CMS DAR File Method Schaie Opticad Action Tools a t C H current method none El File
49. Menu View commands e Zoom operations through the Zoom Mapping facility which is provided in a special window Navigator Panel of the lower panel see 7 3 2 for a full description 80 wey Be Zoom n Schedule e A EA 0 000 1600 3200 4800 6400 Define Blob Define ROI Define noise min area Cancel i l 20 320 40 640 60 961 81 281 101 61 Chromatogram Graphical param Chromatogram I Spectrum analysis 2 80667e 07 07 ra a W N Jo a eit PTY pe a A hy f ve aoa i Figure 7 1 Map Plot Context Menu 0 00000 A Method Mod 760 DP 42 TtR 105 369 2tR 2 113 1tR 105 333 Int 21799 05 The Figure 7 2 shows the Map Plot after Zoom In operation and the representation of the zoomed region in the Zoom Mapping facility This is the same setting of 7 1 after that the first menu item Zoom has been selected 7 2 3 1 Zoom In The Zoom In operation may be performed in three different ways e by selecting a region through mouse dragging with the left button the region will be zoomed as soon as the button is released this is the preferred way e by selecting a region through mouse dragging with the right button and then selecting Zoom from the menu context that will be shown as soon as the button is released this way is slightly more complex and it is provided only to facilitate the user e by using the menu command Vie
50. New method 1 d current method Open method Std Smp Save method Save method as v NIST Spectra Search Engine Figure 4 6 The Menu Method 4 2 1 New Method The command clears the current method and switches the current visualization see 3 2 to the Method Panel The Panel will show an empty Method that is a Method with only some default parameters defined that the user could fill as described in Chapter 6 4 2 2 Open Method The command opens a File Selection Dialog Box which prompts the user for the selection of an existing method file xcm extension As soon as the file is loaded the visualization shifts to the Method Panel that will show all the parameters of the Method just loaded 4 2 3 Save Method The command saves the method that is being currently edited in the Method Panel writing it onto a Method file If this method had been previously loaded from a Method file through an Open Method command the current method will be saved onto the same file and folder 47 If no method had been loaded the software asks the user to use the command Save Method As 4 2 4 Save Method As The command saves the method that is being currently edited in the Method Panel writing it onto a Method file In this case the software always asks the user to select the name and the folder to be used for the Method file The command is typically used when no Method file had been yet defined but it can als
51. View Calibration results See Section 9 1 11 139 9 2 12 Save the Schedule again See Section 9 1 12 93 Quantitation of an Unknown Sample Chro matogram The Quantitative Analysis of an unknown sample also called Quantitation is a very simple operation once that a Schedule file containing a Calibration has been set up 9 3 1 Add unknown sample files to the schedule Execute the following steps e Switch to the Schedule Panel e Add one or more sample chromatograms to the current Schedule using the commands described at Section 9 1 1 3 If the sample chromatograms have already been added to the Schedule when defining the Standards skip to the next Section e Define the chromatograms just added as Samples using the Std Smp combo box In the Schedule Table rows corresponding to Samples have a yellow background whereas Standards have a green background 9 3 2 Load a chromatogram The operation is the same described for Standards See Section 9 1 3 9 3 3 Integrate the chromatogram The operation is the same described for Standards See Section 9 1 4 Since the current Method already contains the identification of the Blobs the integration command should also recognize the Blobs 140 This operations may be skipped if all unknown samples have been added to the Schedule together with the Standards in this case the command Integrate All Chromatograms described at 9 1 7 will also integrate the unknown
52. View Window Surface colorization 160 10 3 Compare subtract Tool tic Be ao A A 162 10 4 The Compare Subtract Window Tool Bar 163 10 5 Defining a Polygonal Region lt 166 10 6 Entering the polygon region name o 167 10 7 The polygonal Blob after integration 167 10 8 Open Dialog Box with Profile Combo Box 169 10 9 The New Profile window 2 20000202 ae 170 10 10Method Panel with Ion Selection 0 172 10 11 The File selection Dialog box of the Assemble case 173 10 12List of the files to assemble o 020004 174 12 10 13Files selected for assembling 10 14Asking for confirmation before assembling 10 15The resulting assembled chromatogram 13 Chapter 1 Foreword 1 1 Contact Information Chromaleont S r l Via Industriale 143 1 98123 Messina Italy Tel 39 3392620612 Fax 39 090 358220 e mail chromaleont chromaleont it Web site www chromaleont it 1 2 Legal Information 1 2 1 Copyright Copyright Chromaleont S r l 2009 2010 All rights are reserved including those to reproduce this publication or parts thereof in any form without the express permission of Chromaleont S r l Information in this publication is subjected to change without notice and does not represent a commitment on the part of the vendor Any error or omissi
53. alculation progress 128 Integrated chromatogram Zoomed view 128 Manual identification of a Blob o 129 Method Panel showing Blob names and Method file name 130 Integrated chromatogram with names of the recognized Blobs 131 Schedule panel ready to insert concentration values 138 Schedule panel while inserting a concentration value 134 11 9 18 The Calibration Results Dialog box 135 9 19 The Calibration Graph window o 137 9 20 The Quantitation Results reported on the Schedule Panel 142 9 21 Method Panel with some Blob ID defined 144 922 hist ofall Blob TDS sas o Acne E AE ars ES 145 9 23 Selection of the Blob IDs o 145 9 24 Method Panel with one Group o 146 9 25 Method Panel with more Groups o a 147 9 26 Integration Results with Named Groups 148 9 27 Integration Results Grouping Results Table 149 9 28 Group Calibration Command 150 9 29 Group Calibration Summary y ls a Ge a RR e 151 9 30 Group Calibration Data ose eke es dew ee a 152 9 31 Group Calibration Grp id ee ea 153 9 32 Group Calibration Quantitation Results Data Analysis Page 154 9 33 Group Calibration Quantitation Results Grouping Page 155 10 1 3D View Window Initial View 2 157 10 2 3D
54. am specified by the row Initially all concentrations are set equal to zero 4 At this point the user can fill in the table this is a two step procedure The first step is the choice between Standard or Sample this is easily done using the combo boxes of the second column The second step is the assignment of the concentrations to the Blob IDs of the chromatogram that should be used as standards for the calibration The user should assign the concentrations filling the table cells according to the following criteria e Chromatograms defined as standards have all concentrations different from zero 66 e Chromatograms to be considered as unknown samples maintain zero concentrations for all Blobs with the exception of the Blobs that are referenced as internal standards which also have concentrations differ ent from zero 5 The process of filling in the schedule table may be a long process espe cially when there are many rows and or many Blobs during this process the software does not check the coherence of data entered to avoid continuous interruptions for the user A global check is performed only when the user gives a Save or Calibrate command if some concentration is not coherent with the preceding schema a message error is shown giving the user the pos sibility to correct the data When everything is right the software accepts the schedule table and redraws it using the following coloring model e Sample Blob IDs i e
55. ations not linked to the current loaded chromatograms The menu is shown in 4 13 4 7 1 Report Template Editor This command calls a special tool the Report Template Editor The Report Template Editor allows the user to prepare a custom Report Tem plate This template will be automatically added to the list of Report Tem plates and appear in the selection panel when the command Action Generate Chromatogram Report is executed 60 a ll ChromSquare License ALL FEATURES File Method Schedule View Options Action a ftl iS Filename Compare Subtract Chromatograms Schedule Report Template Editor Figure 4 13 The Menu Tools The Report Template Editor window is shown in 4 14 Report Name draw map plot draw blobs table all blobs Column TER 2tR 4rea ID Area Yo Area ID Norm Display Decimals lt paper size A4 paper orientation Portrait v Figure 4 14 The Report Template Editor Window The user can choose e paper format and orientation e whether the report should include the Plot Map or not e whether the report should include the Result Table or not e which columns of the Result Table have to be printed and in the case of numerical values how many decimal digits are requested 61 The report name must be unique if accidentally an existing name is chosen the text appears in red Once finished pressing the
56. atogram at a time e or automatically through the main menu command Action Integrate A11 Chromatograms see 4 6 5 The first one may be preferred in tricky cases where may be useful to investigate every single chromatogram for possible anomalies This way lets the user to check the results of every single chromatogram before continuing with the next one The second one is that preferred in the daily routine work This is a powerful command that for each chromatogram of the Schedule performs the following operations e loads the chromatogram e integrates it e saves the results in the xrs file All operation are carried on without further intervention of the user The inte gration of many chromatograms can take a lot of time obviously depending on number and complexity of the chromatograms during which various images will flash in the graphical interface Also various monitor windows will appear and disappear during the process The process cannot be interrupted by the user at any case once started it should be left working and terminated by itself Trying to close the monitor window has no effect on the whole process this only closes the monitor window for that integration a new window will be generated at the next integration step 9 1 8 Assign the concentrations Now you can return to the Schedule Panel Nothing seems to have happened this because when you added the chromatograms the Method was still
57. buttons used to easily perform the most common operations e The Data Analysis context menu it applies to the currently selected Blob and offers options for further investigation of its properties 93 An example of the Data Analysis page is reported in Figure 7 13 Since the table is rather large the Figures 7 14 and 7 15 report an enlarged view for a better readability 3 200 e oy pr se Y e un j e X A p 4 me ha 3 A EAS tice S 1 600 0 000 lame bil ati 20 320 60 961 st Graphical param Chromatogram Spectrum Data analysis Sb a MM gt E Polygon mode Index ID Name TR 2R Area Area ID Area Area ID Norm Area Norm Area ID Conc LRI LRI lib Sim Idx 12517472 1116659119 Neral 29 547 4 830 169894187 8 169894187 8 34 294 34 294 34 294 34 294 1245 1238 97 12779629 1665957737 Carvone 30 091 5 484 102736439 8 102736439 8 20 738 20 738 20 738 20 738 1256 12 97 1382820i 648969502 Geranial 32 221 5 283 222768001 3 222768001 3 44 967 44 967 44 967 44 967 1301 1268 96 Figure 7 13 Data Analysis page Overall view ll j 8 P 5 r gi 0 A pa A yy y Ms del e r Pa 8 T si l s i I ee 0 000 20 320 40 640 Graphical param Chromatogram Spectrum Data analysis 2 am o gt E Polygon mode Index ID Name TR 2R Area Area ID Area 1251747
58. cells with zero concentrations are shown with YELLOW background e Standard Blob IDs i e cells with non zero concentrations are shown with GREEN background 6 When one or more rows have been defined as standard that is all concen trations have been assigned it is possible to proceed with the calibration If calibration succeeds the software automatically calculates the concentra tions of the Blobs of the sample chromatograms and fills the table with the calculated concentration values A Schedule Table can be saved on a Schedule File extension w1s using the Save icon of the Schedule Tool Bar or the corresponding Menu command When a Schedule File is opened the Schedule Table is automatically filled with all information saved on the file The various steps of the process are shown in Figure 5 2 Figure 5 3 Figure 5 4 5 2 1 The Context Menu of the Schedule Table When the number of the cells of the Schedule Table is large the work of manually providing all values can be a long and tiring activity In order to simplify this activity especially when there are replications of the values the user can use a special context menu which allows some copy and paste operations on cells and rows The context menu is activated by clicking with the right button of the mouse on a given cell the menu shows the following items 67 i ChromSquare License GCxGC MS File Method Schedule View Options Action Tools E bed c
59. change very often in these cases information specific to the type of data file could be fully specified only the first time that this file is used for all other instances information could be given as a link to some sort of already defined set of information We call profiles these set of information Profiles are defined and maintained by the user all profiles are stored in a specific XML file ChromaConfig xml which is resident in the installation folder A sample of ChromaConfig xml is automatically generated during installation Important note users must not directly modify the ChromaConfig xml file only the software should do this in the case of improper modifications the entire profile engine could not work properly any more If this happens the best thing to do is to reinstall the software from scratch it is also possible to manually delete the file and manually generate all user defined profiles Profiles change with the kind of data files being used Not all data files are presently linked to a profile The working scheme is however equal for all kind of profiles 10 4 2 Defining a new Profile This Section reports the various steps that the user must execute to define a New Profile 1 Give the File Open command a File Selection Dialog Box will be opened 2 Browse to the desired folder and Select a data file the present example points out the case of GCsolution or GCMSsolution files acquired in ged or qgd
60. clicking with the mouse onto them e The third part regards the Intensity Shift that is the shifting of one chro matogram with respect to the other one along the vertical axis it includes two vertical arrows a text field and a mouse icon J The command shifts the reference chromatogram down along the intensity axis by an amount indicated in the text editor t The command shifts the reference chromatogram up along the inten sity axis by an amount indicated in the text editor sl The command allows the user to shift the reference chromatogram along the intensity axis by selecting the start and the end positions by click ing with the mouse onto them e The fourth part just contains a button Execute subtraction The command executes the subtraction of the two chromatograms previously loaded according to the shifts set through the Tool Bar and displays the resulting chromatogram in the lower panel 164 10 2 3 4 Shifting the reference chromatogram using the mouse Once the user selects the button in order to shift the chromatogram upon the time or intensity axis a prompt to select the first point is displayed in the status bar After the start point is selected a prompt to select the second point is displayed in the status bar After the second click the shift is performed 10 2 4 Operations A typical set of operation 1 2 Open the base chromatogram using menu File Open Base Chromatogram Open the reference
61. corner between the horizontal and vertical slider can be used as a short cut to reset the zoom it has the same effect of the menu item View Reset Zoom 7 4 The Chromatogram page The chromatogram page shows the linear chromatogram that is the plot of inten sity vs modulation time for each modulation 89 The chromatogram displayed in the Chromatogram page represents the modulation corresponding to the mouse pointer position in the main Map Plot As the user moves the mouse in the upper window the plot quickly changes in the lower window following the various modulations the plot changes so much faster than modulation are dense in the main plot Use the zoom for a less dense representation of modulations allowing a more precise identification of a single modulation The linear chromatogram may be shown in 4 different ways activated by corre sponding buttons placed at the top of the chromatogram page see Figure Graphical param Chromatogram Spectrum Data analysis a 2 max int 51l Figure 7 7 The Toolbar of the Chromatogram page 7 4 1 Single modulation in zoom interval This representation shows the plot of the chromatogram concerning the current modulation that is the modulation corresponding to the current mouse pointer position in the 2D Map Plot on a time domain that is a fraction of the modulation time corresponding to the vertical zoom interval in the 2D Map Plot More precisely
62. d 63 5 Schedule Panel 64 ble Schedule Tool Batt e be ered A a a ae G a 64 52 Schedule Table cs A lb tee A eae A 65 5 2 1 The Context Menu of the Schedule Table 67 6 Method Panel 71 6 1 Papel Contents rd cts bth a ee iS 71 6 1 1 Integration PITO oO A E AR a 72 6 1 2 Method Filename iio a E ar a ae 72 6 1 3 Method Parameters ce hd At 72 6 1 4 Blobs ID Table Blob Recognition 72 6 1 5 Buttons for Blob ID Table management 13 6 1 6 Groups ID Table 2 00 E a eS e BR 74 6 1 7 Buttons for Group Table management 74 68 RUE soe sas a sch a e a cee A 75 6 1 9 Ion Selection for Map Display 75 6 2 Shimadzu Spectra Search page 75 6 2 1 Liprary ile ene 5 2 oa igs ein Sed ain eee te eR ee ES 75 6 2 2 Delete duplicates Ai RE A eS 76 a sd A II ee we eK Se YS 76 6 2 4 LRI upper tolerance lower tolerance 76 6 2 5 LRI index in library ora es Gua km EM Ss 76 AA oe av eee oe hs Ge ok mut Me oe Hepat se ee TT 7 Chromatogram Panel 78 Lal Panel Cont ents po fe erate CAE rca ec A ad 78 7 2 The Map Plot Window 2534 xeace a A Oe Eko ee 79 TL Feat res a ees HA ht ge Re Oe eA SE Oe SEO 79 EL lec O de he acc e Beh a Reece he ite ate bse 79 Ble POON oie ted iia the Le tee ob he A Mee ae e Mig dee ele a 80 7 2 4 Map Plot Context Menu ies ur 2 8 6 4 2 4 64 de 83 7 3 The Graphical Parameters Page cba AR AA 86 7 3 1 The Color Mapping Pan
63. d chromatogram will be shown See Chapter 7 for a full description of the Chromatogram Panel See also the various Sections of Chapter 8 1 2 for details about the operations that can be made to better view the displayed chromatogram 126 9 1 4 Integrate the chromatogram To integrate the chromatogram e either use the main menu command Action Integrate whole chromatogram e or in the lower window of the Chromatogram Panel switch to the Data Analysis window and click on the first icon the one with the gear symbol of the Data Analysis Tool Bar 7 6 2 The Menu Action command is shown in Figure 9 10 ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS 8sec xrs ax File Method Schedule View Options Mai Tools DO Integrate ROI Integrate whole chromatogram Quantitate I Remove integration j Integrate all chromatograms Schedule Create Calibration Show Calibration Generate chromatogram report Search selected Blob Search all Blobs Show selected blob Correct Wrap Around Chromatogram Graphical param Chromatogram Spectrum Data analysis 80667e 07 reset to saved values od 136 DP 137 TtR 22 248 2tR 6 893 1tR 22 133 Int 6196 88 Figure 9 10 The Menu Action Integrate Whole Chromatogram Integration may require some time especially in large chromatograms a Calcula tion Progress window allows the user to monitor the status of the integration The Calcula
64. d description of the procedure Since Quantitation requires that a Calibration has already been set and saved on a Schedule file see Section 3 3 3 the first action subsequent to the Quantitate command is a File Selection Dialog Box that will ask for the Calibration Schedule file 4 6 4 Remove Integration The commands clears the current integration The Map Plot will be cleared by Blob marks and labels and the Table of the Result page will be emptied This command is seldom used since each integration both Whole and ROI overwrites the former one 4 6 5 Integrate all chromatograms The command starts a procedure for the automatic integration all the chromatograms in the schedule list The process once initiated cannot be stopped by the user In the case of huge chromatograms the integration of all chromatograms may take a long computation time it is then a good practice when dealing with a new schedule to execute a single integration see 4 6 2 to get an estimate of the overall computation time 4 6 6 Create Calibration The command starts the procedure for the generation of a new Calibration curve based on the current schedule 56 See Section 9 1 10 for a detailed description of the procedure 4 6 7 Show Calibration The command opens a new window where are summarized the features of the current Calibration if it has already been defined 4 6 8 Create Calibration Group The command starts the procedure for th
65. e Section 4 2 1 Figure 9 5 shows the switch to Method Panel icon Figure 9 6 shows the Open Method menu command ChromSquare License GCxGC MS File Method Schedule View Options Action Tools a t 4 m H current method none Filename Std Smp HC1 6sec xrs IHC2 6sec xrs Schedule Chromatogram gt lt Figure 9 5 Switch to the Method Panel 123 License GCxGC MS JE H current method none E Std Smp Figure 9 6 The Method Menu with Open Method command selected 9 1 2 1 Define a New Method After giving the menu command Method New Method the Method Panel should show an empty method that is most fields should be void some fields will contain the value 1 0 which means a default value If you see other numbers or words somewhere a method is already loaded better to close all and start again The Method Panel with an empty Method is shown in Figure 9 7 9 1 2 2 Define Method Parameters In the Method Panel enter Minimum Area Noise and Peak Shift The Method Panel with these fields filled is shown in Figure 9 8 In this case the numbers 100000 10000 6 have been used for the three parameters 124 1 ChromSquare License GCxGC MS File Method Schedule View Options Action Tools method filename min area noise peak shift of Mod Time blob recognition 1D Int Std ID Ton Selection Chromatogram gt Method
66. e View Options Action El Schedule Chromatogram p y Figure 9 20 The Quantitation Results reported on the Schedule Panel 9 4 Group Calibration 9 4 1 Description The Group Calibration is a type of calibration based on groups of Blob IDs a Blob ID in its turn is a group of adjacent Blobs as described in Section 3 1 6 in order to distinguish the two kinds of groups we will call the groups of Blob IDs as Named Groups or simply Groups Whereas a Blob ID is a set of one or more Blobs belonging to a given Region rectangular or polygonal a Named Group is a set of more Blob IDs assembled by the user according to different criteria Both Blob IDs and Named Groups are defined inside the Method whereas Blob IDs are defined by the user through a graphical procedure Named Groups are defined at a second step by manually assembling the Blob IDs defined in the former step The Group Calibration is similar to the normal Calibration also called Blob Cal 142 ibration with the difference that it handles Groups of Blob IDs instead of single Blob IDs This means that in the Calibration algorithm the area of a Blob ID is replaced by the sum of the areas of all Blob IDs included in the Group and the concentration of a Blob ID is replaced by the sum of the concentrations of all Blob IDs included in the Group From the analytical point of view the Named Groups are useful to treat more similar compounds as a single entity
67. e current Schedule i e to move upward or downward Other commands are available in the Schedule Menu to create a new Schedule save the current Schedule or open an existing one see Section 5 for a detailed description of the Schedule management The Schedule Menu is shown in Figure 9 4 120 Pd ChromSquare License GCxGC MS File Method Schedule View Options Action Es Chromatogram VY aN Method t Qts hed current method none Figure 9 2 Initial Window for Quantitative Analysis ChromSquare License GCxGC MS File Method Schedule View Options Action Tools 21J3 h bed current method none Fa Std Smp HC1 6sec xrs Ej IHC2 6sec xrs A o Schedule Chromatogram y7 AN Method Figure 9 3 Schedule Panel with some Chromatograms added 121 ChromSquare License GCxGC MS Schedule vies A aj pp 4 Figure 9 4 The Schedule Menu with Add chromatograms command selected 122 9 1 2 Set up the Method Once the Schedule has been completed switch to the Method Panel using the Method icon at the bottom of the left sidebar If you already have a suitable Method for this calibration load it through the menu item Method Open Method see Section 4 2 2 Continue to Section 9 1 2 2 if you want to modify some parameter otherwise skip directly to Section 9 1 3 If you do not have a Method define it now though the menu command Method New Method se
68. e generation of a new Group Calibration curve based on based on the current schedule See Section 9 4 4 for a detailed description of the procedure 4 6 9 Show Calibration Group The command opens a new window where are summarized the features of the current Group Calibration if it has already been defined 4 6 10 Create Chromatogram Report The command generates a report on a PDF file Reports can containing both the chromatogram map and the table of results with different levels of details which are assigned through a Report Template Some standard Report Templates are supplied at installation time Other tem plates may be created by the user using the tool Report Template Editor see Section 4 7 1 As first action the Create Chromatogram Report command displays a pop up window showing the list of available Report Templates see Figure 4 11 After that the user has chosen the desired template the relative report is generated and displayed in a Preview window A sample of this window is shown in Figure 4 12 3The Report Templates are XSL files that is XML text files which follow the XSL Transfor mations XSLT Version 2 0 rules see http www w3 org TR xs1t20 57 Area Percent a OnlyID m CET Lc Figure 4 11 The Report Template Selector Dialog Box Lavandulol Benzyl acetate Benzyl acetate Borneol Terpinen 4 ol Terpineol lt alpha gt Estragole Nerol Oct 7 enol lt 3 7
69. e name and a folder for the Method File name should have the extension xcm the software assigns it automatically at any case folder may be any but it is recommended to use the same folder which contains the calibration data files After the saving operation the full path of the Method file will be shown in the Method Panel The Figure 9 14 shows the Method Panel with the Blob names and IDs it also shows the Method path name ChromSquare License GCxGC MS Current file HC2 6sec xrs File Method Schedule View Options Action Tools method filename 3154 Data ChromSquare 32 QuantitativelData21Hydroc xcm min area 100000 0 noise 6 0 A peak shift of Mod Time 1000 0 blob recognition Schedule ID Int Std ID Ton Selection C v delete SUPE EIE groups 1D gt Caj ROI 13 201496666666669 25 399096666666665 Method Figure 9 14 Method Panel showing Blob names and Method file name 9 1 6 Integrate again From the main menu select the command Action Integrate Whole Chromatogram again at the end the software switches to the Chromatogram Panel Now since the Method contains a full definition of Blobs the software can recog nize all the Blobs of the chromatogram and show their names in the Map Plot Select the Data Analysis tag in the lower window to see the Data Analysis Table which now contains also the names and the IDs of the Blobs which have been 130
70. e navigated in both directions at any time it is possible to go back to correct some parameter but normally the user advances through a series of Next commands 1 The first window is a Prerequisite window it warns the user about some hardware requirements If the requirements are satisfied select Yes to con tinue with the installation The compatibility dopes not apply to some components which provide the interaction with the instrumentation software which are only compliant with Microsoft Windows systems 19 10 The next window is a Welcome window no answer is required select Next to continue The next window informs the user about the installed Java Environment no answer is required select Next to continue The next window informs the user about the installed Java Environment select Next to continue The next windows is Select Destination Location The software suggests as default the folder ChromSquare in the C root disk It is recommended not to change the default folder Select Next to continue The next windows is Select Start Menu Folder The software suggests as default the folder ChromSquare It is recommended not to change the default value Select Next to continue The next windows is Select Additional Tasks The software asks for creating a desktop icon It is recommended to check the box Select Next to continue Note The installation procedure creates a second ChromSquare icon
71. e switch enables an enhanced representation of the Blob line contour for a better visualization in the linear chromatogram view 4 5 3 2 Draw Names onto Chromatogram The switch enables the writing of the name of the Blob which the peak belongs to in the linear chromatogram view 4 5 3 3 Draw TtR onto Chromatogram The switch enables the writing of the total retention time of the top of the Blob which the peak belongs to in the linear chromatogram view 4 5 3 4 Draw 2tR onto Chromatogram The switch enables the writing of the second dimension retention time of the top of the Blob which the peak belongs to in the linear chromatogram view 4 6 Menu Action The commands of the menu Action correspond to the actions that the user can per form on the software like various kinds of integration Blob searching calibration quantitation and so on The menu is shown in 4 10 Note Figure 4 10 shows a full menu Action some menu items are not implemented in all software versions The items implemented in your license version are those 54 described in the following of the current section 2 ChromSquare License GCxGC MS Current file Shimadzu GCxGC File Method Schedule View Options Emi Tools an a t 4 Integrate ROI Integrate whole chromatogram Filename Quantitate Remove integration f Integrate all chromatograms A Create Calibration Show Calibration Schedule Generate chromatogram repo
72. e types listed in the next Section License types refer to the types of data files that the software can analyze namely e data files in proprietary format generated by various kinds of Shimadzu instruments e data files in open format AIA ANDI There is also a general purpose license type which includes all the other ones 1 3 1 1 License types Presently the following license types are provided 16 1 GCxGC MS amp LCxGC MS version for Shimadzu GCMSsolution and GC solution 2 LCxLC version for Shimadzu LCsolution 3 LCxLC version for Shimadzu LCMSsolution and LCsolution 4 Text and NetCDF Version 5 Text and NetCDF Version with NIST Spectra Search 6 All Features Version The present Manual is specific for GCxGC MS amp LCxGC MS ver sion for Shimadzu GCMSsolution and GCsolutionlicense type Manuals relative to other licenses may be furnished on demand 1 3 2 Outline of the Manual 1 3 2 1 Prerequisites Please read this Instruction Manual carefully before using the product and operate the product in accordance with the instruction given thereby Keep this instruction manual for future reference This Instruction material was written with the assumption that the reader has some knowledge of MS Windows For information on the names and terminology associated with MS Windows please refer to the MS Windows user documenta tion If you are using MS Windows for the first time please read the MS Windows user documenta
73. earch using NIST Spectra Search engine e All Features Version using Shimadzu Search engine or NIST Spectra Search engine The present license provides the Automatic Identification of the Blobs The automatic search includes from one to three steps 1 the first step consists of the command which starts the search 2 the second step is optional and allows the user to modify the identified Blob 3 the third step is also optional and allow the user to add one or more identified Blobs to the Method 115 8 3 2 1 Search the Blobs The automatic Blobs search can be performed in two modes e searching a single Blob at a time e searching all Blobs with one command The first one is preferred in new cases when the user wants to check libraries computing times and so on The second one is the preferred one in the daily analysis routine To search one Blob e Select the row corresponding to the Blob in the Data Analysis table e cither give the command Search Blob of the Data Analysis Context Menu see Section 7 6 3 1 e or the command Search Selected Blob Section 4 6 11 of the Action main menu To search all Blobs e give the command Search All Blobs Section 4 6 12 of the Action main menu 8 3 2 2 Select the Compound This step may be executed when the user wants to modify one or more automatic Blob assignments selecting a different compound from the list generated by the automatic search procedure The st
74. ed in the Method The columns gave the following meaning 1 Index This is just a counter of the rows groups 2 Name The name assigned to the Group in the Method 3 Area The Area of the group corresponding to the sum of the Areas of all Blobs which belongs to the Group 4 Area The Area of the group corresponding to the sum of the Areas of all Blobs which belongs to the Group 5 Area Group The percentage of the Area of the group with respect to the sum of the Area of all Groups It obviously is 100 if only one Group is defined 106 6 Conc The Concentration of the group corresponding to the sum of the Concentrations of all Blobs which belongs to the Group The Figure 7 24 shows a Method Panel with the definition of a Group The example shown refers the same case described in Section 7 6 1 1 a Group named Group AB has been defined as the aggregation of Blob A and Blob B The Figure 7 25 shows the results of the integration in the Grouping page Com paring the results with those shown in Figure 7 13 it can be seen that the Area of the Group AB is equal to the sum of Areas of Blob A and Blob B The same for Area 7 8 The Status Bar The Status Bar see Figure 7 8 shows the current position of the mouse pointer inside the Map Plot window It includes the following information e Mod Modulation counter along horizontal axis e DP Data Point counter along vertical axis e TtR Total Retent
75. el mt 4 a 40 2 ae eS 86 7 3 2 The Navigator Panel oyo ra e ria eR 88 7 4 The Chromatogram Pare RAE AA E 89 7 4 1 Single modulation in zoom interval 90 7 4 2 Single modulation in whole modulation interval 90 7 4 3 Multiple modulation in zoom interval 91 7 4 4 Multiple modulation in whole modulation interval 92 7 5 The Spectrum page 2444044 ack a Se a 93 7 6 The Data Analysis page 02 200222 93 7 6 1 The Data Analysis table 94 7 6 2 The Data Analysis Toolbar wa dto oa watson 99 7 6 3 The Data Analysis Context Menu 100 7 7 The Grouping page gt rca a A spa 106 A AS NA Ge te a Bek ot Stat et a 107 8 Qualitative Data Analysis 108 8 1 Set up the Chromatogram e e 108 8 1 1 Load the Chromatogram io a 108 8 1 2 View the Chromatogram aa 108 8 2 Integrate the ChromatograM 109 8 2 1 Set up the Metodo ira a as YE 110 8 2 2 Integrate Whole ChromatograM 111 8 2 3 Integrate ROL a a 111 8 2 4 Check the Integration Results 112 8 2 5 Change the Integration Parameters 112 8 3 Identify the Blobs soe 4 oe A A AS Be ee 114 8 3 1 Manual Identification of the Blobs 114 8 3 2 Automatic Identification of the Blobs 115 Quantitative Data Analysis 118 9 1 External Standard Calibration 118 9 1 1 Set up the Schedule
76. elected region See 7 2 3 1 for a full description of Zoom In operation 7 2 4 2 Define Blob The menu item command is used to manually identify one or more Blobs that is to assign a Blob ID to the Blob or Blobs contained in the selected region If the region contains more Blobs the same Blob ID will be assigned to all them As described in 3 1 6 a Blob ID is a data structure which contains a numerical Jd a name and a geometrical description Whilst the Id is automatically generated by the software and the description is given by the selected region the name should be explicitly supplied by the user The assignment of the Blob name works as follows e Select with the right mouse button a region containing one or more Blobs as described in 7 2 2 e When the context menu appears select the second item Define Blob a small window with title Insert Blob Name will be displayed The window contains a label which reports the initial and final Total Retention times of the selected region and a text field for entering the name e Enter the desired Blob name in the text field and give Ok o confirm the assignment or Cancel to delete the current assignment The window is shown in Figure 7 3 It is also possible to define a Blob ID whose region is a polygon instead of a simple rectangle this feature is called Polygonal Blob area see Section 10 3 for details A rectangular region becomes in this way a particular case of a polygonal o
77. ented with the same syntax used to select ions for quantitation see Chapter 10 6 Once this parameter has been set 1t is possible to display the Chromatogram map with a color representation given only by the selected ions 6 2 Shimadzu Spectra Search page The window for the Shimadzu Spectra Search is shown in the Figure 6 2 In order to perform the Shimadzu spectra search GCMSsolution LCMSsolution or LabSolutions must be installed Please read the technical note Chapter 11 1 in order to correctly set up the Shimadzu software 6 2 1 Library file n The page contains five edit fields where the user can specify the full paths of the spectra libraries that the Shimadzu software will use to perform the spectra search The assignment of the paths is accomplished by pushing the little buttons rep resenting a folder placed on the right of any text field The buttons cause the opening a File Selection Dialog Box which allows the user to select the library files lib navigating across the Shimadzu software folders Shimadzu software allows to perform the search using up to 5 libraries At least one library must be defined 79 i ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS 8sec xrs EAX File Method Schedule View Options Action Tools Integration Shimadzu spectra search Library File 1 C GCMSsolution Library FFNSC 1 3 FFNSC 1 3 lib I Library File 2 C GCMSsolution Library SHIM2205 SHIM2205 LIB Library File
78. ention time of the single datafiles that should be the same for all files 7 At the end of the procedure the resulting assembled chromatogram is dis played see Figure 10 15 The software will treat this chromatogram like any other chromatogram obtained in the traditional way The user should only consider that in this case the number of modulations is generally much lower that in the other cases this could cause some limitations at zoom level 175 Y ChromSquare License ALL FEATURES Current file LCxGC berg 008 xrs File Method Schedule View Options Action Tools Ey Schedule Graphical param j Chromatogram aphical pa Chromatogram Spectrum Data analysis p p reset to saved values Mod 0 DP 33577 TtR 0 000 2tR 0 000 1tR 0 000 Int 604 36 Figure 10 15 The resulting assembled chromatogram 176 Chapter 11 Technical Notes 11 1 Prerequisites to install ChromSquare with GCMSsolution spectra search 11 1 1 Description Since version 1 5 the spectra search function is deeply integrated with GCMSso lution An agreement with Shimadzu Co allowed to perform a seamless search but a version of GCMSsolution higher than 2 71 is required The steps to upgrade are e Install GCMSsolution 2 71 according to the instructions provided by Shi madzu e Run ShimadzuDliSetup exe to enable the communication API e Install ChromSquare The spectra search function will not work with previous
79. ep is optional in many case the suggested compound is a good choice See Section 7 6 3 6 8 3 2 3 Add Blob s to Method This step may be executed when the user wants to insert one or more Blob iden tifications in the Method 116 The step is mandatory only if the current analysis will be followed by Quantitative Analysis See Section 7 6 3 8 117 Chapter 9 Quantitative Data Analysis In this chapter 1t will be presented a summary of the operations of a routine Quantitative Analysis The summary will be illustrated with frequent screen shots which drive the user along the various steps of the procedure The Chapter contains four Sections 1 External Standard Calibration 2 Internal Standard Calibration 3 Quantitation of an Unknown Sample Chromatogram 4 Group Calibration 9 1 External Standard Calibration 9 1 1 Set up the Schedule Chrom software can be used to build Calibration Plots by processing data analyses performed at different concentration levels using pre measured GCsolu tion or GCMSsolution files acquired in gcd or qgd data formats All files that enter in a Quantitative Analysis must belong to the same folder 118 9 1 1 1 Rename the files This Section reports some hints about the file names of the chromatograms in order to simplify the operations and avoid errors Some file renaming could be then necessary according to the following rules e The software order
80. es otherwise it is expressed in seconds remember that a chromatographic point corresponds to a pair of numerical values i e time and intensity absorbance in 2D chromatography the whole chromatogram is divided into a set of mod ulations according to a modulation time in this representation the time value of the chromatographic point corresponds to two time values the overall time value represented along the z axis and the time value of the single modulation represented along the y axis the intensity value or absorbance corresponds to the third dimension in the 3D representation see this corresponds to the z axis whereas in the 2D representation this corresponds to a color level the whole set of all points generates a Contour Map 7 2 2 Selection When a new chromatogram is loaded into memory the corresponding Contour Map of the full chromatogram is displayed in the Map Plot window The user can then select through the mouse a smaller a smaller region of the Contour Map in a way that it well be represented in the whole Map Plot window this action is typically called Zoom and yields as a result an enlargement of the selected region allowing to see more details Zooming can be handled in different ways and is described in 7 2 3 79 The selection af a smaller region of the chromatogram can be used not only for zooming but also for other operations concerning regions The selection operations are performed
81. es of the chromatograms e the second column is used to define the role of the chromatogram in the calibration procedure quantitative analysis see 9 that is if it is either a standard or an unknown sample the column header reports the abbreviation Std Smp Subsequent columns correspond to the Blob IDs defined in the Method loaded 5 2 0 1 Populating the Schedule Table The population of the table evolves as follows 1 Initially when software starts or the command New Schedule is executed the table only contains the header of the first two columns Filename Std Smp and no rows exactly as shown in Figure 5 1 2 As long as chromatograms are added to the schedule list new rows are added to the table reporting the chromatogram filename in the first column of the table leaving for the moment empty the Standard Sample option of the second column see Figure 5 2 3 When the user defines a Method this may be accomplished in different ways see the Method Panel chapter for details the table is filled with new columns each column corresponding to a Blob ID defined in the Method The new columns have headers that report the names of the Blob IDs The columns are designed to contain the concentrations of the compounds cor responding to the Blob IDs a cell placed at a given row and column will contain the concentration of the compound corresponding to the Blob ID specified by the column in the chromatogr
82. es vs ChromSquare Files Before to proceed with the description of the software operations it is important to understand the File Management engine adopted by Chrom 2ere The File Management is based on the following concepts e The software reads Raw Data Files that is files that are generated by in struments these files may have different extensions according to the kind of instruments and to the software license installed but all have a common peculiarity a Raw Data File is always treated as is it cannot be 34 changed or modified in any way Raw Data Files can only be read copied or moved to a different folder e Chrom 1 e software uses for its purposes various files schedule files result files method files which will be described later we will call them generically Chrom t e files All these files have a common peculiarity they are all XML files even if they do not have xml extension but a specific extension for each kind of file Normally the user does not interact directly with these files 1t is the software that manages them However being XML files means that they are open text files if necessary the user can explore their content using a normal browser like Internet Explorer or Mozilla or Safari A special mechanism based on check sum control prevents the user to modify the file contents the user may inspect the files but only the software may manage them The Chrom 2 0 files are described in the fol
83. etooth Adapter key or simply BT key e demo licenses are based on an expiration date In both cases e any license is supplied together with an activation code that is a sequence of 32 hexadecimal digits e licenses both regular and demo ones are provided in different license types Types are relative to a particular set of instruments See the Section 1 3 1 1 for a list of the available types The first time that the software is executed it asks the user to enter the activation code in the case of demo license it also asks for the expiration date Hexadecimal digits are the 10 digits from 0 to 9 and the 6 characters from A to F 23 If the data entered are correct they are saved internally so that they are non longer required the next time the software is executed and the software session can start If they are not correct the software will be terminated 2 4 2 License management When the software starts it performs the following operations 1 Before all it checks if a BT key is present e if it is present the software assumes that this is a case of a regular license e if it is not present the software assumes that this is a case of a demo license 2 A window is then displayed showing the Software License Agreement and some fields to be compiled by the user e The Activation Code that the user had received together with the soft ware CD it may be entered manually beware of entering
84. etween Blob and Blob ID e Blobs are single objects belonging to a chromatogram Blobs are stored in the ChromSquare Result File xxx e Blob IDs are analytical information stored in a Method e In the first instance Integration finds all the Blobs of a chromatogram Blob IDs are unknown at this time e In the second instance a Method containing Blob IDs description can be applied after this step some Blobs are identified and a Blob ID is assigned to them 3 1 8 Methods A Method is a set of information that are used by the integration process A Method contains three kinds of information e parameters that are used for Blobs recognition see xxx for details e alist of Blob identifiers called also Blob IDs see 3 1 6 e an optional Region of Interest also called ROI Methods can be saved and loaded to from special files called Method Files see XXX 31 3 1 9 Units Time is always represented internally in minutes tables and graphs also show time in minutes except in the particular cases where time is represented in seconds Absorbance is normally represented in micro Volts except in the particular cases where absorbance is represented in milliVolts Anyway please refer of the man ual of the software that generated the data file that has been imported since ChromSquare uses the same unit as it is defined in the original file 3 2 Chrom Graphical User Interface The Chrom 2 Graphical User
85. even if they are placed in different regions of the graph In the following paragraphs are summarized the various steps of a Group Calibra tion 9 4 2 Define Groups in the Method Panel To set up a Group Calibration you should have already set up a normal Calibra tion that is you should have already defined a Schedule a set of Standard and Sample Chromatograms and a Method See Sections 9 1 1 and 9 1 2 To define the Named Groups execute the following steps 1 Switch to the Method Panel in the main window the upper table labeled Blob recognition shows the already recognized Blob IDs the lower table labeled Groups is initially empty A sample of Method Panel with Blob IDs and no Group is shown in Figure 9 21 143 El ChromSquare License GCxGC MS Es Schedule Chromatogram 2 Click on the button add immediately below the Groups table a window showing a list of all Blob IDs will be displayed the window assigns a tempo rary name to the Group being defined that is Group followed by an increasing File Method Schedule View Options Action Tools method filename nd Settings Aqualis Dooumenti ChromSquare My Samples 2010 TestCalib 2010 IS Groups ProvaCalibrezione Group 2 xcm min area 10 0 noise 10 0 peak shift of Mod Time 0 0 blob recognition iD name Int Std 1D 1 Ac Butirrico 21 2 __ Ac Caproico 21 3 Ac Caprilico 21 4 Ac Caprico 21 5 Ac Undecanoico 21 6 Ac Laurico 21
86. ew window is equipped with a Tool Bar which allows to reset the scene to the initial position and to take a snapshot of the current view The Tool Bar contains the following controls e Icon immediately restores the initial position for the surface e Icon I takes a snapshot of the current view The first time that it is pressed a File Selection Dialog Box is shown allowing the user to choose path and file name where to save the image Once selected the path and the first part of the file name will be the same for the whole session until the 3D View window is closed and every time the camera button is pressed a new picture will be automatically taken and saved on a file having the same prefix file name followed by a progressive number For additional information please refer to the specific documentation provided together with the mouse 158 Icon Ea takes a movie of the scene capturing one frame every half second Once the user has started the recording session the icon changes into Li the same button allows the user to stop the recording session the icon changes to the original one Icons actually three icons numbered 1 2 3 switch On and Off a corresponding directional light that enlighten the scene Icon displays a Color Selection Dialog Box which allows to select the color for the background Icon A switches On or Off the colorization of the surface depending on the elevation that is the intensity See Fig
87. exactly 32 hexadecimal digits or may be copied and pasted if the license had been provided though an electronic medium e The License Type to be chosen through the Combo Box e In the case of demo license also the Expiration Date in the format YYYY MM DD should be entered Figure 2 3 shows the Software License Agreement window in the Normal License case Figure 2 4 shows the Software License Agreement window in the Demo License case 24 Fa Enter License code CHROMALEONT SOFTWARE LICENSE AGREEMENT The present document is to be considered a legal agreement between yourself denominated from now as user and Chromaleont The seal on this packet with any terms of the agreement do not break the seal and promptly return this includin ing Upon return you will receive a full refund Grant of License uses permitted 1 Chromaleont grants to the user the right to copy the software program named as ChromSquare from now as Program enclosed in the CD ROM container onto a single computer and the right for the user and others to make use of that copy of the Program on a single computer 2 The user is allowed to relocate the Program permanently This as long as the user does not withhold any copies and that the receiver acknowledges all terms of Figure 2 3 Enter License Code window for Normal License 25 Enter License code CHROMALEONT SOFTWARE LICENSE AGREEMENT The present doc
88. fo required Use a profile Risorse del computer File name Shimadzu GCxGC MS 8sec QGD Risorse direte p Files of type Files QGD GCD CDF AIA ANDI XRS Figure 4 2 File description and features of the Data file selected e the upper part is used to define and select a profile see Section 10 4 since profiles are defined only for some kinds of files this part is not always dis played e the central part that becomes the upper part when profiles are not used is reserved to the user which can specify some additional information about the data file being opened modulation time 2 elution time 1 modulation time e the lower part is used by the software to display information about the data file the software actually reads a little fragment of the file trying to detect all information about its structure and reports the obtained information in this part of the window when this is not possible information should be supplied by the user through a profile in this case the software warns the user with the message further information required use a profile 4 1 1 3 Setting the Modulation Time The Modulation Time must always be defined otherwise the software does not know how to extract the modulations from the chromatogram Modulation Time can be assigned in two ways e Explicitly by directly writing the value in the text field note that the value 42 entered in the field will be i
89. go eo ee 9 Metamorphosis in Separation Science Chrome t 1 5 User Manual GCxGC MS amp LCxGC MS version for Shimadzu GCMSsolution and GCsolution Chromaleont srl September 2012 Contents 1 Foreword 1 1 Contact Information 6 hie e Di A ee a di 1 2 Legal tomate BR Sed A ed ae E ES Bel A Ge NA E22 Warranty e se A a Ee a ee E T3 Introd ti n ce 2 kere diva ere ih Bogie Bed neck eek dle Be a ed 1 3 1 Outline of the Software o 1 3 2 Outline of the Manual eo RAE E a oe O 2 Installation and Startup 2 1 Installing the software lt A AA o e 2 2 Uninstalling the software a oho op A ia O A BS 2 3 Starting ChromSquare software 2 02 0004 2 4 Registering the License 2 4 408 ba a eee 2 4 1 License policy aw eee he ee EM a A See A 2 4 2 License management dar cp AA we Ge eS 3 Chrome Overview 3 1 General Terminology and Concepts o 3 1 1 Chromatogram lt gt ds e a es Eee eS 3 1 2 Chromatographic Map Laa a Be ee ae 3 1 3 Integration te Lo ett be ok ap e A E ILL PEAKS a e A e oboe Gon eee 29 Sulla a VOUS sick shout Ed y ESE STE ANT ek ey Ye 29 p66 BlOb IDS a2 has 28 at AN 30 3 1 7 Blobs vs Blob IDs poca Grid ae ES we Ok Se o 31 Ss Method g s ssi Gee ls ST STARR RA 31 SO TUNICS 0 a e a A den a 32 3 2 Chrome Graphical User Interface 32 3 3 File Management pr o ae Hc e A 34 3 3 1 Raw Data Files vs ChromSquare F
90. gram Panel is made up of two windows horizontally aligned that is one above the other and a status bar in the lower part of the panel e The upper window shows the Chromatogram as a colored Map also called Map Plot or Contour Map this window corresponds to the 2D representation of the chromatogram it is described in Section 7 2 e The lower window contains various sub windows or pages that can be selected using the upper tabs Graphical Parameters page see Section 7 3 this page contains the Color Map Panel the Navigation Panel Chromatogram page see Section 7 4 Spectrum page see Section 7 5 Data Analysis page see Section 7 6 Grouping page see Section 7 7 e The status bar see Section 7 8 displays information about the current po sition of the mouse cursor inside the upper Map Plot The upper and the lower windows are separated by an horizontal floating bar that is a bar that can be dragged up and down with the mouse allowing the resize of the two windows according to current needs of the user 78 7 2 The Map Plot Window 7 2 1 Features The upper Map Plot window contains a Contour Map which corresponds to the 2D representation of the chromatogram namely Let s the abscissa axis represents the Total Retention Time this is always ex pressed in minutes the ordinate axis represents the Modulation Time this is expressed in min utes when its value is greater then 2 minut
91. hen be edited by the user The command does not automatically switch the current visualization to the Schedule Panel 4 3 4 Save Schedule As The command opens a File Selection Dialog Box for the selection of a folder and of a file where the Current Schedule will be saved as a Schedule File w1s The command is used both for defining a new Schedule File and for overwriting an existing one 4 4 Menu View The Menu View contains some commands concerning the visualization of the chro matogram The menu is shown in 4 8 49 ChromSquare License ALL FEATURES File Method Schedule Options Action Tools Reset Zoom Cd current method Zoom In Std Smp Zoom Out 3D view Schedule Figure 4 8 The Menu View 4 4 1 Reset Zoom The command restores the initial visualization of the full chromatogram after one or more zoom operations See Section 7 2 3 for a complete description of Zoom operations 4 4 2 Zoom In This command increases the zoom factor by a fixed amount the effect is to see a smaller region and more details around the current position of the mouse cursor See Section 7 2 3 for a complete description of Zoom operations 4 43 Zoom Out This command decreases the zoom factor by a fixed amount the effect is to see a larger region and less details around the current position of the mouse cursor See Section 7 2 3 for a complete description of Zoom operations 4 4 4
92. her use the menu command Action Integrate Whole Chromatogram Section 4 6 2 e cither use the first icon of the Data Analysis Tool Bar Section 7 6 2 Figure 8 2 shows a samples of whole chromatogram integration L ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS Bsec xrs File Method Schedule View Options Action Tools Graphical param Chromatogram Data analysis com P 80667e 07 re ave jes jod 392 DP 44 TtR 56 304 2tR 2 214 1tR 56 267 Int 17214 93 Figure 8 2 Qualitative Analysis Whole Chromatogram integrated 8 2 3 Integrate ROI To integrate a ROI e Define the ROI if this has not already been defined the ROI is stored in the Method and its definition can be viewed in the Method Panel as the Total Retention Time values of two opposite points see 6 1 8 To define a ROI use the Map Plot Context Menu see Section 7 2 4 e Perform the ROI integration 111 either use the menu command Action Integrate ROI Section 4 6 1 either use the second icon of the Data Analysis Tool Bar Section 7 6 2 Figures 8 3 and 8 4 shows samples of a ROI definition and its corresponding inte gration 8 2 4 Check the Integration Results After the integration both in the whole or the ROI cases the user may check whether the results are correct in the routine analysis this step is not necessary but in a new analysis it may be important To check the a
93. his command opens a new window called Java Console Java Console window is used by the software to record various information that can be useful to the technical assistance in the cases of malfunctioning of the software See Section 3 4 Normally this window is shown initially if the user choose to start ChromSquare in Debug mode the command lets the user to show the window at any moment at run time 4 8 5 About The command displays information about Chrom 2 2 current version and ref erences about the software producer 63 Chapter 5 Schedule Panel The Schedule Panel is shown when the user clicks on the corresponding large icon Schedule of the Side Bar Panel see Figure 3 1 The Schedule Panel contains a Schedule Bar and the Schedule Table 1 ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS Bsec xrs File Method Schedule View Options Action Tools fE bed current method ES Filename Std Smp Schedule Figure 5 1 The Tool Bar of the Schedule Panel 5 1 Schedule Tool Bar The Schedule Tool Bar is placed in the upper side of the Chrom 2 main in terface immediately under the Menu Bar 64 This Tool Bar is shown only when the Schedule Panel is selected The icons of the Tool Bar summarize the most frequent operations some of these operations correspond to menu commands other ones are exclusive of the Tool Bar The meaning of each icon is also shown as a tooltip small informat
94. ide to add the selected Blob to the Blob Table of the Method or not using this command e As soon as this operation is performed the Blob that has been included in the Method is available for quantitation and is redrawn in black 7 6 3 8 Add All to Method The command automatically performs the procedure of adding a Blob to the Method described in the previous Section 7 6 3 7 on all the Blobs of the Blob Table In other words the procedure search for all Blobs of the Table corresponding to rows marked in red and add them to the Method at the end of the procedure all rows are redrawn in black 105 7 7 The Grouping page The Grouping page is the last page of the lower window of the Chromatogram Panel It is activated only when in the Method is defined at least one Group in the Group table blob recognition D name Int Std ID Ton Selection Background Subtrac 1 Blob A 1 O 2 Blob B 1 E 3 Blob C 1 LI _ delete Bgonan sgoffal groups D Name Blobs i Group AB 12 Figure 7 24 Method Panel showing one Group TOA ATEN ES WA T T T T r T 20 320 40 640 60 961 81 281 101 601 Graphical param Chromatogram Spectrum Data analysis Grouping ed x A Index Name Area Area Area Group Conc 1 Group 1 272630627 6 17 285 100 000 Figure 7 25 Grouping page The results The page consists of a table with 6 columns and as many rows as the Groups defin
95. iles 2 34 3 3 2 Chrome Result Files a A O ae eae 35 3 3 3 Chrome Schedule Files A eS 36 3 3 4 Chrome Method Files ir e A E Ge 37 3 3 5 Openin Fles ms edt eine aenga a TE Goa PESE kee 37 oot The Java Console ed be See e Bee ee ae 37 3 4 1 The Java Console buttons 60 4 aaa 38 Menu Bar 40 Asli Menm Fil zesp a dho A a L 2 40 ALL Open Datatles arnt E AE ARE p Bed 40 4 1 2 Olose Datafiles s Sie sere os one ea eee a AES eS 43 4 1 3 Assemble Datafile coo pea we Ree ee es 43 4 1 4 Save Datafile ba Go e oe de hk pete Wg he 43 4 1 5 Export as CDF file 44 4 1 6 Export as XML for Excel sis sr gad ee ew Re Ge 44 4 1 7 Save Map as Image ds E oe gree ge ak hse ee le A 44 Ass Data LO es ms ea dg Be Sod aie oh Sek se era ee 46 AO EIE he din evn eden tees whan ween Bk ce aa ee oe aa Ge ae Y 46 Aca Menu Method TAS AE de ee Godel IIS a 46 AD New Method iragar e raaa ak Sed ae Sed a ag aS AT 4 3 4 4 4 5 4 6 4 2 2 Open Method sence uo amp Geka a elie Ae Epes Ba ee Be AT 4 23 Save Methodi AAA 47 4 2 4 Save Method As mb a4 dt Ne es 48 Menu Sehed la lt td erena Er ae See ee ee o a 48 Md NEW wc ee op yah lo RSE ANTAS A 48 4 3 2 Add chromatograms 0 62 4 sade A A dea le 49 4 3 3 Open Schedule e hap a gt Ong oh gtd e Aas hg 49 4 3 4 Save Schedule As Cu dedi eh dod ee GOS dh ed 49 IMGT SW Sat dg a BS ie ot oop Bde As we oe BL SA Fe ee US 49 AAA Reset Zooni a e A AR A E 50 AMD
96. in the Result file and loads it into memory 4 the software cannot open a Raw Data file if there is in the same folder another Raw Data file with the same file name but different extension in this case a warning message is displayed and the user should move one of the two Raw Data files to another folder before continuing this happens for example when an exported version AIA ANDI or any other version of the same datafile had been generated using the same name 3 4 The Java Console This is a special window that does not belong to the main Graphical User Interface but that is shown in a separate window on the desktop This windows is used by the software to record various kinds of informative messages See Figure 3 2 for a sample of this window These messages contain technical information and are not designed for normal user but for technical assistance 37 The contents of the Java console greatly changes according to the operations made by the user The window automatically scrolls like in traditional Log windows always showing the most recent message It is however possible to look at the oldest messages using the right scroll bar It is also possible to clear the console in the case of too many messages Normally the Java Console is not shown It should be used only in cases of mal functioning of the software There are two ways to show the Java console e Starting the software using the Debug Mode icon e Show
97. ing the window at run time using the proper menu item see 4 8 4 Note that Java Console always is not automatically displayed as a full size window but always starts in an iconized state The user should explicitly maximize it to the normal size 3 4 1 The Java Console buttons On the bottom of the Java Console there are three buttons e Copy to clipboard The contents of the window is copied to the clipboard the clipboard contents can then be pasted on some other document e Mail To The contents of the window is directly mailed to the technical service e Clear The contents of the window is cancelled 38 115 blobs matching minimal area Class name com aqualis chromsquare ChromatogramPanel Calculus Metod name run Line number 2991 183 83 231 119 Class name com aqualis chromalib Integrator Metod name reducetoRect Line number 773 3 blobs matching rectangle Class name com aqualis chromsquare ChromatogramPanel Calculus Metod name run Line number 295 Number of already identified blobs O Class name com aqualis chromalib Integrator Metod name createBlobs Line number 608 13580 blobs detected Class name com aqualis chromalib Integrator Metod name applyMinArea Line number 675 115 blobs matching minimal area Class name com aqualis chromsquare ChromatogramPanel Calculus Metod name run Line number 2991 183 83 231 119 Class name com aqua
98. ing to the type of analysis some columns may be empty For example if Quantitative Analysis has not yet been done the Concentration column is void 95 10 11 12 13 14 15 Area ID Sum of the areas percent of all Blobs having the same ID In the case of Blobs Grouping this is the overall area percent of the Group of Blobs Normalized Area Area percent of the individual Blob normalized with respect to the sum of the areas percent of all identified Blobs Normalized Area ID Sum of the areas percent of all Blobs having the same ID normalized with the respect to the sum of the areas percent of all identified Blobs Concentration This column will be filled after quantitation LRI LRI lib Sim Index 7 6 1 1 A Grouping sample The Figure 7 16 shows an example af Data Analysis table for the ID Grouping case In the sample shown three Blob IDs have been manually assigned Blob A and Blob B are relative to individual Blobs whereas Blob C is relative to an area which include 3 different individual Blobs As can be seen the first element that catches the eye is that the Blobs which belong to a Group are represented with a grey background Examining these 3 Blobs we can see that e The quantity called Area ID is the sum of the Area of the 3 Blobs and is equals for all them The quantity called Area is the fraction of the Blob Area with respect to the Area of all Blobs the sum of all these va
99. involving the automatic Blob Search see Section 8 3 2 and for chromatograms which contain Mass Spectra In these cases the command shows a new window which displays the Mass Spec trum of the currently selected Blob A sample is given in Figure 7 20 The window contains two Spectrum Plots 1 The upper plot displays the mass spectrum at the top point of the Blob 101 2 the lower plot displays the average spectrum computed as the average of all spectra corresponding to the chromatographic points of the Blob Each plot represents the spectrum according to the usual practice that is M Z along the abscissa axis and intensity percent along the ordinate axis Search Max Search Avg Spectrum at TtR 32 221 Average Spectrum Figure 7 20 Show Spectrum window 7 6 3 4 Zoom Blob The command automatically performs a zoom of the 2D Plot Map in such a way that the currently selected Blob is shown at the maximum enlargement filling the whole available space of the window A sample can be seen in Figure 7 21 102 mua mor vevo mu au 29 418 29 609 29 800 29 991 30 182 iraphical param chromatogram Spectrum Data analysis Grouping i 4 2 Ml gt E Polygon mode adex ID Name TR 2tR Area 12517472 1116659119 Neral 13828201 648969502 Geranial 32 221 5 283 222 Figure 7 21 A full zoomed Blob 7 6 3 5 Info Blob The command displays a window which reports a description of the
100. ion Time in minutes 2tR Second Dimension Retention Time in seconds e 1tR First Dimension Retention Time in seconds Int Intensity or Absorbance Note that TtR 1tR 2tR 60 lod 194 DP 113 TtR 29 961 2tR 5 686 1tR 29 867 Int 536446 50 Figure 7 26 Chromatogram Panel The Status Bar 107 Chapter 8 Qualitative Data Analysis In this chapter 1t will be presented a summary of the operations of a routine Qualitative Analysis In order to maintain the description fluent only the key points will be here reported with frequent references to specific sections of the manuals 8 1 Set up the Chromatogram 8 1 1 Load the Chromatogram Use the menu command File Open Datafile see Section 4 1 1 to load a chro matogram into memory For large chromatograms the loading may require some time several seconds or even minutes a progress bar is always displayed giving a rough estimate of the time required to complete the process When the loading operation has finished the progress bar window closes and the chromatogram is automatically displayed in the Chromatogram Panel 8 1 2 View the Chromatogram After a little expertise the user is generally able to view at a first sight whether the displayed Map Plot is correct or not Some anomalous patterns are easily recognized and may suggest corrections to some parameters from the user Modulation Time and Wrap Around are the two parameters to be influenced 108
101. ion license allows to perform an automatic identification auto matic identification is executed through calls to GCMSSolution software 28 3 1 4 Peaks A Peak is the basic object of any kind of chromatography peaks are a significant variation of the absorbance signal along modulation time we have a Peak when it is possible to recognize a maximum point Peak Top a Beginning Point and an End Point 3 1 5 Blobs A Blob is a close geometrical figure obtained by joining together the beginning and the end points of the same Peak in adjacent modulations Due to the discrete nature of Chromatogram data Blobs figures are close polygons In Chrom 1 ere Blobs are data structures which contain various information both geometrical and analytical 3 1 5 1 Blobs geometrical data Geometrical data are defined when the Blob is recognized They include e The description of the Top of the Blob that is the point of the Blob having the maximum intensity it corresponds to maximum of all Tops of the Peaks included in the Blob The Top Blob is identified by the Top Modulation Index that is the counter of the modulation cor responding to the Top the Top Time Index that is the counter of the chromatographic point corresponding to the Top the Total Retention Time also called TtR that is the retention time of the chromatographic point corresponding to the Top the Second Retention Time also Called 2tR that is
102. is an horizontal colored bar which represents the color scale The minimum and the maximum of the color scale are shown as little vertical white lines inside the colored bar the left line obviously representing the minimum and the right line the maximum The user can manually change the minimum and or the maximum values of the color scale in two ways 87 e dragging with the mouse the corresponding vertical white line towards left or towards right e clicking with the mouse near one of the vertical white lines the line will move automatically to the current mouse position 7 3 2 The Navigator Panel The Navigator Panel is a window which always contains a view of the whole chro matogram When the zoom is not activated the window shows the same image of the main Map Plot window even if with less details and different size When the zoom is activated the Navigator Panel window shows the chromatogram with a rectangle inside it representing the zoom area which is displayed enlarged in the main Map Plot window A sample is shown in Figure 7 6 ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS Bsec xrs File Method Schedule View Options Action Tools Ss Schedule Chromatogram Graphical param Chromatogram Data analysis Groupin c 2 8066784 07 reset to saved values Method od 253 DP 24 TtR 37 753 2tR 1 208 1tR 37 733 Int 87406 36 Figure 7 6 Navigator Panel with Zoo
103. ive yellow window which is automatically displayed when the mouse passes over or near the icon Scanning the icons of the tool bar from the left to the right you can find the following commands 1 Plus Sign Adds a Chromatogram to the current Schedule does not open the chromatogram just add it to the list A File Selection Dialog Box ap pears allowing the user to select the chromatogram 2 Minus Sign Removes a Chromatogram from the current Schedule the com mand does not actually delete any file only file reference is deleted from the current list 3 Magnifying Lens Loads the selected chromatogram from the Raw Data file 4 Arrow Up Moves the selected chromatogram one row up 5 Arrow Down Moves the selected chromatogram one row down 6 New symbol Creates a new empty Schedule Corresponds to the menu command 4 3 1 7 Open symbol Opens an existing Schedule Corresponds to the menu command 4 3 3 8 Floppy Disk symbol Saves the current Schedule Corresponds to the menu command 4 3 4 The last item in the tool bar shows the name of the current Method if a Method has already been loaded or defined 5 2 Schedule Table The Schedule Table is a table which shows a row for each chromatogram loaded The Schedule Table is a dynamic table that is its contents vary according to the operations performed on the software The Table has at least two columns 65 e the first column contains the filenam
104. l Blobs which is a time consuming activity which can last for minutes 85 7 3 The Graphical Parameters Page The Graphical Parameters page is divide into two panels e the left panel is used for defining the Color Mapping e the right panel is used for representing and changing the zoomed region with respect to the overall chromatogram it is called the Navigator Panel 7 3 1 The Color Mapping Panel Graphical param Chromatogram Spectrum Data analysis E 0 00000 l reset to saved values Figure 7 5 Graphical Parameters Page This panel contains some controls designed to define with precision the Color Mapping that the software uses to represent the Chromatogram Map Contour Plot described in Section 7 2 Color Mapping is the rule which transforms maps an absorbance or intensity value into a pixel color value Color Mapping is defined by assigning e the Color Map e the minimum and the maximum of the intensity scale e the minimum and the maximum of the color scale The panel is shown in the Figure 7 5 It contains e an upper pair of controls including a slider and a text field these controls are used to define the maximum of the intensity scale as described in 7 3 1 2 e an intermediate colored bar which represents the color scale e a lower pair of controls including a slider and a text field these controls are used to define the minimum of the intensity scale as described in 7 3 1 2
105. l summary of the calculation in a Dialog Box This summary is only a warning that the Calculation has been completed correctly a diagnostic Dialog Box showing the anomalies will be generated otherwise The full reporting of the results will be discussed in the next Section The Figure 9 18 is an example of the Dialog Box which shows the calculation Results Warning i Calibration ok J Calibration Standards 3 Blobs 2 Standard n 1 File 10 ppm xrs CONCENTRATIONS 10 000 10 000 TOTAL BLOB AREAS 414332 917 1 000 Standard n 2 File 50 ppm xrs CONCENTRATIONS 50 000 50 000 TOTAL BLOB AREAS 1860323 692 2857667 329 Standard n 3 File 100 ppm lcd CONCENTRATIONS 100 000 100 000 TOTAL BLOB AREAS 3211522 981 5148867 229 REGRESSION LINES FOR EACH BLOB Blob 1 Area 30913 664 Conc 179997 782 Blob 2 rea 61083 360 Conc 196500 686 Figure 9 18 The Calibration Results Dialog box Press the Ok button to close the Dialog Box If the Calibration terminated without errors the window Calibration Data will open soon after 135 9 1 11 View Calibration results The Calibration Data window is made up by two pages or tabs Calculation and Graphs e The Graph page a sample is shown in Figure 9 19 includes four panels The upper left panel is a List of all Blobs included in the Calibration the List is used to select a Blob at a time whose characteristics will be shown in the other
106. lis chromalib Integrator Metod name reducetoRect Line number 773 3 blobs matching rectangle Figure 3 2 The Java Console 39 Chapter 4 Menu Bar 4 1 Menu File The Menu File contains the main operations concerning chromatographic data files opening closing saving The menu is shown in 4 1 M ChromSquare License ALL FEATURES File Method Schedule View Options Action Tools Open datafile ts ODd current method Close datafile Assemble datafile Save datafile Export as CDF file Export as XML for Excel Save map as image Std Smp Data Info Exit Sz Figure 4 1 The Menu File 4 1 1 Open Datafile This command is used for choosing a chromatogram and loading it directly into memory The same operation can be performed through the icons of the Schedule 40 Panel in that case however the user first choose one or more chromatograms and then load them into memory The command Open Datafile on the contrary makes the two steps in a single operation this command is useful when the user wants to quickly check the contents of some chromatogram the Schedule Panel procedure is recommended in the routine work The operation includes two steps 1 File selection A special File Selection Dialog Box is shown to let the user to choose the file to be loaded 2 File loading The selected file is loaded into memory and shown in the chromatogram Panel if everything goes well
107. lowing paragraphs 3 3 2 Chrom Result Files Chrome Results Files are XML files which have xrs extension Xml Re sult Set They contain all possible information about the chromatogram except chromato graphic data that remain stored in the Raw Data File the reason of this choice is evident chromatographic data can be really huge there is no real benefit in the duplication of this amount of data for each chromatogram being analyzed what is actually important is to maintain in the Result File a link to the Raw Data file Results File is a sort of summary of the relevant chromatographic data useful for qualitative and quantitative analysis It is a small file that can be quickly read avoiding the CPU overload due to reading of large raw data file when this is not necessary A Result File is generated as soon as a chromatographic file has been loaded with the Open Data File command see xxx initially it contains only the link to the Raw Data File and few input information Modulation Period Second Eluition Time that have been set by the user at the opening of the file Subsequently the Result File will be enriched with other information which reflect the progress of the analysis e after the integration completion the Result File will include the list of all Blobs with full details of each Blob 35 e after the integration completion the Result File will also include the full description of the Method used fo
108. lues is then 100 The quantity called Area ID is the sum of the Area of all Blobs having the same ID Whereas for Blob A and Blob B this corresponds to the Area in the case of Blob C this corresponds to the sum of 3 Blobs and it is equal for all them 22 55 8 25 26 80 57 60 The sum of all Area ID must obviously take into account just one value for each Blob ID 10 77 6 51 57 60 this sum is less then 100 because there are unidentified Blobs 96 e The quantity called Normalized Area is the fraction of the Area of the Blob with respect to the Area of all identified Blobs the sum of all these value is 100 e The quantity called Normalized Area ID is the fraction of the Area ID of the Blob with respect to the sum of the Area ID of all identified Blobs the sum of all Normalized Area ID must obviously take into account just one value for each Blob ID the sum of all these value is 100 97 0 0 0 0 0 0 8E8 OE T bee s mouun oo rbereppos echt Suain oog sz r zezezssos tWztestezzp oze te esoor zr_ tlt ZLVE S207 E0 8VS 9 Figure 7 16 Data Analysis page Example showing ID Grouping case 7 6 2 The Data Analysis Toolbar The Toolbar is shown in Figure 7 17 Graphical param Chromatogram Spectrum Data analysis 4a 4 H gt E Polygon mode Figure 7 17 Data Analysis page The Data Analysis To
109. ly e Abscissa axis is retention time ranging within the limits of modulation time set by the current zoom area that is between the lower and upper limits of the zoom rectangle e Ordinate axis is intensity ranging from 0 to an upper limit corresponding to the maximum intensity The limits can be changed as described in Section 7 4 1 92 7 5 The Spectrum page Chrom t are is able to display Mass Spectra when these are included in the chro matographic Data File Mass Spectra when available can be viewed by selecting the page Spectrum in the lower panel As the user moves the mouse in the upper window the spectrum quickly changes in the lower window following the various modulations like in the case of Chromatogram page see 7 4 A sample of Spectrum page is shown in Figure 7 12 Graphical param Chromatogram Spectrum Data analysis Spectrum at TtR 32 191 100 las 69 so 4 604 55 81 404 82 6 95 20 4 al 56 123 70 83 53 la 138 o T L T T T 40 60 80 100 120 140 Mod 211 DP 69 TtR 32 191 2tR 3 472 1tR 32 133 Int 143036 63 Figure 7 12 The Spectrum page 7 6 The Data Analysis page The Data Analysis page shows the results of the integration and identification activities The Data Analysis page is characterized by e The Data Analysis Table table rows correspond to the Blobs table columns report the characteristics of each Blob e The Data Analysis tool bar 1t contains some
110. mation about the cur rent chromatogram A sample of the Data Info Dialog Box is shown in Figure 4 5 This information is useful for checking purposes but is not generally used during the normal routine analysis x Filename C Users Aqualis Documents ChromSquareData 2012 GCxGC MS Shimadzu GCxGC MS 8sec xrs gt Number of chromatograms 762 Total Number of points 121853 Min Value 0 0 Max Value 2 8066724E7 Min Scale 1601 0 Max Scale 2 8066724E7 2nd Elu Time 0 13333333333333333 m 8 0 s Timelnit 4 0 m 240 0 s Time Begin Mod 0 0 m 0 0 s Mod Period 0 13333333333333333 m 8 0 s Figure 4 5 The Current Data Info Dialog Box 4 1 9 Exit This command closes the current session of Chrom 1 a confirmation request is displayed in order to avoid an accidental closure 4 2 Menu Method The Menu Method contains the commands for the management of the Method files xcm see 3 3 4 The menu is shown in 4 6 The commands Open Save Save As act like all traditional file management com mands the only peculiarity is that file names are filtered through the extension xcm and that the software keeps a record of the last folder used for storing 46 method files so that it normally shows this folder when prompts the user for a method file selection y I ClworSquare license All FEATURES File Method Schedule View Options Action Tools
111. med Area The window is called Navigator since it can be used not only for viewing the 88 location of the zoom area but also for moving or navigating 1t across the whole plot area The navigation can be accomplished in two different ways e Operating on the Navigator Panel e Operating on the scroll bars of the main Map Plot 7 3 2 1 Moving the zoom area with the Navigator Panel The user can move the current zoomed area operating on its representation inside the Navigator Panel in two ways e by clicking on the center of the new position in the Navigator Panel as soon as the mouse button is released the rectangle representing the zoom area will be moved and drawn around this new position maintaining its previous sizes e by dragging the rectangle representing the zoom area to the new desired position 7 3 2 2 Moving the zoom area with the Map Plot sliders The zoomed area may also be moved using the pair of sliders placed on the upper side and on the right side on the main Map Plot The sliders show the current position and sizes of the zoom area with respect to the overall Map Plot view The fraction of the sizes of the slider with respect to the corresponding sizes of the Map Plot represents the fraction of the horizontal zoom area with respect to the overall chromatogram axis When the user moves a slider the rectangle on the Navigator Panel is updated as well A special small button placed in the upper right
112. minates the installation procedure If you want to launch immediately the program tick the appropriate check box and then the Finish button 2 2 Uninstalling the software There is no specific uninstall procedure of the software just use the standard Windows uninstall facility as follows e From the Windows Start menu choose Settings 21 e then click Control Panel e double click the Add Remove Programs icon e select ChromSquare e choose Remove The procedure may slightly vary according to the different Windows versions and languages 2 3 Starting ChromSquare software To execute Chrom 1 use the desktop icon or the corresponding item of the M Start menu see Figure 2 1 Ghromsgus Figure 2 1 The Chromsquare desktop icon A splash screen containing information about the software and its producer is shown during the few seconds required to load the application software The splash screen is shown in the Figure 2 2 22 ChrolnSpuere A tool for multidimensionalichromatography Produced by Chromaleont s r I via Industriale 143 Messina Italy 1 98123 tel 39 839 2620612 fax 39 090 358220 www chromaleont it chromaleont chromaleont it Figure 2 2 The Chromsquare initial splash screen 2 4 Registering the License 2 4 1 License policy Chrom 4 software always requires a license this may be a regular license or a demo license e regular licenses are based on an hardware device Blu
113. mmand Prompt window that appears and disappears in few seconds except in the case that other sample files are found in this case the Command Prompt window will ask if you want to overwrite existing sample files even in this case answer Yes At the end a message will inform the user about the folder used for the sample files installation e The second optional installation concerns the manual files manual files are specific for the license purchased Answer Yes if this is the first installation or an upgrade of the software manual files may change between different releases of the software answer No only if you are re installing the software starting from the same media in case of doubt answer Yes The installation of manual files is accomplished through a Command Prompt window that appears and disappears in few seconds except in the case that other manual files are found in this case the Command Prompt window will ask if you want to overwrite existing manual files even in this case answer Yes At the end a message will inform the user about the folder used for the manual files installation Note the windows about the optional installations are shown only if the installation package contains the samples or manual files if you are installing an upgrade of the software it is possible that these files are not present and the corresponding windows are not shown 11 The last window Completing the ChromSquare Setup Wizard ter
114. n the mouse passes over a Blob ID belonging to some Group the list of the Groups is shown as a tooltip 145 ChromSquare License GCxGC MS File Method Schedule View Options Action Tools method filename nd Settings Aqualis Documenti ChromSquare My Samples 2010 TestCalib 2010 15 Groups ProvaCalibrazione Group 2 xcm min area 10 0 S amp 4 peak shift 96 of Mod Time 0 0 lAc Tridecanoico Chromatogram Figure 9 24 Method Panel with one Group 5 It is possible to change the name of the Group assigning a more significant term by directly writing inside the cell table 6 The operation can be repeated for more Groups A more complete example is shown in Figure 9 25 where 3 groups have been defined and renamed 7 At any time the user can delete one or more groups through the button delete subsequent operations of adding and deleting groups will destroy the regular sequence of ID numbers this however is not a problem since IDs are just identifiers and not counters 8 At the end the user must save the Method before continuing with the cali bration and other operations Important Note the definition of Named Groups is an activity that must be performed after the Blob recognition the two activities cannot be mixed in order to avoid complicated side effects For this reason the Blob ID structure should not be changed once one or more Groups have been defined If the user tries to dele
115. nalysis result the user may e View the integration results that is the representation of the integrated Blobs on the 2D Map Plot in order to improve this visualization it is then possible to zoom on particular zones of the plot see Section 7 2 3 to change the details of the Blob representation through the various items of the Menu Options see Section 4 5 to change the Color Map as described in the Section 7 3 1 All operations described above are managed in real time that is the map is automatically updated and re drawn without performing any additional operation no further integration is required e View the integration results as numerical values in the Data Analysis win dow which is fully described in Section 7 6 e View the linear chromatogram profile in correspondence of the single Blobs as described in Section 7 4 8 2 5 Change the Integration Parameters If the results checking described above is not satisfactory the user can change some integration parameter and repeat the steps described in Sections 8 2 1 8 2 2 8 2 3 8 2 4 The integration parameters that can be changed are the Method parameters de scribed at 8 2 1 112 i ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS Bsec xrs File Method Schedule View Options Action Tools Zoom Define Blob Define ROI Cancel Chromatogram Graphical param Chromatogram Data analysis p reset to saved value
116. name Schedule Save Schedule Open Schedule Se New Schedule Ss zi Select Method Panel Figure 3 1 Chrom 2 Main Window 33 Name Function Title Bar The Title Bar displays the program name the license type and the name of the chromatogram file currently loaded Menu Bar The Menu Bar represents the main access to all com mands and options of the software it contains all the operations for loading and saving files and workspaces editing reports viewing and saving images switch on and off various graphical features and interpolation op tions Tool Bar Frequently used commands are assigned to the buttons of the Tool Bar making their use easier For a detailed explanation of the Tool bar see the chapters describing the corresponding commands Side Bar Contains icons for switching among the main Panels Schedule Panel This window is used to display the Schedule Panel for quantitative analysis Chromatogram This window is used to display the Chromatogram Panel Panel which represents the raw chromatogram as entire or as modulation chromatogram of a single fraction In this window it is also possible to display the integration results including retention times compound names and quantitative results subsequent to data analysis Method Panel This window is used to display the Method Panel which lets to assign the method parameters 3 3 File Management 3 3 1 Raw Data Fil
117. ne 83 Insert blob name Start TER 38 053 end TER 41 002 Laurate Figure 7 3 Insert Blob Name window The new Blob Id with all its data is automatically inserted in the current Method The user can at once verify the Blob name and Id by switching the view to the Method Panel Figure 7 4 shows the Method Panel after that the new Blob ID has been defined X ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS 8sec xrs ax File Method Schedule View Options Action Tools Integration Shimadzu spectra search method filename C Documents and Settings Aqualis Documenti ChromSquare My Samples 2010 Test Cases GCMS quantitate xcm Ej min area 1 088 A noise 10000 0 Schedule peak shift of Mod Time 6 0 blob recognition D name Background Subtraction 1 Laurate v gt Chromatogram delete agonal egorran _ 27 262890 34 901460 Figure 7 4 The Method Panel with the new Blob ID The geometry is also stored in the Method but it cannot be seen in the Method Panel due to the complexity arising from the polygonal model 84 7 2 4 3 Define ROI The menu item command is used to manually define the selected region as a Region of Interest ROI The ROI description is maintained inside the Method and is displayed in the Method Panel After this operation is possible to integrate only the Region of Interest see Chapter 9 ins
118. ne Naringin 3000000 0 2500000 0 2000000 0 1500000 0 1000000 0 500000 0 0 0 10 Id Std Conc Hesperidin 414332 916905 10 000000 Hesperidin 1860323 692368 50 000000 Hesperidin 3211522 980831 100 000000 Linear Regression calculated with 3 points Regression Line rea m Conc b Coeff m Slope 30913 664 Coeff b Intercept 179997 782 RF 3 234815514e 05 Linear Correlation Coeff R 0 996505 R squared 0 993023 Figure 9 19 The Calibration Graph window This time in the Schedule file will be also stored the coefficients of all the Re gression lines The Schedule file is now a Calibration file that may be used for a Quantitative Analysis of an unknown sample 137 9 2 Internal Standard Calibration Calibration with Internal Standards is very similar to the one described for the External Standard in the Section 9 1 In this Section only the steps that differ from the External Standard will be dis cussed for all other steps it will only be reported a reference to the corresponding Section 9 2 1 Set up the Schedule See Section 9 1 1 9 2 2 Set up the Method See Section 9 1 1 9 2 3 Load a chromatogram See Section 9 1 3 9 2 4 Integrate the chromatogram See Section 9 1 4 9 2 5 Identify the Blobs See also Section 9 1 5 Once the Blobs have been recognized they are enumerated in the Blob table of the Method Panel
119. nterpreted by the software according to the units minutes or seconds shown in the combo box on the right if the units are changed after that the value has been entered the value will be automatically converted to the new units for this reason the user should always choose the correct units before entering the Modulation Time value e Implicitly by assigning to the data file a filename which contains the mod ulation time according to the following rules The last part of the filename before the file extension should consist by a number followed by a unit unit can be min for minutes or sec for seconds For example 2min or 120sec 4 1 2 Close Datafile This command closes the current data file clearing all references to the chro matogram currently loaded 4 1 3 Assemble Datafile This command assembles more chromatograms into a unique data file it is used when the modulations had been saved onto different files a single file for each modulation with files numbered as a sequence See Section 10 7 for details 4 1 4 Save Datafile This command allows to save various information about the chromatogram being analyzed into a special Chrom 1 file called Result File extension xrs See Section 3 3 2 for details about this file 43 4 1 5 Export as CDF file This command allows to export the current chromatogram in NetCDF format according to the AIA ANDI standards The exported file will have
120. o be used to save an existing Method file with a different name 4 3 Menu Schedule The Menu Schedule contains the main operations concerning the management of the Schedules See Chapter Section 5 for the description of the Schedule Panel and Section 3 3 3 for a description of Schedule Files The menu is shown in 4 7 wl ChromSquare License ALL FEATURES File Method Schedule View Options Action Tools New 1 d current method El Add chromatograms Std Smp Figure 4 7 The Menu Schedule Open schedule Save schedule as 4 3 1 New The command will set up a new Schedule that is an empty one containing no chromatograms that will become the Current Schedule 48 4 3 2 Add chromatograms The command opens a File Selection Dialog Box which allows the user to select one or more chromatograms to be added to the current Schedule Pressing the CTRL key during the selection will allow to select more than one chromatogram The command performs the same action of the Plus Sign icon of the Schedule Toolbar see Section 5 1 As already pointed out in that Section the command does not actually load the chromatogram s but just creates the link s for the subsequent analysis 4 3 3 Open Schedule The command opens a File Selection Dialog Box for the selection of an existing Schedule File w18 see 3 3 3 that becomes the Current Schedule The file contents is copied in the Schedule Table see 5 2 that can t
121. of the selected Blob 100 Data Analysis page The Data Analysis Context Menu 101 10 7 20 7 21 7 22 7 23 7 24 7 25 7 26 8 1 8 2 8 3 8 4 9 1 9 2 9 3 9 4 9 5 9 6 9 7 9 8 9 9 9 10 9 11 9 12 9 13 9 14 9 15 9 16 9 17 Show Spectrum window ooa a 102 A full zoomed Blob ea rr a a 103 The window displaying Blob Info 104 The Assign Compound dialog box ooa o 105 Method Panel showing one Group aoaaa a 106 Grouping page The results ooa aaa o 106 Chromatogram Panel The Status Bar 107 The Method parameters ooo a 110 Qualitative Analysis Whole Chromatogram integrated 111 Qualitative Analysis Define ROI 113 Qualitative Analysis ROI integrated 113 A list of files properly ordered for quantitative analysis 120 Initial Window for Quantitative Analysis 121 Schedule Panel with some Chromatograms added 121 The Schedule Menu with Add chromatograms command selected 122 Switch to the Method Panel rea rada ae ee 123 The Method Menu with Open Method command selected 124 The Method Panel showing a new empty Method 125 The Method Panel with some parameters entered 125 The icon for loading the selected chromatogram 126 The Menu Action Integrate Whole ChromatograM 127 The window showing the c
122. olbar It contains a sequence of buttons most buttons are shortcuts for commands that can be also executed through menu commands the last button perform a specific operation The buttons are 1 gear Integrates the whole chromatogram It corresponds to the menu Action command Integrate Whole Chromatogram of Section 4 6 2 2 gear with red rectangle Integrates only the defined Regions of Inter est It corresponds to the menu Action command Integrate ROI of Section 4 6 1 3 gear with a red delete cross Removes integration It corresponds to the menu Action command Remove integration of Section 4 6 4 4 wrap S red sign on a blue background Correct wrap around It cor responds to the menu Action command Correct wrap around of Section 4 6 13 5 book Search selected Blob in spectra library It corresponds to the menu Action command Search selected Blob of Section 4 6 11 6 book collection Search all Blobs in spectra library It corresponds to the menu Action command Search all Blobs of Section 4 6 12 7 check button Polygon mode This is a switch which controls the mode of defining the Blob regions on the 2D Plot if unchecked the normal mode is active rectangular selection see Section 7 2 3 and 7 2 4 2 if checked the Polygon mode is active see Section 10 3 99 7 6 3 The Data Analysis Context Menu To show the Data Analysis context menu the user must e select a Blob by clicking with the left mou
123. on Integrate all chromatograms Create Calibration Show Calibration Create Calibration Group Show Calibration Group iS Schedule Generate chromatogram report Search selected Blob Search all Blobs Show selected blob Graphical param Chromatog Correct Wrap Around aa A Bc gt lt L_JPolygon mode ec Index ID Name 1638520 unknown 1638523 unknown 1704046 unknown 1704058 unknown 1769583 unknown 1769591 unknown sarenan Figure 9 28 Group Calibration Command 2 An informative window with a summary of the calculations will be shown give Ok to close the window A sample of the informative window is shown in Figure 9 29 3 The software will display the calibration results opening a new window named Calibration Group Data with two pages respectively named Calculation and Graphs The Calculation page reports the calculation details of the Calibration A sample of the Calculation page is shown in Figure 9 30 The Graphs page lets the user to view the calibration curve for each Group The list in the upper left corner of the windows reports all the Groups of the 150 Warning Calibration of Groups ok Calibration Standards 3 Groups 3 Standard n 1 File 6 antiox 50 ppm2 270 xrs CONCENTRATIONS 50 000 50 000 50 000 TOTAL GROUP AREAS 8573995 652 8573995 652 8573995 652 Standard n 2 File 6 antiox 100
124. on which may have occurred in this publication will be corrected as soon as possible but not necessary immediately upon detection Chromaleont makes no claims or warranty concerning the influence of the appli cation of this publication on the operation of the machine Note that Chromaleont does not have any obligation concerning the effects result ing from the application of the contents of this manual 14 1 2 2 Warranty Chromaleont provides for this product the warranty described in the following paragraphs 1 2 2 1 Period Please contact Chromaleont at chromaleont chromaleont it for information about the period of this warranty 1 2 2 2 Description If a product part failure occurs for reasons attributable to Chromaleont during the warranty period Chromaleont will repair or replace the product part free of charge However in the case of products which are usually available on the market only for a short time such as personal computers and their peripherals parts Chromaleont may not be able to provide identical replacement products 1 2 2 3 Exceptions Failures caused by the following are excluded from the warranty even if they occur during the warranty period 1 Improper product handling 2 Repairs or modifications performed by parties other than Chromaleont or Chromaleont designated companies 3 Product use in combination with hardware or software other than that des ignated by Chromaleont 4 Computer vir
125. onding to the Unknowns will be coloured in yellow 133 Pl ChromSquare License GCxGC MS Current file HC2 6sec xrs File Method Schedule view Options Action Tools a t y C fed current method Hydro xcm Filename Std Smp Hydro1 HydroZ Hydro3 HC2 6sec xrs A HC3 6sec xrs Schedule Chromatogram gt Method Figure 9 17 Schedule panel while inserting a concentration value 9 1 9 Save the Schedule At this point you can save the Schedule through the menu command Schedule Save schedule as Choose the same directory of the other files and a proper name for the Schedule Schedule files have a w1s extension 9 1 10 Create the Calibration Now you have all the elements for making the Calibration e the concentration values of all Blobs of all Standards assigned by the user at 9 1 8 are stored in the current Schedule e the area values of all Blobs have been saved in the xrs files corresponding to each Standard generated at 9 1 7 Just select the menu item Action Create Calibration 134 The command performs the following operations e opens sequentially all the xrs files and loads into memory the area data this is a fast operation since the xrs files are relatively small files already containing all essential data e make the calculation of the Regression lines for each Blob correlating the areas as linear functions of concentrations e shows a fina
126. op 97 end 102 area 332753 750 height 41265 750 Figure 7 22 The window displaying Blob Info 7 6 3 6 Select Compound The command allows the user to select a different compound among those re turned by the similarity search performed by the external software component see Section 8 3 2 The command is active only if the automatic Blob Search has already been per formed on the selected Blob through the context menu command Search Blob of Section 7 6 3 1 or the main menu command Search All Blobs of Section 4 6 12 The command displays a dialog window named Assign compound for Blob showing all compounds found by the similarity search The software by default identifies the Blob using the compound with the highest similarity index the first of the list the user can select a different compound and confirm his choice with the button 0k The choice is archived so that the assigned compound will be shown if this window will be opened at a later time in this case it will be however possible to change the former choice The dialog box is shown in Figure 7 23 7 6 3 7 Add Selected to Method Adds the currently selected and identified Blob to the Blob list used in the Method for quantitation The command does not work if the current Blob has been not identified 104 ChromSquare License ALL FEATURES Current file Unknown 8 sec xrs gt E
127. panels The upper rightpanel is a Plot of the regression line corresponding to the currently selected Blob of the List The central panel is a Table which describes the characteristics of the input data used to build the regression of the currently selected Blob of the list the Table contains a row for each Standard chromatogram columns represent 1 The Blob name this is equal for all Standards 2 the Id of the Standard that is a counter 3 the Area of the Standard this is the result of the Integration 4 the Concentration of the Standard this is the value assigned by the user in the Schedule Table see the Section 9 1 8 The lower panel shows a Report of the calculations regarding the cur rently selected Blob of the List the Report includes The coefficients of the regression line the Response Factor x the Linear Correlation Coefficient R and R squared e The Calculation page contains a detailed report of the various calculations and results of the calibration in numerical form In the Graph Page select the Blobs one at a time in the upper left list and check the Regression Line and the numerical coefficients in the Report Panel 9 1 12 Save the Schedule again Finally save again the current Schedule through the menu command Schedule Save schedule as choosing the same file name used before see Section 9 1 9 136 LL Calibration Data Calculation Graph List of Blobs regression li
128. perations are e Integration of a group of chromatograms using the same method this operation is normally finalized to a calibration but not necessarily this operation is activated by the command Integrate All Chromatograms see XXX e Calibration Calibration is performed using the chromatograms of the schedule defined as Standards A chromatogram is treated as a Standard if concentrations have been assigned for all Blobs defined in the method 36 3 3 4 Chrome Method Files Chrom Method Files are XML files which have xcm extension XML ChromSquare Method Method Files are used for storing Method data see 3 1 8 3 3 5 Opening Files When the user executes a command for opening a file using the menu File Open command or the View icon of the Schedule Tool bar the software executes some controls before actually opening the specified files 1 if the user selects a Raw Data file and there is no corresponding Result xrs file in the same folder the software opens it and generates the cor responding Result file 2 if the user selects a Raw Data file and there is a corresponding Result xrs file in the same folder the software displays a Dialog Box which informs the user that opening the Raw Data will destroy the existing Result file 3 if the user selects a Result xrs file the software loads from this file all available information then searches the Raw Data specified
129. ppm2 270 xrs CONCENTRATIONS 100 000 100 000 100 000 TOTAL GROUP AREAS 12727590 572 12727590 572 12727590 572 Standard n 3 File 6 antiox 25 ppm2 270 Icd CONCENTRATIONS 25 000 25 000 25 000 TOTAL GROUP AREAS 3954704 030 3954704 030 3954704 030 REGRESSION LINES FOR EACH GROUP Blob 1 Area 111986 117 Conc 1882906 570 Blob 2 Area 111986 117 Conc 1882906 570 Blob 3 rea 111986 117 Conc 1882906 570 Figure 9 29 Group Calibration Summary Method by double clicking on one of the groups of the list the corresponding graph is shown in the right side whereas the details of the calibration line and other statistical parameters are shown in the lower table A sample of the Graphs page is shown in Figure 9 31 The command Action Show Calibration Group allows the user to view the former windows without perform again all calculations 151 2 Calibration Group Data Calculation Graph Calibration Standards 3 Groups 3 Standard n 1 File 6 antiox 50 ppm2 270 xrs CONCENTRATIONS 50 000 50 000 50 000 TOTAL GROUP AREAS 8573995 652 8573995 652 8573995 652 Standard n 2 File 6 antiox 100 ppm2 270 xrs CONCENTRATIONS Slo Intercept OR l IN 111986 117 1882906 570 0 000008930 0 975578446 111986 117 1882906 570 0 111986 117 1882906 570 Figure 9 30 Group Calibration Data 152 Calibration Group Data calculation regression line Group GA EG 7 7 7 Group Tyr
130. r the files anyway if names are scrambled it will be more difficult to place them in the right order 119 File Back 2 D Search Folders y r EH B C ChromSquarelsamples HC1 6sec gcd HC2 6sec gcd File and Folder Tasks A GC Data File A GC Data File mm 1 791 KB mm 1 931 KB GJ Make a new folder A Publish this folder to the HC3 6sec gcd Web GC Data File 2 Share this folder 2 156 KB Edit View Favorites Tools Help Address Other Places E ChromSquare My Documents iw My Computer Figure 9 1 A list of files properly ordered for quantitative analysis The operation is shown in Figure 9 3 You must add one Standard chromatogram at a time using an increasing order of concentrations This is not mandatory but it will help to write down the corresponding concentrations Alternatively you can add chromatograms to the Schedule by selecting the Schedule Add Chromatograms command from the Menu Bar Files can be removed from the current Schedule using the Minus Button this is the icon with the minus sign at the left side of the Schedule Panel Tool bar Note that adding a chromatogram to the Schedule does not mean loading it adding just creates a reference to the file the very loading will be accomplished later see Section 9 1 3 Like removing a chromatogram from the Schedule does not mean to delete it but just the link Use the Arrows to change the order of files in th
131. r the integration e if quantitative analysis is performed the Results File will also include all results of the quantitation namely the concentrations of the identified Blobs e the Result File also includes visual information like axis scales and color scale so that when the file will be reopened it will reproduce the exact state of the analysis that there was at the instant of last saving The file is overwritten each time a relevant operation is performed on the chro matogram file overwriting may be also forced by the user through the Save Data File command see xxx in order to reflect minor changes 3 3 3 Chrom Schedule Files Chrom 1vere Schedule Files are XML files which have w1s extension Worklist Set A schedule called also worklist is a set of one or more chromatographic files The user defines a schedule by adding files to the table of the Schedule Panel files may be also removed or moved up and down across the table Adding a file to the schedule is not the same thing that opening it opening a file means loading into memory the full raw data allowing the visualization of the chromatogram adding a file to the schedule just means to add an item to a list a special icon can be used to load and view each single chromatogram of the list Setting up a schedule give the user the possibility to plan operations to be ac complished on the whole set of chromatograms with just one command These o
132. rofile Sample file Data027 4sec qgd Figure 10 9 The New Profile window 10 5 Configuration 10 5 1 Description This section describes some technical features of Chrom 1 7 software Its con tent is oriented to software specialists System Administrators IT people How ever some information can be useful also for common users 10 5 2 The file Options xml This file maintains some software options between different executions for ex ample when the user changes one or more visualization options see 4 5 the information is stored in this file The file is a text file coded according to the rules of the XML language 170 A prototype of the options xml is generated in the installation folder by the instal lation procedure At first execution the software creates a copy of the file in the Windows temporary folder all modifications of the original options are stored on the copy of the temporary folder Most options are managed directly by the software so that the user does not normally modify the file wrong changes could make the file unreadable Manual modifications are required for less common options 10 6 Ion selection 10 6 1 Description If the chromatogram under analysis is generated by a mass spectrometer a selec tion of fragments can be used for quantitation instead of the whole intensity This procedure is called Jon Selection Note When data file is generated using GCMSsolution profile SPC
133. rt Search selected Blob Search all Blobs Show selected blob Correct Wrap Around Figure 4 10 The Menu Action 4 6 1 Integrate ROI The command performs the integration on a Region of Interest ROT that is on only a part of the chromatogram plot see Section 3 1 3 for the exact meaning of integration ROIs are useful to speed up the calculation especially in the case of large chromatograms The ROI is stored within the Method see Section 6 1 8 and can be graphically defined through the mouse and context menu command see Section 7 2 4 3 See the next Section 4 6 2 for details about the integration process and results 4 6 2 Integrate Whole Chromatogram The command performs the integration of the whole chromatogram plot see Sec tion 3 1 3 for the exact meaning of integration The integration can take some time during which a progress bar showing the advancement of the calculation may be displayed At the end of the integration the following changes will appear to the user 59 e In the Map Plot window all recognized Blobs are marked and tagged with various kinds of information according to the options set up through the Menu Options see 4 9 e The Table of the Results page in the lower window of the Chromatogram Panel will be filled with the integration results 4 6 3 Quantitate The command starts the execution of the Quantitative Analysis also called Quan titation See Chapter 9 for a detaile
134. rt the creation access and sharing of array oriented scientific data 2 ATA ANDI is a set of standards developed in 1990 by manufacturers of analytical instru mentation through their trade organization the Analytical Instrument Association ATA ATA sponsored the development of a series of standards to interchange analytical data across vendor platforms These standards known as ANalytical Data Interchange ANDI standards are sup ported by many commercial software products NetCDF files are binary files that must read and written by means of specific software libraries they cannot be managed by plain text editors 44 My Documents o vi amp 1535 E My Music Width re My Pictures My Recent Documents Height Font SansSerif plain 8 Leia My Computer My Network Places Files of type all Files Figure 4 3 The Save Map as Image Dialog Box e The text fields Width and Height that can be used to specify the size of the image in dpi e the button with the three dots which in turn opens a second dialog box for choosing the appropriate font and size Figure 4 4 Arial Arial Black Comic Sans MS Courier New Dialog DialogInput Estrangelo Edessa Franklin Gothic Medium Gautami Georgia Impact Latha Lucida Console ABCDEFGHabcdefgh01234 Figure 4 4 The Select Font Dialog Box 45 4 1 8 Data info This command opens a Dialog Box which shows some infor
135. s lod 278 DP 54 TtR 41 112 2tR 2 717 1tR 41 067 Int 13195 91 Figure 8 3 Qualitative Analysis Define ROI L ChromSquare License GCxGC MS Current file Shimadzu GCxGC MS Bsec xrs File Method Schedule View Options Action Tools Schedule Chromatogram Graphical param Chromatogram Data analysis p reset to saved values Method jod 254 DP 81 TtR 37 935 2tR 4 075 1tR 37 867 Int 21580 70 Figure 8 4 Qualitative Analysis ROI integrated 113 8 3 Identify the Blobs The identification of the Blobs that is the operation of assigning a Blob Id to the Blobs can be performed in two ways e Manual identification e Automatic identification 8 3 1 Manual Identification of the Blobs Manual identification is obviously the only identification possible when the current software license does not provide the link with the software which performs the identification see next section for details about this Manual identification may be also recommended when the user is interested to few well known and defined Blobs which are recognized at a first sight and prefers to avoid the automatic process that in some case may be time consuming Manual identification must be made separately for each Blob to be identified The Manual identification is made up by two steps e In the first step the user defines the region which includes the Blob this may be a rectangular region or an arbitrar
136. s the files alphabetically according to their names The names should therefore reflect the logical order of the standards concentra tions and replication numbers e Avoid spaces in filenames use underscore signs _ or minus signs instead It is difficult to distinguish one space from more spaces if the characters are small besides spaces alter the alphabetic order e When using numbers to identify concentrations try to always maintain the same number of digits As an example if you use 025 050 100 the standards will be always ordered correctly if you omit the leading zeroes the order may be wrong In the directory shown in Figure 9 1 the files have been renamed according to the aforementioned rules 9 1 1 2 Define a New Schedule Once your file list has been created open Chrom 2 software using the appro priate license and switch to the Schedule Panel using the schedule icon Schedule at the top of the left sidebar See Figure 9 2 9 1 1 3 Add files to the Schedule Use the Plus Button that is is the icon with the plus sign at the left side of the Schedule Panel Tool bar to add chromatograms to the current Schedule You must insert in the Schedule all chromatograms that should enter in the Cali bration as Standards You can also add one or more chromatograms that should be treated as Unknown Samples the latter ones however may be added in a second time The software actually accepts any kind of name fo
137. se button on the corresponding row the selected row will be shown with a blue background e click with the right mouse button on the selected row Note that the Blob corresponding to the selected row will be also shown in the 2D Map Plot with enhanced boundaries like in Figure 7 18 28 733 30 529 32 325 Data analysis lu gt E Polygon mode ID Name TR 2 1116659119 Neral a l 1 648969502 Geranial Figure 7 18 Enhanced representation of the selected Blob The context menu is represented in Figure 7 19 The various items are described in the following sections 100 Name Neral Geran Search Blob Delete Blob Show Spectrum Zoom Blob Info Blob Select Compound Add Selected to Method Add All to Method m Figure 7 19 Data Analysis page The Data Analysis Context Menu 7 6 3 1 Search Blob This menu item is implemented for the license types involving the automatic Blob Search see Section 8 3 2 The command executes a search of the currently selected Blob using the external software component If the search is successful a Name and a Blob ID are assigned to the Blob and immediately shown both on the 2D Map Plot and in the Data Analysis table 7 6 3 2 Delete Blob The command deletes the currently selected Blob from the list of the integrated Blobs The 2D Map Plot and the Data Analysis table are updated as well 7 6 3 3 Show Spectrum This menu item is implemented for the license types
138. t an area e give the menu command View 3D View e optionally use the buttons of the 3D tool bar to modify the representation and save it as an image The process to create the 3D scene can take several seconds according to the performance of the computer On a last generation PC it should take a few seconds The initial view is seen from the top and the orientation is the same as the view selected in the map plot of chromatogram view as shown in 10 1 156 Orbit left click drag Move right click drag Zoom mid click drag Figure 10 1 3D View Window Initial View 157 10 1 2 Changing the scene The possible behaviours of the scene are obtained clicking and dragging the mouse in the scene e Orbit rotates the plot according to mouse movement is obtained by left clicking the mouse button and moving it e Move pans the plot is obtained by right clicking the mouse and moving it e Zoom increases or decreases the observer s distance is made by clicking the central mouse button and moving it for two button mouses the central button is generally simulated by clicking simultaneously the left and the right ones in wheel mouses third button event is generated clicking the wheel For most of the wheel mouses zoom can also be obtained rotating the mouse wheel The Status Bar at the bottom of the window reports some short hints about how to change the scene with the mouse 10 1 3 The 3D View Tool Bar The 3D Vi
139. t flinch oe ee A De ade he EMC ih rahe teehee E 177 Tht Description ea wh Be A BD HB Oe UE g 177 List of Figures 2 1 22 2 3 2 4 3 1 3 2 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 4 10 4 11 4 12 4 13 4 14 4 15 The Chromsquare desktop icon ooo a 22 The Chromsquare initial splash screen o o 23 Enter License Code window for Normal License 25 Enter License Code window for Demo License 26 Chrome gt Main Window wa edie Oe gy aw Ae ye oe Be 33 The Java Console es fn asc db oc hb ee le AA 39 The WSUS File a E A cao BE a ge BEC 40 File description and features of the Data file selected 42 The Save Map as Image Dialog Box o o 45 The Select Font Dialog Box o 45 The Current Data Info Dialog Box o 46 The Menu Method vara bos a Sed ae Bee a RES 47 The Menu Schedule aaa aa a a 48 The Mena Viewers iea sei e a Ses i i A AA 50 The Menu Options a tos gue eaka Sek A ae a ae E a 51 The Menu Action a a a 55 The Report Template Selector Dialog Box 58 The Chromatogram Report Preview 59 The Menu Tagle oat cee dio deca ri db eee deta Peed ii 61 The Report Template Editor Window 61 The Menu Help wrt a ele el Ge a a BE Eee Oe 62 4 16 5 1 5 2 5 3 5 4 5 5 6 1 6 2 7 1 7 2 7 3 7 4 7 9 7 6 T T 7 8 7 9 7 10 7 11 7 12 7 13 7 14 7 15 7 1
140. te C7 Carbon 7 Heptane etc 2 RT is the retention time of the top of the milestone Blob 3 LRI is the retention index conventionally set to the number of carbon atoms multiplied by 100 The table is fully editable so that the user can enter a new line using the button on the bottom of the table delete a line using button and fill in the values Once the table is filled with all values when an integration is performed the retention times for the Blob identified in the chromatogram are rescaled to the times included in the LRI table and the LRI is computed for each Blob The user can also prepare a chromatogram containing the milestone Blobs for example an appropriate mix of alkanes and integrate it Once this operation is done having the datafile open 1t is possible to import those Blobs in the table by pressing the button Use current Chromatogram to define LRIs The table is automatically filled in and the user must only fill the LRI for each entry At this point the table is anyway completely editable so it is possible to change the names or make corrections Once the method is saved all Blobs which do not have an assigned LRI are deleted from the table since they do not contribute to the computation All the Blobs that are outside the RT interval between the first and the last entry of the table are assigned LRI equal to 0 TT Chapter 7 Chromatogram Panel 7 1 Panel Contents The Chromato
141. te a Blob ID when Groups are present a message will be displayed warning the user that the action will reset the Group structure 146 ChromSquare License GCxGC MS File Method Schedule View Options Action Tools method filename nd Settings Aqualis Documenti ChromSquare My Samples 2010 TestCalib 2010 15 Groups ProvaCalbrazione Group 2x0m m n area 10 0 Ss amp A peak shift of Mod Time 0 0 Schedule blob recognition 1D name Int Std ID 1 JAC Butierico 21 A 2 JAc Caproico 121 E Ac Caprilico 21 __ Ac Coprico 21 5 Ac Undecanoico 21 6 JAc Laurico 21 y iZ lAc Tridecanolco 21 LS Chromatogram delete groups D Name Blobs 1 Group Capr 234 2 Group Palm 1213 3 Group Lino 19 20 LY g 6 4135 62 9 Figure 9 25 Method Panel with more Groups 9 4 3 Integrate All Chromatograms After that the Method has been saved it is possible to integrate the whole set of chromatograms included in the schedule through the menu command Action Integrate All Chromatograms Once the integration has been completed you can view the traditional Results Table clicking on the tab Data Analysis of the lower pane A sample of the Data Analysis table is shown in Figure 9 26 If the current Method defines Named Groups a new tab page named Grouping will appear in the lower pane at the right of Data Analysis By clicking on it a new table with the results of the integration of Groups will
142. tead of the full chromatogram 7 2 4 4 Define noise min area The menu item command is used to manually define the selected region as a background region for the purpose of defining the Method parameters noise and minimum area see Section 6 1 3 These parameters are initially defined by default as 1 meaning that the software automatically calculates an optimal estimate over the whole chromatogram and may be changed by the user in order to obtain a better integration in many cases however the user does not know which values to assign The menu item Define noise min Area can be used for this purpose The user should select a rectangular region that can be considered as background that is a region which does not contain significant Blobs and then give this command The software calculates an estimate of ASTM noise and minimum area throughout the selected area and automatically assign them to the current method The user can then watch the values by switching to the Method Panel and finally perform a new integration to apply these settings The operation may be obviously repeated more times on different regions until a satisfying pair of value has been obtained 7 2 45 Cancel The menu item cancel the context menu without performing any operation The minimum area parameter is very important values too large have the effect of hiding significant Blobs whereas values too small may cause the recognition of thousands of smal
143. the retention time of the chromatographic point corresponding to the Top measured along the modulation i e the second dimension the Blob Index this is an integer number which unequivocally identify the Blob through a special combination of the Top Modulation Index and Top Time Index the Index is useful in the various calculations since it avoids the uncertainty arising from the approximations of decimal numbers 29 e The description of the limits of the Blob in the two directions the First Modulation and the Last Modulation two integer numbers which give the extent of the Blob along the First Dimension the Top Modulation Start and the Top Modulation End two integer numbers which give the extent of the Blob along the Second Dimension that is the first and last chromatographic points of the Blob measured along the modulation passing for the Top e the Area of the Blob that is the area of the corresponding polygon 3 1 5 2 Blobs analytical data Analytical data are computed and stored in the Blob data structure as the various calculation steps are executed e After the completion of the recognition of all Blobs Integration the software can compute the Area percent of any single Blob e During the identification process a Blob ID see next Section may be as signed to the Blobs some Blobs could be remain unidentified and classified as unknown To each identified Blobs an ID and a Name are assigned
144. this is equivalent to a time axis which measures the total time e Vertical Axis is a time axis which measures the modulation time that is the time elapsed from the beginning of each modulation e Colors are used to represent the value of the absorbance at a given point the relation between Colors and absorbance is ruled by a Color map that can be configured by the user The Chromatographic Map is defined on a rectangular mesh of discrete points If the points are too much separated this is frequent especially in zoomed visual ization the graphic result is a set colored rectangles resembling a pixel map too much enlarged For a better visualization it is possible to perform an interpola tion that calculates a color value at each pixel point of the screen and not only at the points of the mesh in this way the result is a smoothed continuous map of colors 3 1 3 Integration Integration is the calculation process which analyzes the Chromatogram in order to recognize and identify Peaks see 3 1 4 and Blobs see 3 1 5 e Recognize means to find the Blob contours on the map Recognition is geo metrical analysis of the chromatogram map that can be tuned on the basis of some parameters e Identify means to assign an identity that is a name to a recognized Blob Identification is a process that is generally carried on manually by the user which selects the Blobs with the mouse on the map and assign them a name GCMSSolut
145. tion prior to reading this Help material 1 3 2 2 Structure The Manual can be considered as consisting of four parts 1 the first part is a general introduction and includes the Chapters 1 3 2 the second part is an analytical description of all parts of the Graphical User Interface the Menu and the three Main Panels it includes Chapters from 4 to 7 3 the third part is a detailed description of the main routine operations namely Qualitative and Quantitative Analysis it includes Chapters 8 and 9 17 4 the fourth part is a description of various advanced operations 1t includes Chapter 10 1 3 2 3 How to use this Manual We recommend that the user carefully read the following key chapters e Chapter 3 Overview this gives an essential introduction to the main con cepts and terminology e Chapter 8 Qualitative Analysis this describes step by step the most com mon usage of the software e Chapter 9 Quantitative Analysis the same as before for users which will use the software for quantitation All others chapters may be used as reference that is they do not need to be read sequentially but more profitably consulted upon the frequent references from the aforementioned key Chapters 1 3 2 4 How to understand the Figures The Figures reproduce screen shots or parts of them taken during software op erations Figures have the main function of supporting the user to better interpret the contents of
146. tion progress window is shown in Figure 9 11 When the integration terminates the Map Plot is redrawn showing the boundaries of the Blobs and other information which are displayed directly on the Map Plot at each Blob position At this point before proceeding with the other steps the user may use the various zoom functions to get a more detailed representation of some regions of the Plot 127 Integration monitor Calculation Progress percent 90 Figure 9 11 The window showing the calculation progress and also use the various items of the Menu Options see Section 4 5 to change the details of the displayed information Zooming also improves the readability of the information displayed on the Map when too many Blobs have been recognized the various info texts overlap zooming has also the effect of spreading the Blobs allowing to distinguish the single texts See also the Section 8 2 4 for various hints about the visualization The Figure 9 12 shows a zoomed region of the chromatogram after the completion of the integration ChromSquare License GCxGC MS Current file HC2 6sec xrs File Method Schedule View Options Action Tools E Schedule Chromatogram Graphical param Chromatogram Data analysis gt 245 000 i Method i 417245 od 148 DP 658 TER 14 888 2tR 5 271 1tR 14 800 Int 69 65 Figure 9 12 Integrated chromatogram Zoomed view 128 9 1 5 Identify the Blobs
147. ument is to be considered a legal agreement between yourself denominated from now as user and Chromaleont The seal on this packet should not be broken before reading the license agreement reported below If you disagree with any terms of the agreement do not break the seal and promptly return this packet including packaging Upon return you will receive a full refund Grant of License uses permitted 1 Chromaleont grants to the user the right to copy the software program named as ChromSquare from now as Program enclosed in the CD ROM container onto a single computer and the right for the user and others to make use of that copy of the Program on a single computer 2 The user is allowed to relocate the Program permanently This as long as the user does not withhold any copies and that the receiver acknowledges all terms of this agreement Copyright Statement The owner of the Program is Chromaleont The Program is No Bluetooth device found A temporary license will be used Code License Type fiv vw Expiration Date YYYY MM DD Figure 2 4 Enter License Code window for Demo License 26 Chapter 3 Chrome Overview 3 1 General Terminology and Concepts This chapter introduces some terms thoroughly used by the software and in the manual text Even if these terms should be already familiar to the software users in the field of bi dimensional chromatography it is important to specify e
148. ure 10 2 Combo Box Shininess allows to change the shininess i e the intensity of the shining of the image selecting among a series of values low medium high and default 159 Orbit left click drag Move right click drag Zoom mid click drag Figure 10 2 3D View Window Surface colorization 160 10 2 Compare subtract tool 10 2 1 Introduction The Compare Subtract tool is available through the menu item Tools Compare Subtract Chromatograms it can be used even if no data is currently loaded This tool allows the user to load two different chromatograms to superimpose them and to subtract them Note that the tools handles the chromatograms as whole linear chromatograms i e without taking into account the modulations The chromatogram Plots therefore represent Total Retention Time along the horizontal axis and Intensity along the vertical axis 10 2 2 Definitions e Base Chromatogram is the first chromatogram it determines the time and modulation boundaries and the allowed sampling rate e Reference Chromatogram is the second one The only restraint is that the sampling rate must be the same of the first chromatogram if it is not the case no operation is obviously possible 161 1 Time shift 100 m 9 Intensity shift 10000 Y Y Execute subtraction 12 061 37628 2 Figure 10 3 Compare Subtract Tool 10 2 3 Description of the Compare Subtract window The Compare Subtract window shown
149. urrent method none Filename Std Smp HC1 6sec xrs HC2 6sec xrs A HC3 6sec xrs Schedule Figure 5 2 The Schedule Panel before loading a Method ChromSquare License GCxGC MS Current file HC3 6sec xrs File Method Schedule Yiew Options Action Tools t Q8 9 iH current method TestCalb xcm Filename Std Smp Peaks PeakB PeakC HC1 6sec xrs o o 10 0 10 0 HC2 6sec xrs o o 0 0 o o A HC3 6sec xrs o o o o 10 0 Schedule Chromatogram Figure 5 3 The Schedule Panel after loading a Method 68 i ChromSquare License GCxGC MS Current file HC3 6sec xrs File Method Schedule View Options Action Tools K AE f urent method TestCalib xem Filename Std Smp PeakA 0 0793 PeakB 0 0792 PeakC 0 0793 0 3966 0 3958 0 3966 HC1 6sec xrs HC2 6sec xrs A HC3 6sec xrs 1 983 Schedule Chromatogram Figure 5 4 The Schedule Panel after the assignment of the Concentrations e Copy Cell the value of the currently selected cell is copied to the clipboard e Paste Cell the value stored in the clipboard is copied to the currently selected cell e Fill Row Only null values the value stored in the clipboard is copied in the cells of the currently selected row which have values equals to zero e Fill Row A11 values the value stored in the clipboard is copied in all cells of the currently selected row
150. uses leading to device failures and damage to data and software including the product s basic software 5 Power failures leading to device failures and damage to data and software including the product s basic software 6 Turning OFF the product without following the proper shutdown procedure leading to device failures and damage to data and software including the product s basic software 15 7 Reasons unrelated to the product itself 8 Product use in harsh environments such as those subject to high tempera tures or humidity levels corrosive gases or strong vibrations 9 Fires earthquakes or any other act of nature contamination by radioactive or hazardous substances or any other force major event including wars riots and crimes 10 Product movement or transportation after installation 11 Consumable items Recording media such as floppy disks and CD ROMs are included 1 3 Introduction 1 3 1 Outline of the Software Congratulations on your purchase of the Chromaleont Chrom GCxGC MS amp LCxGC MS version for Shimadzu GCMSsolution and GCsolution Chrom workstation software is designed for visualizing processing and re porting on data obtained by Two Dimensional Chromatography A license and USB Bluetooth Key is required for execution A trial version with 60 days soft key license is also available at no charge The software and the documentation are released with different licens
151. w Zoom In this is equivalent to select a zoom region having half sizes and the same centre with respect to the current plot 81 6 246 El Schedule 3747 4372 4996 5 621 27 AT 30 574 33 971 37 368 Chromatogram Graphical param Chromatogram I Spectrum I Data analysis Je 2 80667e 07 07 NS S lt lt Method 0 00000 od 180 DP 133 TtR 28 112 2tR 6 692 1tR 28 000 Int 15797 06 Figure 7 2 Map Plot Zoomed Region The Zoom In operation may be performed more times obtaining always more deep enlargements 7 2 3 2 Zoom Out The Zoom Out operation can be performed in only one way e by using the menu command View Zoom Out this is equivalent to select a zoom region having double sizes and the same centre with respect to the current plot 7 2 33 Reset Zoom The Reset Zoom operation can be performed in the following way e by using the menu command View Reset Zoom this causes the redrawing of the whole chromatogram clearing all previous zoom operations 82 7 2 4 Map Plot Context Menu The context menu is displayed after that a rectangular region of the Map Plot has been selected This rectangular region represents the domain to which the operation of the selected menu item will apply The context menu shown in Figure 7 1 contains the menu items described in the following Sections 7 2 4 1 Zoom A Zoom In operation will be performed on the s
152. ways present and optional pages depending on the pur chased license Shimadzu Spectra Search page and NIST Spectra Search page 6 1 1 Integration page The Integration page is shown in Figure 6 1 Its components are described in the following paragraphs 6 1 2 Method Filename This is the full path name of the file corresponding to the Method shown If the Method has been loaded from a file this is the path name of the source file If the Method has been saved on a file this is the path name of the destination file The two files normally coincide This field cannot be edited directly in the table the field is read only the name of the Method file is assigned through the File Selection Dialog Box associated to the command Method Open and Method Save As 6 13 Method Parameters This section contains three parameters these parameters have initially values 1 0 meaning that the integration algorithm will assume default values The parameters are e Minimum Area minimum value of the area of the integrated Blobs Blobs with smaller area will be ignored e Noise value for filtering the noise level e Peak shift percent value per for the peak shift tolerance 6 1 4 Blobs ID Table Blob Recognition The Blobs ID Table contains a list of the Blobs IDs that will be used by the software for the Blobs recognition The Table contains 72 e a row for each Blob ID e the following five columns ID name Int Std ID Ion
153. with an established Method is a straightforward activity which involves just one click 109 8 2 1 Set up the Method Setting up the Method includes the following actions 1 Switch the main view to the Method Panel using the Method command in the Side Tool Bar Method Panel is thoroughly described in Chapter 6 2 Use an existing method loading it by a Method File either create a new Method from scratch e To open an existing Method use the menu command Method Open Sec tion 4 2 2 and select a xcm Method file through the File Selection Dialog Box e To create a new Method use the menu command Method New Section 4 2 1 3 Define the three Method parameters shown in the Figure 8 1 e Minimum Area the minimum value of the area of the Blobs that will be integrated Blobs with smaller values will be discarded e Noise value of the noise level for Blobs filtering e Peak shift value per cent for the peak shift tolerance In the case of a new Method the three fields are initially filled with the value 1 0 which means that a default value is assumed for each parameter In the case of an existing Method the values assigned in a previous work session are taken method filename C ChromSquare samples Collaudo Test 3 2 xcm min area 1 0e8 noise 10000 0 peak shift of Mod Time 6 0 Figure 8 1 The Method parameters 110 8 2 2 Integrate Whole Chromatogram To integrate the whole chromatogram e eit
154. xGC berg 008 0017 gcd Ei i LCxGC berg 008 0005 gcd ri LCxGC berg 008 0018 gcd GCsolution file Desktop mi LCxGC berg 008 0006 gcd y is LOxGC berg 008 0007 gcd No further info available go ei LCxGC berg 008 0008 gcd Documenti 53 LCxGC berg 008 0009 gcd LCXGC berg 008 0010 gcd i LCxGC berg 008 0011 gcd Risorse del LCxGC berg 008 0012 gcd computer f LCxGC berg 008 0013 gcd Nome file LCXGC berg 008 0001 gcd Risorse di rete Tipofile Files GCD QGD QLD LCD TXT CSV AIA ANDI CDF XML XRS 1st Mod Time Figure 10 11 The File selection Dialog box of the Assemble case 2 Select one of the files shown in the window and press Open 3 A new window Select files to assemble with two panes will be shown The left pane will contain the list of all the files having similar names and ending with different sequence numbers the right pane will contain the files that the user wants to assemble See Figure 10 12 4 Use the two central buttons marked with and lt to transfer all files between the panes or double click on a file name in order to transfer just one file See 10 13 173 Select files to assemble LCxGC berg 008 0001 gcd LCxGC berg 008 0002 gcd LCxGC berg 008 0003 gcd LCxGC berg 008 0004 gcd LCxGC berg 008 0005 gcd LCxGC berg 008 0006 gcd LCxGC berg 008 0007 gcd LCxGC berg 008 0008 gcd LCxGC berg 008 0009 gcd LCxGC berg 008 0010 gcd L
155. xactly their meaning since there are some subtle differences that must be cleared to avoid misunderstanding of some descriptions 3 1 1 Chromatogram With this term we mean the data coming from chromatographic instrumentation since data are normally recorded on files the term chromatogram is used both for data loaded in memory and for files containing these data more accurately one should speak of chromatographic data and chromatographic files for brevity often we simply speak of chromatograms Chromatogram is basically a sequence of data each datum being a couple of numeric values corresponding to time and absorbance In bi dimensional chromatography chromatograms are acquired through a cou ple of instruments the resulting data are normally recorded on a unique chro matogram The overall chromatogram is then split into many slices called mod ulations according to a parameter called modulation period in some case the original chromatogram corresponds to a set of files each file corresponding to a single modulation in these case the overall chromatogram is build by the software joining together the single modulations 27 In bi dimensional chromatography chromatograms are represented as bi dimensional Maps 3 1 2 Chromatographic Map A Chromatographic Map is a bi dimensional representation of a chromatogram according to the following criteria e Horizontal Axis is a counter which represents the modulation number
156. y polygonal region e In the second step a dialog box allows the user to write the name to be assigned to the Blob The procedure will be reported in details for the two cases 8 3 1 1 Rectangular Regions To perform the Manual identification of Blobs included in a rectangular region follow these steps e Define the Blob region according to the instructions reported in Section 7 2 2 and 7 2 4 e Enter the Blob name in the dialog box that will be displayed soon after as described in Section 7 2 4 2 If the selected region contains more than one Blob the same name will be assigned to all them 114 8 3 1 2 Polygonal Regions To perform the Manual identification of Blobs included in a polygonal region follow the procedure described in Section 10 3 If the selected region contains more than one Blob the same name will be assigned to all them 8 3 2 Automatic Identification of the Blobs The automatic identification of the Blobs can be performed in the cases in which the current license provides a link with an external software which can execute the task Presently the following licenses provide this feature e GCxGC MS amp LCxGC MS version for Shimadzu GCMSsolution and GC solution using Shimadzu Search engine e LCxLC version for Shimadzu LCMSsolution and LCsolution using Shimadzu Search engine e LCxLC version for Shimadzu LCsolution using Shimadzu Search engine e Text and NetCDF Version with NIST Spectra S

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