Home

C h r o m S t a r 6 M a n u a l

Contents

1. Clicking Retention Times annotates the retention times above the individual ei peaks When using a calculation file with entries in the peak table only the peaks of the peak table are labelled if Annotate Results is entered in the CHRST32 INI file in Report If all peaks are to be annotated the entry or no entry must be Annotate All Page 4 4 6 4 4 1 Reprocess Reconstruction To annotate Peak Names it is necessary to have defined them in the a calculation file which is specified in the data handling file used in the reintegration The peak names to be annotated are entered on page 2 peak table of the calculation file The peaks are defined according to the corresponding peak windows The submenu point Manual allows you to write individual comments horizontally or vertically on the chromatogram in addition to the possibilities already described After clicking Manual a box appears in which the direction Horizontal or Vertical is defined Then with the right mouse button click the area of the chromatogram at which the text is to appear The text is then entered in a text box with a maximum capacity of 30 characters in the horizontal and 20 in the vertical direction A previous annotation can be erased with Delete last annotate All manual annotations can be erased one after the other by clicking the menu points Annotate Manual and Delete last annotate The annotation appears only on the current sc
2. a mino rpt a Qu4 0021 rpt a Qum10003 rpt ja Amino rpt a Qu4 0031 rpt f Qum10004 rpt ia Aral0201 rpt ia Qu4 0041 rpt Qum10005 rpt ja Fmtest rpt i Qu4 0051 tpt Qum10006 rpt a Palm2 rpt ta Qu5 0011 rpt Qum10007 rpt a Qu4 0011 rpt a Qu5 0012 rpt Qum10008 rpt Filename Bumi 0009 rpt OK Remove Filetype Slice Files rpt 7 Cancel If a Report File is clicked more than once and confirmed with OK the error message appears Two identical Report Files selected Attention Only Report Files generated by one point calibrations can be used for multi calibrations After correct file selection and clicking OK a graphical representation of the average calculation appears Page 4 5 8 4 5 2 2 Reprocess Calculations Multi Calibration The yellow here blue bars represent the peak areas of the individual runs the horizontal yellow line in the middle represents the average value the white here black lines represent the standard deviation and the blue lines the confidence range The closer the blue lines are to the average value the more reliable the calculation is The confidence range decreases as more runs are made or if a few well reproducible runs are made J ChromStar DAD GPC Multilevel Calibration olx File Select Window Help 18 x AE 3 am 3 rm emaema A Peak Name Toluol Average Area 1155601 Standard Deviation 10439 64 0 90 Confidence R
3. The following operations function slightly different in the Split window Files Print always prints out the section of the chromatogram which is displayed on the lower sector of the window If the lower sector is empty the entire chromatogram will be printed Files Copy transfers the part of the chromatogram in the lower sector of the window to the clipboard View Show Values displays the retention time and the detector signal at the current position of the cursor in the lower sector Show values can only function when a section is displayed in the lower screen Results displays only the results list of the lower section of the screen ES Clicking again View Split the split representation is cancelled chromatogram in which the retention time and the detector signal of each data point in the chromatogram are displayed The vertical white line shows the position of the mouse cursor Moving the mouse across the chromatogram makes the changing retention time and signal values visible in m 2 See 13 000 We 396 551 the view box Clicking Show values or the button again causes the box to disappear After clicking Show Values a field is displayed in the status line above the Show Gradient In the chromatogram representation the gradient used when recording the chromatogram can be shown only possible when the pump is controlled via ChromStar and the gradient is entered in the LC Procedure file On top of the chromatogram the c
4. 2 4303E 017 0 0000E 000 0 0000E 000 Calculation Report on 17 04 1996 at 11 34 Included 2 Pages Page 6 4 6 1 Print outs in Edit Files 4 Print out of an LC procedure file Current user H Risler Date 17 04 1996 Time 11 35 LC Procedure GRADIENT Created by H Risler 0n 01 29 96 at 08 56 52 Time Function Value ee A 0 00 Solvents 80 0 20 2 00 Solvents 80 20 4 00 Solvents 10 90 6 00 Solvents 10 90 16 00 Solvents 100 0 20 00 Solvents 100 0 22 00 Solvents 80 20 25 00 Solvents 80 20 caa araa aaa cooeooo o oooooo0oo0oo0 oooo0oo0oo0o00 ooooo0oo0oo00 oooooo0oo0o0o End of LC Procedure Report on 17 04 1996 at 11 35 Included 1 Page 5 Print out of the preset table Current user H Risler Date 17 04 1996 Time 11 36 PRESET_SYSTEM_PARAMETERS Flow rate ml min Max press bar Min press bar Wavelength nm Attenuate Screen scale 2 Compress factor Slice width msec Noise Threshold mV Skim ratio Syringe vol ul Bubble check Y N Oven temperature Rep AS Start Y N Emission nm 6 2 Print out of a chromatogram Page 6 5 6 2 Print outs of chromatograms using the report print templates RPF 6 Print out after recording a chromatogram with SHORT RPF AROMO0001 Date 14 03 1996 H Risler Sample Identifier Injected on 26 01 1996 Injected at Data Processing Parameters Slice Width msec 250 Noise Threshold mV 500 Skim Ratio
5. Clicking Print or pressing the button prints out the file cp printer protocols 1 5 Printer Setup opens a dialog box in which the printer setup can be changed Copy copies the file into the clipboard With File Information the author name date of origin and date of the last changes of a file can be viewed Close Method allows you to leave a processed file If changes have been made the question is asked whether the file has to be saved or not Page 4 1 6 4 1 1 Edit Files Method The different pages of the Method file Run Table Autosampler Table Sample Table und Documentation Table are accessed by clicking with the mouse into the cards Access via key entry is possible as well CTRL TAB moves to the next card to the right CTRL Shift TAB moves to the next card to the left The different pages of the data handling file and of the calculation file are accessed in the same way Clicking the Minimise Box in Edit Files Method iconises the application the icon EF appears at the bottom of the ChromStar window and the file can be further viewed and processed later It is not possible to open more than one Edit Files window so only one file at a time may be edited 4 1 1 1 Edit Files Method Run Table Page 4 1 7 4 1 1 1 Edit Files Method Run Table ig Edit Files Method neu Run Table Sample Table Documentation Table Mode Notes Test CS6 0 109 HPLC C GC I Autosampler F Use Line Numb
6. Depending on the type of autosampler the vial number entered against Vial is the start vial all following vials are processed in a continuous sequence in exactly the same way as this first one A calibration run is defined with S against Type one point calibration In the Data Handling File to be used the name of an existing Calculation File must be defined this contains the retention times and standard amounts of the peaks to be calculated If more than one injection is made from a vial which is declared with Type S as standard the average of their response factors will be entered into the calculation file defined in the data handling file against K1 in the regression table In order to perform a multi level calibration the series of vials with different standard solutions is defined in the autosampler table in one line which is marked with S against Type In the Sample Table the number of standard solution in a vial is entered under Conc Level The Calculation File of which the name is defined in the Data Handling File contains the corresponding level entries and in the peak table the amounts in the standard solutions The Multi Level Calibration is performed automatically after recording the chromatograms and the coefficients of the approximation functions in case of more peaks are entered in the regression table It is also possible with this type of calibration to perform repeated injections of the standard solutions Depen
7. PKS PKS Js 2 K3 PKS PKS Js 3 wherein is KO K1 K2 K3 Regression coefficients of the approximation of the Ist 2 or 3rd order for the component 1 PKS area or height of the im peak PKS JS area or height of the internal standard in the actual run in this case normalized Page 5 3 24 5 3 4 Calculation Examples with Formulas 6 Printer Protocols 6 Printer Protocols This chapter contains a series of print outs of chromatograms results lists calculations calibration curves etc obtainable when working with ChromStar The bold printed numbers in the previous chapters refer to the figures in this part 6 1 Print outs in Edit Files 1 Print out of a method file Current user H Rigler Date 17 04 1996 Time 11 36 Method File C CHRSTNEUDATA NEU MET Created by H Risler Last Updated by H Run Tab g on 0J 21 56 at ii J3i 0 Risler en Gb 179E abe lls aos Hotes Test C540 AS LCrr Report sBhort cpt Aucosampler 1 Hormaligation Daca File 1 NE Daca File if Daca Fil iJ Data File Autosampler Tabie Included J Vials Vial co Inj Vol Dela Rur l 5 1 26 0 00 4 000 1 30 0 00 4 000 i Fa 0 00 4 000 ganple Table Included Sa a Id S 3 6 1 8ed S13 6 2 800 BIJ 6 3 5bd 609 6 4 8rd 3 6 5 Std S 4 6 Probel Std 6 Probez LC PROC _DH 1 B ni TEU Tahu T Tu neu T Samples Factor Weight Int
8. Temperature Split Ratio l Notes Detector Temperature Notes Help Close Save Save s 4 1 1 4 Edit Files Method Documentation Table Page 4 1 15 If Mode HPLC is chosen the following entry boxes appear F9 Edit Files Method Untitled Run Table Autosampler Table Sample Table Documentation Table m Mobile Phase Detector Details Type Range Wavelength nm Notes m Column Details Sample Details Type Name Col Length 00 mm Origin Diameter 0 0 mm Preparation Particle Size Injection Yol Solvent Peak 10 00 min Notes He ce ose _ Saves The entries under Column Details against Column Length and Solvent Peak are used for calculation of column coefficients For the calculation the preset column length is 125 00 mm the solvent peak or dead time retention time of a an unretended solute is set to 1 00 min These values can be changed as required For more details about column coefficients see chapter 5 2 6 The Define key opens the User defined details box where the descriptions of the items which appear in front of the entry boxes can be changed Page 4 1 16 4 1 1 4 Edit Files Method Documentation Table In the following example the descriptions in the Mobile Phase box were changed The box now shows the head line Eluent and the box texts Solvent A Solv
9. an average response factor out of the response factors obtained from one point calibrations after several injections of the same calibration sample or a calibration function representing the correlation between amount of a component and its peak area or height this can be done after one point calibration with calibration samples that contain different amounts of the standard components The menu point Calibration with the sub menu points One Point Calibration Multi Calibration permits various ways of calibration which are described in the following sections A further description can be found in section 5 3 1 4 5 2 1 Reprocess Calculations One Point Calibration Page 4 5 5 4 5 2 1 Reprocess Calculations One Point Calibration For a one point calibration the Report File of a calibration chromatogram and a Calculation File are required The Report File contains the retention time the peak area and the peak height for each peak as a result of the integration or reintegration of the calibration chromatogram The calculation file is created as described in section 4 1 3 Page 2 of the calculation file Peak Table is used to define suitable time windows for each peak to be calculated If more than one peak is found within these time windows the greatest peak will be used for calibration If no peak is found in a defined time window the message appears No peak found in time window T1 to T2 If one peak is found in more than
10. 4 1 4 Edit Files LC Procedure The LC procedure file is used for controlling an HPLC pump if this is equipped with an appropriate interface Clicking the menu points Chrom Files and LC Procedure in the Edit Files window or using the pro button and entering a file name as described in 4 1 1 displays the file on to the screen P Edit Files LC Procedure testmix3 LC Procedure Programmable Function Preset Function 0 00 100 0 00 00 00 C 2 Flow Rate 1 00 Flow Bate C 3 Max Pressure 400 4 Min Pressure 0 C Oven Temp 20 C 6 Wavelength 254 C 7 Attenuate 1 00 D O Time Function A X BI Ch DIZ c c c 9 Event 0 00 fi Solvents f100 0 oo oo oo 10 Stop 11 Emission iat Delete Help Close Save Save As In this file file extension PRO the following parameters can be time programmed 1 Solvent This function defines the solvent composition at a certain time Entered here is the percentage proportion from 0 0 to 100 of the solvents A B and C separated by a space The proportion of solvent D is automatically calculated Attention An LC Procedure must contain a solvent programmed at time 0 The programming of a gradient see section 5 1 2 requires only the specification of the start and end conditions A linear solvent gradient is calculated between two different solvent compositions at two different times The percentage proportion of a solvent can either increase or decrease 4 1 4 E
11. About ChromStar DAD GPC xj ChromStar DAD GPC Version 6 3 Build 18 14 03 2006 i Copyright 1994 2006 SCPA GmbH Software fur Chromatographie und Prozessanalytik GmbH Am Weidufer 32 28844 Weyhe Leeste Tel 04 21 7802806 Fax 0421 8903 46 Web www scpa de Mailto Info scpa de 3 6 ChromStar Function keys Page 3 7 3 6 Function Keys The Function keys F1 F12 can be used to accelerate handling when recording data in the Analysis application The application must be active They have the following functions Fl START F2 STOP F3 ABORT F4 CONT F11 SCRN SCALE F12 NORM starts a chromatographic run in a method Ea oy stops the chromatogram which is evaluated and printed up to the time of stopping it interrupts the chromatogram without storing and evaluating the data recorded stops data acquisition and starts integration and printing An LC Procedure is further finished off allows you to change the screen scale of the detector signal in an entry box normalises the screen display of the detector signal so that the lowest value is displayed 5 The following function keys control an HPLC pump F5 F6 F7 F8 F9 F10 SOLV A SOLV B SOLV C SOLV D STOP PUMP FLOW RATE Solvent A is pumped Solvent B is pumped Solvent C is pumped Solvent D is pumped The pump is stopped so that no further solvent is pumped E Le EI The flow rate
12. Reintegration 4 4 4 4 4 9ff 4 4 17 5 2 1 Batch 4 5 18 GPC 4 9 13 6 32 GPC Batch 4 9 26 Rel Relative 5 3 7 Relative Window Update 4 1 24 Remove 4 7 4 Rep AS Start 4 1 36 Report 3 8 4 1 8 4 5 18 4 6 3 4 8 14 5 1 1 Report Editor 1 2 2 4 Report File 3 8 4 1 28 4 2 1 4 3 1 4 4 3 4 4 10 4 4 17 4 5 1 4 5 2 4 5 5 4 5 7 4 5 8 4 5 10 4 5 17 4 5 18 4 5 20 4 9 13 4 9 19 4 9 22 4 9 23 4 9 27 5 1 5 5 2 1 5 2 2 5 2 9 5 3 1 5 3 2 5 3 3 5 3 1 1 Report Format 3 8 Report Print Template RPF RPC RPD RPL 1 2 2 3 3 8 4 1 8 4 4 4 4 4 10 4 5 3 4 5 14 4 5 16 4 5 18 5 1 1 5 2 2 6 5ff 6 14ff RPG 4 9 14 4 9 23 4 9 26 6 32 RMG 4 9 28 6 38 Reprocess 4 4 1 4 5 1 4 6 1 4 6 17 5 2 1ff 5 3 1 GPC 4 9 13ff Reset Range 4 7 6 Reset Region 4 6 5 Resp Fact Res Fct 4 1 27 4 1 29 4 5 2 4 5 5 4 5 6 5 3 1 Response factor RF value 4 5 2 4 5 5 4 5 8 5 3 1 5 3 4 5 3 9ff 5 3 11 GPC 4 9 7 4 9 22 Results 4 4 7 4 4 8 4 4 12 4 4 18 4 5 8 4 5 9 4 5 11 GPC Cal File 4 9 12 GPC Reint 4 9 15 4 9 16 4 9 19 4 9 21 Curve Fitting 4 8 13 Result list Reintegration 4 4 8 4 4 12 6 5 6 6 6 17 Recalculation 4 5 2 6 8 Calibration 4 5 6 6 9 6 10 6 12 Retention time 4 9 9 Retention time Ref Peak 4 9 6 Reverse 4 8 1 RF Calculation File Code 4 1 27 RS232 interface 2 6 4 2 1 4 2 2 4 3 3 5 1 1 5 1 9 Run 4 1 11 Run
13. Spectrum Reprocessing Spectra eesesecooooosocooocsooooosoooooooooosooosoooooooossosoooooooo eesooesocosocooooooooossoosoooooooosoosoooooooossooooooooo Contents Page 1 3 4 8 Transform 4 8 1 Transform Modify seeessesesssesseseesssersseresssseessseessssreesseeessee 4 8 1 4 8 2 Transform Arithmetics ccccccccssestsisenscaseieadascsdeetesatassecasassas 4 8 5 4 8 3 Transform Baseline s0scsssessssssssssssssssssessseeeseeeseeecs 4 8 8 4 8 3 1 Transform Baseline Automatic cccccceceeeeceeceeeeeeeeees 4 8 8 4 8 3 2 Transform Baseline Manual cccccccccceccccecceseeeeeeeees 4 8 9 4 8 4 Transform Curve Fitting 0 cece ccecesseceeeeseecceeeneeeeeenteeeeees 4 8 12 4 9 GPC Processing GPC Chromatograms 5 Chromatography with ChromStar ccccscccceeeeees 5 1 1 5 1 DEIL WATAS LITRI LATO a UA EEE EEE E E EEEE EE 5 1 1 5 1 1 Recording a Chromatogram seesessseesssesesssressseresseressssresse 5 1 1 5 1 2 Programming a Gradient i oscescsgscctcesassncvnewdnncvaestncdesenecievamsearede 5 1 6 5 1 3 Recording a UV spectrum sessesseeessereesssersserresssrressresssseees 5 1 8 5 1 4 Operating an Autosamplet sssessseesessseeesseresssseresseressereessseess 5 1 9 5 1 4 1 Installing the Autosampletr ysicc sccs cscs saececacateseseaaseidecavededevences 5 1 9 5 1 4 2 Programming the Autosampler ccccsecceesssecceeeeteeeeeeeeees 5 1 9 5 1 4
14. 2 Startinjection 1 i Nein Abbrechen 4 2 4 Analysis Chromatogram Method Page 4 2 9 If no autosampler is used a number has to be entered before the start injection under which no chromatogram is stored yet If more than one injection out of the same vial is made by editing the vial in different lines of the autosampler table an error message under Error appears when the second chromatogram is to be saved since the chromatogram with the same name already exists The already existing chromatogram is moved into the TEMP0OO directory cp p 5 1 13 If an autosampler method was completely carried out and is called again the message appears Slice File exists Overwrite all chromatograms Yes No After answering No the method can be cancelled the chromatogram name in the method can be changed After answering Yes the message appears All stored chromatograms of this method will be deleted Continue With OK all chromatograms will be deleted and the method can be started After leaving the Load Method File window with OK the method name is displayed in the line above the signal area in the Draw Baseline window When using a controlled HPLC pump the initial solvent composition of the LC procedure used in the method will be pumped through the whole system On leaving the window with Cancel the Message No Method selected appears in the information line As soon as pressure and detector signal have stabilised and th
15. 2 5 Insert fig Edit Files LC Procedure Gradient LC Procedure m Programmable Function Preset Function 1 Solvents Solvents 80 0 20 0 0 0 0 0 i 4 Solvents 80 0 20 0 0 0 0 0 2 Flow Rate ml min 1 00 l Solvents 10 0 90 0 0 0 0 0 c C 3MaxPress bar 300 Solens 1000 00 00 00 Caines 0 O C 50ven Temp C 20 25 00 Solvents 80 0 20 0 0 0 0 0 C 6Wavelength nm 254 C 7 Attenuate 1 00 c c c c 8 Autozero Time Function Value 9 Event 25 Solvents eo 20 10 Stop 11 Emission nm A ee Help Close Save Save s The LC procedure file GRADIENT can be stored by clicking File and Close or the screen key and then clicking the box Yes against the question Save it Now the file can be used in a chromatographic run and for this purpose it can be entered in a Method File Page 5 1 8 5 1 3 Recording a UV Spectrum 5 1 3 Recording a UV spectrum Using a UV detector with variable wavelength controlled by ChromStar through an interface a UV spectrum can be recorded when the pump is stopped For a UV spectrum in the wavelength range of 200 400 nm for example the LC procedure file in this case named SCAN will be created fig Edit Files LC Procedure Scan LC Procedure Programmable Function Preset i Function C 1 Solvents i Solvents 100 0 0 0 00 00 7 Flow Rate 0 000 C 2 Flow Rate ml min 1 00 i wavelength 400 3 Max Press bar 300 eS 4Min Press bar 0 C 50ven Temp C 20
16. 5 3 4 Calculation Examples with Formulas Page 5 3 19 De 2 2 050 1990622 _ 3 95E 000 0 00 Toluene Totals 3348808 7 97E 000 0 00 Formula The calculation of the unknown amounts Absolute is carried out according to the equation RES KO K1 PKS K2 PKS 2 K3 PKS 3 where RES Result of the quant Evaluation for the i peak Absolute K0 K1 K2 K3 Regression koefficients of the 1 2 or 3 Ordnung for the component i PKS peak area or height of the i peak The coefficients are calculated during multi level calibration 5 3 4 4 Internal Standard Method A After one point calibration One point calibration Chromatogram BENA6121 Chromatogram of the standard solution Sample Identifier B T Toluene is the internal Standard IS Calculation File BENAIS Calculation Method Calibration Peak Win Ret Time Area Resp Std Amt Name No No min Fact AU Std Amt Amount in the standard solution 1 1 1 775 420346 2 621E 001 2 000E 001 Benzene 2 2 IS 2 075 550833 3 631E 005 2 000E 001 Toluene Formula Resp Fact amount peak sta area ys sta area Peak Std For the internal standard the Resp Fact is calculated according to Resp Fact ts amount js sta area qs sta Calculation of the unknown amounts Chromatogram BENA1111 Chromatogram of the mixture with unknown amounts Sample Identifier B T Page 5 3 20 5 3 4 Calculation Examples with Formulas Calculation File
17. 6Wavelength nm 254 7 Attenuate 1 00 8 Autozero Time Function Value c c c 9 Event 4 Wavelength 200 10 Stop 11 Emission nm raed Delets Help Close Save Save s The entry Flow Rate 0 causes the wavelength to be scanned in a continuous linear way wavelength scan and not in steps at times 0 min and 4 min As the absorption in the short wavelength range is usually higher than in the long wavelength range it is recommended to scan from long to short wavelengths and also to adjust the detector signal to zero before commencing the wavelength scan in order to be able to follow the spectrum on the screen If a measurement is to be made with wavelengths greater than 400 nm it is necessary to install the second order filter To carry out the measurement a method with a normal LC procedure and data handling file is started When the peak maximum of the compound to be investigated appears in the chromatogram i e when the highest amount of the compound is in the detector cell the pump is stopped with F9 and the method is aborted via F3 The method SCAN with the LC procedure SCAN and a data handling file SCAN containing a delay time as long as the run time is started by clicking the submenu point Method in the Analysis window entering the file name and pressing F1 The long delay time in the data handling file prevents a senseless evaluation The same can be achieved by using the parameter Integrate Inhibit
18. Chrst6 NeueDemodaten 4ROM112042031 pt Date 26 06 01 Manually changed No Time 10 04 shown to the right After clicking OK the ij Peak Fet Time Pk Start Pk End Height report list appears on Nr min min min the screen Pile iF ae 1 192 2108 nn BAAS an 204 704 2 2 250 2 108 2 442 965515 358 248 Page 4 4 4 4 4 1 Reprocess Reconstruction E Print permits the printing of a chromatogram After clicking Print a selection box appears in which you can select the format of the chromatogram print out pp y g p Report Template File 3 Beispiel rpt scpalce pf 3 Short32 1p a colcoeff32 rpf a scpazcc pf a short5d pf 3 docul 1pf j Rec 22 3 scpa3cc rpf a shorteal rp 3 docu2 pf A reint321 rp 3 short rpf 3 shortml rpf 3 Docu3 rpf A reint321test rpf I short2 rpf 3 table32 rpf B Docu32 rpf B reint322 rpf short3 rpf gt Filename Reconst322 rpf Li Filetype Report Files rpf Cancel 1 Pages The directory containing report template files can be chosen on top of the box The report templates appear in the list to the right The print preview appears in the box to the left On deselecting Watch the preview disappears With Printer Setup printer settings like resolution paper size portrait or landscape orientation can be changed Copy with ist sub menus Chromatogram and Report transfers the chromatogram or the results list to the WINDOWS
19. Kan la Chromatogram ne110032 Sampla Identifier Injected on 21 03 1996 Injected at Data Processing Parameters Slice Width msec 00 Noise Threshold mv soo Skim Ratio Parameter Files Data Handling File NEUL Calculation File Calculation Method Percent Rev Time Pk Start Height min tain av 708 083 383 533 786 3196 1 07 0 285 15972 7 53 0 923 99727 18 19 647 14389 3 70 9 831 93408 407 97 53 970 3778 1 98 0 218 S946 2 83 0 344 6SS6S1 231 57 37 883 vn re ww nwo 1730734 674 84 100 000 Chromatogram ne120032 Sample Identifier Injected on 21 03 1996 Injected at Data Processing Parameters Slice Width msec soo Noise Threshold mV 500 Skim Ratio Parameter Files Data Handling File NEU2 Calculation File Calculation Method Percent Peak Ret Time Pk Start Pk End Height No imin mint min iav 1 317 1 100 1 667 353943 42 36 18 594 1 842 1 733 2 033 908323 329 54 47 718 2 292 2 233 2 375 958 2 43 0 333 2 700 2 567 2 967 635286 190 13 33 374 2903510 564 52 100 000 of 2 6 5 Print outs processed in chapter 5 Page 6 17 6 5 Print outs processed in chapter 5 Chromatography with ChromStar 17 Result list without grouping and summation Chromacoqram TBA0102 Date 18 04 1996 Time 8 05 Sample Identifier Ciba Basel R Injected on 05 03 87 Injected at 00 00 Data Processing Parameters Slice Width msec 500 Noise 800 Threshold mv 500 Skim Ratio
20. Parameter Files Data Handling File CIBA Calculation File Calculation Method Percent Peak Ret Time Pk Start Pk End Area Height Areat twin min tmin mV ec a ee ee re 0 308 0 217 0 350 90829 44 16 0 208 0 467 0 350 1 033 1709127 411 768 3 850 0 988 0 808 4 033 380028 69 21 0 856 1 200 1 033 1 692 5510110 964 3 12 411 1 467 1 358 1 692 32243 109 76 1 199 2 817 1 692 1 954 125395 25 15 0 282 2 260 2 356 2 333 24860 40 0 086 2 700 2 567 2 786 9401 18 62 0 134 2 900 2 758 3 167 206462 2 21 0 645 4 700 4 467 5 000 2184290 403 69 4 652 5 21893 5 000 5 325 184155 29 62 0 415 5 500 5 32S 5 775 218645 29 38 0 492 6 650 6 508 6 808 65799 13 23 0 148 8 042 7 875 217 01145 14 31 0 383 11 663 11 458 11 917 416052 71 26 0 937 12 942 22 758 13 333 794228 110 17 1 789 16 942 16 667 17 225 1022925 148 95 2 304 17 658 17 450 17 042 239382 42 06 0 539 18 708 18 575 18 492 84508 16 68 0 190 19 767 19 367 20 050 9008606 936 60 20 285 20 225 20 050 20 408 102626 19 52 0 232 21 008 20 806 21 225 222143 36 08 09 500 22 292 22 208 22 492 88437 15 54 0 199 22 656 22 492 22 833 167900 28 63 0 378 24 650 24 392 24 925 2811236 2 44 6 332 25 433 25 308 25 567 46131 10 70 0 204 25 925 25 806 26 042 30726 7 67 0 069 26 417 26 242 26 575 96811 19 33 0 218 ly ovevanneaune 9 Re meem e uynve une owo yanna One 29 27 975 27 783 28 192 360845 63 01 0 813 30 28 608 38 500 28 750 474078 11 66 0 108 31 29 483 29 300 29 817 410672 61 97 0 92
21. RPT max 15 levels which contain the results of the One Point Calibrations with the standard solutions to be used for the calibration The Report Files generated with the recording of the chromatograms have to be changed in such a way that they are suitable for a Multi Calibration Therefore after recording the individual chromatograms they have to be evaluated in a one point calibration with the Calculation File containing the exact amounts as described in 4 5 2 1 Attention Only Report Files generated by one point calibration may be used for multi level calibrations A Report File may not be used more than once After clicking into Calibration select Multi Calibration A dialog box appears for selection of the various Results Files cp p 4 5 7 After selecting the desired Report Files by double clicking or multi selecting and Insert their names appear in the list to the right On clicking OK the calculation starts and the approximation function of the first order is displayed for the first peak using the peak areas or heights for the x axis and the quantities entered under Amt in Std in the peak table of the calculation file which are used to generate the Report File for the y axis In addition the coefficients of the regression function and the correlation coefficient are displayed The form of the third order approximation is y KO K1 x K2 x2 K3 x3 EJ ChromStar Multilevel Calibration Lof x EE Fie Select Windo
22. Std 1 000000 0 000000 0 000000 L Gbbhod O 000000 0 000000 1 000000 6 050000 6 000000 1 000000 0 000000 6 660008 1 000000 8 000000 F00000 1 666600 0 000000 0 000000 1 000000 9 000000 O d00000 DHA CH la Cone Lave 1 a 3 4 5 i i Page 6 2 6 1 Print outs in Edit Files Method file cont Page 2 Date 17 04 1996 Time 11 30 Output of C CHRSTNEU DATA NEU MBT Documentation Table User Details Name S 3 6 Origin Alkylbenzole Preparation in CH3CN H20 Injection vol 20 pl Mobile Phase 85 CH3CN 15 H20 Notes Flow 1ml min Column Details Type RP18 Size 125x4 Smm Particle size 5 um Notes Detector Details Type UV Range Wavelength nm 254 Notes End of Method File Report on 17 04 1996 at 11 30 Included 2 Pages 2 Print out of data handling file Current user H Risler Date 17 04 1996 Time 11 31 Data Handling File NEU2 Created by H Risler 0n 03 21 96 at 11 32 08 Last Updated by H Risler on 04 17 96 at 11 31 47 Acquisition Page Slice Width msec Delay Time min Run Time min Calculation File Screen Scale 2 Peak Parameter Integration Page Time Function Value 0 000 Noise 1000 000 0 000 Integrate Inhibit 1 000 3 000 Noise 300 000 End of Data Handling Report on 17 04 1996 at 11 31 Included 1 Page 6 1 Print outs in Edit Files 3 Print out of a calculation file Current user H Risler Cal
23. The peak table of the Calculation File used to generate the first Report File is taken over together with its retention times amounts response factors of the one point calibration and the peak names The parameter Regression for Calculation on the first page method table of the new file is automatically marked so that the approximation functions are used in later calculations instead of the response factors of the peak table Page 5 3 4 5 3 2 Quantitative Calculation Methods 5 3 2 Quantitative Calculation Methods ChromStar offers six different quantitative calculation methods 1 Percent method 2 Normalization method 3 Employing an external standard 4 Employing an internal standard 5 Employing a calibration function obtained by an external standard 6 Employing calibration functions for external and internal standards These methods are to be defined by the corresponding entry in the Method field on the method table of the calculation file and also by the parameter Regression for Calculation and by identifying the internal standard peak in the peak table In quantitative calculations with the first three methods the universal formula applies RES PKS RF MULT DIV 1 in which RESi The result of the quantitative calculation for the i th peak PKSi The peak area or height of the 1 th peak RFi The response factor of the component i RF value which is determined by the method of calibration MULT and DIV depend o
24. allows an enlarged section of the upper chromatogram to be displayed by value entries for the time axis and for the y axis in mV the corresponding calculation appears in the lower field Expand can also be used before executing a calculation Set Default restores the original values After clicking OK the original chromatogram appears together with the calculation 4 8 2 Transform Arithmetics Page 4 8 5 4 8 2 Transform Arithmetics This program point allows you to add or subtract two chromatograms to display the ratio between them or to append them After selecting the menu point Arithmetics a dialog box appears for selection of the chromatograms to be processed these are described as the Reference and the Work chromatogram When selected the Work chromatogram appears in the upper half of the screen and the Reference chromatogram in the lower The title bar now carries the text File Arithmetics and the menubar contains the points Files Alignment Calculation Expand Window Help Only chromatograms recorded with the same slice width cp 4 1 2 or slice widths which are integer multiples of one another can be used in these calculations If chromatograms have been selected that do not meet these requirements the message Slice Width Mismatch appears Clicking OK allows new chromatograms to be selected EJ ChromStar File Arithmetics olx Files Alignment Calculation Expand Window Help lej x s S Sjeme a
25. and Save E If sections of the chromatogram have still not been satisfactorily integrated then the menu point Files Manual Integration can be used to perform a manual integration EJ ChromStar Reintegration OF Xx MM File Expand Annotate View Param Results Window Help la x S S lwl Fl a al e 2 ziemen 2 Au Chromatogram 122 30031 0 0 1 0 2 0 3 0 4 0 5 0 min Press F1 for Help NUM Ui 5 2 2 Integration Parameters Page 5 2 3 5 2 2 A Description of some Integration Parameters 5 2 2 1 The Influence of the Noise Value on the Integration The noise value 1 to 9999 in uV sec determines the minimal increase of the detector signal which is still judged as peak start Thus the first derivative of the chromatogram is examined peak start is determined if the increase is greater than the noise value for 6 consecutive data points The peak start is then plotted to the first of these points The peak end is determined as soon as the increase of 6 consecutive data points is lower than the noise value In this case the peak end is plotted to the last of these points Peak recognition is also influenced by the slice width because the examined time interval of the detector signal function is dependent on the slice width Peak recognition will obviously not function satisfactorily if the slice width is defined so small that peaks are recognised in the noise of the detector The number of points the slope vaue of
26. in which LINp Index of the peaks LINss Index of the next standard peak LINps Index of the previous standard peak RTp reduced retention time of the peaks RTss reduced retention time of the next standard peak RTps reduced retention time of the previous standard peak The calculation is done either in Reprocess Reintegration using a data handling file with the name of the calculation file mentioned above or in Reprocess Calculations The following chromatogram was evaluated with the Internal Standard method mu Chromatogram WACHS2 120 100 80 60 40 20 The standard peaks were annotated manually Page 5 2 14 5 2 5 Calculation of Kovats Indices The results list after evaluation in Reprocess Calculations shows under Win the number of the time window the retention time of the standard peak the Kovats Index entered and under Name the description For all other peaks for which the peak area is greater than the area defined in the Calculation file against Minimum Area the Kovats Index will be calculated ChromStar CALCULATION Iof x File Calculation Calibration Displ Chrom Batch Convert Exit Window Help l x Chromatogram wachs2 Calculation File WACHSI2 Calculation Method Kovats Indices Intern Peak Win Ret Time Kovats Name No No rin Index 10 32 205 3599 064 11 1 33 500 3600 000 C36 12 34 080 3663 614 i 13 2 35 325 3800 000 C38 14 35 875 3863 268 15 3 37
27. parameters can only be changed in the CHRST32 INI file cp p 2 5 Analysis Select does not exist since there is only one data acquisition channel ChromStar Integrator does not contain the Transform module A number of print report templates are copied onto the computer during the installation these can be used to get print outs of the chromatograms The Report Editor is an independent program to create new templates A separate manual describes how to use the report editor program 2 ChromStar Installation Page 2 1 2 Installation 2 1 Description of the Hardware Components For the ChromStar chromatography software a PC with a pentium processor or better with the following individual components is needed RAM as needed by the operating system a floppy disk drive a CD drive and a hard disk a graphic card with a 800x600 resolution VGA Graphic Card ahigh resolution 800x600 graphics colour monitor a keyboard a mouse Microsoft WINDOWS operating system version 3 11 is possible recommended is Windows95 98 or NT which need 16 Mbyte RAM a Microsoft WINDOWS compatible printer The ChromStar software package consists of one or two A D converter boards with plugs depending on the configuration one board for two channels or a one channel board respectively the ChromStar software CD a manual in English or German 2 2 Installation of the A D Converter board Follow these instructions f
28. unless the subpeak is only a shoulder Only in manual integration it is possible to calculate a small peak separated by a valley on the left flank of a larger peak by drawing a skim function 5 2 2 Integration Parameters Page 5 2 7 5 2 2 3 The Baseline Parameter With the parameter Baseline a new start point of the baseline can be defined This function is always necessary when the baseline of the detector signal has changed considerably during a short period of time e g after a change of wavelength a change of eluent or in a gradient run as in the example shown In the example the integration was prevented using Integrate Inhibit during the period of the increase of the detector signal 200 175 150 125 100 75 50 25 Time 8 4 Baseline Page 5 2 8 5 2 3 Manual Integration 5 2 3 Manual Integration If a data handling file with unsuitable parameters for evaluation was used when recording the chromatogram the peak data can be obtained later by manual integration of the chromatogram For that purpose click the menu point Integration and its submenu point Manual Integration in the Reprocess Integration window Select the desired chromatogram by clicking it in the list of stored chromatograms Select a section of this by constructing a rectangle in the upper part containing the peaks to be evaluated EJ ChromStar Manual Integration OF x M File Mode Expand Edit Annotate View Hesults Window Help l x Chro
29. you must again create an enlargement in the lower section by constructing a rectangle Page 4 8 12 4 8 4 Transform Curve Fitting 4 8 4 Transform Curve Fitting The menu point Curve Fitting allows the reintegration of an enlarged sector of the chromatogram by means of an approximation of the peaks with a Gaussian function To carry out this reintegration first select a chromatogram and define the part to be examined by constructing a rectangle The window title bar reads Curve Fitting The menu bar contains the points File Autofit Results Expand Window Help EJ ChromStar Curve Fitting Ioj x File AutoFit Results Expand window Help l x a 3 au m e F Chromatogram ARALO2O1 Fix a Rectangle to zoom 500 400 300 200 100 17 0 17 5 18 0 18 5 13 0 13 5 20 0 20 5 min Press F1 for Help After selecting the point Autofit three approximation algorithms are offered Peaks Single Determination of the peaks by means of a Gaussian function Peaks Double Determination of the peaks with a Gaussian function on both peak flanks Peaks Rational Determination of the peaks with a fractional rational function of the fourth order After clicking one of these three evaluation options a window appears in which the number of peaks to be calculated can be entered The default number of peaks is set to 4 4 8 4 Transform Curve Fitting Page 4 8 13 OK starts the calculation The num
30. 0 01 to 10000 minutes in steps of 0 001 default value 60 000 with a slice width of 500 msec the chromatographic data of a run are acquired If this period is longer than that defined under Run in the method file using the autosampler the data acquisition will stop when the time entered in the Autosampler Table is passed If a shorter Run Time is entered than in the Autosampler Table the data acquisition ends after the time specified here in the Data Handling File Calculation File The name of a calculation file default value name of the data handling file can be defined here It is used to perform quantitative calculations with the integrated peaks data If no name is defined or if the calculation file defined does not exist a percentage calculation is made Screen Scale 2 This parameter defines the scale of the y axis on the screen 0 to 15 The value 10 shows the detector signal from 0 to 313 mV 9 shows the range 0 to 156 mV 8 the half value again etc Peak Parameter This parameter defines the number of successive data points to be used for averaging the slope of the detector signal function lowest value 5 highest value 15 odd values The slope is checked for a certain number of data points for the determination of peak start and peak end After accessing the data handling file the first parameter of the acquisition page appears on a black background and can be now be edited TAB accesses the next box down which now
31. 0 086 2 700 59401 1 000 0 134 2 900 286461 1 000 0 648 128 2557089 1 000 8 760 6 650 65799 1 000 0 148 8 042 81145 1 000 0 183 42 312 1220280 1 000 2 726 16 942 1022925 1 000 2 304 17 658 239351 1 000 0 539 18 708 4808 1 000 0 190 29 767 18601564 1 000 41 099 20 225 102626 1 000 0 331 22 008 222143 1 000 0 500 22 392 08437 1 000 0 199 22 658 167900 1 000 0 378 24 650 4812373 1 000 10 846 Summe2 26 433 46131 1 000 0 104 25 925 30726 1 000 0 069 26 417 96821 2 000 0 218 27 975 360845 1 000 0 813 28 608 47878 1 000 0 308 29 483 410672 2 000 0 925 evraweune wun enn ne we RN we me o owew ne unr Owes Hew Ow 30 883 532241 1 000 1 199 31 225 846682 1 000 1 307 31 758 48735 1 000 6 103 32 883 34203 1 000 0 212 33 167 28748 1 000 0 088 34 425 42755 1 000 0 096 34 625 4070 1 000 0 169 34 867 168361 1 000 0 379 38 388 340144 1 000 0 766 37 375 650035 1 000 1 464 37 542 239331 1 000 0 539 39 033 9813909 1 000 2 144 39 588 45609 1 000 0 103 42 533 210715 1 000 0 475 43 4932 1150770 1 000 2 592 rr e 44396080 100 000 6 5 Print outs processed in chapter 5 Page 6 19 19 One Point Calibration in a current run Chromatogram QUANO021 18 04 1996 Time Author H Risler Notes Sample Identifier Injected on 18 04 1996 Injected at Data Processing Parameters Slice Width msec 500 Noise Threshold mv 500 Skim Ratio Parameter Files Data Handling File QUAN Calculation File QUAN Calculation Method Calib
32. 065 4000 000 C40 16 37 590 4062 922 17 4 38 735 4200 000 C42 18 39 230 4262 309 19 5 40 325 4400 000 c44 20 40 795 4461480 21 6 41 855 4600 000 C46 22 42 095 4632 903 For Help press F1 NUM 5 2 6 Column Coefficients Page 5 2 15 5 2 6 Column Coefficients Various column coefficients are used to describe the performance of a column such as theoretical plate number plate height capacity factors and coefficients of selectivity These coefficients should be tested from time to time by means of a chromatogram of a test mixture The calculation needs the following parameters Column length L in mm Retention time of a non retended substance To in min e g solvent peak RT Solv The following coefficients are calculated Linear velocity u Theoretical plate number N Th Plate No Plates per meter Plate height HETP Capacity factor k Asymmetry factor A Selectivity a Half height peak width WO 5 Resolution R The calculation of the column coefficients is carried out immediately after recording the chromatogram if the CHRST32 INI file contains the entry Calculation Standard in the chapter Colcoeff When the entry is Calculation Disabled or when there is no entry no column coefficients are calculated The preset values for the parameters used for calculation are column length 125mm and solvent peak 1 min These values are entered in the method file in the documentation page and can be changed before recording the chromatog
33. 1 5 1 4 5 1 10 5 4 1 5 4 2 6 1 Method Page GPC Cal File 4 9 6f Method Table 4 1 22f min 4 1 26 Minimum area height 4 1 23 Minimum Time Window min 4 1 25 Min press 4 1 33 4 1 35 Mode HPLC GC GPC 4 1 7 4 9 1 4 9 7 4 9 22 5 4 1 5 4 2 5 4 4 Mode 4 4 14f 3D Plot 4 5 15 Modify 4 4 15 4 8 1 Molecular Weight 4 9 4 4 9 8 4 9 15 5 4 4 Molecular weight calculation 4 1 7 4 9 1ff 5 4 8 Mol Wt 4 9 8 4 9 9 Moments GPC 4 9 15 Multi Calibration 4 1 24 4 1 31 4 5 7 5 3 2 Multi Calibration Parameters 4 1 31 Multi Calibration Graphic 4 1 31 Multi Level Calibration 4 1 31 4 5 10ff 5 3 2 6 12 Multiple selection 4 4 8 Multiplicate 4 8 1 Multiplication 4 8 3 Multi Point Calibration 6 10 N Negative Peak 4 1 20 GPC DH File 4 9 3 New 3 5 4 1 5 4 9 5 4 9 9 Noise 4 1 19 4 1 36 4 2 14 4 3 16 5 2 3 Norm 3 7 Normalization Normalisation 4 1 7 Calculation File 4 1 23 5 3 5 Normalize 4 2 6 4 3 9 4 7 5 Notes 4 1 7 4 9 7 Number Average Molecular Weight 5 4 8 Page 7 6 7 Index ChromStar O Offset 4 2 2 4 3 4 4 5 14 Oligomers 4 9 3 One Point Calibration 4 5 5 5 3 1f 5 3 9 6 9 Open 4 1 5 4 4 3 4 4 10 4 4 17 4 5 3 4 5 6 4 5 8 4 5 10 4 5 14 4 5 16 4 6 2 4 7 1 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 4 9 5 4 9 9 4 9 10 4 9 14 4 9 19 4 9 22 4 9 24 4 9 28 Open File box 4 1 4 Open Next 4 4 3 4 4 10 4 4 17 Open Previous 4 4 3 4 4 1
34. 2 3526E 004 Peak Name Toluene Time Window 2 06 Std Amt Area 1 5000E 002 562232 1 5000E 002 550833 Average Area 5 5653E 005 Standard Deviation 8 0600E 003 1 4 Confidence Region 7 2438E 004 K1 2 6953E 004 Formula Average Area i 1 ton Areai n 5 3 4 Calculation Examples with Formulas Page 5 3 17 n number of chromatograms i number of components in the standard solution Kli Amount of the standard i Average Area i The averaged response factors are entered in the Regression Table of the calculation file BENAEXT CAL as K1 the parameter Regression for Calculation is automatically ticked Calculation of the unknown amounts Chromatogram BENA9121 Chromatogram of the sample with unknown amounts Sample Identifier B T of the solutes Calculation File BENAEXT Calculation Method External Standard Calibration Method Multi Level Peak Win Ret Time Area Resp Absolute Weight Name No No min Fact AU 1 1 758 1358186 2 353E 004 3 20E 002 0 00 Benzene 2 2 2 050 1990622 2 695E 004 5 37E 002 0 00 Toluene Totals 3348808 8 56E 002 0 00 Formula RES PKS Kli Wherein is RES Result of the quantitative evaluation for the i peak Absolute PKS Peak area or height of the i peak Kl Standard amount average area of the component i is calculated for each peak in the calibration run C after Calibration with regression function Multi Level Calibration Multi Level C
35. 2000E 002 1681258 Regression Analysis Average Area 1 6648E 006 Standard Deviation 1 3774E 004 Confidence Region 1 9084E 004 0 0000E 000 7 2079E 005 6 3 Print outs of calibrations Page 6 11 11 Graphical presentation of a multi point calibration here only shown for the first peak Multi Point Calibration Date 14 03 1996 Time 9 06 Peak Name Toluol _ Time window 2 02 Average Regression Analysis Average Area 125128 006 Standard Deviation 9339E 003 0 78 Confidence Region 0542B 004 Page 6 12 6 3 Print outs of calibrations 12 Result list of a multi level calibration here only shown for the first two peaks Multi Calibration Table Date 14 03 1996 Time Calculation Mode External Standard Standard Sample Table Nr Report File Name qu4 0011 1 2 qu4 0021 3 qu4 0031 4 qu4 0041 5 qu4 0051 Peak Table Peak Name Toluol Std Amt Area 1 0000E 001 319709 319709 548247 548247 845240 845240 1180311 1180311 1528649 1528649 Time Window 1 99 2 0000E 001 3 0000E 001 4 0000E 001 5 0000E 001 Regression Analysis KO 1 1818E 000 Kl 3 2584E 005 0 996890 1 245968 Coeff of Correlation Std Error of Estimat K2 0 0000E 000 0 0000E 000 Peak Name Ethylbenzol Time Window 2 26 Std Amc 3 0000E 001 6 Q000E 001 9 0000E 001 1 2000E 002 1 5000E 002 Area 464142 464142 766108 766108 1199684 1199684 1696792 1696792 2193673 219367
36. 4 1 35 Print 4 1 2 4 1 5 4 4 4 4 4 7 4 4 10 4 4 17 4 5 3 4 5 6 4 5 8 4 5 11 4 5 14 4 5 15 4 6 3 4 6 17 4 6 18 4 7 2 4 8 3 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 4 9 5 4 9 9 4 9 11 4 9 14 4 9 19 4 9 21 4 9 28 Print Report GPC 4 9 23 Printer protocols 1 2 4 1 2 4 1 8 6 1ff Printer Setup 4 1 5 4 4 4 4 4 10 4 4 17 4 5 3 4 5 6 4 5 9 4 5 11 4 5 14 4 5 16 4 6 3 4 7 2 4 8 3 4 8 6 4 8 7 4 8 9 4 8 11 4 8 13 4 9 5 4 9 9 4 9 11 4 9 15 4 9 19 4 9 21 4 9 28 Process 4 4 Process Time GPC 4 9 4 4 9 18 Programming an autosampler 4 1 10 5 1 9 Programming a gradient 4 1 32 5 1 Pump control 4 1 32 4 2 1 4 2 4 4 Pump stop 4 1 33 4 2 4 4 3 5 6 3 5 ChromStar 7 Index Quantitative evaluation 4 1 22 4 4 10 4 5 1 4 5 18 5 3 1ff 6 20 GPC 4 9 7 4 9 22 R Ratio 4 8 7 Raw data Raw data file 3 8 4 1 37 Recalculate 4 9 11 Recalibration GPC 4 9 22 Reconstruction 4 4 2ff 4 4 10 4 4 17 4 4 18 Recording a chromatogram 4 2 1 4 3 1 5 1 1 6 5 Rectangle enlargement 4 4 5 Redo 4 4 16 Reference 4 5 14 4 9 28 Reference chromatogram 4 8 5ff 6 22 Reference peak cp RF 4 1 27 4 1 29 GPC 4 9 6 Reference Spectrum 4 3 13 Reference Window 4 4 5 4 4 11 Ref Peak Window 4 9 6 Regression for Calculation 4 1 24 4 5 8 4 5 11 5 3 3 5 3 7 5 3 8 Regression coefficients 4 1 24 4 1 31 4 5 11 5 3 7 5 3 8 Regression Table 4 1 31 5 3 2 5 3 7 5 3 8
37. Abort need to be confirmed after selection with OK in a dialog box before they are executed Continue starts the next injection when using an autosampler Data recorded up to then are evaluated and a print out is made not yet operational Unlimited Runtime After clicking into this sub menu procedure the chromatogram does not end automatically at the elapsed run time it can be stopped manually at any later time A corresponding message appears in the title line When using an autosampler the chromatogram must be stopped using Continue The autosampler run is then continued by carrying out the next injection After the data handling run time is elapsed a scroll bar appears at the lower frame which can be used to show the last recorded data This can also be achieved by using Control Fullsize The function keys are used without calling the Operation menu point from which these functions can also be activated If more Analysis windows are open the function keys operate only on the active window which has a red frame If you try to close an Analysis window or to quit ChromStar or WINDOWS with the control menu box or Exit while a chromatogram is running a box is displayed with the warning Method running Stop first before Ending Page 4 2 14 4 2 7 Analysis Chromatogram Options 4 2 6 Analysis Chromatogram Options Options offers various options in its sub menu Select Channels opens the window for selection of data
38. Analysis Chromatogram Method 4 2 4 Analysis Chromatogram Method After clicking into Method Load you must first enter the name of the desired method in the Select Method File window Load Method File Search in 3 Data m a neu3 MET a neu4 MET a test met Filename marathon met Filetype Method Files met 7 Cancel Start End iep Mil nip Linef You can select the drive and the directory in the box after Search in A method file is selected by clicking a name in the list or by entering the name against Filename If an autosampler is being used the start point for a series of samples to be run can be defined by entering against Line the line number of the autosampler table against Vial the vial number and against nj the injection number If no autosampler is being used the injection number specified in the Sample Table of the Method File can be entered against Inj to define the start point of the analysis A check is made whether the chromatogram already exists If this is the case one of the following messages appears An existing chromatogram can not be ye Ea overwritten To continue this method when using an autosampler a higher start line must be 3 entered or the Data File name must be changed Slice File exist ChromStar will change in the Method File or the existing the folowing entries chromatogram must be deleted Stattline 2 Startvial
39. Arithmetics Page 4 8 7 After selection of one of the points of Operation Addition Subtraction Ratio or Append a calculation takes place and its results are displayed in the lower part of the screen In the Subtraction procedure the Work chromatogram is subtracted from the Reference chromatogram Ratio displays the ratio from Reference chromatogram to Work chromatogram The information box shows behind Operation which procedure has been used If the calculation is only to be performed for a section this must first be enlarged by drawing a rectangle in the lower chromatogram before calculation takes place EJ ChromStar File Arithmetics OY x B File Operation Back Window Help laj x ejaj am rm eln F mU Reference AROMA1213A5051 Work AROMA1213B2031 Operation Addition 1500 1000 500 0 1500 1000 500 3000 2500 2000 1500 1000 500 Press F1 for Help NUM The menu point File now contains the submenu points Save As for storing the results A dialog box enables you to define the name of the result chromatogram which receives the file extension SLI The original chromatogram cannot be overwritten The result of the ratio display cannot be stored Print with its submenu Portrait and Landscape prints out the Reference and Work chromatograms and the result 22 Printer Setup allows the printer settings to be changed Copy transfers the chromatograms a
40. ChromStar User Login box press OK or enter a name and press OK Creating a Method file Press Edit Files button or Edit Files menu point Press met button or Chrom Files Method menu in the Open Method File against Filename overwrite UNTITLED by TEST press Open deselect No Report select Single click downwards arrow after Report choose report print template SHORT RPF press Close key question Save it Yes Creating a Data Handling file Press int button or Chrom Files Data Handling menu int point in the Open Data Handling File box against Filename overwrite UNTITLED by TEST press Open oerwrite number against Run Time by the number for the run time e g 10 Press Close key question Save it Yes Opening the data acquisition window Press Analysis Chromatogram button or Window New Chromatogram menu points in the Selection of Data Channel and Serial Port box press circle after Trace 1 press OK Loading a Method Press met button or menu points Method Load click in the Load Method File box in the list of files TEST MET press Open 5 1 5 Short instruction guide Page 5 1 15 The HPLC pump is started to pump the required solvent wait until the pressure has stabilised and the column is equilibrated autozero the detector Then the sample is loaded into the injector loop Simultaneously to turning the injector to the inject position the chromatogram is started by doing the following Start
41. Clean 4 8 2 4 8 4 Clipboard 4 1 2 4 1 5 4 4 10 4 4 17 4 5 3 4 5 6 4 5 9 4 5 11 4 5 14 4 5 16 4 6 3 4 6 17 4 6 18 4 7 2 4 8 3 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 4 9 5 4 9 9 4 9 11 4 9 15 4 9 19 4 9 21 4 9 28 Close All 3 5 Close 4 1 6 4 1 9 Code Calculation File 4 1 27 5 2 10 5 3 6 5 3 8 Column Coefficients 4 1 14 4 5 2 5 2 15 Column oven 4 1 33 4 1 36 4 2 5 4 3 6 Comment 4 6 14 Compare 4 5 15 Comparison Chromatogram 4 2 10 4 3 12 Composition 4 2 4 4 3 5 Compress ibility factor 4 1 35 Compute Data 4 9 9f 5 4 5 Conc Level 4 1 24 Confidence region 4 5 Configuration 2 7 5 1 Cont 3 7 Continue 4 2 11 4 2 13 4 3 15 5 1 12 Continous Peakgroup 4 1 20 Contour 4 6 7 Convert 4 5 19 Conv oluting Points 4 8 1 4 8 3 Copy 4 1 2 4 1 5 4 1 28 4 4 4 4 4 7 4 4 8 4 4 10 4 4 12 4 4 17 4 4 18 4 5 3 4 5 6 4 5 9 4 5 11 4 5 14 4 5 16 4 6 3 4 6 17 4 6 18 4 7 2 4 8 3 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 4 9 5 4 9 9 4 9 11 4 9 15 4 9 19 4 9 21 4 9 28 Cubic spline approximation 4 8 10 Cumulative GPC 4 9 15 Cumulative Area GPC 5 4 9 Cumulative Area GPC 5 4 9 Curve Fitting Transform 4 8 12 8 9 D D Solv 3 7 4 1 32 4 2 3 4 2 4 4 3 4 4 3 5 2D 4 6 1 2D Data 3 8 4 6 1 2D Process 4 6 2 DAD 3 8 4 3 1 4 3 3 Data Field 4 1 38 Data File s 4 1 8 5 1 2 Data Handling 3 8 4 1 1 4 1 17 5 1 2 Data Handling 1 to 4 4 1
42. Clipboard where it is stored temporarily Accessing Copy a second time will overwrite the previous contents of the clipboard The contents of the clipboard can for instance be viewed with the WINDOWS application Editor This can be done by opening the editor and transferring the contents with the Paste command from the Edit submenu With File and Save you can now create a text document into which the required information from ChromStar is inserted via the clipboard Save as allows a chromatogram to be saved under a new name The corresponding report file RPT is created simultaneously Export opens a file select box where a print report template can be chosen The chromatogram can be saved as WMF file in the representation of this template and can then be represented in an appropriate program for grafic display Reintegration displays the entry box Select Chromatogram and DH File After selecting the files it allows you to make a quick transition to the application Reintegration cp 4 4 2 1 Manual Integration opens the application Manual Integration s 4 4 2 2 at Exit Reconstruction closes the Reconstruction window 4 4 1 Reprocess Reconstruction Page 4 4 5 Expand allows a section to be selected from the entire chromatogram After clicking Expand a submenu appears with the points Reference Window By Values and Default Reference Window allows a section to be selected by drawing a rectangle With Auto range the sel
43. PKS K2 PKSj2 K3 PKS 3 4 in which is RES result of the quantitative evaluation of the i th peak K0 K1 K2 K3 regression coefficients of the Ist 2nd and 3rd order for the component i PKS area or height of the i th peak The coefficients are determined by means of a multi level calibration Herewith is also decided which order is employed for the calculation Depending on the order selected KO through K3 differ The coefficients are stored in the regression table on the 3rd page of the calculation file The Regression for Calculation parameter on page must be marked The peaks on page 2 for which also the multi level calibration was performed are calculated Page 5 3 8 5 3 2 Quantitative Calculation Methods 5 3 2 6 Internal Standard Method with Calibration function In the Multi Level Calibration with internal standard for each substance a regression function is calculated the dependency of the amount in the standard solution on the ratio areapeak to areays The calculation of the unknown quantities is made according to the formula Amount KO K1 PKS PKS Js K2 PKS PKS Js 2 K3 PKS PKS qs 3 in which KO K1 K2 K3j regression coefficients of the 1st 2nd and 3rd order for the component i PKS area or height of the ith peak PKS Js area or height of the internal standard in the actual run If the amount of the internal standard is different in the single standar
44. Parameter Files Data Handling File Calculation File Calculation Method Percent Peak Ret Time Pk Start Pk End Height No min min min mY 1 258 1 138 2443481 677 39 1 729 1 612 1576444 463 47 2 075 1 950 1070014 284 88 2 446 2 317 1272040 328 02 2 796 2 700 33627 10 04 3 250 3 138 105701 26 74 4 633 4 463 156164 29 51 4 967 4 867 11537 2 88 C E a E O E Wn 6669009 1822 94 Page 6 6 6 2 Print out of a chromatogram 7 Print out with LONG RPF The result list of the integration with Mode HPLC GC contains retention times peak start and peak end time peak areas peak heights and area percent of the individual peaks This evaluation is obtained when no calculation file has been defined in the data handling file when the defined calculation file does not exist or when a calculation file is defined in which the parameters Area and Percent s 4 1 3 1 are marked Chromatogram AROM0001 7 08 2001 Time 12 34 Author H Risler Notes Sample Identifier Injected on 21 02 1994 Injected at Data Processing Parameters Slice Width msec 250 Noise Threshold mV 500 Skim Ratio Parameter Files Data Handling File AROMATEN Calculation File AROMATEN Calculation Method Percent Ret Time Pk Start Pk End Height Area min min min mV 758 167 237 979 608 125 892 454 LTL 129 217 529 213 804 950 O71 829 8925 5 27 275 5290 2 275 3585 3 213 2249781 431 804 2
45. Summe2 52 38 125 1 000 1 0000E 000 1 0000E 000 0 0 Summel 1 Time Percent Amt in Std Resp fact Areal Peak Code 5 060 22 500 1 0000E 000 1 0000 000 0 0 Gruppet 7 Insert Paste Delete Graphic Help Close Save Save As Level mi The name of this calculation file is entered into the data handling file CIBA INT The evaluation 18 contains in case of peak grouping the average retention time of the peaks in the Group in case of peak summing the retention time of the first peak of the corresponding Sum is presented In case of summing the individual peaks will not be listed in the report Page 5 2 12 5 2 5 Calculation of Kovats Indices 5 2 5 Calculation of Kovats Indices The Kovats Index represents the relation between carbon content and retention time of a substance in a gaschromatographic system The determination can be performed with an internal standard as well as an external standard The standard to be used is a series of homologous hydrocarbons For this calculation a Calculation File is created which has marked as method on the first page Kovats intern or Kovats extern Time windows are defined in the peak table The Kovats index of the standards are entered against Kovats Ind and the retention times of the standards are entered against the following Time field The name of the Calculation File is entered in the Data Handling File to be used Peak T able WACHSI2 BE Code EDI
46. Table 4 7 7f Run Time 4 1 18 4 1 37 4 2 12 4 2 13 4 3 14 4 9 2 5 1 2 Page 7 8 7 Index ChromStar S S Method File Type 4 1 11 4 9 7 5 1 10 5 3 1 5 3 2 Calculation File Code 4 1 25 5 2 10 Sample Id Sample Identifier 4 1 3 4 1 12 4 4 2 4 4 9 Sample Info 4 1 13 Sample Table 4 1 13f 4 2 8 4 3 10 Sample volume 4 1 10 Save as 4 1 5 4 1 9 4 1 22 4 1 36 4 4 10 4 4 17 4 6 3 4 7 2 4 8 3 4 8 7 4 8 9 4 9 5 4 9 10 5 2 2 5 2 9 5 4 5 Save Multiple Spectra 4 6 14 Save Raw Data 4 1 37 Save Results 4 5 8 4 5 11 5 3 1 5 3 3 Save Scan Parameters 4 3 12 Scaling 4 6 16 4 7 6 Scan Parameters 3 8 4 1 1 4 1 37 Scan Parameters File 4 1 37 4 3 11 Screen Scale 4 1 18 4 1 35 4 2 6 4 3 8 4 9 3 5 1 5 Scrn Scale 3 7 Search for Sample Id 4 1 3 Select Channels 4 2 2 4 2 14 4 3 3 4 3 16 Select Multi Calibration 4 5 9 4 5 11 GPC Man Int 4 9 17 Select Batch File Name GPC Manual Int 4 9 25 Select points 4 8 11 Select Raw Column 4 6 13 Select Range 4 7 6 Select Region 4 6 5 Selection 3 8 4 2 2 4 3 3 5 1 4 Selection List 4 6 13 Settings 4 6 10 4 7 7 Setup 2 3 Short Report GPC 6 32 Show Chrom Spec 4 6 20 Show Color Palette 4 6 20 Show Column Coefficients 4 5 2 5 2 15 Show Comparison Chromatogram 4 2 7 4 3 8 Show Comparison Spectrum 4 6 20 Show Connect 4 1 2 Show Gradient 4 2 7 4 3 8 Show Grid 4 6 20 Show Peaks from File 4 4
47. The method is i Do defined in the oie acl e Tl Relative Window Update d field Calculation Units fz secon 1e1d 1n Number of Conc Levels fi this case External Standard Help Close Save Save As Minimum area height and Link to other Calc remain unchanged The unit of concentration is entered against Calculation units in this case 5 3 3 Simple Quantitative Calculation Page 5 3 11 The other parameters remain unchanged The retention time and the amount for the substance to be determined against Amt in Std are entered on the peak table The retention times of the chromatogram recorded first can be inserted in the peak table in different ways 1 This is done by clicking the point Import Peaks under File and selecting the corresponding file QUANO011 from the list of Report Files 2 The same is done by reconstructing the chromatogram in Reprocess Reconstruction clicking Results marking the appropriate peaks and pressing the Copy key The marked peaks are copied into the clipbaord They now can be entered into the peak table of the Calculation file by using the Paste key 3 Click into Grafic select the chromatogram QUANOO11 by double click drag the mouse with left key pressed over the peaks Standard amounts and peak names can also be entered in this box Quit with OK Ea ChromStar 5 x File Edit Zoom Window Help x fag Edit Files Calibration Graphic Windowname Naphth
48. These are also updated after using the submenu point Reference Window to zoom out Default returns to the display of the entire chromatogram Annotate Automatic allows the user to mark peaks in a chromatogram by numbers by retention times or by names When Peak Numbers is clicked the chromatogram with its peaks numbered is displayed Clicking Retention Times annotates the retention times above the individual peaks cp p 4 4 6 To annotate Peak Names it is necessary to have defined them in the calculation file which is specified in the data handling file used in the reintegration The submenu point Manual allows you to write individual comments horizontally or vertically on the chromatogram The annotation appears only on the current screen presentation on the print out and on the presentation transferred by means of Copy The menu point No Annotate deletes an existing annotation After clicking View the sub menu Param Results Show Comparison Chromatogram Split Show Values Show Gradient and Audit Trail appears The menu point Param Parameter permits direct modification of the integration parameters After clicking the menu point the data handling file appears as a dialog box The integration parameters can now be modified as described in 4 1 2 2 Exiting with OK starts the new calculation and the chromatogram is shown with the new integration results The card index Acquisition can be used to access the first page
49. Total amount 4 1 12 4 5 1 4 9 7 Type 4 1 11 4 9 7 5 1 10 5 3 1 5 3 2 U Undo 4 4 16 4 6 5 Universal Calibration GPC 4 9 6 Unlimited Runtime 4 2 11 4 2 13 Use Peaks 4 6 15 User Level 3 4 UV detector 4 1 31 4 1 33 4 2 5 4 3 6 5 1 8 UV spectrum 5 1 8 Vv Valley 4 4 14 Valley Valley 4 1 20 Vial 4 1 10 4 1 12 4 2 3 4 2 8 4 3 4 4 3 10 5 1 9 Vial number 4 1 10 4 1 12 4 2 8 4 3 10 5 1 9ff View 4 4 6 4 4 12 4 4 18 4 7 8 4 8 4 GPC 4 9 12 4 9 16 4 9 18 4 9 19 View Angle 4 6 11 4 6 12 4 7 7 View Cursor 4 6 10 4 6 20 4 7 8 View Document Page 4 6 4 View File Information 4 6 4 View Scan Parameter 4 6 4 Viscosity 4 9 8 5 4 8 Viscosity Average Molecular Weight 5 4 9 Viscosity Number 5 4 9 Vol 4 1 10 W W Method Autosampler Table Vial 4 1 10 Wave 4 2 3 4 3 4 Wavelength 4 1 31 4 1 33 4 1 35 4 1 38 4 2 2 4 2 5 4 3 44 3 6 Wavelength scan 4 1 31 5 1 8 Weight 4 1 12 4 5 2 5 3 5 Weight Average Molecular Weight 5 4 8 Whitewash 4 5 15 Window 3 5 Windowname 4 1 28 Windows GPC Reint 4 9 11 WINDOWS operating system 1 1 2 1 2 3 WIN INI File 2 7 Work chromatogram 4 8 5ff 6 22 Z Z Average Molecular Weight 5 4 8 Z 1 Average Molecular Weight 5 4 8 Zero Value 4 5 12 Page 7 10 7 Index ChromStar
50. accessed either with the F11 key or via the menu option Display and the submenu Screen Scale Select Screen Scale Chi C by Scale iby Value Scale fho low value 20 00 high value 500 00 Cancel After clicking By Scale an attenuation factor can be entered in the box after Scale 10 corresponds to a range of 0 313 mV 9 corresponds to 0 156 mV etc After clicking By Value a fixed mV range for the representation on the screen can be defined by entering the relevant values against low value and high value These entries are saved in CHRST32 INI and are used when opening the application again When using more than one data recording channel the box for channel 1 is the first to appear Enter the desired value and then click OK or strike the enter key and the box for channel 2 appears in which you can enter the scale of the vertical axis for the representation of the second channel etc Cancel aborts the operation chromatogram recording sets the lowest data point at the lower border of the display provided that the screen scale is defined by Scale In this way the chromatogram is still visible on the screen even if the baseline has drifted away Clicking the command Normalize or function key F12 during a Fullsize re establishes the chromatogram in full size e g after using the Unlimited Runtime option cp sect 4 2 5 While recording a chromatogram an enlargement can be made by using the mouse With Fullsize all recorded dat
51. appears on the screen EJ ChromStar Manual Base Line Correction Ioj x Ba File Order Subtract Select Points Expand Window Help la x AE ojm meee F Chromatogram BAN30201 Operation Cubic Spline 0 0 5 0 10 0 15 0 20 0 25 0 min Press F1 for Help NM Z The menu bar contains the points File Order Subtract Select points Expand Window Help Order allows the order of the approximation function to be changed Subtract deducts the calculated baseline from the complete chromatogram The menu point Order is now no longer accessible 4 8 3 Transform Baseline Page 4 8 11 The menu point File allows various options again Open allows you to select a new chromatogram The result of the baseline correction can be stored with Save as and the definition of a file name extension SLI If Subtract has not yet been clicked the message No Result File appears The original chromatogram cannot be overwritten File Information displays a box with entries about Author Sample Identifier Recording date Changes Number of datapoints Runtime Minimum and maximum mV value Print with its submenu Portrait Landscape and Chart Speed prints out the chromatogram Printer Setup allows the printer settings to be changed Copy transfers the screen contents to the clipboard Exit returns you to the File Transformation window Select points allows the entry of new points for plotting of the baseline To do this
52. can be altered in an entry box If you prefer to use the mouse instead of the function keys you can carry out all operations described here by clicking the various commands in the menus and submenus Page 3 8 3 7 ChromStar File extensions 3 7 File Extensions When recording and evaluating data with ChromStar various types of files are created to which automatically a file type description is added as an extension separated from the file name by a dot These file extensions and the files they belong to are listed below A detailed description of the file contents will be found in Chapter 4 where the ChromStar menus are described Method MET chromatographic method Data Handling INT integration parameters Calculation CAL quantitative evaluation calibration LC Procedure PRO pump control Preset INI default parameters Scan Parameters DAM parameters for the use of a diode array detector Selection RSE selection of data acquisition channels Error ERR error messages during data acquisition Slices SLI chromatogram raw data Report RPT results of the last calculation is always created together with the SLI File 2D Data DAD 2 dimensional set of data recorded by means of a diode array detector Spectrum DAS UV spectrum extracted from the 2D data set Report Format RPF report print templates RPD RPC RPL RPG GPC RMG GPC RPA for DAD data RPS for spectra GPC GIN parameters of the molar weight evaluation Data Handling GP
53. channels and serial ports cp section 4 2 1 Error The function of this menu point is to make a reconstruction if an error occurs Errors occurring during an automated analysis are registered and stored in a file ERR The field Error View can be accessed through the menu point Ervror A list with errors that may have occurred can now be printed Using Open other error files can be opened An error file can be deleted by Delete Cancel closes the Error View window When another method is opened and started the error window is automatically emptied With Automatic Noise an automatic measurement of the noise value can be performed It is necessary for this evaluation to have acquired enough data points in the computer memory If this is not yet the case the message appears Not enough data points acquired After a successful evaluation a dialog box appears with the determined value The noise value is determined so that no peak recognition occurs during this period Clicking Ok enters this value to the preset file and in all data handling files were it is entered as preset value This value is used in the evaluations if no defined Noise value is entered in a data handling file integration page at time 0 Clicking Cancel prevents this value to be entered into the parameters files 4 4 Reprocess Integration Page 4 4 1 4 4 Reprocess Integration Reprocessing of Chromatographic Data The application Reprocess offers the fa
54. chrstar data Preset directory for 2D data Param c chrstar data Preset directory for the selection of the data channels and for the report templates Program c chrstar Preset directory for the ChromStar program Printing logo no No logo appears on print outs in Transform GLPReport yes With GLPReport yes an extended protocol is created during printing in the GLP version it can be switched off with GLPReport no Pen x x 0 1 2 pen width in some print outs Preset aoe Contents of the preset table Configuration eventbox no No if an event box is not to be used Alternatively change to yes COM1 LC 1 AS 3 This part shows the configuration of LC devices In this example the instruments are connected to the RS232 interface COMI LC1 in the selection The pump LC has the device number 1 the autosampler AS device number 3 The LC devices are connected by a nine pole flat cable DISABLEAUTOSAMPCOM 1 no An autosampler is in use AllowMulti SelectForMulti 1 multiple file selection using Shift or Strg SCANPRESET Preset parameters of the diode array detector PARAM22COM 1 Instrument parameters for a controllable LC pump Document table Entries in the documentation page of the method file cp 4 1 1 4 2 ChromStar Installation COLORS Calibration pO p4 mms range normal wide ultra Board Channel 1 amp 2 1 Channel 3 amp 4 7 WindowPositions Channeldef Colcoef
55. file BENA with Method Percent Peak Win Ret Time Area Resp Area Name No No min Fact 1 0 600 904605 1 000E 000 46 68 2 0 917 41248 1 000E 000 2 13 3 1 1 750 429783 1 000E 000 22 18 Benzene 4 2 2 042 562232 1 000E 000 29 01 Toluene Totals 1937868 100 00 Formula RES PKS 1 100 PKS Wherein is RES Result of the quantitative evaluation for the i peak Area PKS Peak area or height of the i peak 5 3 4 2 Normalization Method Calculation Chromatogram C CHRSTAR DATA BENAII11 Sample Identifier B T Data Handling file BENA witht Calculation file BENANORM Calculation file BENANORM witht Method Normalization Peak Win Ret Time Area Resp Area Name No No min Fact 3 1 1 750 429783 1 000E 000 43 32 Benzene A 2 2 042 562232 1 000E 000 56 68 Toluene Totals 992014 100 00 Formula RES PKS RFj_ 100 gt PKS RF 5 3 4 Calculation Examples with Formulas Page 5 3 15 Wherein is RES Result of the quantitative evaluation for the i peak Area PKS Peak area or height of the i peak In the Normalization method only peaks mentioned in the Peak table of the Calculation file are used for the calculation 5 3 4 3 External Standard Method A after one point calibration Ein Punkt Kalibration Chromatogram BENA2111 Chromatogram of the standard solution Sample Identifier B T Calculation File BENAEXT Calculation Method Calibration Peak Win Ret Time Area R
56. fixed to far backward and the peaks areas are too large 5 2 2 Integration Parameters Page 5 2 5 5 2 2 2 The Use of the Skim Ratio The integration parameter Skim Ratio is used to perform a differentiated integration of two overlapping peaks with a common valley First the ratios a b and c b are determined in which a and c are the heights and b is the common valley height of two adjacent peaks This relationship is analysed for a b gt gt Skim Ratio gt gt c b If the skim ratio value lies between these two values an exponential skimming is carried out i e the lower of the two peaks is treated as being on the flank of the higher The skim function plots the presumed shape of the higher peak The area calculation of the peak on the flank is performed up to this line If the Skim Ratio value is outside of both values the peaks are separated by a perpendicular drop line A Skim Ratio value of 0 also results in peak separation by perpendicular drop lines The following figures show the effect of the value of the Skim Ratio parameter on the integration of two peak groups consisting of overlapping peaks with different height ratios The value is changed from 0 to 8 Skim Ratio 0 Skim Ratio 1 Page 5 2 6 5 2 2 Integration Parameters Skim Ratio 4 Skim Ratio 8 Skim Ratio values from 5 to 7 show the same result as for 4 A subpeak situated on the left rising flank of a main peak is always separated by a perpendicular drop line
57. has a black background Shift TAB accesses the previous box The entry fields can also be accessed with the mouse cursor after over striking with a depressed mouse button a black background appears and now a new entry can be made Clicking the check boxes switches between selecting and de selecting 4 1 2 2 Edit Files Data Handling Integration Page Page 4 1 19 4 1 2 2 Edit Files Data Handling Integration Page The Integration Page appears after clicking the corresponding index card fag Edit Files Data Handling testmix Acquisition Integration Time Function Value Time Programmable Function Preset 1 Noise 500 2 Threshold m 500 C 3 Skim Ratio 3 C 4 Baseline 5 Valley Valley 6 Horizontal 7 Integrate Inhibit Time Function Value c c c 8 Negative Peak 0 000 fi Noise x 100 000 9 Event 10 Cont Peak Group Insert Delete Help Close Save Save s The parameters of this page can be programmed on a run time basis and have the following function 1 Noise This parameter controls the recognition of peak start and peak end The value of the noise Microvolt second defines the maximum slope of the detector signal which is not yet recognised as peak start The noise value can be set between 0 001 and 10000 and can be changed at any run time 2 Threshold mV With Threshold a height of the detector signal is defined above which no new baseline is allowed to be se
58. identified as S the same number The parameter Minimum Time Window does not work when peak groups or sums are being computed further explanation see section 5 2 4 After clicking the Peak Table index card the table appears with all entered values and an entry line The cursor blinks in the first box of the entry line After editing the next box is accessed with TAB or by clicking in with the mouse The line is inserted into the table and sorted chronologically by clicking Insert Retention times which are part of the peak data in existing Report Files file extension RPT can also be imported into the peak table with the Import Peaks command from the menu point File Selecting of a report file and clicking OK copies the retention times into the peak table Clicking the Import Peaks command again deletes the imported data from the peak table If a line in the table is to be deleted or changed click it with the mouse It then appears in the entry line below It can now be changed using TAB and entering the desired parameter value It is entered in the table again by using Insert or deleted by clicking Delete If more than 11 lines are required the scroll bar is displayed at the right side allowing you to scroll through the table The number of the next calibration solution can be selected by clicking in the scroll bar of the box down left behind LEVEL or by entering its number Here after only the amounts of the components in the ca
59. in the chromatogram header Selection of Data Channel and Serial Port Channel Caption Dim Polarity Factor Offset Tracel C Off On cht mv T Invert roo pooo Trace Off C On Cn v M Invert hooo ooo Cancel os oa Trace Off C On mv T Invert 1 000 o 000 mv Invert 1 000 focoo Load Traced Off On Save C None HP LaserJet 4L on LPT1 Default PERE Printer Selection Instrument Control For each data channel the name can be defined under Caption and the dimension under Dim The detecor signal can be multiplied by a factor Factor and an offset Offset can be added If a detector signal is expected to have a negative polarity it can here be switched over by clicking into Polarity and Invert These entries are saved in the CHRST32 INI file in the section channeldef1 etc Also a connected LC device via RS232 COM 1 or COM 2 can be selected here The printers installed are shown in a list after clicking the downwards arrow in the Printer Selection box now the printer to be used can be selected j After clicking Options Select or when first opening the Analysis window the desired options are marked with the mouse cursor The selections can be stored in a file RSE with Save and recalled later with Load A selection can be saved as DEFAULT RSE by clicking into Default This selection is automatically carried and the Draw Baseline window is shown when Ana
60. mB A Select Files to Convert Suchen in E Demodata l c ee EE i mino sli a Qu4 0021 sli a Qum10003 sli a Qum1000 a Qu4 0031 sli fa Qud 0041 sii a Qu4 0051 sii Qu5 0011 sli a Qu5 0012 SL1 Qum10004 sli fa Qum10005 sli Qum10006 sli Qum10007 sli a Qum10008 sli Dateiname Dateityp Slice Files sli nd Abbrechen Calculation Files cal rpt Data Handling Files int rpt LC Procedure Files pro rpt Method Files met Peak Data Files rpt Result Files rpt Slice Files sli Press F1 for Help A maximum of 25 Files can be selected for conversion The original ChromStar files remain of course unchanged The conversion starts after pressing the Open key The window is automatically closed The converted files are in the same directory as the original files They can be opened e g by the windows application Notepad Page 4 5 20 4 4 5 Reprocess Calculations Convert Depending on the file type different file extensions are allocated to the converted files they are summarised in the following table File type ChromStar ASCII File extension File extension Raw data Slices SLI SLT Calculation CAL CAT Data Handling INT ITT Method MET MTT LC Procedure PRO PRT GPC Calibration GCA GCT GPC Data Handling GIN GIT Reportfile RPT Peaks Data File after Integration PKT Result File after calculation with Cal file RET GPC Di
61. of all the peaks with their names and retention times cp p 4 5 9 Clicking the respective line of a peak which is then displayed on a black background allows the calibration function of this peak to be selected and displayed as a graph Select and Order allows you to define another approximation order for this peak It is therefore possible to use different approximation orders for all the peaks of a peak table Get Concentration Table displays a table with the concentrations of the individual components for each calibration run In case of multiple injections of one calibration sample the average area is displayed as first entry above those of the individual runs Individual values in the table can be marked with Disable Then after clicking OK the calibration function is evaluated without this particular value This allows you to exclude incorrect results from a calculation initially and later to recheck this concentration by re recording the chromatogram In a multi calibration where a series of standard solutions with different standard amounts is injected more than once a regression without averaging the areas of the single injections can be carried out with the entry MultiCalNoAverage 1 in the section Calibration in CHRST32 INI If there is no entry or the entry is 0 an average value of the single areas is calculated and then the regression is carried out using the average values When executing a multi level calibration the zero leve
62. of the Data Handling File so that the parameters of this page can be changed Page 4 4 12 4 4 2 1 Reprocess Reintegration Cancel aborts the calculation with the modified parameters With Save the changed parameter Reintegration Parameter PALM2 x values are stored in Acquisition Integration the data handling Time Function Value T ime file otherwise they p Programmable Function Preset 100 Nae SSCL are lost after leaving 11 Noise 500 30 000 Noise 5 000 the reintegration 2 Threshold mv 500 C 3 Skim Ratio 3 With Results A i the results aside f 5 Valley Valley list 1S 6 Horizontal displayed on the Pee screen The table ERAT Time Function Value 1 C 8 Negative Peak 0 000 i Nase zl 50 000 ciara peak C Seven NUMOS On 10 Cont Peak Group 1 i Insert Delet times peak areas ome __ Delete and peak p i Help OK Cancel Save result calculation under Abs Rel and peak names The scroll bar at the right of the table allows you to scroll through the list if it is longer than the screen page Marked peaks can be copied into the clipboard with the Copy key cp p 4 4 6 The box disappears when OK is clicked Show Comparison Chromatogram shows or hides the comparison chromatogram The entry CompBase 1 in the section Reprocess in CHRST32 INI displays the peak baselines as well Split Split divides the window displaying the chromatogram into two sections EA The upper par
63. of the data handling file AROMATEN appears on Acquisition Integration Slice Width 10 10000 ms 250 the POTEO The value applied up till now of the Delay Time 0 99 999 min 0 000 parameter Slice Width is Run Time 0 001 10000 min i2 shown on a black ground m and can be overwritten Sn olla Here 250 should be entered Screen Scale 2 0 15 this is an appropriate value for a minimal half height peak width of 5 sec He _ Lose Swe __Saveds After this the desired run time in this case 12 minutes has to be entered This is done by striking the TAB key twice to reach the parameter Run Time and entering the value 12 Peak Parameter 5 7 15 5 1 1 Recording a Chromatogram Page 5 1 3 The name AROMATEN is automatically entered against Calculation File This file does not have to exist Now the file is saved as described for the method file If your HPLC instrument is not controlled by ChromStar or instead of an HPLC separation a gas chromatogram is to be recorded then skip the following section file name AROMATEN Now the field as shown in figure 90 appears but without entries in which the solvent composition to be pumped must be defined pro After clicking Chrom Files and its point LC Procedure enter here as well the The cursor is blinking in the Time box With the key sequence TAB defining the time TAB selects the function Solvent 60 space 40 solvent A 6
64. of the retention times of the components contained in the calibration solution by recording a chromatogram line 1 in the Autosampler Table of the Method File QUAN 3 Creating the Calculation File QUAN 4 Determination of the response factors after recording a chromatogram of the calibration sample in order to calculate amounts out of peak areas one point calibration line 2 in the autosampler table 5 Determination of the unknown amounts after recording chromatograms of the sample line 3 in the autosampler table The Method File QUAN created in Edit Files contains on the first page only one entry which is the name of the chromatogram to be recorded in this case as first characters QUAN also the Autosampler is marked and the print out Short Report is selected ig Edit Files Method quan Run Table Autosampler Table Sample Table Documentation Mal to ii volj Run Toe G Proceckes Report Fi DH 1 1 20 7 000 short rpf 1 20 7 000 short rpf 1 20 7 000 short rpf g Vial to Inj Yol Run Type LC Procedure Report File E ia w fan e x DH Files DH DH 2 Overwrite quan x Zl aa aia Lose Delete Help Close Save Save s The Autosampler Table contains the entries necessary to carry out the steps described before The run time is 7 minutes The solvent entries for the pump are defined in the LC Procedure QUAN Also the Data Handling File QUAN has to be used Page
65. of this list changes according to the selection made under Mode The following operations can be selected in the Mode field Reconstruction for presentation of chromatograms Reintegration for recalculation of peak areas Recalculation for quantitative evaluations List as Reconstruction One Point Calibration for carrying out one point calibrations It is also possible to select a part of the y axis of each chromatogram with the Low mV and High mV values Start time and End Time allows the start and end time of Page 4 5 18 4 5 4 Reprocess Calculations Batch each chromatogram to be selected The entire chromatogram is presented if all values remain 0 When a recalculation is being carried out the values for factor weight and Int Std can be re entered For one point calibrations the appropriate level number of the standard chromatogram must be entered under Level The type of print out is selected in the field Report A print preview can be seen in the box to the right With No Report no print out is generated at all After selecting a chromatogram the required operation the necessary Data Handling or Calculation File the type of Print out and the required section all entries are transferred into the Batch List with Insert Chromatograms can be removed from the list with Delete A scroll bar appears when more than 5 entries are made The chromatograms can be entered in the list quickly by double click Selection of se
66. same units as used for the standards in the Calculation File section 4 1 3 2 4 1 1 3 Edit Files Method Sample Table Page 4 1 13 Int Std The quantity of the internal standard If this differs from the quantity used in the calibration this new value can be entered here Entries must be in the same units as those of the Calculation File The parameters Factor Weight and Int Std are saved together with the chromatogram they are used in later calculations but also can be changed before carrying out a calculation Conc Level Number of the calibration sample with defined standard concentration for Multi Level Calibrations The level number entered here must correlate with the concentration defined under the same level number in the peak table of the calculation file cp p 4 1 26 Sample Info Additional sample information can be entered here for each line in the table Entry of parameters is described in 4 1 1 2 Several lines of the table can be marked by using the mouse The Up and Down key in the Shift Selected items box are used to move the lines up or downwards The Copy Cut und Paste key in the Edit Selection box can be used to copy cut or paste marked lines Insert inserts the edit line into the table Delete deletes a marked line Delete all deletes all lines The various entries in the Sample Table are used during an analysis when carrying out a number of sequential injections When a method is recalled later
67. solution Amount JS Std Amount of the internal standard in the standardlsolution PKSJS Std peak area or height of the internalstandard in the calibration run PKS i Std peak area or height of the substance 1 in the calibration run 5 3 2 Quantitative Calculation Methods Page 5 3 7 The amount of the sample is calculated according to Amount Resp Fact PKS PKS Js Amount JS Resp Fact 15 PKS js Menge IS Std Menge IS Inj in which is PKS peak area or height of the substance i PKS Js peak area or height of the internal standard Amount Inj Amount of the standard in the current run defined in page 2 of the calculation file against Amt in Std or against Int Std before calculation in the box Update Input Values if an amount other than that in the standard solution is present In a current run the value of Amt in Std in the calculation file for the internal standard will be overwritten by the value of Int Std in the sample table of the method file if this has a value other than 0 The result list of the quantitative calculation with Method Internal Standard contains also the ratio between amount of the determined substance and amount of the Internal Standard Rel For the internal standard is Rel amount J5 amount Inj 5 3 2 5 External Standard Method with Calibration function The calculation of the unknown quantities is made according to the equation RES KO K1
68. unknown amounts of the substances used for the calibration At choice the quantitative evaluation of a chromatogram can also be performed later cp section 4 5 1 5 3 4 Calculation Examples with Formulas Page 5 3 13 5 3 4 Calculation Examples with Formulas The following sections contain examples and formulas of calibrations and calculations Arbitrary standard amounts were used in the calibrations The quantitative results are mere fiction Therefore the calculations 5 3 4 3 B and 5 3 4 3 C show different results for the same chromatogram 5 3 4 1 Percent Method A with only a Data Handling file Calculation Chromatogram C CHRSTAR DATA BENAII11 Sample Identifier B T Data Handling file BENA no name of a Calculation file is entered Calculation file Peak Ret Time Pk Start Pk End Area Height Area No min min min mV 1 0 600 0 283 0 833 904605 159 17 46 68 2 0 917 0 833 1 108 41248 9 49 2 13 3 1 750 1 658 1 908 429783 203 89 22 18 4 2 042 1 908 2 217 562232 249 50 29 01 Totals 1937868 622 05 100 00 Formula RES PKS 1 100 2 PKS Wherein is RES Result of the quantitave evaluation for the i peak Area PKS Peak area or height of the i peak Page 5 3 14 5 3 4 Calculation Examples with Formulas B with a Calculation File Method Percent Calculation Chromatogram C CHRSTAR DATA BENAII11 Sample Identifier B T Data Handling file BENA with Calculation file BENA Calculation
69. windows Amt in Std In this column a known amount 1 0000E 009 to 1 0000E 009 in the unit of measurement defined under Calculation Units default value 1 can be entered of a component of a calibration mixture with the retention time specified in column 1 Herewith the response factor Resp Fact cp next column for this peak can be calculated after a calibration run Resp Fact This column contains the response factor 1 0000E 009 to 1 0000E 009 default value 1 for a specified peak or peak group The default value of 1 is used during a calibration run After a calibration run the response factors are automatically transferred to or updated in the calculation file Areal Here the user can enter a peak area obtained from a calibration run to use it for a Standard Elevation Method The area entered here is subtracted from the total peak area before the unknown amount is calculated The entry Recovery Yes in the CHRST32 INI file in the chapter Calibration changes the column header to Recovery and enables a quantitative evaluation under respect of a recovery rate The recovery rate is entered in It can be different for the different peaks The default value is 100 for a recovery of 100 Peak In this column a peak name of up to 14 characters can be entered which belongs to the retention time at the start of a line The name is printed in the results table behind the corresponding retention time Code In this column a one or t
70. 0 4 4 17 Operating elements 3 2 Operation 4 2 11 4 3 1 4 4 4 3 4 8 4 8 7 Options 4 1 2 4 2 2 14 4 3 3 4 3 16 4 6 20 8 Order 4 5 11 4 8 10 1 Oven 4 2 5 4 3 6 Oven Temp erature 4 1 33 4 1 36 Overlay Plot GPC 4 9 27 6 38 Overlayed 4 2 7 4 3 7 Overwrite 4 6 10 2 7 9 11 P Param 4 4 128 Parameter 4 8 1 4 Password 2 4 2 7 Paste 4 1 28 4 4 8 Peak 4 1 29 Peak area 4 1 23 4 1 28 4 5 1 4 5 4 4 5 5 4 9 22 5 1 5 6 6 Peak end 4 1 18 4 1 23 6 6 Peak grouping 4 1 27 5 2 10f 6 17f Peak height 4 1 23 4 1 28 4 5 1 4 5 4 4 5 5 4 9 22 5 1 5 6 6 Peak name 4 1 27 4 1 29 4 4 6 4 4 11 5 2 2 Peak Parameter 4 1 18 Peak Picking Options 4 6 16 Peak Purity 4 6 18 Peak Purity Options 4 6 19 Peak start 4 1 18 4 1 23 6 6 Peak summation 4 1 29 5 2 10f 6 17f 8 3 3 4 Peak Table 4 1 26ff 4 6 22 Peak Window GPC Cal File 4 9 7 Peaks 4 6 18 Peaks data 4 5 1 Peaks Group 4 4 15 Percent 4 1 23 6 6 Percent Absolute Defined Time Window 4 1 24 Percent method 4 1 23 Percent Window 4 1 24 GPC Cal File 4 9 7 Plate number 5 2 15 Plate height HETP 5 2 15 Plot 4 45 GPC 4 9 15 4 9 19 Points 4 6 10 Point Display 4 5 15 Polarity 4 2 2 4 3 3 Polydispersity 4 9 16 5 Polymer Standard 4 9 6 Portrait 4 8 3 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 Preset Table 2 6 4 1 1 4 1 35f 4 2 14 5 2 3 6 4 4 8 6 40 Pressure limit 4 1 32 4 1 33
71. 0 and B 40 the eluent composition is defined On clicking Insert the line is transferred into the table above No further entries are necessary in the LC Procedure file ee Edit Files LC Procedure aromaten Biel x LC Procedure Programmable Function Preset Time Function Value 1 Solvents 0 00 Solvents 65 0 35 0 0 0 0 0 2 Flow Rate ml min 1 00 3 Max Press bar 300 4 Min Press bar 0 5 Oven Temp C 20 6 Wavelength nm 254 7 Attenuate 1 00 8 Autozero Time Function Value C 9Event 2 00 Solvents s 35 C 10 Stop 11 Emission nm Delete Help Close Save Save s The file is saved by clicking File and Close and answering Yes against the question AROMATEN PRO is a new file Save it The Edit Files Window is left with Exit Edit Files Page 5 1 4 5 1 1 Recording a Chromatogram All the necessary files are now created and the method can be started as described in the following section Therefore click the menu point Analysis Chromatogram in the ChromStar window To choose the data acquisition channel and if available an HPLC instrument click click Trace 1 and in case of instrument control COM 1 Against Printer Selection the selected printer is shown The box is left with OK Selection of Data Channel and Serial Port Analog Input Dim Polarity Factor Offset Tracel Cof em mv l invert foo ooo Trace Off en mv T invert fi coo ooo Trace3 of C en v I inver
72. 0 0 C 0 0 D 0 0 Elt 7 1 Press F1 for Help The window now carries the title Data Acquisition Channel 1 The names of the Method and the currently recorded chromatogram are displayed above the data field The time elapsed since chromatogram start is displayed against Elt in the line below the data field Stop initiates the premature evaluation of data recorded up to then and generates a print out depending on the report print template e g 6 The chromatogram and the Report File are stored If an LC procedure is being used with entries in the time table processing is terminated with Stop and the current eluent composition will continue to be pumped Attention A chromatogram must not be terminated with Stop Recording will normally continue for the time specified in Run Time of the data handling file In this case the LC procedure is also normally processed until the end 4 2 5 Analysis Chromatogram Operation Page 4 2 13 Stop Int causes the integration to stop at the time this command is executed The chromatogram recorded up to this moment is stored and printed out If an LC procedure file is used with a time table it will normally be processed until the end Abort stops the data acquisition No raw data are stored no evaluation takes place and no print out is made The processing of the time table in the LC procedure is aborted and the current eluent composition will continue to be pumped The commands Stop Stop Int and
73. 031 SLI 38 aromal219e3031 SLI a aromal214b3041 SLI a aroma1219c3041 SLI aa aromal219e3032 SLI a aromal215a1021 SLI a aroma1219d1021 SL1 aa aroma1219e4041 SLI fa aromal219a1021 SLI a aroma1219d2031 SLI a aroma1221a1041 SLI a aromal219b1021 SLI aroma1219e1021 SL1 aa aroma1221a2021 SLI ja aroma1219c1021 S REMME a aroma1221a3031 SLI Filename aroma1219e2061 5L1 Filetype Sice File sli 7 Cancel EACAEAEACAE EA The drive and directory can be chosen in the top The chromatograms are listed in the middle After clicking on one of the names a preview of the chromatogram and the Sample identifier if any was entered when recording the chromatogram appear in the box to the left If another name is clicked the preview changes accordingly thus offering immediately information about the chromatogram and the Sample identifier EJ ChromStar Reconstruction olx MR File Expand Annotate View Window Help l x S 8 H ale al m z S a m e2 0 3 eee reeset min 40 Press F1 for Help NUM 7 4 4 1 Reprocess Reconstruction Page 4 4 3 After selection of its name the chromatogram appears in the full area of the application window with the integration carried out last which is saved in the corresponding RPT file The title bar carries the application name Reconstruction the menu bar contains the following menu points File Expand Annotate View Windo
74. 09 10 Mix2 260405 0052 26 04 2005 12 12 11 Mix2 260405 0062 26 04 2005 12 16 12 Mix2 260405 0061 26 04 2005 12 14 m Regression Analysis Con Coeff Std Est Error Phenol 3 4890E 001 4 9264 006 O 0000E 000 Q 0000E 000 9 9886E 001 8 9112E 002 Anisol 2 9055E 001 4 3151E 006 O 0000E 000 O 0000E 000 93 9915E 001 7 7103E 002 Ej Help Close Save Save s After determination of the calibration functions KO K1 K2 and K3 are the regression coefficients of the equation y K3 x3 K2 x2 K1 x KO where y amount of a substance x peak area or peak height of this substance These coefficients are generated in a multi level calibration cp section 4 4 2 3 and they cannot be altered manually The Multi Calibration Parameters index card of the calculation file shows the chromatogram names of the standard solutions used in the Multi calibration The table beneath shows the regression analysis of averaging the response factors or of the multi level calibration respectively The Multi Calibration Graphic index card shows a grafic display of the multi calibration By clicking with the right mouse key in the grafic display a peak list appears in which another peak to be displayed can be chosen In Reprocess Calculations a calculation file with a multi calibration can be loaded and edited via the menu procedures File and Load Multi Calibration Page 4 1 32 4 1 4 Edit Files LC Procedure
75. 18 997 M 8 18 225 137195 20 703 M 9 19 383 302324 41 051 M 10 19 742 905964 118 364 M 11 20 333 1001690 142 096 M x Show Comparison Chromatogram shows or hides the comparison chromatogram The entry CompBase 1 in the section Reprocess in CHRST32 INI displays the peak baselines as well With Show Values the retention time and the corresponding detector signal at the cursor position in the lower sector of the chromatogram are displayed in a box in the information bar At the same time the submenu points of Mode for defining peaks can be accessed View can only be accessed when a section is displayed in the lower sector Audit Trail opens a list showing all events which have happened during recording the chromatogram with date and time In the simplest case Start Chromatogram and Stop Chromatogram are mentioned here 4 5 1 Reprocess Calculations Page 4 5 1 4 5 Reprocess Calculations Quantitative Calculations Calibrations Comparisons Batch Operations This program part allows you to carry out a quantitative recalculation of a stored chromatogram to execute different ways of calibrating to make up a calculation file to carry out batch operations on a series of chromatograms to compare up to 10 chromatograms and file conversions Reprocess Calculations contains the following menu points File Calculation Calibration Displ Chrom Batch Convert Exit Window Help The buttons in the Toolbar to the left allow quick ac
76. 2 10 Height Alignment 4 8 6 GC Method Run Table Mode Help 3 6 4 1 9 4 1 7 4 1 14 Help Index 3 6 4 1 9 Gel permeation chromatography GPC 4 9 1ff 5 4 1ff 6 29ff Get Concentration Table 4 5 9 4 5 12 Get Peak Table 4 5 9 4 5 12 GLP Options 3 1 3 4 GLP Version 2 4 2 6 2 7 3 1 3 4 3 5 GPC 4 9 1ff 5 4 1ff 6 29ff Method Run Table Mode 4 1 7 5 4 2 GPC Batch 4 9 24 GPC Batch Reintegration 4 9 26 GPC Batch Manual Integration 4 9 24 GPC Calibration 4 9 6 4 9 20 5 4 4 GPC Calibration File 4 9 5 4 9 6ff 4 9 13 4 9 17 4 9 20 4 9 22 5 4 2 5 4 4 6 29 6 36 6 37 GPC Data Handling 4 9 1 GPC Data Handling File 4 9 1ff 4 9 13 4 9 15 4 9 17 4 9 26 5 4 2 5 4 6 6 29 GPC Edit Files 4 9 1ff GPC Integration DH File 4 9 3 GPC Manual Integration 4 9 17 GPC Overlay Plot 4 9 27 6 38 GPC Reintegration 4 9 13ff 5 4 7 6 32 6 33 GPC Reprocess 4 9 13ff GPC Regression Analysis 6 30 6 36 Gradient Programming 4 1 32 5 1 6 Graphic s 4 1 28 4 5 8 4 5 9 4 5 1 1 4 6 3 4 9 1 1 4 9 15 4 9 19 5 4 5 Grid 4 6 20 4 7 8 4 9 28 Grid visible 4 6 10 Help Mode 3 6 Help pages 2 3 3 6 Horizontal 4 1 20 GPC DH File 4 9 3 HPLC Method Run Table Mode 4 1 7 4 2 4 4 3 5 I Icon 2 4 2 6 3 5 Import Peaks 4 1 28 4 9 9 Information 4 7 2 Inj 4 1 10 4 1 12 Inject 4 1 33 Installation 2 1f Autosampler 2 6 5 1 9 Pump LC 22 2 6 4 2 4 Eventbox LC 427 2 3 2 6 Inte
77. 2 11 4 2 13 4 3 14 5 1 11 Absolute 4 5 2 Absolute Window min 4 1 24 Absorbance 4 1 36 Acquisition Page 4 1 17f 4 4 12 5 1 2 GPC DH Datei 4 9 2 Activate 4 7 3 Actualize 4 2 10 Addition 4 8 7 6 6 22 A D converter board 2 2 4 2 2 Align Intensity 4 7 6 Align Wavelength 4 7 6 Alignment 4 8 6 alpha Mark Houwink constant 4 9 3 4 9 4 5 4 9 Broad Standard Calibration 4 9 21 Amount in Standard GPC Cal File 4 9 7 4 9 22 Amt in Std 4 1 27 4 1 29 4 5 5 4 5 10 5 3 6 Analog Input 4 3 3 Analysis start 4 2 8 4 2 1 1 4 3 13 5 1 5 5 1 11 Analysis 4 2 1 4 3 1 5 1 4 5 1 1 1 5 1 12 5 2 3 5 3 10 5 4 2 Angle 4 5 14 Annotate 4 4 5 4 4 1 1 4 4 17 4 6 21 5 2 2 GPC 4 9 15 4 9 19 6 33 Append 4 8 7 Area Height 4 1 23 Area 0 4 1 27 4 1 29 Area GPC 5 4 9 Arithmetics 4 8 5ff Arrange Icons 3 5 ASCII File 4 5 19 Attenuate 4 1 33 4 1 35 Attenuation 4 2 5 4 3 6 Autofit 4 8 12 Autoincrement 4 1 37 Automatic Noise 4 2 14 4 3 16 5 2 3 Autorange 4 4 5 4 4 1 1 Autosampler 2 6 4 1 7 4 1 10f 4 1 36 4 2 8 4 2 1 1 4 2 12 4 3 10 4 3 15 5 1 9ff 5 3 1 f Autosampler Table 4 1 10f 4 2 8 4 3 10 Autozero 4 1 33 4 2 5 4 3 6 Average 4 1 38 Average calculation of response factor 4 1 31 4 5 7f 5 3 1 6 10 Average Molecular Weight 4 9 16 5 4 8 5 4 10 B B Solv 3 9 4 1 32 4 2 3 4 2 4 4 3 4 4 3 5 Baseline 4 1 19 4 3 13 4 4 14 5 2 7 Transfor
78. 260352 419 467 4408528 813 183 1684454 274 883 1655972 173 WW Ne Pe oO SN WNNRPRrR RP OC sa B WNNH FH OS 1 2 3 4 5 6 7 8 12276886 2124 6 2 Print out of a chromatogram Page 6 7 7 Cont Chromatogram AROM0001 Date 7 08 2001 Time 12 34 s 8 8 8 8 8 28 s 7856 O 125 ov o9 ri og 4 Page 2 of 2 Page 6 8 6 2 Print out of a chromatogram 8 Print out of the results list of a quantitative evaluation calculation file with Method External Standard using the report print template TABLE RPL Chromatogram qu5 0012 7 08 2001 Time 12 38 Author H Risler Notes Test CS 3 2 Quant Auswertung Sample Identifier Testmix5 Injected on 14 12 1993 Injected at Data Processing Parameters Slice Width msec 500 Noise Threshold mV 500 Skim Ratio Parameter Files Data Handling File Qul Calculation File QU1 ML Calculation Method External Standard Calibration Method Multi Calibration Peak Win Ret Time Area Resp Absolute No No min Fact Arb u 992 928661 1278E 05 31 361 Toluol 250 1288667 4635E 05 91 193 Ethylbenzol 853263 8070E 05 59 352 Cumol 896905 5173E 04 184 034 Butylbenzol 636728 2879E 04 848 Hexylbenzol 4604224 788 6 3 Print outs of calibrations Page 6 9 6 3 Print outs of calibrations 9 Result list of a one point calibration Chromarogram qu4 0011 Date 14 03 1996 Time 9 05 Auchor
79. 3 Regression Analysis KO 4 3413E 000 Kl 6 7764E 005 K2 0 0000E 000 K3 0 0000E 000 0 995767 4 360044 Coeff of Correlation Std Error of Estimat 6 3 Print outs of calibrations Page 6 13 13 Graphical presentation of the Ist order calibration function here only shown for the second peak MULTILEVEL CALIBRATION Date 14 03 1996 Time 9 12 Peak Name Ethylbenzol Time Window 2 26 w z Y E lt 1400 0 1600 0 1800 0 Area x1000 Coefficients of Least Square Fit to a LINEAR equation KO 4 34138 000 Kl 6 7764E 005 Coefficient of Determination 0 991551 Correlation Coefficient 0 995767 Page 6 14 6 4 Print outs of multiple chromatograms 6 4 Print outs of multiple chromatograms 14 3 Dimensional presentation of several chromatograms Print template RPD Display Chromatograms 3 15 03 1996 qu4 0011 qu4 0021 qu4 0031 qu4 0041 qu4 0051 6 4 Print outs of multiple chromatograms Page 6 15 15 Presentation of 3 stacked chromatograms Print template RPC Compare Chromatograms 17 04 1996 Chromatogram qu4 0011 Sample Identifier Testmix5 Chromatogram qu4 0012 Sample Identifier Testmix5 Chromatogram qu4 0013 Sample Identifier Testmix5 Page 6 16 6 4 Print outs of multiple chromatograms 16 Print out of chromatograms with multi channel data acquisition Print template RPD Date 18 04 1996 Timer 12 21 Author H Risler Notes Test CS40 AS LC22 2
80. 3 4 4 10 4 4 17 Show Status 4 6 20 4 7 8 Show Values 4 4 6 4 4 7 4 4 12 Sight 4 6 11 Single 4 1 8 Skim Ratio 4 1 19 4 1 36 5 2 5 Slice Width 4 1 17 4 1 35 4 1 37 4 9 2 5 1 2 Smoothing 4 8 1 Solv A B C D 3 7 4 2 4 4 3 5 Solvent 4 1 32 Solvent composition 4 1 32 4 2 4 4 3 5 Solvent gradient 4 1 32 Source 4 2 2 Spectrum 3 8 4 3 9 4 7 1 4 7 2 Spectrum 4 6 16 Spectrum Info 4 3 9 Split 4 4 6 4 4 12 Split Screen 4 6 9 Stacked 4 2 7 4 3 7 4 6 10 Standard 3D Plot 4 5 15 Standard deviation 4 5 8 Standard elevation method 4 1 27 Standard Report Print template 2 8 4 1 8 6 5ff Start 3 7 4 2 11 4 3 13 Start Line 4 2 8 4 3 10 Start Time GPC DH File 4 9 4 Start Value GPC DH File 4 9 4 Status Line 4 3 16 Status visible 4 6 10 Stop 3 7 4 2 11 4 2 12 4 3 14 5 1 5 Stop Int 4 2 11 4 2 13 4 3 14 Stop Pump 3 7 4 2 4 4 3 5 Subtract 4 8 9 4 8 10 Subtraction 4 8 7 Swap 4 8 2 4 8 4 Syringe Vol ume 4 1 36 ChromStar 7 Index Tangential Baseline 4 4 14 Tangential Skim 4 4 15 Threshold 4 1 19 4 1 35 4 6 7 Tile 3 5 Time 4 1 24 GPC Cal File 4 9 8 Time 1 Time 2 4 1 26 4 1 27 Time Alignment 4 8 6 Time Unit 4 1 37 Toolbar 3 2 4 1 37 Toolbar buttons 3 2 4 1 2 Transform 4 8 1ff 6 21 Transform Arithmetics 4 8 5ff Transform Baseline 4 8 8ff Transform Curve Fitting 4 8 12 Transform Modify 4 8 1ff Transmission 4 3 13 Trapezoidal 4 9 3 to 4 1 10 5 1 10
81. 3 525924 4 0000E 000 2014605 503651 5 0000E 000 2410053 482010 For benzene the regression function of the 1 order for the dependency of the amount in the standard solution on the ratio areapeak to areays is calculated The regression coefficients are saved in the Regression Table of the calculation file BENAISML CAL as KO and K1 In the Method Table the parameter Regression for Calculation is automatically ticked Toluene is the internal standard no regression function is calculated for this If the amount of the internal standard is different in the single standard solutions as here it is the area of the internal standard is normalized to the amount of the internal standard in the first calibration solution PKS IS Norm PKS Js amount Js 1 amount JS Calculation of the unknown amounts Chromatogram BENA8121 Chromatogram of the mixture with unknown amounts Sample Identifier B T Calculation File BENATISML Calculation Method Internal Standard Calibration Method Linear Fit Internal Standard 3 00 In the sample solution the internal standard was added in the three times amount Peak Win Ret Time Area Resp Absolute Rel Weight Name No No min Fact AU 1 1 1 758 1102628 _ 3 104E 000 1 03 0 0 Benzene 2S 2 2 050 1558244 _ 2 772E 000 0 92 0 0 Toluene Totals 2660872 3 104E 000 0 0 Formula The unknown amounts are calculated according to Amount KO K1 PKS PKS Js K2
82. 3 Carrying out an Analysis with the Autosamplet 5 1 11 5 1 4 4 Trouble shooting and error messages ceeeceeeeeeeeeeeeeteeeeees 5 1 12 5 1 5 Short Instruction Guide ayes crass ccrane ace wossedecuntnactenenatanessmbenzoaness 5 1 13 5 2 Evaluating a Chromatogram cccccccessseceeeeeeeeeeeseeeeeeeeeees 5 2 1 5 2 1 Automatic Reintegration wascacccesacecegend ivevendes eee seeiatiowseateceenes 5 2 1 5 2 2 A Description of some Integration ParameterS eee 5 2 3 5 2 2 1 The Influence of the Noise Value on the Integration 5 2 3 5 2 2 2 The Use of the Skim Raion asa cata tae Cel ale te 5 2 5 5 2 2 3 The Baseline Parameters caw veiserctesestsseeeneontncscae cee saveeevae 5 2 7 5 2 3 Manual Intesrationia esnnwatainctiastin rane 5 2 8 5 2 4 Grouping and Summation of Peaks 20 0 0 eee eeeteceeeenteeeees 5 2 10 5 2 5 Calculation of Kovats Indices ccccecesseceeeseeceeeeteeeeeneees 5 2 12 5 2 6 Calculation of Column Coefficients ccceeeeeeeeeeteeeeeees 5 2 15 5 3 Quantitative Calculation ccccccccccccccccccececceeeeeeeeeeesssseeeeees 5 3 1 5 3 1 Calibration Methods iscscstestneannstonssssrecceandsecwneetinendatacnessientneauats 5 3 1 5 3 1 1 One Point Calibration ssesseeeeseeeessesessseressserssssressseessseresssseess 5 3 1 5 3 1 2 Averaging a number of One Point Calibrations 5 3 1 5 3 1 3 Multi Level Calibration ccecc cee ceeeccesecc
83. 4 4 1 1 1 Edit Files Method Run Table ssseeeeenseeeeseesssssreessserssse 4 1 7 4 1 1 2 Edit Files Method Autosampler Table 0 00 0 eee 4 1 10 4 1 1 3 Edit Files Method Sample Table 2000 eee eeeseeeeeeeeees 4 1 12 4 1 1 4 Edit Files Method Document Table eee eeeeeeeees 4 1 14 4 1 2 Edit Files Data Handling oe ceeessecceestneeeeetnteeeeees 4 1 17 4 1 2 1 Edit Files Data Handling Acquisition Page 00 4 1 17 4 1 2 2 Edit Files Data Handling Integration Page 4 1 19 4 1 3 Edit Files Calculation sa ccxessencteeacnved aos snesausxscabcceterdaassatisceaees 4 1 22 4 1 3 1 Edit Files Calculation Method Table 00 0 0 eeeeeeeeees 4 1 22 4 1 3 2 Edit Files Calculation Peak Table 0 eee eeeeeeeeeeteeees 4 1 26 4 1 3 3 Edit Files Calculation Regression Table eeee 4 1 31 4 1 4 Edit Files LC Procedure scccacriczascoscenusnsenteapesbsnmiaseeee bepetexesaes 4 1 32 AS Edit Fl s Preset srren haia a a ete 4 1 35 Page i 2 Contents 4 2 4 2 1 4 2 2 4 2 3 4 2 4 4 2 5 4 2 6 4 3 4 4 4 4 1 4 4 2 4 4 2 1 Reprocess Reintegration 4 4 2 2 Reprocess Manual Integration 4 5 4 5 1 4 5 2 4 5 2 1 Reprocess One Point Calibration 4 5 2 2 Reprocess Multi Calibration Analysis Chromatogram Data Acquisition Instrument Control eeeeeeeeereeees Analysis Chromatogram Options Select 0 0 0 ee Analysi
84. 4 2 5 4 3 6 End Time GPC DH File 4 9 4 End Value GPC DH File 4 9 4 Enhancement 4 8 2 4 8 4 Error 3 8 4 2 14 4 3 16 4 9 12 5 1 13 Evaluation range GPC 4 9 4 5 4 6 Event 4 1 20 4 1 33 Eventbox 2 3 2 5 2 6 4 1 20 Expand 4 4 5 4 4 11 4 4 13 4 4 17 4 5 14 4 5 16 4 8 4 4 8 6 4 8 8 4 8 9 4 8 15 GPC 4 9 17 GPC Overlay Plot 4 9 28 Exponential Baseline 4 4 14 Exponential Skim 4 4 15 Export 4 4 4 4 4 10 External Standard 4 1 23 5 3 5 6 8 GPC 4 9 7 4 9 22 External Standard Method 5 4 5 External Standard Method with calibration function Regression 5 4 7 F Factor 4 1 12 4 2 2 4 3 3 4 5 2 4 8 1 4 8 3 5 2 16 5 3 5 File s 4 1 5 4 4 3 4 4 7 4 4 10 4 4 17 4 5 3 4 5 6 4 5 8 4 5 11 4 5 14 4 5 16 4 6 2 4 7 1 4 8 3 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 GPC 4 9 5 4 9 9 4 9 10 4 9 14 4 9 19 4 9 21 4 9 22 4 9 24 4 9 28 File Arithmetics 4 8 5 6 22 File extensions File suffix 3 8 4 5 19 File Information 4 1 5 4 4 3 4 4 10 4 4 17 4 8 3 4 8 6 4 8 9 4 8 11 4 8 13 GPC 4 9 5 4 9 9 Filled 4 6 10 File Name 1 1 3 8 Filter 4 8 2 4 8 4 Filter 4 7 5 Flow 4 2 3 4 3 4 Flow Rate 3 7 4 1 30 4 1 33 4 2 4 4 3 4 4 3 5 Fluorescence detector 4 1 33 4 1 35 4 3 6 Fullsize 4 2 6 4 2 13 4 3 9 Function keys 3 7 4 2 4 4 2 11 4 3 5 4 3 13 Page 7 4 7 Index ChromStar G H G Calculation Peak Table Code Hardware components 2 1 4 1 27 5
85. 5 32 30 883 30 692 31 042 3224 115 08 1 199 33 31 225 31 042 31 592 846682 1249 00 1 907 34 32 758 31 5932 31 867 45738 11 54 9 103 3 32 342 32 075 32 683 5857524 922 32 13 194 3 32 683 32 683 32 992 94283 38 43 0 212 37 33 167 33 100 33 342 28748 12 62 0 058 3 34 933 33 667 34 256 2001137 319 59 4 507 39 34 425 34 325 34 508 42755 10 79 0 096 40 34 628 34 508 34 733 84070 17 28 0 189 41 34 867 34 733 35 100 1686362 30 87 0 379 42 35 358 35 100 35 875 340244 49 60 0 766 43 37 375 37 167 37 483 650035 122 26 1 464 44 37 542 37 483 37 758 233331 64 92 0 539 45 38 125 37 092 38 583 3738438 820 18 6 422 46 39 033 38 792 39 317 952909 132 26 2 144 a 39 558 39 467 39 717 45609 42 32 0 103 48 42 833 42 267 42 750 210715 27 06 0 475 43 892 43 525 44 300 1150778 120 38 2 592 1a aaaaaaaairirai a 44396080 7284 22 100 000 Page 6 18 6 5 Print outs processed in chapter 5 18 Result list with grouping and summation Chromarogyam IBAO101 Date 18 04 1996 Sample Identifier Ciba Basel R Injected on 05 03 87 Injected at Rara Processing Parameters Slice Width msec 00 Noise Threshold mV 500 Skim Ratio Paramecer Files Data Handling File Calculation File CIBA3 Calculation Method Percent Peak Win Ret Time Resp Areat No No min Pact 0 308 90829 1 000 0 208 0 467 1709127 1 000 3 850 0 258 380028 1 000 0 656 1 200 5 510 0 1 000 12 411 1 467 32243 1 000 1 199 2 617 125398 1 000 0 282 2 250 24880 3 000
86. 5 3 10 5 3 3 Simple Quantitative Calculation The Data Handling File QUAN contains the Edit Files Data Handling quan Acquisition Integration integration parameters and the name of the Slice Width 10 10000 ms Calculation File to be used Delay Time 0 99 999 min Run Time 0 001 10000 min Calculation File Screen Scale 2 0 15 Peak Parameter 5 7 15 Help Close Save s Once these files except the Calculation File have been set up and saved the method QUAN can be started from the point Analysis Chromatogram in the ChromStar window First the chromatogram here QUANOOI1 is recorded in order to determine the retention times It is not important for the evaluation of the first chromatogram QUANO011 that the Calculation File does not yet exist a percent method will be performed Following upon this the Calculation File QUAN is created Edit Files Calculation quan geg 1 eh field on e rs age Method Table Peak Table Regression Table of P r Calculation Base Time Window P Pi Calculation file Percent Absolute C Defined QUAN is defined Method z5 whether eak C Percent Normalization Parent areas a a C Intemal Standard External Standard Mesa Taian mo keehi areto be C Kovats Intern C Kovats Extem a use as Univ Resp Factor 11 0000E 000 calculation basis Minimum Area Height 100 I Regression for Cale
87. 7 97 Low mi 10 32 m Window 3 Window 4 Start Time 0 00 Start Time 0 00 End Time 3 00 High m 239 92 Low mi 1 14 19 Cancel 4 5 4 Reprocess Calculations Batch Page 4 5 17 4 5 4 Reprocess Calculations Batch The menu point Batch of the Reprocess Calculations window allows the reprocessing of whole groups of chromatograms After clicking Batch a dialog box appears in which all operations to be carried out have to be defined Batch Arge s prej Selec Fier ui hod Seeman File ete ater aea oat Aimi i k Aum hal r HGE oh _ FT Se aje A Presi oe sateen OTT i HET ph Pa m ra ET d sectors F Pla Herding Fig TERTHIEJ ke a ra E H Hih m rre HOE okt al aA AR l ji aij iL i t a brie HTT k ro betes Tes Thal a Flew Bek int Sd O Racine fi if fii C Rerisgimn i fecabdisen Repat Fie raei T w TO ke Fo Foa The i ta Ta M ii beir HE i wri a y 1 te im ti r Sa ab ab o ab ibelia a Cande Batch Lit 7 i T Hakem BF wh The current directory is displayed after Search in it can be changed via the downwards arrow to the right The chromatogram to be processed is selected under Slice File the corresponding Report Files must be available there is no separate indication here Under Second File the Data Handling File required for reintegration or the Calculation File required for recalculation are selected The title
88. 75 xl 4 4 1 Reprocess Reconstruction Page 4 4 7 Show Comparison Chromatogram shows or hides the comparison chromatogram The entry CompBase 1 in the section Reprocess in CHRST32 INI displays the peak baselines as well Split divides the chromatogram display window in two areas with a 1 2 He proportion The upper area is used to display the entire chromatogram the lower area is used to display enlarged fragments of the chromatogram To do this first open a rectangle in the upper area Its contents are displayed in an enlarged form in the lower area The area covered by the rectangle is shown on a light background in the upper chromatogram EJ ChromStar Reconstruction Iof x IA Fie Expand Annotate View Window Help l x gja lla lie xl m rm ez ea 2 my Chromatosram FMTEST 800 600 400 200 10 00 11 25 12 50 13 75 15 00 16 25 17 50 18 75 20 00 min Press F1 for Help NUM Ui A further enlargement is now possible it can be done in one of two ways 1 The enlarged section displayed on the lower window sector can again be enlarged by opening a rectangle as described above and confirmed with the right mouse button In the upper chromatogram the area on a light background is changed accordingly 2 A new section can be defined in the upper chromatogram Clicking the upper chromatogram with the right mouse button allows you to erase the section Page 4 4 8 4 4 1 Reprocess Reconstruction
89. 8 Data Handling file 4 1 17ff 4 4 9 4 4 12 4 5 17 5 1 2 5 2 1 5 3 10 5 4 1 6 2 Data Handling file GPC cp GPC Data Handling Datei 4 9 1 ff Data Point 3D Plot 4 5 15 Data Points 4 7 3 Data acquisition 4 2 1 4 3 1 5 1 1 6 5 GPC 5 4 1 Data recording channel 4 1 8 4 2 2 4 3 3 ChromStar 7 Index Default 4 2 2 4 3 3 4 4 5 4 4 11 4 4 17 Define Details 4 1 14 Delay 4 1 10 Delay Time 4 1 17 4 9 2 Delete 4 1 5 4 1 11 4 1 21 4 1 28 4 1 34 4 4 15 4 5 7 4 5 18 4 9 4 4 9 5 4 9 9 4 9 26 Density Plot 4 3 8 4 6 8 Ist 2nd Derivative 4 8 1 6 21 Detector signal 4 1 18 4 2 1 4 2 2 4 2 6 4 3 1 DH 1 to DH 4 4 1 11 Differential 4 9 14 Differentiation 4 7 6 Dilution factor 4 1 12 Dim 4 2 2 4 3 4 3 Dimensional representation 4 5 13 6 14 Diode array detector 3 8 4 1 37 4 3 1 4 6 1 Display 4 2 6 4 3 7 4 6 6 4 7 6 GPC Overlay Plot 4 9 28 Displ Chrom 4 5 13ff Display Data Density 4 6 11 Display Settings 4 6 10 4 7 7 Distribution Plot Mn Mw Mz Mz 1 4 9 13 4 9 19 4 9 27 5 4 7 5 4 9 6 33 6 35 6 39 Documentation Table 4 1 14 3D Plot 4 3 7 4 5 13ff 4 6 8 4 6 10 6 14 Draw Baseline Channel etc 4 2 2 5 1 5 E Edit 4 4 16 4 6 5 Edit Files 4 4 1ff 5 1 1 5 1 6 5 1 9 5 3 9 6 1 Edit Files GPC 4 9 1ff Edit Scan Parameters 4 3 12 Effective molecular weight 4 9 8 elt elapsed time 4 2 3 4 2 12 4 3 4 4 4 13 Emission 4 1 33 4 1 36
90. 8 NT Windows 98 2 edition will work but is not recommended The PCI A D converter boards require as operating system Windows NT 2000 XP or Me The maximum user comfort offered by the windows technology is hereby achieved such as the use of easily accessed menus and dialogue boxes and above all the possibility of carrying out various jobs simultaneously for instance recording of a chromatogram while reintegrating an existing file and documenting the results It is therefore advisable although not essential to be acquainted with WINDOWS when using the ChromStar software this operating manual describes the steps necessary in sufficient detail Normally the most simple input method is described a combination of keyboard and mouse entries This operating manual is not intended as an introduction into chromatography Beginners are advised to consult the relevant literature The windows operating system exists in different languages Depending on the language in use some menu procedures appear in other languages than described in the manual ChromStar always uses easy understandable menu items in English The on line help pages are availanble in English or in German This manual describes the ChromStar version 6 Changes made in later versions will be recorded in supplements to the manual Chapter 2 deals with the installation of the ChromStar software and the accompanying A D converter card Chapter 3 describes the structure of th
91. 8 events per channel can be switched 10 Continous Peakgroup This parameter defines a time window in which a peaks group is evaluated as one peak All parameters are programmable on a time basis Enter the start time against Time in the time table the number of the requisite function against Function and the end time against Value or the desired value for the functions 1 3 Program as follows Click the Time box at the right in the entry line with the left mouse button and enter the desired time in units of 0 001 min from 0 to the time defined against Run Time on the Acquisition Page Jump to the next box with TAB Select the desired function by entering its number as given in the table at the left or click the function in the table itself Jump to the last box with TAB and enter the value for the defined function for the functions 4 to 8 a time in minutes For the Event function programming against Value is done by entering the event number space and on 1 or off 0 The line is inserted into the table above by clicking Insert If a line needs to be deleted from the 4 1 2 2 Edit Files Data Handling Integration Page Page 4 1 21 table click it with the mouse The line now appears in the table on a black background and in the edit line it can now be deleted with Delete Entries in the table are sorted according to their time Attention A total of 40 values of the same parameter and 75 values of different parameters can be def
92. A D converter board of the ChromStar Integrator SCPA LC42PCl i 02 50 20 23 01 3 pupi Oupsi Oust i e 3 pim mi o oos fi fy nc no connection Page 2 4 2 ChromStar Installation 2 3 Installation of the ChromStar Software Be sure that before you install the ChromStar software you first make a copy and store the original diskette or CD in a safe place Before installing the ChromStar software the WINDOWS operating system must have been installed Follow the instructions for installation of this in the WINDOWS manual Insert the ChromStar CD in the CD drive and start the installation by double click on setup exe The ChromStar Setup window appears The Next Back or Cancel keys lead through the installation The installation program is self explanatory In the GLP configuration window you can choose wether you want GLP support or not In the GLP version the user s name and a password need to be entered The password appears during entering as The user whose name is entered at this moment will be the superuser In every following ChromStar start the user s name and his password are asked for so that no unauthorised persons can use ChromStar System Configuration x Please select your System Configuration ChromStar Version ChromStar DAD 6 3 GPC x m Board Type C ISA C PEI ENB Program Path C Chrom
93. Analysis application the Toolbar shows besides the buttons to the right for the ChromStar main applications a number of buttons for quick and easy operation These are at the beginning of the work in Analysis mainly grey In the following sections the menu points and buttons are described in the sequence in which they are normally used when carrying out an analysis If independent chromatograms should be recorded the application Chromatogram can be opened once more via Window and New After entering the data channel and printer if necessary to be used in the second Analysis window in the Selection box a method can be started via Method and entering a file name Up to four independent Analysis Chromatogram applications can be opened Using multi channel data acquisition in one Analysis Chromatogram window the data handling files entered in the method file starting with DH 1 DH 2 etc are used for data acquisition and integration All chromatograms are recorded with the slice width and the run time entered in DH 1 Page 4 2 2 4 2 1 Analysis Chromatogram Options Select Channels 4 2 1 Analysis Chromatogram Options Select Channels With Options Select Channels the data recording channel to be used Trace 1 to Trace 4 is defined whereby a maximum of 2 or 4 channels depending on the number of A D converter boards installed can be selected to operate in parallel The serial number of the A D converter board is stored
94. BENAIS Calculation Method Internal Standard Calibration Method One Point Peak Win Ret Time Area Resp Absolute Rel Weight Name No No min Fact AU 3 1 1 750 429783 2 62E 001 2 003E 001 1 00 0 0 Benzene 4 IS 2 2 042 562232 3 63E 005 2 041E 001 1 02 0 0 Toluene Formula The amounts under Absolute are calculated according to the formulas Amount peak Resp Fact peak areapeax area rs Amount js Resp Fact js area ys amount jg sta AMOUNT 1s tnj If another amount of internal standard is injected in the sample mixture than in the calibration mixture the area js is multiplied with the ratio amount js sta amount 1s mj In this example the amount of internal standard is in the standard solution amount js sta and in the solution with unknown amounts amount js mj the same 20 For the internal standard the ratio Rel of calculated amount to injected amount is calculated Rel amount js amount 1s jy B after calibration with average values Calibration with averaging Calculation Mode Internal Standard Nr Result File Name Chromatograms of the standard solution a y b enal11l e The same standard solution is injected twice 2 bena6121 Toluene is the internal standard IS Peak Table Peik Nemet Benzon a Time Window 1 75 Std Amt Area Area IS Area 2 0000E 001 0 764 429783 2 0000E 001 0 763 420346 Average Area Area IS 7 6377E 001 Standard Deviation 9 2812E 004 0 1 5 3 4 C
95. C Calibration GCA calibration data for molar weight evaluation GPC Batch GBA evaluation of series of GPC chromatograms 4 1 Description of the ChromStar Software Edit Files Page 4 1 1 4 Description of the ChromStar Software 4 1 Edit Files Creating and Editing of Files The application window Edit Files permits you to create new or change existing files necessary for recording and evaluating a chromatogram or a 2D data set The files required for carrying out the analysis integration and quantitative calculation have the following function Method The Method File contains the necessary entries for the execution of an analysis Registered here are the name of the chromatogram the processing HPLC GC or GPC the type of print out and the names of the LC Procedure and Data Handling files When an autosampler is used the sequence and vial handling used are specified here Further on entries can be made here for each injection about the type and quantity of the sample It is also possible to write notes concerning the analysis in a document table Data Handling In the Data Handling File parameters are defined for recording of the raw data and for the integration The page with data acquisition parameters contains for example the run time of a chromatogram the slice width time between the data points and the data storage parameters For quantitative calculations a Calculation File can be specified here The integration parameters such a
96. ChromStar 6 Manual SCPA Software fiir Chromatographie und Prozess Analytik GmbH Am Weidufer 32 D 28844 Weyhe Tel 0421 802806 Fax 0421 890346 Email info scpa de Internet www scpa de Edition March 2006 for ChromStar 6 2 and higher versions Contents Page 1 1 Contents 1 MME O CUE IO Mofo stencs 3 eiact eater recm eatin dascth ee Geoateasaatonmeneceumenness 1 1 2 Installation as cco Se acters esac eh age ere ede aaa 2 1 2 1 Description of the Hardware Componentts cceeeeeees 2 1 2 2 Installation of the A D Converter Board cceeceesceeeeeeees 2 2 2 3 Installation of the ChromStar Software cccceesssccceeeeeeees 2 3 3 Description of the ChromStar Window 00 3 1 3 1 ChromStar Structure ssessseseesseeeseseesserssereressreesssresssreessseesse 3 1 3 2 Operating Elements 29 nccncesrostorpuacediaeearunantenaccpsemmndcanuronccindooes 3 2 3 3 Menupoint GLP Options sssssssseesssesessserssseressssressereessseeessressee 3 4 3 4 Menupoint Window 3 06 eee ae 3 5 3 5 Menupoint Helpts on anesthe naa Cases 3 6 3 6 Function IE YS ea ena E AE A 3 7 3 7 Pale EXE MSI ONS nna anae eo aera R r R EE 3 8 4 Description of the ChromStar Software 000 4 1 4 1 Edit Files Creating and Editing of Files seeensseeessesseesssreessrerssseresssses 4 1 1 4 1 1 Edit Files Method sseseeensseseessseesseeesssseeesserssseressseresseeessssee 4 1
97. H Risler SCp Notes Test CS 3 2 Quant too mans eth Coan Auswertung Sample Identifier Testmixs Injected on 7 12 1993 Injected at Rata Processing Parameters Slice Width msec 500 Noise Threshold mV 500 Skim Ratio Parameter Files Data Handling File QUX Calculation File QU4 Calculacion Method Calibration Win Ret Time Area Resp Std Amt No min Pact 008 319709 12788 05 10 000 Toluol 308 464142 46358 05 30 000 Ethylbenzol 600 293817 8070B 05 20 000 Cumol 329533 1 51738 04 50 000 Butylbenzol 625 233212 4 2879E 04 000 Hexylbenzol 1640412 Page 6 10 6 3 Print outs of calibrations 10 Result list of a multi point calibration here only shown for the first two peaks Multi Calibration Table Date 14 03 1996 Time Calculation Mode External Standard Standard Sample Table Nr Report File Name qum10003 1 2 qum10004 3 qum10005 4 qum10006 5 qum10007 Peak Table Peak Name Toluol Time Window 2 02 Std Amt Area SSS SSSSST TTP SSSSSSSTCSHMASASSSREHAHSKKAKERRERBRBTESS SCH SEAKSoK BOBS ESBSERESESESEIE TS 0000E 001 1148872 0000E 001 1137723 0000E 001 1154783 0000E 001 1152789 0000E 001 1162010 Regression Analysis Average Area 1 1512B8 006 Standard Deviation 8 9339E 003 0 8 Confidence Region 1 0542E 004 0 0000E 000 3 4745E 005 Name Ethylbenzol Time Window 2 30 Std Amt Area 2000E 002 1650572 2000E 002 1650355 2000E 002 1671387 2000E 002 1670598
98. Height over the current baseline above which detector signals of zero slope are not evaluated as new baseline Skim Ratio Ratio of the heights of two adjacent peaks to that of their common valley 4 1 2 2 Syringe vol ul defines the syringe volume of the autosampler LC 51 to 50 or 250 ul respectively according to the syringe used in the autosampler Bubble check defines whether the LC 51 autosampler has to perform an air bubble check after each injection or not Oven temp C regulates the temperature of the column oven Rep AS Start defines whether a run has to be continued or not after an error message during an autosampler run Emission nm Emission wavelength of the fluorescence detector With File and Save the altered parameter values are set as default values a With Close Preset or the button the preset table is closed and the Edit Files window reappears on the screen Before the changes are saved the question appears Save current changes which is answered with yes Then the preset table is written to the CHRST32 INI file into the section Preset 4 2 Analysis Chromatogram Page 4 2 1 4 2 Analysis Chromatogram Data Acquisition Pump Control In the application Analysis Chromatogram the detector signal is displayed on the screen Analysis Chromatogram allows you to record a chromatogram and to control HPLC devices The devices to be controlled are connected to the computer via the RS232 port If menu p
99. If a series of vials have been entered in the first two columns the number of injections specified here are first carried out from the same vial before the next vial is processed 4 1 1 2 Edit Files Method Autosampler Table Page 4 1 11 Vol In this column the sample volume to be injected is entered in ul The smallest sample volume which can be entered is 1 ul the largest 50 or 250 ul depending on the syringe type installed in the autosampler and specified in the Preset File Section 4 1 5 The injection volume is stored in the Slice File Header and can be printed out object Inj Volume cp Report Editor Manual Run Run time in minutes three decimals of a chromatogram after this time data acquisition is stopped Type If one or more samples are used as a calibration standard they must be marked in this column with S LC Procedure Entered here is the name of the LC Procedure File with which the samples of this line are to be analysed DH 1 bis DH 4 This name specifies the data handling files to be used on channels 1 to 4 for the samples of this line Entries are made in the entry bar beneath the table The next field is accessed with TAB or by clicking with the mouse Shift TAB moves the cursor back to the previous box File names can be accessed using the downwards arrow Clicking Insert inserts the entry line to the table If a line already present in the table is clicked it appears on a black background and in the entr
100. K causes the menu list to change accordingly Tile The Tile command arranges the open windows in smaller sizes to fit next to each other on the screen Cascade Cascade causes the open windows to overlap so that each title bar is visible Arrange Icons Arrange Icons arranges the icons of opened applications at the lower edge of the ChromStar window Close All Close All closes all applications within ChromStar Log out Clicking Log out causes the application to be blocked Keyboard entries are no more effective In the menu list only the menu point Log on appears which can be used to reactivate the application In the GLP Version entry of the password again is necessary A list of all opened applications is shown in the bottom of the submenu of Window The active application is marked An application can be activated by clicking into this list Page 3 6 3 2 ChromStar Help 3 5 Help pages With Help the help pages can always be accessed from every ChromStar application The language of the help pages is defined during installation of the ChromStar software With Help Context the help page for the currently used application appears With Help Contents the first page with explanations about the main menu points is opened With About ChromStar a dialog box is displayed which shows the information about the version number The More key addionally shows version number and date of origin of the installed ChromStar files
101. Method File field Chapter 4 1 1 After entry of the file name the menu bar appears with the points File Window Help The data handling file has two pages The first Acquisition Page contains the parameters for data recording the second Integration Page contains the integration parameters which can be programmed on a run time basis 4 1 2 1 Edit Files Data Handling Acquisition Page Edit Files Data Handling nov01 e x Acquisition Integration Slice Width 10 10000 ms Delay Time 0 99 999 min 0 000 Run Time 0 001 10000 min 2 500 Calculation File nov28 Screen Scale 2 0 15 Peak Parameter 5 7 15 Help Close Save Save As The parameters of this page have the following functions Slice Width ms This parameter defines the time in ms range 10 to 9990 ms in steps of 10 ms before each next data point is recorded data acquisition rate Page 4 1 18 4 1 2 1 Edit Files Data Handling Acquisition Page Delay Time min During this period entries from 0 to 99 999 minutes in steps of 0 001 default value 0 1 after the start of an analysis chromatographic data are recorded and stored but not integrated This parameter can be used to prevent integration of solvent peaks which appear at the start of a run Data acquired during the delay time can be integrated later in a reintegration run by defining Delay Time 0 Run Time min During this period entries from
102. N Capacity K T To To Asymmetry A Wp We Selectivity a 1 1 K ap K o Resolution Ria Tin T 2 Wi Wi wherein is L column length in mm To Retention time of solvent peak in min T Retention time in min W Half height peak width in min wr Peak width in 10 of peak height from the ascending peak flank to the perpendicular Wp Peak width in 10 of peak height from the perpendicular to the descending peak flank wp and w are calculated in 5 of peak height if CHRST32 INI shows the entry Calculation FDAComp in the section Colcoeff Tis Tj Retention times of two adjacent peaks F Factor as specified in CHRST32 INI in Colcoeff against Factor preset is Factor 2 5 3 Quantitative Calculation Page 5 3 1 5 3 Quantitative Calculation 5 3 1 Calibration methods The various calibration methods allow you to determine the response factor for calculating an unknown amount from a peak area or height or to define a calibration function for the relation between amounts and peak areas 5 3 1 1 One Point Calibration In the one point calibration a chromatogram is recorded of a standard solution with known quantities of all compounds which have to be determined later Retention times peak areas and heights of the compounds are obtained by integration The calculation file to be used for the calculation contains entries on the first page defining whether areas or heights are to be used and
103. Star 6 3 DA m DAD Settings Board Type RAO and 1 0 Address CISA PI IRo Ef 1 0 Addr aco DAD Device SpectraFlow 600 m Help File GemanHelp English Help Back Net Cancel In the System Configuration window you can choose the ChromStar version downwards arrow on the right the type of the A D converter board ISA PCI or Ethernet Box LC421 E the program path the type of the diode array detector DAD and the language of the help pages Depending on the ChromStar version chosen the setup changes a little In case of the DAD ChromStar detector details and parameters are to be entered After choosing ChromStar Integrator the Printer Port Adapter can be installed When the Ethernet Box LC421 E is used the IP address must be entered in the next window 2 ChromStar Installation Page 2 5 In the Configuration ChromStar window the default data path and the default parameter and templates path are defined Configuration ChromStar xj Please select your Configuration for Chromstar Program Data and Templates Default Data Path C ChromStar 6 3 Data oe m Default Parameter and Template Path C ChromStar 6 3 T emplates Plugs m Do you want to install an Event Box C Yes No m Do You use the Game Port for external Start C Yes No Back Next Cancel The use of the event box or of the game port is also defined here Us
104. T FILES CALCULATION Kovats Ind Time Areal Peak Time Percent 33 500 3 000 3 6000E 003 1 0000E 000 0 0 C36 35 330 3 000 3 8000E 003 1 0000E 000 0 0 C38 37 080 3 000 4 0000E 003 1 0000E 000 0 0 c40 38 740 3 000 4 2000E 003 1 0000E 000 0 0 C42 40 320 3 000 4 4000E 003 1 0000E 000 0 0 C44 41 850 3 000 4 6000E 003 1 0000E 000 0 0 C46 43 320 3 000 4 8000E 003 1 0000E 000 0 0 C48 44 720 3 000 5 0000E 003 1 0000E 000 0 0 c50 46 080 3 000 5 2000E 003 1 0000E 000 0 0 c52 47 400 3 000 5 4000E 003 1 0000E 000 0 0 c54 Time Percent Kovats Ind Time Areal Peak Code zm 3000 5400 fiooo0E 000 oo cee O oo ma Ce In the Internal Standard Method the standard is added to the sample and a chromatogram is recorded The retention time of the standard must be obtained before In the External Standard Method a chromatogram of the standard is recorded first the retention times obtained in this way are entered into the Calculation File After this the sample is chromatographed The dead time is determined by a first order linear regression through the standard points 5 2 5 Calculation of Kovats Indices Page 5 2 13 The Kovats Indices of the peaks found in the time windows are calculated according to the following formula LINp 1g RTp lg RTss LINps LINss lg RTps lg RTss LINss For a peak before the first standard applies LINp lg RTp LINss lg RTss For a peak after the last standard applies LINp lg RTp LINps lg RTps
105. a points are represented on the screen again This can also achieved by using the right mouse key in the data acquisition window 4 2 3 Analysis Chromatogram Display Page 4 2 7 When 2 or more channels in one analysis are being used for recording data the representation of the chromatograms on the screen can be done in two ways The submenu point Stacked divides the screen so that the lower half displays the channel 1 chromatogram in yellow and the upper that of channel 2 in green If 4 channels are being used channel three is displayed in blue channel 4 in cyan blue EJ ChromStar Draw Baseline Ch1 Ch2 OF x Method Operation HPLC Display Options Window Help laj x mal AlSel glalBlc p g iJe il omme a No Method selected Ch2 0 020 Ch1 0 442 mY Flow 1 00 100 0 B 0 0 C 0 0 D 0 0 Press F1 for Help NUM YW Selecting the submenu point Overlayed displays the chromatograms in a superimposed graphic with a common x and y axis The channel 1 chromatogram is yellow that of channel 2 is green Show Comparison Chromatogram is used to show or hide the comparison chromatogram which had be loaded under Method The Slice Width parameter must be the same in Edit Files Preset and in the chromatograms Show Gradient When a method with a gradient in the LC procedure file is loaded this menu procedure can be used to show or hide the gradient in the data acquisition window Page 4 2 8 4 2 4
106. access to the main operations in this application The are described together with their corresponding menu points In addition a two line information bar is visible The first line contains the chromatogram name and a description of the actual work stage The second line contains information to support the user in the handling of the individual menu points The manual integration is carried out as follows First the area in which the integration is to be made must be defined by constructing a rectangle as described in 4 4 1 EJ ChromStar Manual Integration iof x MMR File Mode Expand Edit Annotate View Window Help 18 x Hal x N S TE Hal x w i 3 omme F Chromatogram FMTEST MY Select the operation mode or perform Fine selection 800 600 400 200 Ne ah te Aha A a 14 0 21 0 Press F1 for Help NUM Z Before this you can use the menu point Expand to enlarge the displayed chromatogram by direct number entries Page 4 4 14 4 4 2 2 Reprocess Manual Integration With Auto Range in the entry field Expand by Values the selected section is automatically normalized to the highest peak The chromatogram section selected is displayed in the lower part of the window in its enlarged form and in the upper part it appears on a white background The information field displays the message Select the operation mode or perform fine selection i e further enlargements of the expanded section can be
107. according to the formulas Page 5 3 22 5 3 4 Calculation Examples with Formulas amount peak K1 peak area peak area qs amount js Resp Fact js area js amount js sta amount 1s mj If another amount of internal standard than in the standard solution is injected the area js is multiplied by the ratio area js gig area 1s mj In the example the standard solution contains 20AU internal standard the sample solution contains 40AU internal standard For the internal standard the ratio Rel of calculated amount to injected amount is calculated Rel amount js amount 1s mmj C after calibration with regression functionfunktion Multi Level Calibration Multi Level Calibration Calculation Mode Internal Standard Nr Result File Name Chromatograms of the standard solutions 1 benal111 5 standard solutions with 5 different amounts 2 bena2111 are injected 3 bena3111 Toluene is the internal standard IS 4 bena4111 5 bena5111 Peak Table Peak Name Benzene Time Window 1 77 Std Amt Area Area IS 1 0000E 000 0 764 2 0000E 000 1 450 3 0000E 000 2 121 4 0000E 000 2 742 5 0000E 000 3 212 KO 3 101 1E 001 Coeff of Correlation 0 997609 K1 1 6084E 000 Std Error of Estimat 0 109281 K2 0 0000E 000 K3 0 0000E 000 Peak Name Toluene Time Window 2 07 Std Amt Area normalized area 1 0000E 000 562232 562232 5 3 4 Calculation Examples with Formulas Page 5 3 23 2 0000E 000 1096267 548133 3 0000E 000 157777
108. al peaks the corresponding amounts and eventual peak names One Point Calibrations are performed in Reprocess Calculations with the peak data of a calibration solution and the Calculation File after entry of the corresponding level The Report File belonging to the chromatogram of a calibration solution contains for each peak the amount the corresponding area and the response factor A series of one point calibrations can be carried out in Reprocess Calculations via menu point Batch The mode is set to One Point Calibration the standard chromatograms the calculation file and the appropriate levels are entered into the Batch List The calibrations are carried out on clicking OK Calculation of the calibration functions is carried out in the Reprocess Calculations 7 window by clicking Calibration and Multi Calibration The Report File obtained as described above is selected from the table After clicking OK the approximation of the first order for the first peak appears on the screen in a graph showing the correlation between the concentration and peak area Select and Order allows you to display another approximation order File and Save Results stores the current approximation order displayed for all the peaks Therefore enter a new name against Calculation File The coefficients of the approximation functions are stored in the regression table on the third page of the calculation file as well as the retention times of the individual peaks
109. alculation Examples with Formulas Page 5 3 21 Confidence Region 8 3413E 003 K1 2 6186E 001 Peak Name Toluene Time Window 2 06 Std Amt Area Area IS Area 2 0000E 001 562323 2 0000E 001 550833 KO 0 0000E 000 Coeff of Correlation 0 000000 K1 1 0000E 000 Std Error of Estimat 0 000000 K2 0 0000E 000 K3 0 0000E 000 Formula Average Area Area IS i 1 bis n Area i Area IS n n number of chromatograms i number of components in the standard solution Kli amount of the standard i Average Area Area IS i The averaged response factors are saved as K1 in the Regression Table of the calculation file BENAISMP CAL in the Method Table the parameter Regression for Calculation is automatically ticked For the internal standard no average calculation is carried out Calculation of the unknown amounts Chromatogram BENA7121 Chromatogram of the mixture with unknown amounts Sample Identifier B T Calculation File BENAISMP Calculation Method Internal Standard Calibration Method Multi Level Internal Standard 40 00 In the sample solution the internal standard was added in the double amount Peak Win Ret Time Area Resp Absolute Rel Weight Name No No min Fact AU 1 1 1 758 792307 2 62E 001 3 819E 001 0 95 0 0 Benzene 2 IS 2 2 050 1086671 3 866E 001 0 97 0 0 Toluene Totals 1878978 3 819E 001 0 0 Formula The calculation of the amount under Absolute is carried out
110. alculation Method External Standard Calibration Method One Point Factor 1 000000e 001 Peak Win Ret Time Area Resp Absolute Weight Name No No min Fact 0 003 4 1 2 625 978015 1 0140E 06 9 917 0 000 Coffein 978015 9 917 Press F1 for Help NUM Us File has a submenu with the following functions Open or Calculation allows a further report and calculation file to be Ej opened for calculation Save saves the result Save As saves the result in a new file Load Multi Calibration shows a Multi Calibration once carried out and saved in a calculation file Print opens a dialog box in which a print report template can be chosen cp p 4 4 4 and a print out be made 8 The print template should obtain the object Table see also p 4 6 in the Report Editor manual Using the downwards arow after Fileformat the format of the print report template can be changed Printer Setup allows the changing of printer settings Copy transfers the results table to the clipboard Page 4 5 4 4 5 2 Reprocess Calculations Calibration 4 5 2 Reprocess Calculations Calibration Before starting a quantitative calculation as described in section 4 5 1 a calibration with one or several standard solutions must be carried out in order to determine the response factors for peak areas or heights with the sample amount In a one point calibration only one standard solution is used The multi calibration calculates
111. alibration Nr Result File Name Chromatograms of the standad solutions 1 benal111 5 Standard solutions with 5 different amounts 2 bena2111 are injected 3 bena3111 4 bena4111 5 bena5111 Peak Table Peak Name Benzene Time Window 1 77 Page 5 3 18 5 3 4 Calculation Examples with Formulas Std Amt Area 1 0000E 000 429783 2 0000E 000 794832 3 0000E 000 1115697 4 0000E 000 1380790 5 0000E 000 1548442 KO 4 5045E 001 Coeff of Correlation 0 998627 K1 7 6158E 007 Std Error of Estimat 0 082819 K2 1 3722E 012 K3 0 0000E 000 Peak Name Toluene Time Window 2 07 Std Amt Area 1 0000E 000 562232 2 0000E 000 1096267 3 0000E 000 1577773 4 0000E 000 2014605 5 0000E 000 2410053 KO 1 0376E 001 Coeff of Correlation 0 999994 K1 1 4682E 006 Std Error of Estimat 0 005601 K2 2 3303E 013 K3 0 0000E 000 The regression function of 2nd order is calculated The regression koefficients are saved as KO K1 and K2 in the Regression Table of the calculation file BENAML CAL in the Method Table the parameter Regression for Calculation ia automatically ticked Calculation of the unknown amounts Chromatogram BENA9121 Chromatogram of the sample mixture Sample Identifier B T with unknown amounts Calculation File BENAML Calculation Method External Standard Calibration Method Multi Calibration Regression of the 2nd Order Peak Win Ret Time Area Resp Absolute Weight Name No No min Fact AU
112. alin Load Files Peaknamefs z Insert Cancel Amount in Std 100 Resp Factor fil x2 He orig Area 0 pO o O OOOO M Int Std T Ref Peak Press F1 for Help NUM Us The values of Resp fact and Factor remain 1 The name of the component can be entered against Peak The values of Resp fact and AreaO remain unchanged Page 5 3 12 5 3 3 Simple Quantitative Calculation The Calculation File QUAN is saved Edit Files Calculation quan Filed Ea Method Table Peak Table Regression Table Time Percen Amt in Std Resp fact Areal 1 789 10 972 1 0000E 002 1 0000E 000 0 0 Acetophenon 4 768 5 176 0 0 Naphthalin Time Percent Amt in Std Resp fact Areal Peak Code 4 768 5 176 foo f1 0000E 000 0 0 Naphthalin 7 Insert Paste Delete Graphic Help Close Save Save s Save Level om The analysis is continued in the Analysis window The method QUAN does not have to be recalled again with Method Load before the next injection If it is recalled anyway a 2 must be entered against Start line The new response factors are automatically transferred into the Calculation File after recording the chromatogram of the calibration sample here QUANO021 They also appear on the print out of the result list 19 By using the obtained response factors the amounts are calculated and printed immediately after recording the chromatogram QUANO0031 20 of an injection of a mixture with
113. ame or by using the screen keys After changing a file and clicking File and New Open or Close a question is displayed asking whether the changed file has to be save or not With File and Open or the button another file can be selected and opened With File and Close or with the button ea the Edit Files Calculation window is closed 4 1 3 1 Edit Files Calculation Method Table On the first page of the Calculation File parameters are to be entered concerning the type of calculation the type of time window and a second calculation file if required can be specified Pg Edit Files Calculation Mix2 2 Method Table Peak Table Regression Table Multi Calibration Parameters Multi Calibration Graphic rm Calculation Base Time Window Height Percent Absolute C Defined p e 5 Percent Wn Percent Window 5 Internal Standard External Standard Minimum Time Window min 10 100 C Kovats Intem C Kovats Extern Univ Resp Factor f1 0000E 000 Minimum Area Height 100 IV Regression for Calc Link t e eels Relative Window Update Calculation Units jau Number of Conc Levels E Help Close Save Save As 4 1 3 1 Edit Files Calculation Method Table Page 4 1 23 The following parameters can be defined In the Calculation base box Area Height This parameter defines whether areas or heights are to be used in the calculation Click the optio
114. and password to change this password Change User With Change User another user who was registered before with name and password under User Administration and New User can take over the ChromStar control Forbidden Passwords Forbidden passwords can be determined here The points User Administration and Forbidden Passwords are only accessible for the Super User and the Administrator After installing ChromStar in the GLP mode the Lab Manager has all rights and the user almost none The access rights can be changed in the User Administration window as required In the GLP mode the program is frozen after a certain time and can only be reanimated by using the Log on key and entering the password Immediate freezing of the program can be achieved by pressing Window and its sub menu Log out The interval after which the program is automatically frozen can be defined in the CHRST32 INI file as well as the time after which the password is no longer valid Entries in CHRST32 INI in the section GLP PwdTimeOut xx defines the time in days after which the password expires TimeOut yy defines the time in minutes after which the program is frozen 3 4 ChromStar Window Page 3 5 3 4 Menupoint Window The menupoint Window allows you to choose how to display a number of opened applications on your screen Meaning of the submenu New New enables you to open another ChromStar application Selection of the desired application and clicking O
115. and started the Sample Table commences again at injection or the first vial respectively When using an autosampler the SampleTable contains the correlation of the sample name identifier to the vials Before starting the analysis a defined line number of the Sample Table or the Autosampler Table can be specified as start line chapter 4 2 Vials which are already analysed are marked x Chromatogram is recorded SLI and RPT files exist 3 Appears when using an autosampler and injecting several times out of one vial where the injections are not yet finished Page 4 1 14 4 1 1 4 Edit Files Method Documentation Table 4 1 1 4 Edit Files Method Documentation Table Access this table by clicking into Documentation Table Here you can make notes about the sample preparation the column the eluent and the detector being used for the method If in the Run Table Mode GC is chosen GC specific entries as shown below can be made here TAB accesses the next field ShifttTAB accesses the previous field Edit Files Method neugc Run Table Sample Table Documentation Table Sample Details Name TY Origin TY Preparation SSS Injection fs Carrier Gas Pressure Flow Rate Notes Oven Temperature Temp Prog Notes r Column Details Type Col Len d 0 0 mm Diameter 0 0 mm Particle size Solv Peak 0 00 min Notes Injector
116. arameter values can be allocated which appear in the LC procedure and data handling files as default values The threshold values of the individual parameters are given in the LC procedure and the data handling file Only one preset file exists Ta After clicking into Chrom Files and Preset or using the button the Preset Table appears on the screen ig Edit Files Preset Preset Parameter Flow Rate ml min Noise p s Max Pressure bar Threshold m Skim Ratio Min Pressure bar Loop Yolume pl M n pos Wavelength nm Attenuate Bubble Check Screen Scale 2 Oven Temp C dds Rep AS Start Emission nm Help Close Save Compress Factor Slice Width ms oO o The parameters of the Preset Table have the following meaning Flow Rate ml min Flow rate Max Pressure bar Upper pressure limit Min Pressure bar Lower pressure limit Wavelength nm Wavelength of the UV detector or excitation wavelength of the fluorescence detector respectively Attenuate Attenuation of the UV detector Screen Scale 2 Attenuation or amplification for the display of the detector signal on the screen Compress Factor Compressibility factor Slice width ms Defines the period after which a new data point is recorded Page 4 1 36 4 1 5 Edit Files Preset Noise u V s Maximum increase of the detector signal which is not recognised as the start or end of a peak Threshold mV
117. arch in Files of Type Method Files met Sample Id max 20 letters Acetophenon I Match Case Found in File s nov23 4Kanal met 11 E Search File no no 23 4Kanal met 12 nov23 4Kanal met 13 11 The Menu points Window and Help are already described in the chapters 3 4 and 3 5 The Edit Files application window can be reduced to an icon by clicking its minimize button third field at top right corner You quit Edit Files with Exit Edit Files Page 4 1 4 4 1 1 Edit Files Method 4 1 1 Edit Files Method Clicking Chrom Files and Method or using the button accesses the dialog box Open Method File for entering the name of an existing or of a new file Open Method File Search in a Test2000data Es a Test DAD2 met Filename amp romaten Filetype Method Files met hd Cancel Out of the list of all existing files a file is chosen or a new name is entered in the entry field after File Name The box on top after Search in shows the chosen directory The downwards arrow shows the list of all directories The upwards arrow leads to the next higher directory If a new file is to be created its name up to 23 characters is entered in the entry box against File Name It is not necessary to define the file type An existing file can be selected from the list by clicking it whereupon its name appears in the entry box and on a black background in the list You can overwrite a file na
118. art ChromStar again switch on the devices and activate the RS 232 interface again via Analysis Chromatogram and Options Select Channels When a pump and the autosampler is connected to the computer via RS 232 but the autosampler is not in use the autosampler must be disabled in the chrst32 ini file in the section Configuration by the entry Disableautosampcom1 Yes or Disableautosampcom2 Yes After this change ChromStar must be started again When the autosampler is not in use and not disabled it must be switched on and set to serial mode If that is not the case no method can be chosen the menu procedure Method appears in light grey In the Selection box the Comm port can not be chosen Whether an autosampler is used in a method file or not can be seen in the Select Method File box If an autosampler is used entries can be made in the Start box against Line and Inj and in the End box against Line If a method without autosampler is used only entries in the Start box against Inj are possible 5 1 4 Operating an Autosampler Page 5 1 13 When in an autosampler table a vial is programmed in more than one line chromatograms with the same name are created To avoid overwriting of existing chromatograms these are moved into a sub directory of the data directory with the name TEMP and the number e g 001 of the line in which the vial is used first In the information line the message ERROR appears The full error message can be seen
119. atograms can also neu1129 e3111 C be used in the print out when in Edit Files Colors the Ser Print Colors Channel 1 10 are the same as the Screen ap aa Colors Channel I 10 and the object Multi Chromatogram in the report print template rpd has the property Use ChromStar Colors marked 4 5 3 Reprocess Calculations Displ Chrom Page 4 5 15 The menu point Mode allows you to display the chromatograms in one of the four following modes Standard The entire chromatogram is displayed i e the section of the three dimensional display overlaid by another chromatogram is shown as well Whitewash The section overlaid by another chromatogram is not displayed Data Point The entire area of the chromatogram is displayed in colour i e each individual data point is shown as a dash If only one chromatogram is being displayed in enlargement this mode provides a graph of the individual data points Point Display the data points are displayed as dots 4 5 3 2 Reprocess Calculations Compare The submenu point Compare allows a stack of up to four chromatograms to be displayed each with its own time axis After selecting the menu point Compare a dialog box is displayed in which the chromatograms to be displayed are selected The names of the raw data Files are selected by clicking them in the list at the centre which displays the raw data files of the selected directory The selection is terminated with OK T
120. ave As _ Hee oe sae _ Saveas_ Level fi The following columns are used Time In this column the retention time 0 001 to 999 999 min is entered for each peak or group of peaks This column is used for chronologically resorting of the table A new entry at an existing time causes the original values to be overwritten The time entered in this column should be as close as possible to the time of the peak maximum or in the case of a peak group to the time in the centre of the group If defined time windows are being used this column is cancelled time values are then entered against Time and Time 2 see below Percent This column is available if the percent time window has been selected to enter another percent time window for each individual peak retention time than that defined on page 1 The range limits of the entry are the same as those of Percent Window min If the Absolute time window has been selected the column heading reads min A different time window than the absolute time window defined on page 1 can be entered here for each peak 4 1 3 2 Edit Files Calculation Peak Table Page 4 1 27 Time 1 This parameter 0 001 to 999 999 min defines the start time of the time window for an individual peak Time 2 This parameter 0 001 to 999 999 min defines the end time of the time window for an individual peak This time must be greater than Time 1 The following columns are valid for all types of time
121. ave Results herewith the calculation file used for the one point calibration can be overwritten or a new file can be created Automatically entered in the new Calculation File are the complete peak table of the calculation file used for the one point calibration and the averages of the response factors in the regression table against K1 First a warning appears if the original file is to be overwritten The file is overwritten after clicking Yes 5 3 1 3 Multi Level Calibration The multi level calibration establishes a correlation between the concentration of a substance and the peak area and from this to compute an approximation function Later with its aid an unknown amount can be calculated from a peak area The multi level calibration can be performed in a current run as well as afterwards The execution in a current run is carried out either with a real or a virtual autosampler Autosampler is marked on the first page of the Method File The autosampler table contains in one line the series of vials with the calibration solution the start number against Vial the last number against To a continuous series must be available The number of injections from each vial is entered against Inj and S is entered against Type The Data Handling File containing the name of the Calculation File to be used is entered against DH Method External Standard is marked on the first page of the Calculation File the units of concentration are
122. ber of peaks calculated can be set between 1 and 20 whereby the calculation time will be considerably longer the larger the number of peaks to be evaluated Depending on the sector the number of peaks which can be evaluated is limited The peaks are calculated in descending order of their heights The calculation can be interrupted by Cancel After the calculation has been carried out the result is displayed on the screen File and its submenu offer various file operations File Information displays a box with entries about Author Sample Identifier Recording date Changes Number of datapoints Runtime minimum and maximum mV value Open allows a new chromatogram to be selected analysis or the results list either in landscape Landscape or in portrait Portrait format or with an individually selected chart speed Chart Speed Printer Setup allows the printer settings to be changed Copy transfers the sector to the clipboard Print allows you to print out the chromatogram sector with its calculated peaks Exit returns you to the File Transformation window a EJ ChromStar Curve Fitting iof x B File AutoFit Results Expand Window Help lej x 34 omme a Chromatosram ARAL0201 Press F1 for Help NUM 7 Page 4 8 14 4 8 4 Transform Curve Fitting The menu point Results offers the possibility of checking the result Show Fit redraws the individual peaks so that after using the following subm
123. ce chromatogram which plots a line between both points This position is confirmed by clicking the left mouse button This causes the mouse cursor to return to the point defined in the Work chromatogram which can be adjusted if required The alignment is performed after clicking the right mouse button Normalisation of the y axis Height Alignment is performed as follows After clicking the left mouse button in the Reference chromatogram a vertical white line appears which can be positioned by moving the mouse Confirm the position with the right mouse button Upon this a factor is calculated so that the intensities of both chromatograms are identical at the marked position The entire Work chromatogram is now multiplied with this factor and displayed again Both before and after the alignment an enlargement can be made in the lower chromatogram This is always automatically normalized to the highest data point Clicking the right button in the lower sector restores the original scale The alignment is not affected Expand allows an enlarged section to be displayed by value entries for the time axis Also here an automatic normalisation to the highest datapoint is performed Calculation After selecting the menu point Calculation both original datasets are displayed in the upper half of the screen the lower half is reserved for displaying the results The menu bar now contains the points File Operation Back Window Help 4 8 2 Transform
124. cent points is enhanced by multiplication and devision respectively The parameter Slope Width in min determines the width of the neighbourhood of a point The parameter should be smaller than the peak width 4 8 1 Transform Modify Page 4 8 3 Clean When Autoclean is clicked for removing spikes a maximal peak width in min must be defined against Max Time After clicking OK and activating Calculation Clean all peaks smaller than the peak width entered are removed Without Autoclean the cursor appears as rubber After activating Clean a peak is removed by clicking into it Only integrated peaks can be removed If more than one peak is to be removed the result can be moved in the upper part by using Swap and then be calculated again The resultant chromatogram can be saved via Save as and then be reintegrated Swap moves the resulting chromatogram from the lower box into the upper one where it can be calculated again The result of the calculation is displayed in the lower window when the calculation is finished The calculation method is displayed in the information box Ej ChromStar Modify ile ES File Calculation View Expand Window Help lj x slal alaala colexial alt ommen A mY Chromatogram 0121 10031 Operation 1st Derivative 1500 _ 1250 1000 750 500 250 o o 10 20 3 0 4 0 5 0 6 0 7 0 min o o 1 0 20 3 0 4 0 5 0 6 0 7 0 min Press F1 for Help NUM h Enlargement of a se
125. cess to the main operations in this application The are described together with their corresponding menu points 4 5 1 Reprocess Calculation Calculation performs a quantitative evaluation of a chromatogram with a x calculation file using the Report File extension RPT that was generated during recording of the chromatogram This Report File contains the peak data obtained after integration of the chromatogram like retention times peak heights and peak areas In addition there must be a calculation file extension CAL containing the response factors of peak areas or heights with the corresponding quantities for calculation of response factors by calibration see cp 4 5 2 The quantitative evaluation of a chromatogram in a current run is described in section 5 3 3 After clicking Calculation a dialog box appears for selection of a report and a calculation file Select Calculation File and Report File Search in a Data a Caf44011 rpt a mara01140051 rpt a Caf45011 rpt a mara01150011 pt New0003 rpt a mara01140011 pt mara01150021 pt a Neu0004 rpt a mara01140021 rpt a mara01150031 pt a Neu0005 rpt a mara01140031 rpt mara01150041 rpt Neu0006 rpt a mara01140041 rpt fa mara01150051 rpt a test0001 rpt Filename kaffee cal Filename Caf4501 1 rpt Filetype Calculation Files cal z Filetype Report Files rpt z Cancel Calculation File After choosing a
126. cility of reprocessing chromatographic data Individual chromatograms can be reconstructed in full size or in detail chromatograms can be reintegrated either automatically or manually The Reprocess Calculations window offers still more possibilities to handle chromatographic data These are described in section 4 5 the mouse or by using the button opens a new applications window with the im Selecting Reprocess in the menu bar of the ChromStar system window with title bar Reprocess The individual points of the menu bar Reconstruction Integration Exit Reprocess Window Help can again be accessed with the mouse arrow and selected by clicking the left button of the mouse which briefly underscores the menu point selected in black with the exception of Integration where a submenu appears ChromStar olx Reconstruction Integration ExitReprocess Window Help aj a 3 u frm esa 7 fi Reprocess Press F1 for Help An icon of the application can be created by clicking the third box from the top at the right icon box The individual menu points are described in the following sections Page 4 4 2 4 4 1 Reprocess Reconstruction 4 4 1 Reprocess Reconstruction Clicking the menu point Reconstruction opens the dialog box Select Ez Chromatogram for selection of a chromatogram to be displayed on the screen Select Chromatogram Search in 3 Data m ja aroma1214b2031 SLI 8 aroma1219c2
127. constructing a perpendicular drop line through their valley Before doing this the baseline for the whole peak group must have been defined as described above Then the submenu point Valley is selected or the button is pressed upon which in the information bar the message appears Operation Mode Valley After clicking the left mouse button a vertical white line appears at the cursor position in the area of a peakgroup with a defined baseline Move the mouse arrow to the deepest part of the valley between the peaks A click on the right mouse button fixes a red drop line Clicking the left mouse button again brings back the white line a new valley can now be searched and a drop line can be defined again with a click on the right mouse button Various valley points also in different peak groups can be defined one after another The submenu points Tangential Skim or Exponential Skim should always be employed for overlapping peaks when their heights are so different that the smaller peak can be regarded as being a rider peak on the descending flank of the main peak 4 4 2 2 Reprocess Manual Integration Page 4 4 15 Before using this menu point a baseline must have been constructed for the whole peak group The procedure is as follows Select one of the Skim methods from the submenu The information bar now reads Operation Mode Tangential Exponential Skimming In the case of a Tangential Skim the start and end point have to be defined ba on
128. csecccscccececcccceneece 5 3 2 Page 1 4 Contents 5 3 2 Quantitative Calculation Methods ccccsssssseeeeeeeeeeeeees 5 3 4 Oi Percent MEINOd faincucs cadterne dean dete nob ae fine hak 5 3 4 5 3 2 2 Normali ao etic ee n e an i tind EEAO EEE REE 5 3 5 5 3 2 3 External Standard Method eseeeeeeeeeeeeesesesesscesrrrrrrrrrrerererereree 5 3 5 5 3 2 4 Internal Standard Method eeeeeeeeeeeeeeeeeeseeesesscrrrrrcrrrrrrrrerereree 5 3 6 5 3 2 5 External Standard Method with Calibration function 5 3 7 5 3 2 6 Internal Standard Method with Calibration function 5 3 8 5 3 3 Simple Quantitative Calculation ee cceceeeessecceeenteeeeeeees 5 3 9 5 3 4 Examples of Calculations and Formulas ccseeeeeeeeees 5 3 13 5 4 Gel Permeation Chromatography cceeeeeeceeeesteeeeeeeeeees 5 4 1 6 Printer Protocols jcc c c8aiienieandskles anand 6 1 1 Introduction Page 1 1 1 Introduction The chromatography data system ChromStar together with an PC containing the ChromStar A D converter board is capable of recording chromatographic data and operating chromatographic equipment such as solvent delivery systems a UV detector or an autosampler Some diode array detectors can be controlled and 2D data of these can be recorded and reprocessed The ChromStar software runs under the operating system of Microsoft WINDOWS and can therefore only be used in this environment Windows 3 11 95 9
129. ction of the upper chromatogram is possible by constructing a rectangle as already described The enlarged section then appears in the lower window Another section can be enlarged after clicking a point anywhere in the upper sector The original picture can be regained by clicking in the upper range with the right mouse button Page 4 8 4 4 8 1 Transform Modify File and its submenu offer various file operations With Open a new chromatogram can be selected for evaluation With Save as the result of a smoothing file extension SLI can be stored The original chromatogram may not be overwritten The Ist and 2nd derivatives cannot be stored File Information displays a box with entries about Author Sample Identifier Recording date Changes Number of datapoints Runtime Minimum and maximum mV value y The menu point Print with its options Dump and List allows the lower window contents to be printed 21 Printer Setup allows the printer settings to be changed Copy transfers the presentation to the clipboard B Exit returns you to the File Transformation window mfe with the retention time and detector signal value or the value of the 1st or 2nd derivative depending on the position of the mouse cursor in the chromatogram which is indicated by a perpendicular white line The box disappears after clicking View again By clicking the menu point View Show Values an information box appears Expand By Values
130. culation File QU4 ML Created by H Risler Last Updated by H Risler Method Table Calculation base Method Minimum area height Link to other Calc Calculation Units No Concentration Level Regression for Calc Relative Window Update Y N Time Window Percent Minimum Time Window min Peak Table Time Percent Amt in Std Resp Fact Area0 Peak Code Level 1 1 990 260 530 300 500 NUNN Level Level Level Level 000 000 000 000 000 NNN 2 3 4 5 Regression Table End of KO 8 8172E 001 9 9585E 000 1 3344E 001 1 3878E 001 3 6462E 001 en OF we nwo w NEP RAN PUN amp WNnNrFP em Window 0000E 001 0000E 001 0000B 001 0000E 001 0000E 002 9000E 001 0000E 001 0000E 001 0000E 002 0000E 002 0000E 001 0000E 001 0000EB 001 5000E 002 0000E 002 OO00E 002 2000E 002 0000E 001 0000E 002 0000E 002 0000E 001 5000E 002 0000E 002 5000E 002 0000E 002 K1 3 2721E 005 9 4171E 005 1 3113E 004 1 5595E 004 4 1729E 004 Y N Date 17 04 1996 Time 11 34 0n 12 14 95 at 09 39 55 0n 12 22 95 at 08 43 28 Area External Standard 100000 5 Y N Percent 5 0 100 3 1254E 005 6 4538E 005 6 7848E 005 1 5072E 004 4 3209E 004 K2 0 00005 000 9 9287B 012 7 2747B 011 0 0000E 000 0 0000E 000 0 Toluol 0 Ethylbenzol 0 Cumol 0 Butylbenzol 0 Hexylbenzol K3 0 0000E 000 0 0000E 000
131. d a calculation file must be generated During calibration there must be no entry against the Link parameter the calculation method is Internal Standard For the calculation the name of the second and more if necessary calculation file is entered against the Link parameter The results are listed in one table Calculation Units This parameter 8 characters defines the unit of measurement in which the results are specified This unit is printed as the column heading for the results table Number of Conc Level Number of different calibration standards max 15 which are used in the Multi Level Calibration Regression for Calculation This parameter defines whether the regression coefficients generated from a previous multi level calibration see sections 4 4 2 3 5 3 2 5 and 5 3 2 6 and stored on page 3 of the calculation file are to be employed in a quantitative calculation Clicking the box switches between on and off Relative Window Update This parameter defines whether the time window used for defining peaks or peak groups is to be updated shifted at the end of each run The reference peak is specified as RF against Code on the second page of the calculation file An update does not take place if no mark is clicked into the box or if no reference peak is defined in page 2 of the calculation file Attention Do not use with calibrations 4 1 3 1 Edit Files Calculation Method Table Page 4 1 25 In the Time Window box Perc
132. d and lower sides of the ChromStar window scroll bars appear which allow operations outside the window area to be carried out Individual windows can be closed by clicking Exit or use the Exit button Applications and submenus displayed in light colours are not accessible at the present stage of operations After clicking the Minimise Box next to the ChromStar title bar ChromStar appears at the bottom of the screen in the task bar All opened sub windows are included in it A click on the task icon restores the previous status Page 3 4 3 3 ChromStar GLP Options 3 3 Menupoint GLP Options GLP Options is active in the GLP Version and controls the accessibility This menu procedure only appears when ChromStar is installed in the GLP mode It allows new users and passwords to be introduced User Administration In the User Administration window the users access rights are determined In the window to the right the activities can be chosen by clicking into the appropriate item or sub item Using the Apply key the changes become active With the New User key a new user with limited accessibility can be entered After entry of name and password a user level is chosen Administrator Lab Manager or User The Super User had installed ChromStar previously and has no further rights With the Delete key a user can be removed from the users list Change Password Change Password allows the user who already opened ChromStar with his name
133. d solutions as here it is the area of the internal standard is normalized to the amount of the internal standard in the first calibration solution PKS IS Norm PKS Js amount Js 1 amount Ss The coefficients are determined by means of a multi level calibration In this method the internal standard must be present in the standard solutions in varying quantities When calibrating takes place the internal standard does not yet have to be defined During the calibration is decided also which order will be used for evaluation Depending on the order selected KO through K3 differ The coefficients are stored in the regression table on the 3rd page of the calculation file On page 2 the standard peak must be defined by IS against Code as the internal standard when the analysis run takes place It must also be present in a known quantity in the solution to be analysed This value is entered in the Update Input Values box against Int Std at the start of the calculation if the amount of the internal standard in the solution to be analysed is different to that of the first standard solution Calculated are the peaks on page 2 peak table for which also the multi level calibration has been performed 5 3 3 Simple Quantitative Calculation Page 5 3 9 5 3 3 Simple Quantitative Calculation The quantitative calculation is performed in a number of steps 1 Creating the necessary files Method Data Handling and LC Procedure File 2 Determination
134. data handling file The result of the integration is stored in a temporary file with a name where the first character of the original file name is substituted by a sign The result of the integration can be saved in the original report file a warning appears that the file is overwritten or in a new file On leaving ChromStar the temporary files are deleted Only one temporary file SLI RPT is created for all subsequent reintegrations when an appropriate entry in the CHRST32 INI file is made The last integration performed can be repeated with Reconstruction or in Reprocess Calculations with Batch and Mode Reconstruction 2 The noise value should be suitable for evaluating the chromatogram If nothing is known about it a value between 100 and 500 is recommended The integration parameters can be changed during the reintegration via the menu point Param The noise value in the Preset File obtained by Automatic Noise determination and also the default value in the Data Handling File could lead to an unsuitable value being entered Because of this the entries should be tested The noise value can be modified at a later stage by means of Param i Select a chromatogram and a data handling file from the directories In a preview the chromatogram is displayed in the small box to the right showing the on top Sample Identifier if an entry in the Sample Table of the method file had been made After clicking OK the calculation is carried ou
135. ding on the type of autosampler an empty position must be left in the sample tray after the vial s with the standard solution in order to avoid the following vials to be treated as calibrations The further processing is entered in a new line in the Autosampler Table Depending on the type of autosampler the Run Time in the method file must be at least 2 minutes If the Run Time of the data handling file is greater than the Run Time of the method file the chromatogram will be terminated after the Run Time defined in the method file If the Run Time of the data handling file is less than the Run Time of the method file the chromatogram will be terminated after the Run Time defined in the data handling file but the analysis will continue for the Run Time defined in the method file Only at the end of this a next injection is made It is essential that all data handling files and if required LC procedure and calculation files to be used in the autosampler table are existing 5 1 4 Operating an Autosampler Page 5 1 11 5 1 4 3 Carrying out an Analysis with the Autosampler Select the menu point Method Load in the Analysis window and recall a met method with autosampler marked The method must be transferred to the autosampler this takes about 15 seconds time you have to wait until this is finished If the pump is not already pumping the end of the transfer time can be recognised by the fact that the pump starts to pump the solve
136. dit Files LC Procedure Page 4 1 33 2 Flow Rate ml min This function defines the flow rate 0 0 30 0 ml min at a given time It is used either to program the flow rate independently of the preset value at time 0 0 or to change it at any time during the course of an analysis The flow rate should not be altered during the course of a solvent gradient 3 Max Press bar This function is used to define the pressure 0 490 bar above which the pump automatically switches off 4 Min Press bar This function is used to define the pressure 0 490 bar below which the pump automatically switches off 5 Oven Temp This function is used to specify the temperature 20 100 C of the column oven if installed 6 Wavelength nm This function is used to select the required wavelength 190 600 nm of a UV detector spectrum scan cp 5 1 3 7 Attenuate 2 This function is used to modify the attenuation 1 14 of a UV detector Attention The attenuation only affects the Remote Control output of the detector 8 Autozero This function can be used to zero the detector signal from the UV detector Time programmed changes with the last three functions can cause a major change in the detector signal which may negatively effect subsequent data handling integration Integration should be inhibited during changes of these 3 functions this can be done with the Integrate Inhibit function in the data handling file 9 Event This func
137. during the run time in the data handling file SCAN 5 1 4 Operating an Autosampler Page 5 1 9 5 1 4 Operating an Autosampler 5 1 4 1 Installing the Autosampler The autosampler interface is plugged into the backside of the autosampler In case of using a controllable pump the RS 232 input on the interface is connected with a nine pole cable to the RS 232 output of the pump this in turn is linked to the RS 232 interface of the computer Alternatively the autosampler is linked directly with the computer The instrument number device address is set with a screwdriver at the orange screw of the autosampler interface The small notch indicates the selected instrument number This must be different to that of the pump Under Configuration the CHRST32 INI file must contain the entry LCn LC m AS k in which n is the number of the computer s RS 232 output m the instrument number selected at the pump and k the instrument number of the autosampler The same entries can be made in the ChromStar Configuration window which can be found in the system configuration window steuerung machen After changes in the configuration or in the INI file ChromStar must be started again cp fig p 5 12 Switch the pump and the autosampler on one after the other Depending of the model the autosampler executes a test routine this can take approximately 20 seconds Wait until this is finished Attention The instruments must be in serial mode After this t
138. e ChromStar system the various window techniques and the use of the keyboard Chapter 4 describes the ChromStar software the menus and their submenus The sections are named accordingly Chapter 5 shows how the various stages of chromatography are carried out with the help of the ChromStar system All print outs that can be made with the ChromStar system chromatograms results lists distributions plots etc are to be found in Chapter 6 Bold printed numbers in brackets in the text refer to these print outs The following characters are not permissible when naming a new file and the space bar Avoid using national characters such as 4 6 ti All letters and numbers from a to z and 0 to 9 are permitted The file extension also called suffix which is separated from the file name by a point is allocated automatically Page 1 2 1 Introduction ChromStar exists in different versions ChromStar ChromStar with DAD ChromStar with GPC ChromStar with DAD and GPC DAD and GPC are additional options to the ChromStar full version The data systems ChromStar light and ChromStar Integrator are less comprehensive compared with ChromStar ChromStar light does not contain the Transform module ChromStar Integrator does not contain Edit Files Method Autosampler Table and Edit Files LC Procedure since ChromStar Integrator is not able to control any instruments ChromStar Integrator does not contain Edit Files Preset the preset
139. e after the other by moving the mouse cursor in the appropriate area of the chromatogram and clicking the left mouse button This causes a white line to be drawn Confirmation of the start and end points follows after clicking the right mouse button After this a green line is drawn from the start to the end point In the integration this area is subtracted from the main peak area The Exponential Skim function always leads from the graph curve to the Dea baseline The highest signal value always must be clicked first After this a white line is drawn from the graph curve to the baseline this line is dragged with the mouse to the lowest signal value The end value is clicked and the cursor jumps back to the start point Clicking the right mouse button confirms the skim function as a green line These functions also can be used consecutively for a number of peak groups Using Modify baselines perpendicular or exponential peak separations can be changed afterwards The cursor appears as an outstretched hand As soon as the cursor is in a position where a change can be carried out it appears as a gripping hand The information line shows which kind line will be changed e g Baseline Valley or Tangential Exponential Skim In the following the procedure for changing a baseline is described By clicking the left mouse key the baseline appears as white line It can now change ist porition by moving the mouse Clicking again the left mous
140. e are always used if they have not been defined in one of the files mentioned above The specified parameter values can be changed and stored in the CHRST32 INI file they are automatically activated after the next system start Page 4 1 2 4 1 Description of the ChromStar Software Edit Files The menu bar of the Edit Files window contains the points Chrom Files Options Exit Edit Files Window Help Chrom Files with its submenu Method Data Handling Calculation Scan Parameter LC Procedure Preset Colors Show Connect allows entry to the individual file types These are described further in detail in their own chapters Buttons in the toolbar allow quick access to the files pet Pall ond re Colors opens the Colors window in which for the various representations on the screen Screen Colors or for the print out Print Colors the colors can be individually defined by the user After clicking a function on the left a color can be selected either under Basic Colors or in the mixing area The color selected in the mixing area is shown in the Color Solid box at the bottom to the left Some functions can only be represented in a Solid color The selected color is immediately shown in the box on the right to the function Using OK all selected colors are saved in the CHRST32 INI file and are used in ChromStar for the various representations The Set Default key restores the original ChromStar colors Using Cancel the window is closed withou
141. e column is conditioned the system is ready for recording a chromatogram The method can be started from the menu point Operation Start cp next section the injection has to be carried out simultaneously Injection and start of the chromatogram can be synchronised via start input on the A D converter board cp p 2 2 by a signal from the autosampler or the injector respectively When using a controlled autosampler the injection of the autosampler is initiated by the control interface Method Actualize If changes are made in the method file in Edit Files after starting the method and recording a chromatogram these changes can become active by using Actualize when the following injections are carried out Analysis In the method file Run Table the following changes can be made Notes Normalization and Report after Save and Actualize the changes are active in the same run A change of Use Linenumber is only active in the next run Attention A change of Data File S name of the chromatograms is not possible Page 4 2 10 4 2 4 Analysis Chromatogram Method In the Autosampler Table after the line in process a line can be inserted an existing line can be changed or a line can be appended After Save and Actualize the changed method will be carried out Attention The line of the autosampler table in process may not be changed In the Sample Table the parameters Sample Identifier Sample Info Factor We
142. e key moves the cursor to the other end of the baseline By clicking the right mouse key the change is confirmed the baseline appears in green In order to change the perpendicular the left mouse key is clicked the line is moved By clicking the right mouse key the change is confirmed When the cursor appears as an outstretched hand the right mouse key switches from the Modify function into the Delete function Using Delete a baseline a perpendicular or a skim can be deleted The cursor appears as arrow with a cross As soon as the cursor is in a position were a X green line can be deleted it appears as arrow with a rubber The 4 information line shows which kind of line will be deleted e g Baseline Valley oder Skim By clicking the left mouse key the line is deleted ig Page 4 4 16 4 4 2 2 Reprocess Manual Integration The integration of the peaks defined with Mode is effected by moving the mouse into the light area of the upper chromatogram and clicking the right mouse key This transfers the graphic representation of the peak evaluation from the lower window to the upper At the same time the light area disappears Now a new area of the chromatogram can be selected and defined with a rectangle The peak definition can again be made in the lower sector of the chromatogram as described above Negative peaks also can be integrated by defining a baseline Their areas are shown as positive values EJ ChromStar Manual Integrati
143. e stored together with all parameters used for recording and evaluation The recording of the chromatogram is now finished and the pump stopped with the function key F9 Page 5 1 6 5 1 2 Programming a Gradient 5 1 2 Programming a Gradient To program a gradient with two solvents and the following shape 56 6 16 26 t min and the time table 0 min A 80 B 20 2 min A 80 B 20 4 min A 10 B 90 6 min A 10 B 90 16 min A 100 B 0 20 min A 100 B 0 22 min A 80 B 20 25 min A 80 B 20 an LC Procedure file is created by first clicking Edit Files followed by pro Chrom Files and LC Procedure A desired name in this example GRADIENT is entered in the entry box above the directory After clicking OK the entry page of an empty LC procedure file appears The cursor is blinking in the entry box Time to show that entries can be made The following entries are necessary for the gradient described above TAB TAB TAB 8 0 space 2 0 Then Insert is clicked or the Return key Enter is pressed The line appears on a black background in the table above The box under Time also appears on a black background to show that further entries can be made Now 2 is entered and Insert is clicked 5 1 2 Programming a Gradient Page 5 1 7 To complete programming this example the following entries still must be made 4 TAB TAB 1 0 space 9 0 Insert 6 Insert 1 6 TAB TAB 1 00 Insert 2 0 Insert 2 2 TAB TAB 8 0 space 2 0 Insert
144. ected section is automatically normalized to the highest peak An enlargement by setting up a rectangle is made as follows The upper left hand corner is defined by moving the mouse arrow to the point required and clicking with the left button The lower right hand corner is defined by moving the mouse which draws a rectangle the position is confirmed with the left button This causes the mouse arrow to return to the upper left hand corner which can now be corrected and I Auto Range intensity confirmed by clicking the left mouse button The rectangle is confirmed by clicking the right button of the Reference Time Axis mouse The section of the chromatogram appears in the Start window Clicking the chromatogram with the right button again restores the previous size The submenu point By Values allows you to zoom out Reference Intensity Axis by entering the time and mV values in a dialog box The Stat 1740 initial values in this dialog box represent the current size of the chromatogram These initial values are also End 165 593 adapted after utilising the Reference Window procedure Default or clicking with the right mouse button restores the original display of the entire chromatogram End OK Cancel Annotate Automatic allows the user to mark peaks in a chromatogram by numbers by retention times or by names When Peak Numbers is clicked the chromatogram with its peaks numbered N is displayed
145. ection 4 1 2 1 In this case the chromatogram is printed exactly as it appears on the screen Clicking the check box results in switching between Normalisation and No Normalisation Report No report or selection of a report template This entry defines how the chromatogram is to be printed A report print template is chosen from the list of standard templates or templates created by the user Protocols 6 8 section 6 2 for GPC cp section 6 8 Entering No Report means that no print out is produced the chromatogram however is saved In a multi channel data acquisition each chromatogram is printed out on one page if single is clicked In the other case a print report template can be chosen for the representation of several chromatograms RPD or RPC 14 15 16 LC Procedure This field allows entry of the LC procedure file which is used for the analytical part of a method and which controls an LC pump If no LC pump is being used it is not necessary to enter a name here The name of the LC procedure file of up to 8 characters automatically receives the name of the method file by default You can however use any other LC procedure file or enter an other name by over striking the default name with the cursor while keeping the left mouse button depressed A black background appears and now the new name can be entered Data Handling 1 to 4 Here the names up to 8 characters of the data handling files are specified The default name f
146. ed by individual entries E The results list appears on the screen by clicking Results EJ ChromStar Manual Integration oO xi M File Mode Expand Edit Annotate View Results Window Help amp x sjaj ale apclalm fn se l ole omme a Chromatogram RALO2ZO1 Operation Mode Exponential Skimming Peak Report RALO201 Peak Ret Time Area Height Abs Rel Mode Name 6 973 19 375 1137208 136 983 ox ae N L S 17 0 Press F1 for Help The results can be presented or viewed in various ways With File and Print the chromatogram and the results list can be printed after choosing a report print template Save stores the result of the manual integration as a Report File RPT A warning appears that the original report file will be overwritten The chromatogram and the integration saved in the Report File can be displayed in Reprocess Reconstruction The Report File can be used for quantitative evaluations Page 5 2 10 5 2 4 Grouping and Summation of Peaks 5 2 4 Grouping and Summation of Peaks It is possible to evaluate peaks in a chromatogram in groups i e the areas of several peaks are summed this total is used to calculate area percentages or amounts Peak grouping is used to add adjacent peaks The identification of these peaks is done in the peak table of a calculation file by creating a time window around a central peak and marking against Code with G The areas of all peaks occurring in t
147. ed on the next page Select the desired method by clicking the appropriate box Kovats Intern Kovats Extern Kovats Indices can be calculated in GC chromatograms after internal or external calibration The standard values are entered on the second page against Kovats Ind for details see chapter 5 2 5 Seite 4 1 24 4 1 3 1 Edit Files Calculation Method Table Univ Resp Factor Universal response factor only valid when Method Percent and entries in the Peak Table are made The universal response factor is used for all peaks in the Peak Table Minimum area height This parameter 0 to 1000000 default value 100 defines the level of peak height or area obtained from an integration or reintegration below which a peak is excluded from the quantitative calculation Attention When peak heights are used in the calculation the value entered here must be considerably lower Link to other Calc In this field the name of a Calculation File can be entered 8 characters default is blank for a further quantitative calculation of the current run This can be used to perform various types of calculation e g firstly calculation with peak areas and then with peak heights During a calibration run it is nor possible to carry out a further calculation with this parameter The Link parameter can be used to carry out calculations with more than one internal standard For each internal standard and only for the peaks evaluated by using this standar
148. ed out under Batch with Mode One Point Calibration 4 5 2 2 Reprocess Calculations Multi Calibration Page 4 5 7 4 5 2 2 Reprocess Calculations Multi Calibration The submenu point Multi Calibration allows you to calculate the average value of the response factors after a series of one point calibrations or to calculate a calibration function and store this in a Calculation File To carry out a multi calibration with average calculation first record and integrate a number of chromatograms of one standard solution in order to establish the peak data A calibration solution may contain more than one standard component These peak data in the Report File are used to carry out One Point Calibrations with a Calculation File that already contains the retention times and injected amounts through this peak areas are linked to standard amounts and stored in the Report File The peak areas stored in this way can be used to calculate average values After clicking Calibration and Multi Calibration the Report Files to be used A for calculating average values can be selected max 25 in the directory in the middle field which displays the content of the current directory By double clicking or multi selecting and nsert their names appear in the list to the right A file can be deleted from the selected list by clicking its name in the field Selected Files and clicking Delete Select Report Files 27 xi Search in E Demodata
149. eft mouse button calls up a list of operations which can be carried out e g clicking Minimize causes the window to become an icon clicking Maximize expands the window over the entire screen clicking Close shuts down the window A sub window can also be closed down by a double click on the Control Menu box The Control Menu Box functions can also be executed with a combination of keys If none of the functions of this box are accessed it can be exited by striking ESC or by clicking any part of the screen outside the Control Menu box A sub window can be transformed to an icon by clicking the Minimise Box the second box from the right The icons of the sub windows are to be found at the bottom of the ChromStar window A double click onto an icon restores its window to the screen A single click recalls the Control Menu box The Maximise Box at the top right hand corner of the screen allows you to expand an application up to the full size of the screen The title bar then contains the title of the application besides the ChromStar symbol The switches are now in the title bar and in the menu bar appears a switch with an up down arrow Pressing this switch restores the application to its previous scale It also allows you to switch between a large and a reduced size window Clicking and holding the title bar allows you to move a window so that a part of it gets outside of the ChromStar window and becomes invisible At that moment at the right han
150. egion 9 762453e 003 Press F1 for Help NUM 7 In the field above the graph you will find first the components name of which the peak areas have been averaged below this the average peak area as well as the standard deviation and the borders of the confidence range The menu bar contains the points File Select Window Help The menu point File and its submenu point Open returns you to the selection of other Report files and to a repeat multi calibration Load Multi Calibration shows a Multi Calibration once carried out and saved in a calculation file The File submenu point Save Results is used to calculate response factors with the average peak areas these response factors are then saved under K1 in the regression table of a calculation file In the selection box the name of the calculation file appears which is defined in the selected Report File This name can be overwritten with a name that indicates that the calculation file contains an averaged response factor Save Results causes the averaged response 4 5 2 2 Reprocess Calculations Multi Calibration Page 4 5 9 factors of all the peaks to be stored The parameter Regression for Calculation at the first page of the Calculation File is automatically marked E With Print and Results the results are printed 10 Graphic causes the graphical representation 11 to be printed Printer Setup allows the printer settings to be changed With Copy and Result
151. ent Absolute Defined This parameter default value percent defines the type of time window calculation to be performed in identifying peaks If the preset type time window the retention time time in percent is not required click one of the following boxes Absolute time window the retention time time in min Defined each peak on the next page has a time window with precisely start and end times The last two entry fields of this page and the column headings of the table on page 2 vary according to which type of time window calculation has been selected After selecting the time window option Absolute the last parameter of this field is cancelled Selecting the option Defined means that the last two parameters of this field are cancelled Percent Window If the time window option Percent has been selected this parameter integer in the range 1 to 99 default value 5 defines the percentage size of the time window for all peaks Different time window sizes can be entered for individual peaks on page 2 of the calculation file Absolute Window min If the Absolute option has been selected this parameter 0 001 to 9 999 minutes default value 0 05 defines the time window in minutes for all peaks Here also it is possible to define different windows for individual peaks on page 2 Minimum Time Window min If the Percent option has been selected this parameter 0 001 to 9 999 minutes default value 0 01 defines an additional mi
152. ent B Solvent C Solvent D and Flow Rate The user defined descriptions can be saved in the Chrst32 INI file in the chapter Document Table by using the Set as Default key When creating a new method file the ChromStar descriptions are then overwritten by using the Load Default key User Defined Details m Mobile Phase Detector Details Header Eet Header Detector Details A SovetA Tele B Sovente Range Range C Sovente Wavelength nm Wavelength rim D Solvent D Notes Notes Notes FowRatl r Column Details Sample Details Header Column Details Header Sample Details Type lye tt t S Name Name Col Length Col Length Origin loin Diameter Diameter Preparation Preparation Particle size Particle Sze Injection vol finjectionv Solvent Peak Solvent Peak Notes Notes IV Enable User Defined Cancel Load Default Set As Default 4 1 2 Edit Files Data Handling Page 4 1 17 4 1 2 Edit Files Data Handling The data handling file extension INT is necessary for defining data acquisition and integration parameters Recording a chromatogram requires a data handling file one data handling file can be used for recording a number of chromatograms After Data Handling has been selected from the submenu of Chrom Files or using the button the dialog box Open Data Handling File appears for entry of a lint name it is used exactly the same as the Open
153. entered against Calculation Units and the number of different calibration solutions against Conc Level this corresponds with the number of vials On the second page the different amounts of each peak in the calibration samples are entered Therefore it is necessary to know the retention times of the substances to be determined 5 3 1 Calibration Methods Page 5 3 3 The calibration samples are injected also repeated depending on the entry against Inj in the autosampler table one after the other after recalling the method in Analysis Calculation of the coefficients of the approximation functions follows automatically These are transferred to the regression table of the Calculation File and used for the quantitative evaluation of the next sample also if no autosampler is used in the autosampler table If the Multi Level Calibration is to be performed afterwards first a number of chromatograms to be evaluated have to be recorded with calibration samples containing different amounts also multiple injections of one calibration solution are permitted These are integrated in order to obtain peak data Also a Calculation File is created which contains on the first page the Method External Standard against Conc Level the number of different calibration solutions and against Calculation units the unit of concentration By using the Level Entry in the peak table on the second page entries are made concerning the retention times of the individu
154. enu points the original graph is restored without having to repeat the calculation Sum shows the sum of the individual peaks This appears as a white line in the chromatogram and shows how the approximation corresponds to the chromatogram Difference subtracts the sum of the peaks from the chromatogram This difference also appears as a white line and shows the degree to which the baselines differ Report displays the results list of the chromatogram sector with peak numbers retention times peak areas area percentage and peak heights The peak areas and heights calculated can be compared with values obtained by automatic integration Peak numbers are only allocated for the sector of the chromatogram selected Because of the possible long calculation time always a small sector only should be selected for peak analysis by Curve fitting Expand allows an enlarged section of the upper part to be displayed by value entries for the time axis and for the y axis 5 Chromatography with ChromStar Page 5 1 1 5 Chromatography with ChromStar 5 1 Data Acquisition 5 1 1 Recording a Chromatogram In this section the user will be instructed how to quickly record a chromatogram without reading the whole manual first Shown here is how to set the parameters for solving the next task A chromatogram has to be recorded of a test mixture using an isocratic mobile phase of 60 methanol in solvent container A and 40 water in solvent container B a
155. er MV Normalization he r Report LC Procedure reu Short32 rpt 7 Data File s Data Handling 1 red neu l No Report DataHanding 2 an SS M Single Data Handling 3 DAD Method File Data Handling 4 7 cs Help Close Save Save As The parameters on this table have the following meaning Mode HPLC GC GPC determines how the chromatogram has to be evaluated HPLC and GC mode lead to a calculation of the peak areas heights and retention times The GPC mode performs a molecular weight calculation and requires an other data handling file Section 4 9 1 1 A chromatogram for calibrating the GPC column must be recorded in HPLC Mode Once you have clicked in to GC the parameter LC Procedure can no longer be accessed as these are used to control an HPLC device In addition the Documentation Table of the method file changes and permits the entry of specific GC notes The report forms under Report change according to whether you have clicked HPLC GC or GPC The mode required is chosen by clicking the appropriate button with the mouse Notes Several lines for notes concerning the analysis e g sample preparation column eluent etc can be entered here Autosampler This parameter defines whether a with ChromStar controllable autosampler is used or not for autosampler control see also section 5 1 4 If this box is selected the parameters LC Procedure and Data Handling 1
156. esp Std Amt Name No No min Fact AU 1 1 1 758 794832 1 258E 004 1 000E 002 Benzene 2 2 2 033 1096267 1 368E 004 1 500E 002 Toluene Totals 1891099 Amount in the standard solution Formula RF Amount area of the component i is calculated in the calibration run for each peak Calculation of the unknown amounts Chromatogram BENA3111 Chromatogram of the mixture with unknown amounts Sample Identifier B T Calculation File BENAEXT Calculation Method External Standard Calibration Method One Point Peak Win Ret Time Area Resp Absolute Weight Name No No min Fact AU 1 1 1 750 1115697 1 258E 004 1 40E 002 0 00 Benzene 2 2 2 042 1577773 1 368E 004 2 16E 002 0 00 Toluene Page 5 3 16 5 3 4 Calculation Examples with Formulas Formula RES PKS RF Wherein is RES Result of the quantitative evaluation for the i peak Absolute PKS Peak area or height of the i peak RF Standard amount area of the component 1 is calculated for each peak in the calibration run B after calibration with averaging the peak areas Calibration with average calculation Nr Result File Name Chromatograms of the standard solutions 1 BENAI111 The same standard solution is injected twice 2 BENA6121 Peak Name Benzene Time Window 1 75 Std Amt Area 1 0000E 002 429783 1 0000E 002 420346 Average Area 4 2506E 005 Standard Deviation 6 6725E 003 1 6 Confidence Region 5 9968E 004 K1
157. ess Calculations 3D Plot Clicking the menu point 3D Plot displays a dialog box in which up to 10 chromatograms can be selected A list of all directories is displayed on the left in the centre the raw data files of all the chromatograms of the selected directory The chromatograms are selected by double clicking or multi selecting and Insert If more than 10 chromatograms are selected and confirmed with OK the message is displayed Too many data files After correct selection and clicking OK the chromatograms are displayed in a three dimensional form the first selected chromatogram is at the bottom The scaling of the X axis is determined by this chromatogram The individual chromatograms are displayed on the screen in different colours ChromStar Display Chromatograms of x File Expand Reference Mode Window Help 2 x SE am mm ea lt 3 3004 mv 250 4 2004 1504 1004 504 0 0 0 5 1 0 1 5 2 0 rin Press F1 for Help NUM A The menu bar contains the menu points File Expand Reference Mode Window Help Page 4 5 14 4 5 3 Reprocess Calculations Displ Chrom File allows various file operations The menu point Print transfers the displayed sections of the chromatogram to the printer after selecting a convenient report print template 14 16 Printer Setup allows the printer setting to be changed With Open further chromatograms can be selected to disp
158. f Calculation enabled Factor 2 Colors in ChromStar can be changed in Edit Files Chrom Files Colors Coefficients of the calibration function for calculating the wavelength using the diode number of the diode array individual diode array detector parameters these are supplied with each detector Wavelength range of the diode array detector 200 400 nm 200 640 nm 200 1020 nm A D Board in use Example LC421 is installed No A D convertert board is installed This entry is necessary for 1 amp 2 when only a DAD is used On leaving ChromStar the window positions are stored Representation of a detector signal from channel 1 to 4 Calculation of column coefficients Factor for the calculation of the resolution After modification the entries can now be stored by clicking Files and Save Exit the application by clicking into Close ChromStar must be restarted before some of the modifications can take effect In the ChromStar start dialog of the GLP RU yR Ts eis version the user s name and password has to be entered in agreement with those entered during the installation In the none GLP version only the user s name or nothing can be entered The appearance of this window can be switched off if always the same user works with ChromStar This is done as follows click on the program icon with the right OK oneal mouse key and in the menu Properties Ses Extend the Command Line Please enter you
159. gral 4 9 14 Integrate Inhibit 4 1 20 5 2 7 Integration 4 4 1 4 4 9ff 4 7 6 Integration Mode GPC DH File 4 9 4 Integration Page 4 1 19ff GPC DH File 4 9 4 Integration parameters 4 1 19 4 4 12 5 2 2 5 2 3 Integration Time 4 1 38 Intensity Range 4 6 6 Internal Standard 4 1 23 5 3 6 5 3 7 5 3 8 Internal Standard Method 5 3 6 Internal Standard Method with calibration function Regression 5 3 8 Int St an d 4 1 12 4 1 29 4 5 2 IS Calculation Peak Table Code 4 1 27 5 3 6 5 3 8 ChromStar 7 Index K Mark Houwink Constant 4 9 3 4 9 6 5 4 9 Kovats Indices 4 1 23 5 2 12 Kovats Intern Kovats extern 4 1 23 5 2 12 L Landscape 4 8 3 4 8 5 4 8 7 4 8 9 4 8 11 4 8 13 LC Proc 4 1 11 LC Procedure File 4 1 1 4 1 2 4 1 8 4 1 11 4 1 32ff 4 2 4 4 2 10 5 1 3 5 1 5 5 1 6 5 3 9 5 4 2 6 4 Level 4 1 13 4 1 30 4 5 5 5 3 2 Link to other Calc 4 1 24 List 4 7 3 Load 4 2 8 5 1 4 Load Multi Calibration 4 5 3 4 5 6 4 5 8 4 5 11 Log on Log out 3 5 Log Scale 4 9 15 Low M Wt Region 4 9 11 M Manual Integration 4 4 4 4 4 11 4 4 13 5 2 8 GPC 4 9 17 GPC Batch 4 9 24 Mark Houwink Constants 4 9 3 4 9 6 Mark Houwink Const K 4 9 3 4 9 6 Mark Houwink equation 4 9 6 5 4 9 Max Press 4 1 32 4 1 35 Method Calculation File 4 1 23 5 3 4 Method 4 2 8 4 3 10 5 1 4 5 1 11 5 3 12 5 4 1 Method File 4 1 1 4 1 2 4 1 4ff 4 2 1 4 3 4 4 3 10 5 1
160. h the TAB key you step to each next field Shift TAB allows you to step backwards Wherever in a ChromStar file a file name is entered as parameter this can be done in two ways Either the name is entered in the entry box or the downwards arrow to the right of the box is clicked and the file name is chosen out of the list of the existing files The LC procedure Data Handling Files and Scan Parameter file specified in the Method File must first be created in the same directory as the Method File before the analysis specified in the Method File can be started The Close Save Save as and Help key correspond to the menu procedures Close Save Save as and Help Index and allow quick file editing These keys are present on all pages of the ChromStar files and have the same function as described here After creating its four pages the Method File first needs to be saved before executing an analysis Pay attention that only entries on the first page are necessary to carry out an analysis saving the default entries is enough The file can be saved with Save or Save as commands under the menu point File or by using the appropriate screen keys After carrying out modifications and clicking the submenu commands New Open or Close Method a message appears asking whether the changes have to be saved or not Using File and Open or the button k opened Using File and Close or the button EJ Page 4 1 10 4 1 1 2 Edit Files Method Autosample
161. he chromatograms are displayed in windows on top of each other The menu bar contains the menu points File Expand Window Help EJ ChromStar Compare Chromatograms Iof x File Expand Window Help lel x e camee Window 1 neu1129 1111 Window 3 neu1129 3111 Window 2 neu1129 2111 Window 4 neu1129 4111 min Press F1 for Help NUM WY Page 4 5 16 4 5 3 Reprocess Calculations Displ Chrom A sectional enlargement in each chromatogram window is possible by constructing a rectangle Clicking the right mouse button in the respective window re accesses the full display of the chromatogram Here too a sectional enlargement can be further enlarged The menu point File allows various file operations With Open further chromatograms can be selected to display on the screen With Print a print out is made after selecting a convenient report print template 15 Printer Setup allows printer settings to be changed Copy copies the screen display to the clipboard Exit returns you to the Reprocess Calculations window Expand allows you to change the start and end time values x axis and the mV minima i m window 1 Window 2 and maxima y axis for each window Start Time 0 00 End Time 3 00 High m 86 86 Start Time 0 00 End Time 3 00 High m fi 39 81 End Time 3 00 High mv 190 62 Low m 12 35 Low mv
162. he menu point Options Select Channels can be clicked in the ChromStar Analysis window In the selection box the data recording channel is clicked as well as COM 1 or COM 2 according to the RS 232 interface being used even if no pump is being used 5 1 4 2 Programming the Autosampler sampled are made in the Method File Click the menu point Chrom Files and Method in the Edit Files window and create a method in which the Autosampler parameter is selected on the first page Run Table In the field down right all entries concerning LC Procedure and Data Handling File appear in grey i e they are no longer accessible Against Data File Name 4 characters can be entered these are the first characters of the chromatogram name The following 4 characters are determined by the vial number from 0 to 999 and the injection number hexadecimal entry from 1 to F Section 4 1 1 2 describes how to generate an autosampler table Entries concerning the position of the vials and the order in which they are met The vial number entered against Vial in the first line of the autosampler table is the start number of operations A vial entry against the parameter To is the last vial number of the processing sequence All vials between start vial and the vial under To are processed with the Page 5 1 10 5 1 4 Operating an Autosampler same conditions Missing vials generate an error message cp sect 5 1 4 4 the processing sequence is continued
163. his time window are summed A chromatogram can contain several time windows identified with G Peak summing allows you to select non adjacent peaks in a chromatogram for joint evaluation These peaks are identified in the peak table of the calculation file against Code with S1 A further peak area sum of other peaks can be obtained after identification of these peaks as S2 etc In the following example chromatogram CIBAO101 results list without summing or grouping 17 the peaks from 4 0 to 6 12 min are evaluated as one group and the peaks from 10 71 to 13 76 min are evaluated as a further group The peaks at 19 77 32 34 and 38 13 min are evaluated as Sum 1 and the peaks at 24 65 and 34 03 are evaluated as Sum 2 The annotation in the chromatogram was done manually mu Chromatogram CIBAQ101 J vr nm au r Summe 1 Summe 1 Summe1 29 483 Lar ies P 337 1873 E Ys he 37 542375 au Y ao w 0D W oo P nm o _ h 43 092 Eaa 608 gt 42 533 5 2 4 Grouping and Summation of Peaks Page 5 2 11 Method Table Peak Table Regression Table Time Percen Amt in Std Resp fact Areal Peak Code 5060 22500 Gruppe 12 200 13 000 1 0000E 000 1 0000E 000 0 0 Gruppe2 G 19 767 1 000 1 0000E 000 1 0000E 000 0 0 Summel 51 24 650 1 000 1 0000E 000 1 0000E 000 0 0 Summez 52 32 342 1 000 1 0000E 000 1 0000E 000 0 0 Summel 51 34 033 1 000 1 0000E 000 1 0000E 000 0 0
164. ible when controlling a pump Display Options Window and Help can be accessed When acquiring data of more independent channels you must open more than one 2 3 or 4 Analysis application window Page 4 2 4 4 2 2 Analysis Chromatogram HPLC 4 2 2 Analysis Chromatogram HPLC The menu point HPLC is only accessible when a pump is controlled via RS 232 and an appropriate interface The sub menu points or the buttons respectively are used for the pump control Stop Pump stops the pump immediately No dialogue box appears The pump 2 can be stopped even quicker by striking the function key F9 Flow Rate determines the flow rate in ml min and can be varied between 0 0 and 30 0 The flow rate can also be defined with the F10 function key After clicking of Solv A B C or D the pump starts pumping the solvent chosen Also the function keys or the buttons can be used for choosing the solvent without clicking the Pump menu F5 Solv A F6 Solv B F7 Solv C D F8 Solv D Click the submenu point Composition if an isocratic mixture from two different bottles is to be pumped The percentage amount of each solvent is entered in an entry box the portion of D is automatically calculated After clicking Ok the pump starts pumping the defined solvent composition In the status line below the Draw Baseline window the specified solvent composition is displayed behind A B C and D Solvent Composition An LC procedure 4 1 4 m
165. ight and Int Std can be changed After Save and Actualize the changes are active for the next injection Changes in the Documention Table are active in the same run after Save and Actualize When the last line of the autosampler is being processed no changes can be made On doing so the message appears Can t actualize If modifications are made in the data handling file and saved these are active from the next injection without Actualize Changes in the calculation file are active in the same run without Actualize Changes in the LC procedure file are active from the next line of the autosampler without Actualize After changes in the various files the method can be stopped activated again and be restarted by entering the line number the vial and the injection number Comparison Chromatogram An existing chromatogram can be loaded for comparison reasons The parameter Slice Width must be the same in Edit Files Preset and in the chromatogram The chromatogram is shown in the Analysis window and the menu point Show Comparison Chromatogram under Displayis is marked Method Exit the button or the x Box close the data acquisition window The question appears Analysis 7 Do you really want to close the Analysis Window The question is answering as desired 4 2 5 Analysis Chromatogram Operation Page 4 2 11 4 2 5 Analysis Chromatogram Operation The Operation submenu contains the following commands to carry
166. in the Error View box accessable via Error When a vial programmed in the autosampler table can not be found in the sample table of the autosampler the message ERROR appears in the information line and in the Error View box The error messages are saved in an ERR file The file can be printed via Print Use Open to open other error files Delete deletes an error file Cancel closes the Error View box When opening a method the Error View box is automatically emptied Error View ChromStar Error File Hame 010411 C ERR Method TEST MET Path CACHRSTARS DATA User H Risler Vial 2 Inj 1 Ho vial found in Autosampler Vial 1 Inj 1 Moved existing TES10011 SLI to TEMP directory Vial 1 Inj 1 Moved existing TES10011 RPT to TEMP directory Page 5 1 14 5 1 5 Short instruction guide 5 1 5 Short instruction guide The short instruction guide describes how ChromStar acquires data records chromatograms calibrates and evaluates ChromStar and the A D converter board must be installed in the computer A printer must be connected to the computer The detector signal must be connected to sockets 9 and 10 of the A D converter board The HPLC instruments must be ready The shortest possible entries and the filename TEST are used in the following description Recording Chromatograms Program Start Double click on the program icon Ba or Start bottom to the left Program ChromStar click into ChromStar in the
167. in which unit the concentration has to be stated The second page contains entries about the retention times with the corresponding time windows the amount of the compound in the standard solution and the peak names The calibration is performed in the Reprocess Calculations window by clicking Calibration and One Point Calibration and entry of the Report File and Calculation File The result appears in a table on the screen The obtained response factors appear against Res Fct and are automatically transferred to the calculation file Also a compound which is later to be used as an internal standard must have its response factor established as described This compound has to be identified as an internal standard When carrying out a one point calibration in the current run the name of the Calculation File must be entered in the Data Handling File The injection is identified as a calibration run by entering S against Type in the Method File If standard solutes may not be mixed in one solution and one point calibration are to be carried out with chromatograms of the single standard substances in CHRST32 INI in the section Calibration there must be the entry Calculation additive so that the response factors of the substances not found in a standard solution is not set to 0 This is only valid for one point calibrations 5 3 1 2 Averaging a number of One Point Calibrations The response factors obtained from a series of in
168. ined After creating the two pages of a data handling file it must be stored before carrying out an analysis This is done with one of the appropriate submenu points of the menu point File or using the screen keys It is not essential to fill out the second page The default values are used if no entries have been made Using File and Open or the button a file can be selected and opened Customize Function Names allows you to change the descriptions of the programmable functions 1 10 and to save these in CHRST32 INI in the section Edit Files Atention The new descriptions appear in all Data Handling files The original descriptions reappear on deleting the entries in CHRST32 INI in Edit Files Using File and Close or the button 1 a file is closed Seite 4 1 22 4 1 3 1 Edit Files Calculation Method Table 4 1 3 Edit Files Calculation The menu point Calculation allows the calculation file file extension CAL necessary for calibration and quantitative calculations to be created cal After clicking Calculation in the Chrom Files submenu or using the button a file name must be selected as described in 4 1 1 or a new name entered After selection the following points appear in the menu bar File Window Help and the first page of the calculation file appears the parameters of which will now be described The calculation file can be stored with File and Save or with File and Save As in the last way under a new n
169. ines a time window during the data acquisition in which a baseline is plotted between all adjacent valleys regardless of the Threshold and Skim ratio values set This allows a better integration of peaks which appear in the chromatogram upon a broad and unresolved peak The sharp rise of the baseline caused by column bleeding in temperature programmed GC can be compensated for here 6 Horizontal This parameter defines a time window during the data acquisition in which the baseline is plotted horizontally from the height of the signal at the beginning of the time window until the end of the time window This parameter serves to generate a horizontal baseline over a negative peak If this were not done the baseline would be incorrectly calculated 7 Integrate Inhibit This parameter defines a time window during the data acquisition in which no integration takes place During this period the chromatographic data are recorded and stored the baseline setting is continued at the time window borders This parameter allows the integration to be interrupted when the peaks are insignificant or when the detector signal alters as a result of a change of wavelength 8 Negative Peak This parameter defines a time window during the data acquisition in which a negative peak is regarded as positive and integrated accordingly 9 Event The output channels of the Event Box LC 427 can be time switched On and Off with the help of this function 0 Off 1 On Up to
170. ing ChromStar with a DAD the instrument specific coefficients for calculation of diode number to wavelength must be entered in the Calibration Coefficients window In the Scan Presets window some DAD specific preset values are entered Click Finish to complete the setup The ChromStar program files can either be stored in the WINDOWS directory or in a special directory default C CHRSTAR as desired The CHRSTAR FON CHRSTAR HLP CHRGLP DAT and CHRST32 INI files are copied automatically into the WINDOWS directory The CHRSTAR32 EXE COMSERV EXE REPORT32 EXE and ADINT DLL files are stored in the ChromStar directory The DDEML DLL file must be in the Windows SYSTEM directory The INIEDIT CPL and COMMDLG DLL files are also written into this directory After clicking in the program icon with the right mouse button and choosing submenu Properties the directory into which ChromStar has been stored is shown When using the default directory the display states C CHRSTAR chrstar32 exe Page 2 6 2 ChromStar Installation Some parameter settings for ChromStar are stored in the CHRST32 INI file These are accessed and modified as follows Call up the application Notepad in Accessories select File and Open with your mouse and enter the file name CHRST32 INI After confirming with OK the contents of CHRST32 INI appear in the window These are as follows Directories Data c chrstar data Preset directory for ChromStar data files 2DDATA c
171. ing the chromatogram Press Green light button or menu points Operation Start or F1 function key The chromatogram TESTO001 will be recorded Wait until the run time has elapsed A print out of the chromatogram will be printed Recording the next chromatogram Inject the sample and simultaneously press the green light button or menu points Operation Start or F1 the chromatogram TEST0002 is recorded and the print out be printed after the run time has elapsed Recording the next chromatograms Inject the sample etc TESTO003 etc Calibration and quantitative Evaluation All steps are carried out no window is closed and a standard solution had been injected Creating a Calculations file Click menu point Window line Edit Files Press cal button or menu point Chrom Files Calculation in the Open Calculation File box overwrite UNTITLED after filename with TEST click Open Select External Standard enter the concentration unit of the standard solution against Calculation Units Click index card Peak Table click Graphic key In the Select Report Files box double click the name of the standard chromatogram in the list of files press OK Mark peaks by dragging the mouse with pressed left key press OK Click the line of the peak table enter the standard amount in the box under Amt in Std enter the name of the solute under Peak Press Insert key repeat for all peaks Press Close key Save it Yes Page 5 1 16 5 1 5 Sho
172. ix Testmix 1 000000e 000 1 000000e 000 1 000000e 000 1 000000e 000 1 000000e 000 1 000000e 000 1 000000e 000 1 000000e 000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 6 1 1 1 1 a Shift Selected item down m Edit Selection Copy Vial Sample Id max 20 letters Factor Weight IntStd Conc Level fiz Testmix 1 000000 0 000000 0 000000 fi Operation Cut Preset Delete Delete all Sample Info max 250 Letters Benzol T oluol N aphthalir Help Close Save Save As Inj Vial Injection number or vial number when using an autosampler entries from 1 to 499 Sample Id The name up to 20 characters entered here appears on the chromatogram print out behind Sample Identifier It is stored in the raw data file SLI with the chromatographic data The following parameters are used in quantitative evaluations Factor This factor is used as a dilution factor in a quantitative evaluations with the internal or external standard methods The result of the quantitative calculation is multiplied with this factor Weight Total quantity of the injected sample In a quantitative calculation the quantities found for the peaks to be calculated are also calculated as a percentage of this total sample weight The quantity must be entered here in the
173. jections of the same standard solution can be averaged in a current run as well as afterwards When calculating averages during a current run mark Autosampler on the first page of the Method File also if no autosampler is used It is always necessary in calibration runs to use a virtual autosampler The injection of the calibration sample is identified in the autosampler table with S against Type the number of injections to be performed is entered against Inj The name of the Calculation File containing the Page 5 3 2 5 3 1 Calibration Methods Peak Table is entered in the Data Handling File to be used The response factors established after each injection are stored until all injections of the calibration sample are carried out After this the average values of the individual standard are calculated and entered into the Calculation File The Calibration Table 11 one receives contains a report of the average calculation To calculate the averages afterwards first One Point Calibrations have to be y performed with the Report File in the Reprocess Calculations window FA Then click the point Calibration and Multi Calibration Select the files obtained before from the list of Report Files and click OK The result of the average calculation with the standard deviation appears on the screen for the first peak of the Peak Table The averages of all other peaks are similarly calculated The results must be stored by means of File and S
174. l can also be used as a calibration point Click Select and Get Concentration Table and the table with the calibration concentrations and corresponding peak areas appears in which you can now mark the Zero Value box down left The value in the entry box at the right now reads 0 After clicking OK the new calculated calibration curve appears in which zero is used as a data point and is drawn in the graph Concentration Table Toluol Nr Std Amt Area If the component to be evaluated is eluted together with an interfering substance at a constant level a blind value may be entered in the entry box at the right once Zero Value has been clicked This blind value must previously have been determined as a peak area by recording a chromatogram without the calibration component 1 1 0000E 001 319709 enabled a A 2 0000E 001 548247 enabled 2 2 0000E 001 548247 enabled A 3 0000E 001 645240 enabled 3 3 0000E 001 845240 enabled A 4 0000E 001 1180311 enabled 4 40000E 001 1180311 enabled A 5 0000E 001 1528649 enabled x disable Zero Value 0 Cancel 4 5 3 Reprocess Calculations Displ Chrom Page 4 5 13 4 5 3 Reprocess Calculations Displ Chrom In the program section Displ Chrom chromatograms can be displayed in different ways in order to be compared The different possibilities offered by the submenu points 3D Plot and Compare to display chromatograms are described in the following sections 4 5 3 1 Reproc
175. lay on the screen Copy copies the screen display to the clipboard E Exit returns you to the Reprocess Calculations window Selecting the menu point Expand allows you to modify the size of the Angle and the Offset value as well as the start and end times and the maximum and minimum signal value all of which influence the three dimensional representation Expand by Yalues The following limits apply when selecting these parameters Angle 0 and Offset 0 gives a two dimensional display of the overlaid chromatograms Angle eo Offset fi 0 Time Axis Angle 90 and Offset gt gt 0 gives a two Start 0 00 End 299 dimensional display of the chromatograms ae stacked parallel along the y axis Intensity Axis The Offset value determines the distance between the baselines of the individual chromatograms Start 714 End 342 06 omea An enlarged section of the display of all the chromatograms can be made by constructing a rectangle which always has the lowest chromatogram as its base This enlargement can be repeated at will The original display of the full length chromatograms is re accessed by clicking the right mouse button Selecting the menu point Reference displays a dialog box which shows a list of the individual chromatograms and their colours used in the screen display The box is closed Chromatogram Color neu1129 c1111 C uw neu1129c2111 C The colors in used for the different chrom
176. libration samples can be entered in the entry line against Amt in Std The user must enter this for each peak in the calibration solution The Paste key transfers the content of the clipboard into the table Previously the relevant peaks must be selected in the Peak Report Table and copied into the clipboard by using the Copy key in Results in Reprocess Reconstruction Reintegration or Manual Integration cp p 4 4 8 4 4 12 4 4 18 If more than one peak is in a peak window a peak can be selected for the calculation by entering in the chrst32 ini file Calibration PeakDetection 1 default value all peaks in the window are evaluated 2 the largest peak in the window is evaluated 3 the peak in the middle of the window is evaluated 4 the first peak in the window is evaluated 4 1 3 2 Edit Files Calculation Peak Table Page 4 1 29 The Graphic key allows graphic peak window definition After clicking Graphic a file select box is opened where chromatograms can be chosen These appear in an overlay mode representation An entry box appears where detailed information about the peak windows can be made Windowname xcetophenon Load Files Peakname s Acetophenon nsert Cancel Amount in Std 3 Resp Factor 2 3499e 006 2 ae orig Area 0 fo T Int Std l Ref Peak A peak window is defined by moving the mouse with pressed left button from the peak start to the peak end either to the left or to the right This a
177. lues in other words that the chromatograph is ready to inject 5 1 1 Recording a Chromatogram Page 5 1 5 The sample is brought into the injector then turn the injector to the inject position and z at the same time press the function key F1 The running chromatogram appears now in the Draw Baseline window with a time axis according to the Run Time selected Above this next to the name of the method appears the name of the chromatogram being recorded in channel one here AROMOO01 In the line with status information the time elapsed is displayed against Elt In order to have an optimum screen display of the data the display was changed in this case with F11 Select Screen Scale clicking By Values low value 10 high value 800 and OK EJ ChromStar Method Operation HPLC Display Options Window Help nel plola lalele amp dA E g r ee ea Draw Baseline Chi Op x Method aromaten Slice Ch1 aromO001 2600 min No 1 Flow 1 00 A4 100 0 B 0 0 C 0 0 D 0 0 Elt 7 2 Press F1 for Help After 9 minutes the data acqisition is stopped After the calculation Calculation in Process the data are sent to the printer and the chromatogram is printed out on one DIN A4 page in portrait format below it the results table 6 appears The data are saved under the name AROMO001 SLI In the Report File which is created at the same time the results of the integration retention times peak areas and heights ar
178. lysis is used later on The Default key is only visible when in Chrst32 INI in the section Configuration the entry is DefaultButton 1 The entry DefaultButton 0 or no entry causes the button to be invisible the default rse file will be deleted Quit with OK Prior to doing this the HPLC equipment should be switched on 4 2 1 Analysis Chromatogram Options Select Channels Page 4 2 3 Now a window with the title Draw Baseline Channel 1 appears and the baseline of the detector attached to this channel is displayed on the screen The digital display of the mV signal appears in the right top corner of the Draw Baseline window behind Ch1 Ch2 etc When HPLC devices are controlled details of their status are displayed in the line below the draw baseline window In this display the number of the vial analysed in the autosampler is shown behind Vial the number of the injection from this vial behind No and the rate at which the eluent is being pumped behind Flow The percentages of the solvents are shown against A B C and D Wave indicates the wavelength of the detector in nm and elt the time elapsed since the start of the chromatogram EJ ChromStar Draw Baseline Ch1 Iof Xx Method Operation HPLC Display Options Window Help x net iis mlalaiclp fl z i e CAA Ch1 0 420 Flow 1 00 4 100 0 B 0 0 C 0 0 D 0 0 Press F1 for Help NUM After ending the selection the menu points Method HPLC only access
179. m 4 8 8ff GPC 4 9 3 GPC Man Int 4 9 17 4 9 18 Baseline Automatic Transform 4 8 8f Baseline Correction Transform 4 8 8f Baseline Manual Transform 4 8 9f Baseline Mode GPC DH File 4 9 13 5 9 9 Batch 4 5 17f GPC 4 9 24ff Batch File Name GPC 4 9 25 beta GPC Broad Standard Calibration 4 9 21 Broad Standard Calibration GPC 4 9 20 Bubble check 4 1 34 By values 4 4 5 4 4 11 4 4 17 C C Solv 3 7 4 1 32 4 2 3 4 2 4 4 3 4 4 3 5 Calculation 4 1 22 4 5 1f 4 8 1 4 8 6 Calculation base 4 1 23 Calculation File 4 1 1 4 1 18 4 1 22 4 5 1 4 5 5 4 5 7 5 2 2 5 2 10 5 2 12 5 3 1ff 5 3 9 6 3 Calculation examples 5 3 13 Calculation Units 4 1 24 5 3 10 Calibration 4 5 4 4 5 7 5 3 1 GPC 4 9 6ff 5 4 4 Page 7 2 7 Index ChromStar Calibration File GPC 4 9 3 Calibration function GPC 4 9 11 4 9 20 Calibration methods 5 3 1ff Calibration range GPC 4 9 17 Calibration run 4 1 7 5 1 10 Calibration standard 4 1 11 Calibration Table GPC Cal File 4 9 8 5 4 4 6 29 Cascade 3 5 Chl Ch2 4 2 2 Change Password 3 4 Change Time 4 9 3 Change User 3 4 Chart Speed 4 4 4 4 8 9 4 8 11 4 8 13 Chrom Spec 4 6 10 4 6 13 Chromatogram 4 2 1 Chromatogram 4 6 16 Chromatograms 4 3 7 ChromStar Integrator 1 2 2 2 2 8 ChromStar light 1 2 2 8 ChromStar Setup 2 3 ChromStar window 3 1 CHRST32 INI file 2 5 4 1 14 4 1 34 4 2 2 4 3 3 4 6 2 4 6 7 5 1 9
180. mY Reference AR0MA1213A5051 Work AROMA1213B2031 No Operation 1600 1400 1200 1000 0 1600 1400 1200 1000 800 600 400 200 7 1 7 7 0 0 to 20 3 0 4 0 5 0 6 0 min Press F1 for Help NUM 7 The menu points have the following meaning Files with its submenu points permits new chromatograms to be selected Open File Information displays a box with entries about Author Sample Identifier Recording date Changes Number of datapoints Runtime Minimum and maximum mV value Page 4 8 6 4 5 2 Transform Arithmetics Print Portrait or Landscape allows Work and Reference chromatograms to be printed Printer Setup allows the printer settings to be changed Copy transfers the chromatograms to the clipboard Exit closes the File Arithmetics window Alignment In the next work stage which can be carried out before the calculation an alignment of the Work chromatogram along the time axis Time Alignment and an internal normalisation on a data point Height Alignment for both chromatograms can be effected After selecting the point Time Alignment the positions in the Reference and Work chromatograms which are to be matched after the alignment must be defined In the information box the message Align the chromatograms appears This is done as follows A time point is defined in the Work chromatogram by clicking the left mouse button The mouse cursor is moved to the appropriate point of the Referen
181. made in the lower part with rectangles before the final processing Attention Shift ESC cancels an existing enlargement Further processing in Manual Integration is done via the menu points Mode and Edit which will now be described The menu point Mode is used to define the baseline and calculation mode First selected is always the Tangential Baseline The information box lA displays the indication Operation Mode Tangential Baseline To define the start point of the tangential baseline the mouse cursor is moved to the desired position this point is fixed with a click on the left mouse button Moving the mouse arrow along the graph draws a white line Click the left mouse button again at the desired end point of the baseline The mouse arrow returns to the beginning where corrections can be made as necessary Clicking the right mouse button confirms the baseline The white line then changes to a green line on which start and end are marked The baseline for a number of peak groups can be defined one after another If no further peak separation is accomplished by one of the following menu points then the total area from baseline start to baseline end will be evaluated as one peak even when the chromatogram is crossed by the baseline Exponential Baseline not yet operational is only used to separate a group of peaks positioned on the tail flank of a peak The second submenu point Valley is used to separate two peaks by FA
182. matosram ARALO201 mY Select the operation mode or perform Fine selection 500 400 300 200 100 a 14 0 15 0 15 0 17 0 18 0 19 0 20 0 21 0 22 0 min Press F1 for Help NUM h the enlarged chromatogram section by clicking the left button at the desired baseline start drawing it clicking the left button again and confirming with the right button The baseline is drawn as a straight line across the selected range This can be done more times in succession Further peak separations can now Wie the submenu point Tangential Baseline in Mode and draw the baseline in be made in the enlargement by constructing perpendicular drop lines with Valley or by drawing of overlapping peaks with Exponential Skim For a simple peak it is only necessary to draw the baseline In order to have a precise check on the retention times the View function can a be employed when defining peaks with its aid the retention time and the mV signal at the cursor position are displayed 5 2 3 Manual Integration Page 5 2 9 The evaluation of the peaks is started by clicking the right mouse button in the white area of the upper chromatogram This completes the manual integration in this section The peak separations made are drawn in the upper part and the lower part is cleared After constructing a new rectangle the following operations can be performed With Annotate peaks can be shown with retention times In the lower section peaks can be annotat
183. me entered in the entry box by moving the cursor with the left mouse button depressed over the box The name appears on a black background and can now be overwritten The file selection is confirmed with Open or RET Now the selected Method file Run Table cp fig on page 4 1 7 is displayed Cancel closes the file select box without opening a file 4 1 1 Edit Files Method Page 4 1 5 The menu bar contains the menu points File Window Help The individual pages of the Method File accessible via index cards are described in Sections 4 1 1 1 to 4 1 1 4 The menu point File corresponds to the button and appears for all files which can be edited with Edit Files The menu points Window and Help appear on all ChromStar applications detailed description see chapter 3 4 and 3 5 File contains the submenu New Open Save Save as Print Printer setup Copy File Information Close and allows file operations With New the first page of a Method File with the name Untitled appears This can now be edited and with Save As be stored under a new name With Open a file can be opened to make changes or to create a new file To secure the file after changing the question appears Save file after answering yes the selection field Save File As appears where the desired filename can be selected from the file list Save allows a changed or new file to be stored Save as allows a changed file to be stored under a new name
184. mmable functions are on the left a time table on the right and at the bottom right an entry line The cursor blinks in the Time box Enter the desired time and key TAB to reach the box under Function Enter the number of the programmable function then jump with TAB to the Value box You can also select a function by clicking it in the list at the left Enter the composition of solvents in percent for each component separated by a space With Insert the selection is inserted into the table above If more than 11 lines are entered the scroll bar appears to the right of the table If a line of the table is to be modified or deleted it is first clicked with the mouse Then the line appears in the entry bar at the bottom where it can be deleted or edited and inserted back into the table The table can contain a maximum of 75 lines The file can be stored with File and one of its appropriate submenu commands A new file can be selected with File and New or the button or Open EA Customize Function Names allows you to change the descriptions of the programmable functions 1 11 and to save these in CHRST32 INI in the section Edit Files Atention The new descriptions appear in all LC Procedure files The original descriptions reappear on deleting the entries in CHRST32 INI in Edit Files With Close or the button the window is closed 4 1 5 Edit Files Preset Page 4 1 35 4 1 5 Edit Files Preset In the Preset File p
185. n one peak can be defined fa Edit Files Calibration Graphic When dragging the mouse along the chromatogram the cursor shows different shape lt IP Peak start and peak end can be moved by pressing the left mouse button and moving the mouse to the right or to the left The whole range of the peak window can be moved by pressing lt gt the left mouse botton and moving the mouse Load Files leads back to the file select box With OK or File Return the determined peak windows are transferred into the peak table The peak table reappears with the new entries With Cancel the procedure is aborted The peak table reappears 4 1 3 3 Edit Files Calculation Regression Table Multi Calibration Page 4 1 31 4 1 3 3 Edit Files Calculation Regression Table Multi Calibration The Regression Table of the calculation file contains the function coefficients of the Ist 2nd or 3rd order regression analysis of a multi level calibration for the peaks defined in the peak table of the calculation file or respectively under K1 the average value of the response factors after multi calibration with the Result Files of a multiple injected calibration sample fe Edit Files Calculation Mix2 2 Method Table Peak Table Regression Table Multi Calibration Parameters Multi Calibration Graphic m Files used Path C ChromStar 6 3 Data T est260405 9 Mix2 260405 0051 26 04 2005 12
186. n required In the Method box Percent Normalization Internal Standard External Standard This parameter defines the type of calculation to be carried out cp 5 3 2 description of calculation methods and formulae The default method is percentage this is carried out when the calculation file specified in the data handling file is not found or when no calculation file is specified In the percentage method the results list shows for each peak the retention time peak start and end time peak area and height and area respectively height in percent When using the percent method with entries in the peak table cp p 4 1 24 the percentages of all peaks are calculated for the peaks in the peak table the response factors are used in the calculation the peak names are annotated if any names are entered In the normalization method the results list shows the retention time the area and height of all peaks and also the area percent and names of the peaks which are defined on the second page Peak Table Select Internal Standard when this method is to be used for the evaluation A calculation of areas and heights of all peaks takes place as well as a quantitative calculation of all peaks defined on the next page On that page peak table the internal standard peak must be marked against Code with IS The External Standard leads to a calculation of the areas and heights of all the peaks as well as a quantitative calculation of all peaks defin
187. n the method employed and are described in the next sections 5 3 2 1 Percent Method In the percent method a percent calculation is performed of all peaks found The factors of the formula 1 have the following values RF 1 MULT 100 DIV X PKS The formula now reads RES PKS 1 100 PKS No entries are required for this method on pages 2 and 3 of the Calculation File All peaks are calculated whose areas or heights are greater than the value against Minimum area height in the method table of the Calculation File Carrying out the calculation with stored data is done in Reprocess Calculations Calculation by clicking the Report File and the Calculation File 5 3 2 Quantitative Calculation Methods Page 5 3 5 5 3 2 2 Normalization In this method only those peaks are calculated which are defined in the peak table on page 2 of the Calculation File and which are within a defined time window The RF value response factor is defined for each peak on page 2 of the Calculation File The default value is 1 This can be manually adjusted for each peak or defined in the calibration run The other factors have the values DIV 2 PKS RF MULT 100 RF amount area of the i th peak defined in the calibration run The formula 1 now reads RESj PKS RF 100 X PKS RF 5 3 2 3 External Standard Method For quantitative calculations according to this method Calculation File with Method External S
188. nd the report print template SHORT RPF is chosen It is not necessary to change all other parameters Page 5 1 2 5 1 1 Recording a Chromatogram It is of course possible to enter a name and other appropriate information in the Notes field fg Edit Files Method Aromaten The necessary Data Handling and LC Run Table Sample Table Documentation Table Mode Notes HPLC CGC Procedure files I Autosampler IT Use Line Number automatically M Normalization l receive the default gt Report name LC Procedure Jaromaten X short rpf X AROMATEN 3 Data File s Data Handling 1 Aromaten rom I No Report The chromatograms Data Handing 2 x 2 Single Data Files are Data Handling 3 ft H r ei on the Data Handia A o a a on characters The file is closed with the screen key Close In a small Hel Close Save Save As ee ea ser BELA dialog box the message appears AROMATEN is a new file and the question Save it which is answered with Yes Again the Edit Files Window appears in which a data handling file can be created after clicking Chrom Files and Data Handling In this case where it is line important to build an analysis with a minimum number of steps the data handling file must get the the same name as the method file The name AROMATEN is entered in the entry line and confirmed with OK gg Edit Files Data Handling aromaten Of x Now the acquisition page
189. nd the result to the clipboard E Exit returns you to the File Transformation window Back returns to the display of the original chromatogram Here other Work and Reference chromatograms can be selected with Files and Open Page 4 8 8 4 8 3 Transform Baseline 4 8 3 Transform Baseline The menu point Baseline with the points Automatic and Manual allows you to correct the detector baseline of a chromatogram either automatically or manually 4 8 3 1 Transform Baseline Automatic iJ After clicking Baseline and Automatic and selecting the chromatogram this appears in the upper third of the screen The title bar reads Automatic Baseline Correction The menu bar contains the points File Baseline Subtract Expand Window Help The information box reads Select rectangle to zoom Expand allows an enlarged section in the upper window to be created by value entries To carry out the baseline correction first construct a rectangle The created enlargement appears in the lower part of the display After clicking Baseline a corrected baseline is calculated This is shown as a red line in the lower display Ed ChromStar Automatic Baseline Correction olx File Baseline Subtract Expand Window Help 18 x AE ap mee ea lt 3 4 0 6 0 8 0 10 0 12 0 14 0 16 0 18 0 20 0 22 0 min Press F1 for Help NUM 4 8 3 Transform Baseline Page 4 8 9 With Subtract the area beneath the corrected baseli
190. nded With the Gaussian curve approximation small parts of a chromatogram can be integrated The menubar of Transform contains the points Modify Arithmetics Baseline Curve Fitting Exit Window Help 4 8 1 Transform Modify After clicking Modify a file select box appears where a chromatogram can be chosen The chromatogram is shown in the upper part of the window The menu bar displays the menu points File Calculation View Expand Window Help The next step Fix Calculation Mode is displayed in the information box The following operations are available under Calculation y Smoothing a chromatogram 1 Derivative constructing the 1st derivative a 2 Derivative constructing the 2nd derivative T Smoothing digital filtering and constructing of derivatives are done according to the Savitzky Golay method The number of points used in this algorithm are defined as Convoluting Points 5 7 9 11 13 15 under Parameter Multiplicate multiplies the mV values of a chromatogram with the factor defined under Parameter in the box Multiplication against Factor Reverse turns the chromatogram so that the starting point is now at the end of the time axis Page 4 8 2 4 8 1 Transform Modify period in the chromatogram After clicking Parameter the maximum time period of a spike can be defined against Max Time and the minimum signal height against Min Height in the Clean box Nis Filter smoothes the chromatogram in a different wa
191. ne is subtracted from the chromatogram File and its submenu offer various file operations Open returns you to selection of a further chromatogram for automatic baseline correction J Save As stores the result Ensure that you do not overwrite the original chromatogram when doing this File Information displays a box with entries about Author Sample Identifier Recording date Changes Number of datapoints Runtime Minimum and maximum mV value Print with its submenu points Portrait Landscape and Chart Speed prints out E the lower section of the screen Printer Setup allows the printer settings to be changed Copy transfers the result to the clipboard y Exit returns you to the File Transformation window According to the status of the operation the menu points Baseline and Subtract and the submenu point of File Save As are not accessible and appear in light grey 4 8 3 2 Transform Baseline Manual 7 Manual correction of the baseline is performed by clicking Baseline and Manual and selecting a chromatogram The title bar reads Manual Baseline Correction The menu bar contains the points File Order Expand Window Help Here too an enlargement must first be constructed After this select a maximum of 25 points in the lower screen part by clicking with the left mouse button to points through which the new baseline has to be plotted A point can be deleted by clicking with the right mouse but
192. nimum absolute time window If the percentage time window calculated for a specified retention time leads to a value lower than that defined here the time window is defined as being retention time minimum time window The parameter values can either be accessed with the TAB key or Shift TAB the entries appear on a black background The same happens after striking over an entry box with the cursor while the left mouse button is depressed A new value can now be entered The next box to be edited can again be accessed with the mouse or TAB Page 4 1 26 4 1 3 2 Edit Files Calculation Peak Table 4 1 3 2 Edit Files Calculation Peak Table The parameters of the second page of the calculation file are used to define peaks or groups of peaks and their various data e g quantity response factor name or code On this page data are entered into a table which is automatically sorted chronologically when new peaks are entered The column headings of this table depend on the method previously chosen to define time windows egjEdit Files Calculation Mix2 2 Method Table Peak Table Regression Table Multi Calibration Parameters Multi Calibration Graphic Time Petcen Amt in Std Resp fact Areal Peak Code 1 278 11 579 1 0000E 000 3 6021E 006 0 0 Anisol Time Percent Amt in Std Resp fact Areal Peak Code or23s 15423 fooooe o00 f3 3730E 006 f oo Phenol zx a Insert Paste Delete Graphic zi Help Close Save S
193. nt composition of the first procedure as soon as the column is conditioned Depending on the type the autosampler either picks out the first vial programmed and brings it out for sampling or it moves the sample tray into the corresponding position Then the injection follows and the chromatogram appears on the screen Before the next injection takes place the evaluation and a part of the print out must be awaited The display against Vial shows from which vial the injection was made No shows the injection number this is consecutively numbered if repeated injections from one vial are made The chromatogram name also shows from which vial the injection has been made Depending on the type of autosampler any vials in the sample tray following after the defined vials will be processed until no more vials are found When using the pump you will notice that the method has not been correctly finished by the fact that the pump continues to pump solvent The next line of the method is transferred The number of the next vial programmed appears on the screen against Vial The injection takes place shortly after this After the injection of the last vial of a series provided that no further entries have been made in the method a print out is made The pump does not stop Start may only be pressed again after reactivating the method In any case the entry against Data File in Edit Files Chrom Files Method must be changed in order to prevent
194. nt the following formula is used RES PKS RF 100 RC Wherin is RES Result of the quantitative evaluation for the component 1 PKS Peak area or peak height of the ith peak component 1 RF Response factor of the component i RC Recovery rate in for the component i 5 3 2 4 Internal Standard Method In this method which does not depend on an exact volume of the injection sample a known quantity of a substance internal standard is added both to the calibration standard with known quantities and to the sample to be analysed The calculation which follows is based on this internal standard It is necessary in this method to define the Standard Peak as internal standard with IS against Code in the calculation file Method Internal Standard If this definition is not found the following message appears No internal standard peak defined The largest peak found in the time window defined with IS will be appointed as the Standard Peak If no peak is found within this time window the following error message is printed No internal standard peak found Each of these two errors leads to a calculation according to the percent method In calculations according to the Internal Standard method the response factors are determined as follows Resp Fact Amount Std PKS IS Std PKS i Std Resp Fact Ss Amount JS Std PKS IS Std in which is Amount Std Amount of the substance i in the standard
195. ntration in the units Col Type RPS defined in the calculation file Absolute the Soh Peck E weight percentage of the total sample weight if amp s entered Weight and peak names for the peaks defined in the calculation table l Changes of these Parameters will be recorded in Slice File Header If more than one peak is found in a peak window a peak selection can be made by entries in the Chrst32 ini file s page 4 1 26 When a calculation is carried out after multi point calibration average value of the response factors entered as K1 in the Regression Table the response factor in the results list shows the K1 value When a calculation is carried out after a multi level calibration the response factor in the results list shows For a correct documentation the print report template should contain the object Calculation File in which all except the Regression Table can be removed In this display the grey line between table head and body can be moved up or down by pressing the left mouse key and moving the mouse as soon as the cursor appears as double arrow 4 5 1 Reprocess Calculations Page 4 5 3 Quantitative calculations of a series of chromatograms can be carried out under Batch with Mode Calculation EJ ChromStar Calculation OF Xx File Calculation Calibration Display Chrom Batch Convert Exit Window Help l x sjaj 4 ma oE omme A Chromatogram Caf10031 Calculation File KAFFEE C
196. ograms recorded by means of gel permeation chromatography Molecular weight and the specific GPC calculations on GPC chromatograms can be carried out here Under GLP Options in the GLP version new users and new passwords can be registered The menu procedure Extras Options opens a window where optional settings for some ChromStar applications can be made These settings have a corresponding entry in the CHRST32 INI file The meaning of the settings is given in the appropriate places in this manual The menu procedure Extras Options does not appear when the CHRST320 DLL is not present or has been deleted Page 3 2 3 2 ChromStar Operating elements 3 2 Operating Elements With a double click on the program icon the Start dialog is activated After entering the correct information the ChromStar Window fig page 2 7 appears on the screen The title bar contains the name of the application If the window is active it can be moved around the screen by clicking in the title bar with the mouse and holding the button down whilst moving the mouse The full picture cannot be moved however A light blue title bar marks an inactive application Clicking any position within an inactive application activates it whereupon the title bar is shown in dark blue The menu bar contains the current submenus available After a submenu has been selected its menu bar appears above the relevant box The procedure option Window and its submenu New allow you to
197. oints and commands in this application appear in light grey print they cannot be accessed This happens when the necessary preceding steps of an application have not yet been carried out In order to record a chromatogram further details see section 5 1 1 you must first in the Edit Files window have created and stored a method file and a data handling file and if an HPLC pump is to be controlled an LC procedure file The method file should contain on the first page the entry Mode HPLC or GC and the name of a data handling file for the first data recording channel The further entries to be made in the method file are explained in section 4 1 1 The parameters defined for the data handling file are described in full in section 4 1 2 Attention The files required for carrying out an analysis method file data handling file and the LC procedure file when using a controlled pump must be stored in the same directory This is also the directory where recorded chromatograms slice files SLI and report files RPT are stored j After clicking the application Analysis Chromatogram a dialog box appears for selection of the data acquisition channel and pump control see next section The menu bar contains the menu points Method Operation HPLC Display Options Window Help in which only the menus Method Options Window and Help can be selected The sub menus of the points not yet to be used appear in grey letters In the
198. olor references to the solvents A D are shown For the print out of the gradient the item Show Gradient must be marked in the object Chromatogram c p Report Editor manual Audit Trail opens a list showing all events which have happened during recording the chromatogram with date and time In the simplest case Start Chromatogram and Stop Chromatogram are mentioned here 4 4 2 Reprocess Integration Page 4 4 9 4 4 2 Reprocess Integration With Integration chromatograms can again be integrated After clicking the menu point Integration a submenu appears with the points Reintegration and Manual Integration With Reintegration the evaluation is carried out with a data handling file containing the integration parameters With Manual Integration peak recognition is done manually Both methods are described in the next chapters and in sections 5 2 1 and 5 2 3 4 4 2 1 Reprocess Reintegration file The result of the integration is stored in a temporary file with a name where the first character of the original file name is substituted by a sign The files RPT files and SLI files are shown in the file select box On leaving ChromStar these files are deleted The result of the integration can be saved in the original report file a warning appears that the file is overwritten or in a new file Files in Reintegration are immediately deleted by the entry DestroyTemp 1 in Reprocess in CHRST32 INI DestroyTemp 0 the file
199. on of x M File Mode Expand Edit Annotate View Window Help 8 x sla alela elal tel dal ole 3 qu fm ele A Chromatosram FMTEST Operation Mode Tangential Baseline MY Continue integration or acknowledge 800 600 400 200 Press F1 for Help Operations can be made undone or be re activated by the menu Edit and ist sub menu The menu point Undo can only be chosen when a section of the chromatogram is presented in the lower part of the display and after a manual peak separation Was carried out The operation carried out last will be deleted The separation line in the display disappears In this way several operations can be made undone stepwise The menu point Redo reactivates the operations stepwise Redo can only be chosen when Undo was used before 4 4 2 2 Reprocess Manual Integration Page 4 4 17 The other menu points are fully described in section 4 4 1 and will therefore only be mentioned here briefly File has the following sub menu Open to select another chromatogram Ey Open previous selects the next lower numbered chromatogram Open next selects the next higher numbered chromatogram File Information displays various file information Comparison Chromatogram loads a comparison chromatogram Show Peaks from File displays a Report File Print prints the chromatogram and or result table Printer Setup opens the printer setup window Copy copies the chromatogram or re
200. one window the error message appears Peak Ret time found in window T1 to T2 again When calibration samples are used with different amounts then the second page of the Calculation File also contains the entries of these amounts under different Level Numbers The Response Factor is initially set to 1 In the calibration the Response Factor is determined by the equation RF Amt Std PKS where RFi Calculated response factor of the i th peak Amt Std Amount of the standard entered against Amt in Std on page two of the calculation file PKSi Area or height of the i th peak in the calibration chromatogram The calculated response factor is stored on page 2 peak table of the calculation file against Resp fact and can be used later in a quantitative evaluation After clicking the menu point Calibration and its submenu point One Point Calibration a dialog box cp p 4 65 is displayed in order to select the report and the calculation files Selecting and clicking OK displays a second dialog box in which the number of the calibration sample of which the injection generated the selected peak data file is entered against Level The calculation starts after clicking OK Level ja Cancel Page 4 5 6 4 5 2 1 Reprocess Calculations One Point Calibration The established response factors are entered into the calculation file being used and are available for quantitative calculations In addition the
201. open further sub windows The contents of the menu box change according to the window activated A series of windows can be displayed side by side or in an overlay pattern by the Window submenus Tile or Cascade The toolbar contains buttons to facilitate the use of the most important functions of ChromStar Each button corresponds to a menu or submenu procedure The buttons for the main applications of ChromStar are situated to the right The exit button closes the active application and leads to the next higher application The meaning of the buttons is as follows Edit Files Analysis Chromatogram Analysis DAD Reprocess Integration Reprocess Calculations Reprocess 2D Reprocess Spectrum Transform GPC The meaning appears in the information line at the bottom of the ChromStar window as soon as the mouse cursor is moved over a button 3 2 ChromStar Operating elements Page 3 3 The frame of a window has various functions too Moving the mouse over the frame brings a slanting double arrow at the upper and lower rim of the frame and a horizontal double arrow at its right and left side A window can now be enlarged or reduced by depressing the left button of the mouse moving it in the required direction and then releasing it When the window is reduced other windows previously opened become visible In the top left hand corner of the screen is the Control Menu Box Placing the mouse arrow in this field and clicking the l
202. or channel 1 is that of the method file When two or more channels are being used to record data the data handling file of the second and further channels must receive different names to avoid overwriting the chromatograms of the other channels When using more than one channel the entries must be made one after another i e no data handling box may be passed over with the TAB key The channel numbers of the A D boards do not necessarily have to correspond to the numbers 1 to 4 here The Data Handling Files must have been specified before the analysis is started Data File s Behind the Data Handling file for each channel the name of the chromatogram up to 19 characters different names for each channel is entered Each injection receives automatically an increasing number starting from 0001 If an 4 1 1 1 Edit Files Method Run Table Page 4 1 9 autosampler is used the digits 5 until 7 are used to store the vial number Digit 8 is used to count the number of injections from the same vial expressed in hexadecimal numbers maximum 15 F Chromatograms SLI and at the same time Report Files RPT are saved under these names DAD Method File When using a diode array detector the name of the scan parameter file to be used is specified here This file contains the parameters for data acquisition by the diode array detector Entry of parameters is done again after clicking the selected fields with the mouse or by using the TAB key wit
203. or installing the A D converter board in your computer 1 Switch off the computer and all attached devices e g printers and monitors 2 Remove the top of the unit If necessary refer to your computer user s manual 3 Remove the cover of a free slot 4 Plug the converter board with its gold plated connector into the socket The second converter board for 4 channel recording requires its own slot 5 Screw the metal holder of the A D converter board to the rear of the computer 6 Close the top of the main unit Reattach all cables you removed in step 2 and switch on the computer and its peripherals Page 2 2 2 ChromStar Installation The PCI A D converter boards require as operating system Windows NT 2000 XP or Me Windows 98 2 edition will work but is not recommended Pin assignment of the 2 channel PCI A D Converter Board ea scra S LEL Chi N gD 3 GND z nan i SCPA 4 GND THN fe Start 1 3 LEEEEEEETEEE 02 90 20 29 01 i fe Ls Start 2 4 T mmm GND Pin assignment of the 2 channel ISA A D converter board SCPA S Jeonan Le 421 on 2 GND 4 Serr O04 50 01 16 95 Start 1 3 GND Start 2 4 GND 2 ChromStar Installation Page 2 3 Pin assignment of the 1l channel ISA A D converter board of the ChromStar Integrator SCPA 7 1994 SCPA rs Analog LC 42M Signal Ser Nr 01 50 25 27 94 Pin assignment of the one channel PCI
204. out a chromatographic run Start Start the method and therefore the recording of the data also using funktion key F1 Stop End of data acquisition F2 i Stop Int Stop integration if no autosampler is used also stop recording Abort Abort recording F3 Continue Continue the method only operational if an autosampler is used Unlimited Runtime allows the runtime of a chromatogram to be increased These commands are activated by clicking with the mouse Before the method is started only the Start and Unlimited Runtime function can be accessed After the method has been started the functions Stop Stop Int Abort and Continue only when using the autosampler can be accessed Page 4 12 4 2 5 Analysis Operation The Start command is given at the same time as the injection This can for instance be done automatically initiated by switching of the injector with aid of the LC 427 Event Box When an autosampler is used an automatic start is also performed at each injection There is no need for the analysis window to be activated in the case of an automatic Start The processing of the time table of an LC Procedure with a solvent gradient begins at the start time EJ ChromStar Draw Baseline Ch1 Iof x Method Operation HPLC Display Options Window Help l x plola RABI C D S A E omme z Method marathon Slice Ch1 mara01150021 Testmix Chi 2 873 mY min Vial 2 No 1 Flow 1 00 4 100 0 B
205. overwriting of the already existing chromatograms Before pressing the Start key wait until the pressure and detector signals have re stabilised When aborting an analysis by Abort or stopping the analysis by Stop the autosampler aborts the run and the needle is moved to the home position The method is no longer active After Stop the recorded chromatogram is stored and printed The method must be activated again before the next start can be carried out By entering a line number of the autosampler table a vial and injection number the method can be continued Stop Int When using an autosampler causes the chromatogram to be stopped and saved bewirkt only after the autosampler run time is elapsed the next injection will be carried out Continue can be used during an autosampler run to stop a chromatogram before the programmed end of the run time The question Stop data acquisition is answered by Yes the chromatogram is stored and the autosampler carries out the next injection Page 5 1 12 5 1 4 Operating an Autosampler When Unlimited Runtime was switched on during the autosampler run the chromatogramm will be stopped and saved by Continue or clicking Unlimited Runtime again the next injection will be carried out 5 1 4 4 Trouble shooting and error messages If the system hangs up for any reason the best thing to do is to switch off the pump and autosampler than reset your computer and or start WINDOWS once again Then st
206. r Table 4 1 1 2 Edit Files Method Autosampler Table The order in which the samples are to be processed is specified in this table The table can only be accessed if the parameter Autosampler is marked in the Run Table Edit Files Method nov08 Pile Ea Run Table Autosampler Table Sample Table Documentation Table ET E A E R a e E T 3 000 nov Beispiel rpf Beispiel rpf Beispiel rpf Beispiel rpf Beispiel rpf 2 Vial to Inj Vol Run Type LC Procedure Report File a DH Files DH 1 DH 2 Overwrite novon 7 J Insert DH 3 DH 4 Delete Help Close Save Save s The parameters of this table have the following function Vial The number of the vial to be processed entry starting from 1 up to a maximum value which depends on the available autosampler With W a washing procedure is executed without an injection from a vial also depending on the type of autosampler The duration of the washing procedure is entered in column 5 Run and the solvent composition is specified in the LC Procedure File mentioned in the LC PROC column to Depending on the available autosampler a vial number can also be entered here 2 or higher In this way a continuous series of vials is processed starting from the vial number entered in the first column until the vial number entered here Inj The number of injections 1 15 depending on kind of autosampler in use to be made from a vial
207. r User Name Name fH Risler C chrstar32 exe with the user s name This name appears later with every recorded chromatogram as author s name Page 2 8 2 ChromStar Installation A double click on the ChromStar icon and answering of the ChromStar start dialog causes ChromStar to start with its title page 23 ChromStar DAD GPC 15 x Edit Files Analysis Reprocess Transform GPC Extras Window Help pla 3 frm esa co aes 4 Press F1 for Help Num 3 ChromStar Window Page 3 1 3 Description of the ChromStar Window 3 1 ChromStar Structure The ChromStar system consists of 7 main applications with the following functions With Edit Files the parameter files necessary for recording and reprocessing of chromatograms are edited and stored Analysis controls the recording of chromatographic data by e g a UV detector Chromatogram or by a diode array detector DAD Reprocess allows chromatographic data to be reprocessed and _ re presented Integration allows the quantitative evaluation of chromatograms the reprocessing of groups of chromatograms the comparison of series of chromatograms and data conversion Calculations DAD data 2D and spectra Spectrum can be represented and reprocessed Transform allows you to add or subtract two chromatograms to calculate derivatives to correct baselines and to deconvolute peaks GPC accesses all files necessary for evaluating chromat
208. ram as required The calculation can be carried out at a later time in Reprocess Calculation Factor f1 000000e 000 Weight 0 0000 After choosing the chromatogram and a ay ere calculation file the values for column length and Int Std 0 0000 solvent peak are entered in the box Update Input Values After clicking Show Column Coefficients Sample ld CoffeinSth Berg the results list showing the column coefficients Col Len 125 00 appears in the Calculation window For the print out an appropriate print report template Col Type RPS must be used For details cp Chapter 4 in the s tt Report Editor manual objects Column Details Linear Velocity Solvent Peak and Table AN l Changes of these Parameters Available Items will be recorded in Slice File Header Page 5 2 16 5 2 6 Column Coefficients B CALCULATION of x Chromatogram ts610041 Calculation File TEST Sample Identifier Coffein Lsq Solvent Peak 1 500 min ColumnType RPS Column Length 125 00 mm Calculation Method External Standard Calibration Method One Point PeakNo Ret Time Area Height Asymmetry Platenumber K Name min 2 3 067 957529 183 179 1 500 1877 1 044 Coffein a 957529 183 179 The column coefficients are calculated by using the formulae Linear velocity u mm s L To 60 Plate number N 5 545 T wW Plates per meter Nm N 1000 L Plate height HETP or H H um L 10
209. ram can be presented in high resolution as vector grafic or in a desk top publishing program Reconstruction switches to the application Reconstruction with the reconstructed chromatogram Manual Integration opens the application Manual Integration and offers the possibility to execute a manual correction of the integration The selected chromatogram is displayed in the upper section of the screen The peak groups defined with reintegration are transferred to manual integration and can for instance 4 4 2 1 Reprocess Reintegration Seite 4 4 11 be erased there with Delete The further execution of manual integration is described in section 4 4 2 2 Exit Reintegration closes the application Reintegration Expand allows you to select a section of the entire chromatogram After clicking Expand the submenu appears with the points Reference Window By Values and Default Reference Window Auto range allows you to construct a rectangle in the entire chromatogram in order to select a section In the line above the chromatogram the message appears Select rectangle to zoom cp 4 4 1 The selected section is automatically normalized to the highest peak The submenu point By Values allows you to zoom out by entering the time and mV values in a dialog box Here Auto Range can be marked to in order to normalize the selected section to the highest peak The values in the dialog box always correspond to the current size of the chromatogram
210. ration Peak Win Ret Time Area Resp Sstd Amt No No min Fact mg 00ml 1 592 694182 7 2027E 05 50 000 Cumol 2 442 531493 2 8222E 04 150 000 Hexylbenzol 1225675 Page 6 20 6 5 Print outs processed in chapter 5 20 Quantitative evaluation after a current run Chromatogram QUANO0031 Date 18 04 1996 Time Author H Risler Notes Sample Identifier Injected on 18 04 1996 Injected at Data Processing Parameters Slice Width msec 500 Noise Threshold mV 500 Skim Ratio Parameter Files Data Handling File QUAN Calculation File QUAN Calculation Method External Standard Calibration Method One Point Peak Win Ret Time Area Resp Absolute Weight No No min Pact mg 100ml 1 633 808698 7 2027B 05 58 248 0 000 Cumol 2 475 633015 2 8222E 04 178 652 0 000 Hexylb nzol 1441714 236 900 6 6 Print outs in Transform Page 6 21 6 6 Print outs of chromatograms after File Transformation 21 Ist derivative of a chromatogram Current user H Risler Date 17 04 1996 Time FILE TRANSFORMATION Operation 1 Derivative Chromatogram AROM0001 Page 6 22 6 6 Print outs in Transform 22 Addition of two chromatograms Current user H Risler Date 17 04 1996 FILE ARITHMETICS Operation Addition Reference Chromatogram N2000011 Work Chromatogram N2000012 Time 11 52 ChromStar 7 Index 7 Index A A Solv 3 7 4 1 32 4 2 3 4 2 4 4 3 4 4 3 5 Abort 3 7 4
211. rder Then click File and Save Results enter a suitable name for storing the calibration function which is then available for further calculations For each peak another order can be chosen Print and Results prints out the calibration table 12 Print and Graphic the graph of the calibration function 13 Printer Setup allows the printer settings to be changed Copy and Results or Copy and Graphic copy the calibration table respectively the graph to the clipboard ea Exit returns you to the Reprocess Calculations window Select contains the submenu points Order Get Peak Table and Get Concentration Table Clicking Order allows the permissible approximation functions i e of the first second and third orders or all together of the calibration to be displayed on the screen This screen display can be printed out with File Print and Graphic At least 5 Report Files are necessary to calculate a third order approximation function If all calibration functions first to third order are selected for presentation after clicking Order and the results stored via File and Save Results only the coefficients of the first order of approximation will be Select Regression Type entered in the regression table of the ao calculation file For each peak another C First Second C Thid C All Orders order can be chosen Page 4 5 12 4 5 2 2 Reprocess Calculations Multi Calibration Clicking Get Peak Table displays a list
212. rea appears high lighted In the entry box the following entries can be made Windowname Peakname will be added under Name in the Peak Table Peakname s If a calculation with peak names was carried out earlier the name used appears here If more than one peak name is found in the window all the other names appear by clicking into the downwards arrow By clicking into one of these names the name will be transferred into the box after Peakname It will be inserted in the Windowname box by using Insert Amount in Std Amount in the standard solution corresponds to Amt in Std in the peak table and will be inserted there Resp factor Response factor corresponds to the Resp Fact in the peak table and will be inserted there Area 0 corresponds to the same item in the peak table By clicking into nt Std or Ref Peak one of the peaks can be specified as internal standard or reference peak do not use with calibrations x 2 2 orig multiplying or dividing the retention time by 2 or re establishing the original value These entries can be made in a box which appears by clicking the right mouse key in a peak or via menu point Zoom By using Edit Delete or the Delete key or pa a peak window can be deleted After using x2 a scroll bar appears at the bottom of the chromatogram tus enabling all ranges of the chromatogram to be displayed Page 4 1 30 4 1 3 2 Edit Files Calculation Peak Table As described above more tha
213. reen presentation on the print out and on the presentation transferred by means of Copy The menu point No Annotate deletes an existing annotation This can be done easier by clicking the appropriate pressed button View with ist sub menus Results Show Comparison Chromatogram Split Show Values Show Gradient and Audit Trail allow additional displays With Results the results list is displayed on the screen in the same The table Z contains peak numbers retention times peak areas and peak heights the result of the last calculation under Abs Rel and peak names Manually integrated peaks are marked by an M under Mode The scroll bar at the right of the table allows you to scroll through the list if it is longer than the screen page The dialog box may be moved at choice over the screen by placing the mouse cursor into its blue title bar pressing the left mouse button and dragging it After clicking one line of the table and using the Shift or Control key the lines marked in this way can be copied into the clipboard by using the Copy key These lines can now be entered by using the Paste key in Edit Files Calculation in the peak table The box disappears when OK is clicked Peak Report FHTEST Peak Ret Time Area Height Abs Rel Mode Name 2 017 80364 20 396 8 2 350 407266 136 132 2642 9 2 758 24226 4 568 2 858 10 2 992 145406 39 944 3 258 11 3 675 74389 19 353 3 950 12 4092 224749 54 277 4275 13 4 400 100532 22 128 45
214. report file RPT the corresponding chromatogram appears together with the Sample Identifier in the box to the left Page 4 5 2 4 5 1 Reprocess Calculations After both names have been entered and this box quitted with Open a box appears in which the values for Factor Weight and Int Stand and Sample Id can be updated cp sect 4 1 1 3 New values can be entered here for factor multiplication for the total sample weight and for the amount of internal standard in case a run with internal standard has been selected For the calculation of column coefficients the Column Length and dead time Solvent Peak can be entered here These parameters as well as the column description Column Type can also be entered in the Documentation Table of the method file cp sect 4 1 1 4 before recording the chromatogram The box is closed with OK the calculation is carried out and the results table is displayed on the screen ndata A If the column coefficients are to be shown the box Show Column Coefficients must be Factor 1 000000e 000 marked For more details concerning column coefficients calculation cp sect 5 2 6 Weight 0 0000 Cancel The results table show details of the peak data Int Std 0 0000 Report and calculation files used the peak number window number Win corresponding Sample Id Coftein Std with peak number in the calculation file Collen o retention time peak area response factor 7 Res Fct the conce
215. results are entered in the Report File This then contains the amount peak area or height and response factor for each peak defined in the Calculation File The link between peak area and amount is used later in the Multi Calibration see next section After the calculation is finished a results list with new response factors Res Fct appears on the screen The table also contains details of the amount of the substance in the standard solution and the name if this was defined in the peak table of the calculation file Ea ChromStar Calibration OL x File Calculation Calibration Display Chrom Batch Convert Exit Window Help x S S 4 mi Clery Sm m ee a Chromatogram Caf10011 Calculation File KAFFEE Calculation Method Calibration Peak Win Ret Time Area Resp Std Amt Name No No min Fact 0 003 3 1 2 645 986217 1 0140E 06 1 000 Coffein 986217 Press F1 for Help NUM Ai File has a submenu with the following functions Open or Calibration One Point Calibration allows selection of another Report and Calculation file Save saves the result Save As saves the result in a new file Load Multi Calibration shows a Multi Calibration once carried out and saved in a calculation file Print prints out the results table 9 Printer Setup allows changing of printer settings Copy copies the table to the clipboard One point calibrations with a serial of chromatograms can be carri
216. returns you to the chromatogram and data handling file selection Open Previous selects the chromatogram in a series of chromatograms with the next lower number at the end of the chromatogram name Open Next opens the chromatogram with the next higher name Both menu points are light grey when no chromatogram with a higher or lower number is available With File Information details concerning the chromatogram are displayed Show Peaks from File opens a selection box from which an existing Report File can be selected The results list of this file appears on the screen Print permits the print out of a chromatogram With Printer Setup printer settings like resolution paper size portrait or landscape orientation can be changed Copy opens a further submenu with the options Chromatogram and Report which allow you to select whether to copy the chromatogram or the results list into the clipboard Using Save the re integration can be saved A warning appears that the original report file will be overwritten N This Command will Overwrite your Report File Using Save as the report can be saved under a new name Automatically the chromatogram will be saved under the same name ii Abbrechen Using Revive an unfinished chromatogram e g caused by a computer break down can be revived Export saves the chromatogram in a WINDOWS meta file WMEF after having selected an appropriate print report template Thus the chromatog
217. rt instruction guide One point calibration E Press balance button or menu point Window New Calculations Press Red point button or menu point Calibration One Point Calibration in the Select Calculation File and Report File box choose in the list under Calculation File TEST CAL and in the list adjacent the name of the standard chromatogram Test000x RPT press Open in the Select Level 1 box press OK Press the printer button for the print out of the results E list or menu point File Print in the Report El Template File box choose e g SHORT RPF in the list of files press Open Calculating the unknown amount Press Vx button or menu point Calculation In the Select Calculation File and Report File Box click in the list under Calculation File TEST CAL in the liste to the right click the chromatogram name of the sample Test000y RPT press Open in the Update Input Values box press OK for the print out of the results list press the printer button or menu point File Print in the Report Template File box click e g SHORT RPF in the list of files press Open 5 2 Evaluating a Chromatogram Page 5 2 1 5 2 Evaluating a Chromatogram 5 2 1 Automatic Reintegration A stored chromatogram can be evaluated by automatic reintegration at a later stage It is necessary to pay attention to the following when selecting the data handling file to be used 1 Using Reintegration chromatograms are re integrated with a
218. s Chromatogram HPLC 0 eee eeeeeecceeesteeeeeeees Analysis Chromatogram Display ccessseeeeeeeeeeees Analysis Chromatogram Method cseceeesseeeeeesteees Analysis Chromatogram Operation ccesseeeeeeeeees Analysis Chromatogram Options ccceeesseeeeeeeteees Analysis DAD Data Acquisition Instrument Control Reprocess Integration Reprocessing of Chromatographic Data ccceeeeeeeees Reprocess Reconstruction 0 ccceceeeseseceeessteceeeseneeeeeeeaes Re process Integration cs ccecselacaccotivisecedeasestanetereesesees Reprocess Calculation Quantitative Calculations Calibrations Comparisons MB ALC EECCA OID 5 sso car doses pion atepuipssin toe nse T EASE oiia Reprocess Calculation sc sccciesseccensesesancevwadeevevedevnorieteacevevaes Reprocess Calibration heres stadu dust laadetecseyecdetonsanecensaysoueasaes Cece ere crc ccc ccccccccccccccccccccccecccccccecceccees Pere rr ccc ccccccccccccccccccccccccccccccces Peer err cccccccccccccccccccccccccccccs Pere rr ccc ccccccccccccccccccccccccccccceccccs 4 5 3 Reprocess Displ Chrom sxcieesn Sain atone cade eandaeee 4 5 13 4 5 3 1 Reprocess 3D Plot 4 5 3 2 Reprocess Compare 4 5 4 4 5 5 4 6 4 7 Reproc ss Batch cicsi yc hrcca sees teaevesxavctuasytes avanleaatemaaaese R pr cess S CONV OR oinera ieena aa Reprocess 2D Reprocessing 2D Data Reprocess
219. s are deleted on leaving ChromStar Using Reintegration chromatograms are re integrated with a data handling When the submenu point Reintegration is selected a dialog box appears in order to select the chromatogram to be integrated and the data handling file Chromatogram DH File i Amino SLI a Aral0201 SLI a Bam30101 sli Bam30201 sii a Bambasauto1 sli a Ciba0101 sli ja Ciba0201 sli a Fmtest sli j Filename PALM2 Filename Palm2 SLI Filetype Data Handling Files int Filetype Slice Files sli 7 Cancel A preview of the chromatogram and the Sample identifier if any was entered when recording the chromatogram appear in the box to the left Exit the dialog box with Open After this the reintegrated chromatogram is displayed in the complete application window Page 4 4 10 4 4 2 1 Reprocess Reintegration If a data handling file has been used for reintegration which has an existing calculation file defined against Calculation then also the quantitative evaluation takes place according to the method defined there An enlargement can be made by setting up a rectangle with the mouse cp p 4 4 5 The menu bar now displays the menu points File Expand Annotate View Window Help The File submenu offers various options for file processing The menu points are fully described in section 4 4 1 and will therefore only be mentioned here briefly Open
220. s or Copy and Graphic the results respectively the graphics are copied to the clipboard Exit returns you to the Reprocess Calculations window Select contains the submenu points Get Peak Table and Get Concentration Table Get Peak Table allows you to produce a graph of E Tare Gide a further peak if more peaks of a chromatogram a have been evaluated After clicking Get Peak Table as 2 Ethylbenzol 230 a window appears with the title Peak Window 3 Cumol 2 57 Table This table lists all components contained in parse z the standard solution and defined in the Peak Table of the Calculation File Clicking a peak in this table and OK generates the graph of the averaging of the areas for this peak Cancel Get Concentration Table displays a list of the peak areas used for obtaining the average value One or more values in this list can be excluded from the calculation by Disable or restored by Enable Concentration Table Toluol Nr Std Amt Area 4 Q0000E 007 1748872 4 0000E 001 1137723 enabled 4 0000E 001 1154783 enabled 4 0000E 001 1152789 enabled 4 0000E 001 1162010 enabled 4 0000E 001 1167247 enabled 4 0000E 001 1165786 enabled Ol amp OMB disable Cancel Page 4 5 10 4 5 2 2 Reprocess Calculations Multi Calibration For calculating a calibration function Multi Level Calibration in Multi Calibration a number of Report Files are necessary max 75 file extension
221. s skim ratio noise and threshold can be programmed chronologically i e they can be modified during the course of a run Calculation The Calculation File contains the necessary parameters and entries for calibration and quantitative evaluation On the first page of this file the calculation method percent normalisation internal or external standard is specified the type of time window absolute or relative retention time the calculation basis peak area or height and the number of calibration samples The second page contains the peak table with the retention times response factors and peak names Also the amount of the individual components of calibration samples is entered here On the third page regression coefficients for a multi level calibration are stored LC Procedure In the LC procedure file the parameters for the control of HPLC equipment via interface as solvent composition flow rate oven temperature detector wavelength etc are defined in a time table Each parameter can individually be adjusted at different times for instance to generate solvent gradients to change the flow rate or to record a UV spectrum Scan Parameter In the scan parameter file the name of the 2D data set parameters of the diode array detector as wavelength range and resolution and the run time of the data acquisition are defined Preset In the preset file the default values of certain parameters like flow rate or wavelength are specified Thes
222. stribution Plot GDT The content of the Data Handling Calculation LC Procedure GPC Data Handling or GPC Calibration files used when recording a chromatogram is stored in the report file These files can also be converted As ASCII file they get the name of the report file and the extension ITT CAT etc Multiple file selection according to WINDOWS is done as follows Click the start file name press the shift key and hold it depressed click the end file name causes all files between starting and ending file to be marked Click the start file name press the CTRL key and hold it depressed click any other file names causes only the files clicked to be marked Slice File to Andi converts chromatograms into Andi files cdf Chromatograms can be saved in the Andi format CDF at the same time as in the ChromStar format SLI RPT with the entry DoAndi 1 in chrst32 ini in Analysis Andi to Slice File converts Andi files into ChromStar kompatible chromatograms sli rpt The report file must be fitted by a reintegration 4 8 Transform Page 4 8 1 4 8 Transform The program module Transform allows various chromatogram transformations The first or second derivative of a chromatogram can be calculated A chromatogram can be smoothed multiplied or reflected Spikes in a chromatogram can be removed Peaks can be enhanced Baselines can be corrected manually or automatically Two chromatograms can be added subtracted divided or appe
223. sult table to the clipboard Save saves the evaluation in the Report File Save as allows the chromatogram to be saved under a new name At the same time the corresponding report file containing the result of the manual integration is created Reconstruction transfers the chromatogram to the application Reconstruction Reintegration opens the dialog box Select chromatogram and DH file which allows after selection of a Data handling file a quick transfer to the application Reintegration cp 4 4 2 1 Exit Manual Integration quits this program item Expand permits the enlargement of the upper chromatogram by number entries By Values or to restore its original display Default Annotate with its submenu points Automatic Peak Numbers Retention Times Peak Names Manual and No Annotation operates only on evaluated peak groups in the main chromatogram An enlarged part of the chromatogram in the lower section can only be annotated by means of Manual Annotation in the lower sector is possible as soon as integrated peaks are existing these can in addition be a annotated manually I Page 4 4 18 4 4 2 2 Reprocess Manual Integration View offers the sub menu procedures Results displays the results on the screen The peaks obtained by manual E integration are marked by an M under Mode Peak Report FHTEST Peak Fet Time Area Height Abs Rel Mode Name 5 888893 124 079 M 6 17 133 866952 122 499 M 7 17600 212605
224. t a flow rate of 1 ml min and a run time of 12 min with an RP 18 column and a UV detector at 254 nm First switch on the computer the monitor the printer the detector and the pump The detector signal is connected to channel 1 of the A D converter board The pump is controlled via the RS232 Comm Port 1 The operating system is loaded in case of automatic ChromStar loading the title page of ChromStar appears The next step is to create the files necessary for the analysis Method Data Handling LC Procedure If a pump is used which is not controlled via ChromStar it is not necessary to create an LC Procedure file All entries concerning the solvent can be skipped For that purpose click Edit Files in the menu bar of the ChromStar E application Edit Files Analysis Reprocess Transform Window Help Now the Edit Files Window appears on the screen with the menu bar Chrom Files Options Exit Edit Files Window Help y After selecting Chrom Files and the submenu point Method a dialog box appears with on the left a list with already existing Method Files and an entry field and on the right a field for changing the subdirectory Because creating a new method is explained here first a new name max 23 characters and umlauts are not allowed is entered in this example AROMATEN the window is left by clicking OK Following upon this the method file appears To obtain a print out after recording the chromatogram Report is selected a
225. t and the reintegrated chromatogram appears on the screen The name of the chromatogram starts with the tilde sign To perform an automatic reintegration click the menu point Integration followed by Reintegration in the Reprocess window m Several options are available to present the result With Annotate peak numbers retention times individual descriptions or peak names from the relevant calculation file can be written into the chromatogram If the chromatogram is to receive the peak names defined in a peak table of a calculation file the name of this calculation file must be entered in the data handling file against Calculation File The method of calculation specified in the calculation file will be carried out A manual annotation can be saved in the report fila via Files Save Page 5 2 2 5 2 1 Evaluating a Chromatogram EJ With Files and Print and selecting a report print template RPF the chromatogram can be printed A preview of the print out appears in the box to the right In order to judge the reintegration an enlarged section of the chromatogram Fa can be displayed by means of View and Split or Expand If the result is not satisfactory the time programmed integration parameters can be redefined via the menu point Param The calculation with the changed parameters is executed after clicking OK If suitable parameters are obtained in this way these can be entered in the Data Handling File by clicking Param
226. t changing colors Show Connect shows all further used files of a selected Method File like Data Handling LC Procedure and Calculation Files except for Mode GPC When an autosampler is used all files belonging to the individual vials in the Autosampler Table can be viewed with the switches Next Vial and Previous Vial Comrarctod Files m C uChenevS tae E Jilan HT Dati aiaa Gute Harding Fis i aan Fiai Tiempebade E LE Precediane DAD Mathcad fis l E dabang Table line 1 Wale 1 Beie Helin ce 4 1 Description of the ChromStar Software Edit Files Page 4 1 3 Options contains the submenu Print Copy Search for Sample Id With Print or the amp button one of the file types can be printed print outs of all file types protocols 1 5 with Copy one of the file types can be copied into the Clipboard A submenu with a list of the different file types appears after clicking Print or Copy The desired file type is selected here After this a dialog box appears from which a file can be selected After clicking it the file name appears on a black background and in the box above Clicking OK starts printing clicking Cancel breaks off this action Search for Sample Id can be used to find a Sample Identifier cp Method File Sample Table either in a method file or in a chromatogram Search for Sample Id in Method Files Search directory is c chrst6 november Directories Back Se
227. t displays the entire chromatogram the lower enlarged sections of it as described in 4 4 1 With Show Values a field is displayed in the status line above the chromatogram in which the retention time and the detector signal of each data point in the chromatogram are displayed Show Gradient In the chromatogram representation the gradient used when recording the chromatogram can be shown only possible when the pump is controlled via ChromStar and the gradient is entered in the LC Procedure file On top of the chromatogram the color references to the solvents A D are shown For the print out of the gradient the item Show Gradient must be marked in the object Chromatogram c p Report Editor manual Audit Trail opens a list showing all events which have happened during recording the chromatogram with date and time In the simplest case Start Chromatogram and Stop Chromatogram are mentioned here 4 4 2 2 Reprocess Manual Integration Page 4 4 13 4 4 2 2 Reprocess Manual Integration Selecting the menu point Manual Integration accesses a part of the program iat which allows manual peak recognition The chromatogram to be integrated is selected by means of the dialog box described in section 4 3 1 After selection the entire chromatogram appears in the upper part of the application window The menu bar contains the following points File Mode Expand Edit Annotate View Window Help The buttons in the Toolbar to the left allow quick
228. t during a slope of the signal higher than the noise value This parameter is used to prevent a broad valley between two peaks being evaluated as a baseline In the same way the parameter Threshold ensures that a peak which has had its top cut off overloading of the detector is correctly integrated and is not treated as a double base line offset Its value can be between 1 and 1000 and can be modified at any runtime 3 Skim Ratio This parameter compares the ratio of the heights of two adjacent peaks to the height of their common valley It determines whether they should be separated by a perpendicular line or whether the second peak is to be regarded as riding on the first in which case its baseline runs exponential from the valley between the peaks to the peak end Its value can be set from 0 to 20 Page 4 1 20 4 1 2 2 Edit Files Data Handling Integration Page 4 Baseline This parameter allows a baseline for the integration to be set manually at a defined time during the data acquisition for example after a disturbance of the baseline caused by a change of solvents of the flow rate or of the wave length Further explanations concerning Noise Skim Ratio and Baseline can be found in chapter 5 2 2 Attention Be careful with the definition of the baseline when the retention times are unreliable If the baseline is set during a peak the area and height of the peak will be incorrectly calculated 5 Valley Valley This parameter def
229. t hoo focoo Load Traced Off C cha mv invert fi coo ooo Save Printer Selection Instrument Control Cancel Default PEER WSCHULZ LASERJET on Nell bi goseosesessesessesseg In the lower part of the Draw Baseline window a line appears with details about the flow rate and eluent composition The detector signal is displayed in the black area and the value of the detector signal is shown in the right top corner The previously created method AROMATEN can now be recalled by clicking Imet Method Load and selecting AROMATEN by clicking it in the directory with all method files which now appears Leave this box with OK Load Method File 27x In the line above the black i ice area the message Method Searchin Dat S ele AROMATEN appears a aroma MET Aromaten met a neu2 met This shows that the method a ireua ET is activated in the line Ea E Boon aoe below a 1 appears against nahone No ie the next injection a neu MET receives number 1 When a controlled LC instrument is used the pump starts to Filename Aromaten met Open pump the eluent Le composition as specified in Filetype Method Fies mey g Cancel the LC procedure file Start End AROMATEN in this case Tie Line 60 methanol and 40 water Wait until the column is equilibrated and the detector signal and the pressure have reached stable va
230. tandard the RF values response factor of the substances to be analysed are determined first in a calibration run cp 4 4 2 1 with a sample containing known amounts Standard Only those peaks are calculated which are within the defined time windows on page 2 of the calculation file The factors of the formula 1 have the following values RF amount area of the component 1 defined in the calibration run for each peak MULT 1 DIV 1 The formula 1 now reads RES PKS RF In a current run the entry against Factor in Sample Table of the method file is directly the value of the multiplier MULT in 1 This multiplier keeps the value 1 if no entry is made If Weight in the sample table of the method file has a value other than 0 the proportional weight of each component of the solution compared to the total amount of the sample is calculated according to formula 2 RES RES 100 Wspl 2 in which Wspl is the total amount of the sample as entered against Weight The entry Recovery Yes in the CHRST32 INI file in the section Calibration enables a quantitative evaluation under respect of a recovery rate The calculation method should be External Standard The recovery rate is entered in the calculation file in the Peak Table under Recovery in This can be different for the different peaks The default value is 100 for a recovery of 100 Page 5 3 6 5 3 2 Quantitative Calculation Methods For the calculation of the amou
231. tion is used to switch ON or OFF the 4 available external events of the LC 21 pump from Bruker which may in turn be used to activate external devices such as switching valves fraction collectors etc Having selected this function via 9 enter against Value the number of the external event required 1 4 followed by a space and the desired status OFF 0 or off ON 1 or on The table above always contains the number against Value followed by ON or OFF 10 Stop This function allows you to stop the pump when injecting manually or semi automatically For example you can program Stop to a time which will never be reached when conducting a series of injections as the procedure is always being restarted at 0 by Start However once the injection series has finished this time will be reached in the LC procedure and the pump will stop pumping solvent It is not a must to program the Stop function The pump can also be stopped manually with the function key Stop Pump once a chromatogram has been recorded 11 Emission nm This function specifies the emission wavelength of a fluorescence detector The excitation wavelength is defined using function 6 Page 4 1 34 4 1 4 Edit Files LC Procedure After clicking LC Procedure in the submenu of the Chrom Files the Open LC Procedure File box appears for selecting of an existing file or entering of a new name s 4 1 1 After selection of a file name the file appears in a window in which the progra
232. to Data Handling 4 appear in a light grey print which means that they cannot be accessed The names of the data handling and LC procedure files to be used for each individual vial are now entered in the Autosampler Table cp next section Page 4 1 8 4 1 1 1 Edit Files Method Run Table Autosampler must be clicked in a calibration run when the calculation is to be carried out immediately after recording the chromatograms even when no autosampler is in use The standard is marked in the autosampler table under type as S cp P 4 1 11 For more details cp p 5 10 and 5 27ff Use line number When this item is marked the line number of the autosampler table is used to build the name of the chromatogram Data Files s below namely the 5th character of the file name corresponds to the line number 1 9 A line 10 B line 11 etc up to Z The AS table may only contain max 35 lines the 6th and 7th character is the vial number the AS may only contain up to 99 vials The 8th character is the injection number max hexadecimal 15 F The item Use Line number should be marked if a vial is entered in different lines and the chromatograms are used later on for a common calculation e g multi level calibration Normalization This parameter defines whether the print out of the chromatogram should be normalised to the highest peak or not No normalization means that the scale of the y axis is determined by the Screen Scale factor s
233. ton a The menu point File and Open returns you to chromatogram selection Ej 1 Exit returns you to File Transformation File Information displays a box with entries about Author Sample Identifier Recording date Changes Number of datapoints Runtime Minimum and maximum mV value Expand allows an enlarged section of the upper part to be displayed by value entries for the time and mV axis Page 4 8 10 4 8 3 Transform Baseline Calculation of the approximation function for the new baseline is effected in the menu point Order which offers the possibility of conducting an approximation of the first through the fifth order and of a cubic spline approximation The options offered are determined by the number of baseline points which have previously been defined If only two points in the chromatogram have been defined it is only possible to plot a straight line between them which is later subtracted from the chromatogram A minimum of four points are necessary for a quadratic approximation and five for a cubic approximation For approximations of the Ist to 5th order approximation functions are calculated based on the method of least squares The points selected need not necessarily lie on the approximation function For the cubic spline approximation a function is calculated in which all points are on this approximation function After the desired approximation has been clicked the complete chromatogram with the calculated baseline
234. ust be written if a solvent gradient or a stepwise change of flow rate is to be pumped After this a method must be a le le 60 aoj 0 0 procedure selected 4 2 3 and started which contains this LC The programming and operating of a slow solvent change is described in section 5 1 2 4 2 2 Analysis Chromatogram HPLC Page 4 2 5 The wavelength autozero and the attenuation factor of the UV detector or the excitation and emission wavelength of a fluorescence detector respectively are programmed with Wavelength Emission Autozero and Attenuation With Oven the temperature of the column oven is selected Necessary for this are a UV detector and column oven which can be controlled These instruments have to be equipped with the appropriate interface Pump status The status line at the bottom of the Analysis window shows the information about the pump control Vial Vial number when an autosampler is in use No Sample number or injection number respectively Flow Flow Rate in ml min A B C D Eluent composition in Page 4 2 6 4 2 3 Analysis Chromatogram Display 4 2 3 Analysis Chromatogram Display The menu point Display allows you to change various screen display parameters The vertical axis of the screen display of the detector signal can be changed by 3 defining an other attenuation factor Screen Scale or an other mV value These changes are entered in the Select Screen Scale box which is
235. veral chromatograms can be made by using Shift or Ctrl key The reprocessing is started after clicking OK in the Execute Batch File box The results of the calculation are added to the Report File as last entry In the GLP mode chromatograms are not overwritten when reporcessed New files are created chromatograms SLI and report files RPT with inserted characters in the filename After calculation _C_ is inserted in front of the last 4 characters current number of the chromatogram after reintegration _I_ is inserted and after one point calibration _O_ is inserted 4 5 5 Reprocess Calculations Convert Page 4 5 19 4 5 5 Reprocess Calculations Convert The menu point Convert in the Reprocess Calculations window with its sub menu To ASCII Slice File to Andi and Andi to Slice File enables you to convert ChromStar files in ASCII files und to convert ChromStar chromatograms SLI into Andi files CDF and vice versa E After clicking Convert To ASCII a dialog box appears in which the File type List Files of Type down left and the names of the files to be converted can be selected by clicking and multiple marking Only files of the same type can be selected for conversion The marked file names in the list appear under File Name one after the other in such a way that only the last two are visible E ChromStar Calculation iof x File Calculation Calibration Display Chrom Batch Convert Exit Window Help l x m
236. w Help The buttons in the Toolbar to the left allow quick access to the main operations in this application They are described together with their corresponding menu points In the line above the chromatogram the message appears Select rectangle to zoom An enlargement can be made by setting up a rectangle with the mouse cp p 4 4 5 By clicking with the right mouse key in the chromatogram the original size is restored The menu point File permits the following file operations Open returns the user to file selection in order to display another chromatogram Open Previous selects the chromatogram in a series of chromatograms with the next lower number at the end of the chromatogram name Open Next opens the chromatogram with the next higher name Both menu points are light grey when no chromatogram with a higher or lower number exists Comparison Chromatogram loads a comparison chromatogram the slice width must be the same as in the reconstructed chromatogram With File Information the following entries concerning the chromatogram are displayed Author Sample Identifier entered in the sample table of the method file which is used during recording of the chromatogram date of recording changes number of data points run time lowest data point and highest data point Show Peaks from File opens a file select box where a report file can be chosen the preview on the A Show Peaks chromatogram is Report File C
237. w Help 18 x a gu rm ea A Peak Name Toluol Calculation Mode External S Coefficients of Least Squares Fit k0 1 1818E 000 k1 3 2584E 005 k2 0 0000E 000 k3 0 0000E 000 Correlation Coefficient 0 996890 p J as EA rt a y gt oT c 200 0 1000 0 1200 0 1400 0 Area x1 000 Press F1 for Help NUM 7 4 5 2 2 Reprocess Calculations Multi Calibration Page 4 5 11 The menu bar contains the points File Select Window Help File and Open allows you to select alternative Report Files for a Multi Level Calibration Load Multi Calibration shows a Multi Calibration once carried out and saved in a calculation file Using File and Save Results the regression function can be stored in a calculation file default name in the entry box is that of the calculation file belonging to the first selected Report File This name should be overwritten with a name showing that this calculation file is obtained by a multi level calibration The regression coefficients are entered on the third page of the calculation file Regression coefficients of all peaks in the calibration solution are stored In this newly generated file the parameter Regression for Calculation on the first page is marked owing to this the regression coefficients of the third page are used in later calculations If a calibration function of the second or third order is to be stored first select the required order by clicking Select and O
238. which is to be averaged can be increased by the Peak Parameter max 15 Then even in a great signal noise ratio peaks can be recognised The application Analysis offers the option to determine the noise value automatically with Control and Automatic Noise in the draw baseline window This avoids peaks to be detected in the detector baseline This value is automatically transferred to the preset table and is used for all integrations if no other noise value has been defined in the data handling file for time 0 During automatic noise determination the slice width defined in the preset table is used If possible this should be set at the same value as selected for recording the chromatogram If the determined noise value proves to be suitable for the integration it should be stored in the time table of the data handling file at time 0 With that it is available for further evaluations and will not be overwritten by new Automatic Noise determinations 300 Noise 100 300 700 600 300 400 300 200 100 15 0 17 5 20 0 22 5 min Page 5 2 4 5 2 2 Integration Parameters 3900 300 700 600 300 400 300 200 100 Noise 1000 In the example on the left integration has taken place with noise values of 50 100 and 1000 Obviously the value 1000 is too high to allow satisfactory peak recognition The value 50 prevents the recognition of a correct peak end because of this the baseline end is
239. wo character code can be assigned to a specified peak The following codes may be used IS This code defines a peak which is to be used as the internal standard If more than one peak is assigned this code only the first one is used as the internal standard RF This codes defines a peak which is to be used to update the time windows after each run It is necessary that the parameter Rel Window Update on page 1 of the calculation file is switched on G For peak grouping a peak time window is marked with G The areas of all peaks appearing in this time window are added In this calculation the retention time is the average of the retention times of the peaks in the time window In the chromatogram only the centre of the peak group is marked with its retention time or peak number In the report the peak group appears on only one line identified as G for a group of peaks Several peak windows can be marked with G in a chromatogram which leads to an equivalent number of peak groups being calculated Page 4 1 28 4 1 3 2 Edit Files Calculation Peak Table S Number For peak summing various non adjacent peaks can be marked which are added in the evaluation A number of different peak sums can be defined with S1 S2 S3 etc The retention time and the name of the first peak in the current sum are used The peak sums appear in the report in one line identified by S Number The area is the sum of all the peak areas of the peak windows
240. y as under Smoothing z The Clean function removes spikes high mV values for a very short time Enhancement causes a peak enhancement After clicking Parameter the Y frequence can be entered against Filter Cutoff Frequ in the Filter box For this frequence the intensity values in the chromatogram are set to 0 after Fourier Transformation Slice Width Conversion allows the slice width to be changed only increased ruy Selecting the menu point Parameter displays a dialog box where the al parameters for the different calculations can be changed Exit by clicking OK Convoluting Points Convoluting Multiplication Slice Width Conversion Points 5 00 i A 2000 00 V5 Factor Slice Width Caa 7 Filter Enhancement I 3 7 a ri Cutoff Freq 0 60 Hz Slope Width 0 10 min 13 Clean i 15 IV Auto Clean Max Time 0 220 min After this a new calculation can be performed by clicking Calculation and selecting one of its submenu points Parameter to be changed Convoluting Points Number of points which are used as averaging basis values from 5 15 odd numbers Multiplication Factor This factor is used for multiplicating the chromatogram values without limit Filter Cutoff Frequ All frequences greater than Cutoff Frequ e g high frequent noise is filtered off thus smoothing the chromatogram Enhancement Slope Width In this calculation the difference between adja
241. y line below It can now be edited and re entered in the table by clicking Overwrite By clicking Insert the line will be inserted after the old one A marked line can be deleted with Delete If the autosampler table has more than 10 lines a scroll bar appears to the right with which you can move through the table An empty table appears if the autosampler table is selected in a new file The cursor is blinking in the first field of the entry bar Entering 1 TAB TAB 2 TAB 20 TAB 8 TAB TAB new TAB Downwards arrow Short32 rpf TAB new TAB and clicking Insert accomplish the execution of 2 successive injections from vial 1 of 20 ul These injections are carried out immediately once the preparation of the autosampler is completed The pump starts pumping the solvent mixture as specified in the LC procedure file New The chromatograms are stored under the name specified in the Run Table which is extended by 0011 and 0012 and are printed out using the short32 report print template Page 4 1 12 4 1 1 3 Edit Files Method Sample Table 4 1 1 3 Edit Files Method Sample Table When Sample Table is clicked a table appears in which parameters with the following function can be entered ig Edit Files Method nov08 Run Table Autosampler Table Sample Table Documentation Table vist Samnio iden Factor L Weight __ Int Std Cone Level a Acetophenon Acetophenon Acetophenon Acetophenon Acetophenon Acetophenon Testm

Download Pdf Manuals

Related Search

Related Contents

Télécharger en pdf – 3 408 Ko  Suncast BMSA3B Instructions / Assembly  I Ecrire en littérature au cycle 3 - Inspection de l`Education nationale  Agilent Technologies 81637B User's Manual      ASHRAE 62.2 and Ventilation Philosophy  

Copyright © All rights reserved. Failed to retrieve file