Step 1 ‘Setup and Configuration Log-in ‘When:the Login function is enabled, and a CLASS-VP module (j.e., Method Development, Instrument) is opened, a dialog box appears. Enter the name and assigned password. When a name ‘or password is entered that is not recognized by the system, an error message appears, ‘System Configuration ‘The system must be configured when used the first time, or when changes are made to the instruments, detectors, or connections . Your copy of the software has been factory determined to work for the number of instruments and detectors which were purchased, ‘System Configuration involves entering information for instruments, detectors, Analog to Digital converter board(s), optional pump control, optional BCD reader, and printers connected to the system. For details on system configuration, see the CLASS-VP Instruction Manual NOTE: Once system configuration settings have changed, exit the software and restart to Implement the changes. NOTE: Setup the instrument conditions (either GC or LC) with guidance from the Tutorial chapter of the CLASS-VP Instruction Manual. Verification of Analog Signal Using Preview to Check Detector Signals Preview of the signal without performing a run using the PREVIEW function. To access Preview: 4. Click on the appropriate instrument icon from the Main Menu box. 2, Select the Preview command on the Command ribbon to display the real time detector output from the instrument. 3. When finished, select Stop on the Command Ribbon.Setup Method Acquisition Parameters Step2 a Before the first sample is acquired, some basic information about how to acquire the data must be speeified Acquisition Setup Set up the method parameters necessary to acquire the first standard sample. Select Acquisition Setup on the Method menu to display the dialog box for setting up acquisition parameters which include items such as run time and sampling rate. When the choices are complete, select OK. Channel Click on the channel for which the acquisition parameters will apply. ‘Sampling Frequency ‘This is the frequency at which data points are taken. Click on the down-arrow to display a ist of the frequencies available for system configuration. When a sampling rate is selected, the message below the selection box indicates the narrowest peak width (at base) that can be adequately ‘nlegrated using the-selected frequericy. NOTE: in multi-channel methods, the sampling frequency for each channel (with a channel status of ON) must be an integer multipte of the other channel sampling frequencies. Run Time Run Time determines the length of time data are sampled. Acquisition Delay ‘Acquisition Delay is the interval between the start of run (Trigger) and the time when sampling starts for this channel, ‘Analyze After Aca Select this item to analyze data automatically after acquisition. ‘Channel Status Determines whether the channel is enabled. if a channel is off, no data is collected for the channel. ‘Trigger Click on the diagram matching the way the system Is set up for starting data sampling. Non ‘Sampling starts immediately after clicking on Start. Batch acquisitions do not pause between runs. Manual: Contact Normally Open: Contact Normally Closed: User must press Enter to start the run. Batch acquisitions pause for confirmation between runs. ‘Sampling starts on opening of the contact. ‘Sampling starts on closure of the contact,Step 3 Acquire Preliminary Standard ‘To use the data system's graphic aids for method generation, inject a standard mixture that contains ‘the peaks to calibrate, After this run is saved on disk, use it to optimize integration parameters and fill in the calibration tables. Start Single Run To initate a single run, click on the Run Single button in the Instrument window. A dialog box appears where a Sample ID, the name of the method to use, and the file name where the ‘chromatogram is to be stored (Save Run As) are specified 1. Enter a Sample ID for the run in numeric and/or alpha characters. When using Autosampler Pretreatment, enter the path and file name. 2. Enter the name of the method to use, including the path and extension. 3, Enter a file name to save the data under Save Run As (e.g., CAL1.DAT). 4, Click on Start when ready to inject the standard sample.‘Step 4 Optimize Integration Using the just acquired standard sample saved on disk, review the default integration baselines, and use the system's graphic aids to optimize integration for the sample, as follows. Integration Timed Events Click on Anatyze to integrate the chromatogram. When a baseline is not visible, select Annotation ‘on the Options menu to verify that the Baseline box is checked. The baseline should appear drawn the chromatogram. When a baseline is still not visible, verity that the color selected for drawing the baseline shows on the screen (select Colors on the Options menu to review color choices). ‘When the baselines are unsatisfactory as they appear, the integration timed events do not need to be adjusted, and you can go on to the next step. When the peaks are not satisfactorily integrated, optimize the integration parameters as described below. ‘The two required parameters used for detecting peaks are Width and Threshold. When peaks are fairly consistent in width and height throughout the run, the initial values may be adequate for all peaks. However, certain portions of the chromatogram may require changes to these values, entered as timed events, as they will change at specified retention times in the run. ‘When set property, Width helps discriminate noise from true peaks, This parameter effectively smoothes noise by bunching data so that there are 20 bunched points across the narrowest peak. This does not alter the raw chromatographic data stored on disk. Atleast one Width event is required. in most circumstances, an initial Width value based on the narrowest peak in the chromatogram is adequate for proper integration of all peaks. However, whenever ‘the chromatographic peak width doubles, it is recommended that a new Width event be entered. Noise Threshold is defined as'a change in the voltage per unit time, and is used tc determined the peak start and stop. A peak is confirmed when three consecutive thresholds of the chromatogram exceed this value. The Noise Threshold remains in effect until another Noise Threshold event is encountered or until the run ends. To access the graphical event setting portion of the method, select Graphical Events Programming on the Method menu to display all parameters available to change events graphically. In this step, only Width and Threshold are determined. Width Select With on the Graphical Events Programming menu. When prompted on the Status Bar, point the mouse on the start of the narrowest peak and click the left mouse button. When prompled ‘again, move the mouse to the end of that peak and click the left mouse button again, The sysiem displays a Width value for the method integration timed events table based on the selected peak. Click on Reintegrate Now to accept and view the results of the new Width. Threshold Select Threshold on the Graphical Events Programming menu. When prompted, click the left ‘mouse button on one point of that section of the chromatogram where no peaks elute to calculate a noise Threshold value. When prompted again, move the mouse to the right of that section and click the left mouse button, The system enters a Threshold value into the method integration timed events table based on the selected section. To view the integration timed events table that now contains the two new values, select Integration Timed Events on the Method menu. Notice that the new values appear in the table. Use the same procedures listed above to change the values at other points in the chromatogram. ‘When a value is changed, it applies to the rest of the chromatogram from the inserted retention time. To close the integration Timed Events table, double-click on the Control menu box at the upper left comer of the integration Timed Events table.Step5 Analyze Chromatogram ‘Now that the integration timed events for the sample have been changed, reintegrate and review the new baselines. Analyze To reintegrate and display the resulting new baseline for the chromatogram, select Analyze on the Analysis menu, or click on the Analyze button on the Command Ribbon. Review the baselines, and when necessary, adjust the integration timed events again until the baselines are correctly assigned.Step6 Setup Calibration ‘Now that the integration for the method is correctly set, prepare the method for calibration. Create Peak Table (Define Peaks) The easiest way to create a list of the peaks in the standard mixture is graphically using a stored standard data file. CLASS-VP automatically enters the retention times. Manually enter the peak names, concentration levels, and the reference and/or internal standard peaks (when necessary) into the peak table. 1. With the standard file displayed and integrated in the chromatogram window, select Graphical Events Programming on the Method menu. 2. Select Define Peaks(s) on the Graphical Events Programming menu, or on the Tools display, 3, Click the mouse once to the left of the first standard peak in the chromatogram, then click the mouse once to the right of the last standard peak in the chromatogram. 4. A dialog box appears prompting for a peak window (in % of RT) and a unit designator. Enter a peak window value (or keep the default), and enter the units for the standard mixture, €.9., mg/ml CLASS-VP displays the list of detected peaks within the specified range, along with retention ~ times for each. Each row of the table represents calibration information for a given peak, including Peak Name, Peak Identification Window (based on the % specified), intemal Standard Peak ID #, Reference Peak #, Concentration Levels, and Concentration Units. ‘Additional information used for QC reports is also displayed in the table (do not change these columns). ‘Assign peak names by typing each component name next to its retention time. For each peak, enter the ID# of the peak to be used as a reference peak (when necessary). ‘When using an intemal standard method, enter the ID# of the internal standard for each peak. eae 1n the column labeled Level 1, enter the concentration of each peak in the standard mixture. Notice that CLASS-VP has filled in the units previously specified. ‘8° When the list of component names and concentrations is complete, exit the peak table by ‘double-clicking on the Control menu box at the upper left comer of the peak table window. 10. Save the method.Step 7 ‘Single Level Calibration When the method calibration section is complete, rerun the calibration standard. Or, select Start Single Run on the Analysis menu to calibrate. 1. Click on the Run Single button on the Command Ribbon. CLASS-VP will prompt for information to be used in the calibration run. 2. Enter a Sample ID for the run in numeric and/or alpha characters, e.9., CALIB1. 3. Enter the name of the method used, including the path and extension. When using Autosampler Pretreatment, enter the path and file name. 4, Enter a file name to save the data under Save Run As (e.g., CAL1.DAT); then click on the Calibrate box and enter 1 for Calibration Level. 5. Click on Start when ready to inject the standard sample. CLASS-VP updates the method after the standard sample run is complete, entering the area values (and heights) into the calibration for calculation of response factors for each component.} Step 8 Review Calibration Curve After the method has been calibrated, the calibration curve can be viewed for each component. This is applicable for multi-level calibrations. 41, Select Review Peak Calibration Curve on the Method menu to display a screen with a list of the method components in the Peak Library list box. 2. To view the calibration curve for any component, click on the component name in the Peak Library list box. Since the method is currently calibrated for a single level, only a single point on the calibration curve will be visible. For more details on viewing calibration curves, Review Peak Calibration Curve in Chapter 6, Method and in Appendix A of the CLASS-VP Instruction Manual.Step8 Acquire Single Sample ‘An unknown sample can now be acquired and a report generated which contains results based on ‘the calibration just performed. 1 Click on the Run Single button, or select Start Single Run on the Controt menu to display the Acquisition dialog box prompting for information used in acquisition and processing of the run 2. Enter a Sample ID for the run, e.g., UNKNOWN‘. 3. Enter a name for the unknown sample data file in the Save Run As field. 4, Enter the method name under which the method was saved. When the Method field is left blank, the current method is used to acquire and process the data. For this run, verify that the Calibrate box is NOT checked, 5, Indicate the Pretreatment path and file name when appropriate. 6. When ready to inject the sample, click on Start.Step 10 Review Reports Reports can be viewed on-screen or printed during data acquisition, Batch Reprocessing, or Method Development To view reports on-screen: ‘A report can be viewed on-screen without sending it to the printer. This is a convenient way of evaluating the effects of method changes on the chromatography. 1. Click on the Reports button, or select Report on the Analysis menu to display a list of report ‘types for review. 2, Select Area% to display an Area % report for the current chromatogram in a separate window. 3. After reviewing the report, double-click on the Control menu box upper left corner of the report ‘window. To generate a hard-copy of a report: 1. Click on the Print button, or select Print on the Analysis menu to display a dialog box with a list of items to print, 2, Select the Report option, followed by the type of result report to print.Step11 Create Batch Sequence Create a Batch Sequence to automate data acquisition. A Batch Sequence is simply a worklist that specifies what samples are to be run, the order in which they are to be run, and any special instructions (such as calibration). Batch Sequences can be used for automation of data acquisition, or for reprocessing data in batches after it has been saved on disk. ‘The procedure is shown below for creation of a Batch Sequence for running 3 samples - a ‘calibration standard and two unknowns. 1. Click on File followed by New Batch. A dialog box will appear where you can enter paths, file names, values, and the number of uns in the batch, When you finish the dialog box and click on Ok, a batch table wil be created using the information you specified in this dialog box. 2. Type ina Sample ID to be used for the runs. This ID will be stored with the raw data, If you want each Sample ID to be unique, include a starting number, enclosed in parentheses, in the ‘Sample ID. For example, Calib(01). This will cause each ID number to be incremented in the batch. 3. Under Method Name, type the name of the method you are using in this acquisition, 4. Under Filename, type a filename to be used for data storage on disk. To create a unique filename for each acquisition run, include @ number (enclosed in parentheses) somewhere in the filename or filename extension. For example, DATA.(001), or DATA(01).DAT. This will ‘cause each row of the batch to have a unique filename. 5. Under Number of Runs, enter 3. This will cause 3 rows to be created in the batch sequence table, 6. Click on Ok. A batch table containing the information you entered will appeer. Each row of the table represents one sample, or run, and the rows are numbered onthe left side under the Run heading. The columns to the right are for entering or editing information ‘on how each run is to be acquired and/or processed. Note: To ensure existing data is not inadvertently overwritten, always specify unique filenames when creating Batch Sequences for acquistion. Click the first row in the the Run Type column, and select Calibration from the drop-down list. In the Level column, enter a 1. This specifies that this sample is to be the level 1 calibration standard. For calibration standards, designate the sample as a Calibration Run Type, and enter the calibration level number. If the sample is not a calibration mixture, designate the sample as ‘an Unimown Run Type, and enter a 0, or leave the "Level" blank. 8. Edit the information for run numbers 2 and 3. Designate these as Unknown Run Type, as these samples will not be calibration standards. 9. Click on Fite. 10. Click on Save Sequence As. 11. In the dialog box shown, enter a filename for the batch sequence and click on OK. ‘You can give the sequence any name with any extension. However, CLASS-VP looks for the extension .SEQ when it searches for sequence names. Uniess otherwise specified, CLASS-VP will save the sequence in the CLASS-VP\Sequences directory created when the software was installed. 14. Close the batch table by double-clicking on the upper left corner of the table.Step 12 Run Batch Sequence You can now run the batch sequence from the instrument session. Make sure the autosampler contains the standard mixture, followed by two unknown samples. Or, prepare to inject a standard and two unknown samples manually. NOTE: When using batch acquisition, itis crucial to inject standards and samples in the exact order specified in the batch table. To start batch acquisition: 1. Click on the Run Batch button (Control / Batch Acquisition). A dialog box will appear where you enter the batch name and designate which runs in the batch are to be acquired. 2. Make sure the batch name and path match the batch file name under which you have saved the batch sequence. 3. If you want CLASS-VP to wam you if it detects duplicate data files already on the disk, click the box next to Confirm Overwriting Existing Files. If this boxis selected, CLASS-VP will check for existing duplicate file names before the: batch proceeds and prompt you to confirm if you want to overwrite these files, 4. When you are ready to start the batch acquisition, click on Start, then proceed to inject the first sample, or start the autosampler.70 Time erin)we + GES, File : ¢:\class-vp\chrom\Naftal Method ¢:\class~vp\methods\Ipa3.met Sample ID: naftalene Acquired Oct 06, 1997 18:15:45 Printed Oct 06, 1997 19:07:02 User system ‘\elass-vplohrorriNaftal — Channel A, PDA Channel 1 200] 150 Oa 7 — 76, 6 Minutes — 254 nm Band = 4 nen Channel A Results -- PDA Channel 1, 254 nm, 1 nm Band Peak Time Area Area $ a 2,19 649427 1,128 2 2,35 8734920 15,175 3 2,93 1983387 3,446 4 4,46 11015106 19,137 5 5,60 3653258 6,347 6 6,82 2079587 3,613, 7 8,57 8129079 14,123 8 9,81 346721 0, 602 8 12,34 6785649 11,789 10 13,13 11268552 19,577 11 14,27 2050302 3,562 12 15,86 680517 1,182 13 17,93 183591 0,319 57560092 100, 000Page 1 Method \class-vp\metheds\Ipa3.met Printed 1 ot 6, 1997 18:56:32 Creation Date : Oct 6, 1997 18:14:20 Last Changed : Oct 6, 1997 18:14:20 Muovo metodo per naftalene @ altri IPA, lo chiamerd TPA3 (200-500) per il benzo-a-pirene vedi metodo 2 (21 panna Channel : A Acquisition Setup BDA Acquisition ‘Channel status on Ran ‘Time + 30 Min Aoquisition Delay: 0 Min Start wavelength + 200 nm End Wavelength + 500 nm ‘Time Constant (sec): 0,64 Lamp Type pa/w Storage Option 30 Data ‘Analysis Channel 1 Analyze After Reg. : Off Background Correction : off Interpolate Spectra: off Spectral Filtering : None ‘Trigger Type Contact Close Library Search Simflarity Index Threshold : 0,000 Maximum Nonber of Hits =: 0 Wavelength From 200 nm to 500 nm Peak Purity Up Slope 154 Down Slope 258 Wavelength From 200 nm to 500 nm Multi-Chromatogram Definition Channel Wavelength (nm Bandwidth (nm) 1 254 Integration Events Eve Event Start Tine stop Time Value 1 ‘Threshold, 0,000 0,000 100 2 width 0,000 0,000 0,2 3 Minimum Area 0,000 0,000 ° 4 Integration off 5,251 6,394 ° Peak TablePage 1 Internal Standard Report -- Channel A File + e:\elass-vp\chrom\Naftal Method ¢:\class-vp\methods\Ipa3.met Sample ID : naftalone Acquired : Oct 06, 1997 18: User system 0,000 ppm 649427 40386 Totals 0,000 49427 40386naftalene wD: 1 RT:13,131 Wnd:0,657 Ref: 0 ITD: 0 Units:ppm Fit Type: Point-to-point Scale: None Quantitate: area 189 Weighting: Wone Fit Through Zero: No & calib Margin: 08 Update RT after run: No after calib: No Replace Peak Replicates Std Peak TD: 0 Std Peak Mult: 0 Tow Conc: 0 High Conc: 0 Spike 1 Conc: 0 Spike 2 Conc: 0 Tow Spike Limit: 0% High Spike Limit: 08 Check Std, Cone: 0 Check Std. SRD: 0% Dup RD Limit: oF RE SRSD Limit: 0 Level mount Area 1 10, 00000 0 calibration setup Time Unit Uncalibrated Peaks attribute Unealibrated Peaks RF STD Amount, Sample Amount Mult. Factor Group Table Hot specifica External Events Not Specified Display options Start Tim 10 stop Time +30 start anp. 10 Stop Amp. 2 Attenuation 1 512 Export options Page 2Page 1 Normalization Report -- Channel A File : ¢:\class-vp\chrom\Naftal Method 1 ¢:\class~vp\methods\Ipa3.met Sample ID : naftalene Required : Oct 06, 1997 18: User system Pkno Name Mig. Time Cone Area Height 1 naftalene 1,195 0,000 8 649427 40386 Totals 0,000 649427 40386Page 3 Wot specified Performance Options Caloulate Performance —: No Unretained Peak Time + 0,000 Column Length + 0,000 User Programs Before Run After Ron Baseline File Not Specified custom Parameters Not specifieare File 1 e:\class-vp\chrom\Naftal, Method c:\class-vp\methods\Ipa3.met Sample ID naftalene Acquired Oct 06, 1997 18:15:45 Printed Oct 06, 1997 18:58:20 User : System e\clase-vptchremiNatal ~ Channel A, PDA Channel 4 200] 150 U4 c o & y 6 = = 75, Mirutos — 284 nm Band = 1 nm Channel A Results -- PDA Channel 1, 254 nm, 1 nm Band Peak ‘Time Area Area 8 1 1,19 649427 1,128 2 2,35 8734920 15,175 Ss 2,93 1983387 3,446 4 4,46 11015106 19,137 5 5,60 3653258 6,347 6 6,82 2079587 3,613 7 8,57 8129079 14,123 8 9,81 346721, 0, 602 9 11,34 6785649 11,789 10 13,13 11268552 19,577 1. 14,27 2050302 3,562 12 15,86 680517 1,182 13 17,93 183591 0,319 Totals : 57560092 100, 000 i aPage 2 Page: 2 Run 76 Type: Unknown Fail Act: Continue Level 10 Rep rn Sample Amt : 1 ISTD amt : 1 Malt Fact : 1 Sample ID : test,006 Method: multical.met Filename : multical,006 Vial 2 W/A Volume = N/A Pretreat : (None) Batch ¢:\class-vp\sequence\deno. seq Printed : Oct 6, 1997 18:56:49 *** End Printing Batch **+File Method Sample ID Acquired Printed User ¢:\class-vp\chrom\Naftal c:\class-vp\methods\Ipa3.met naftalene Oct 06, Oct 06, system 1997 18 1997 18 5:45, ‘enhelace yplehremNiaftal ~ Channal A, PDA Channel + Page 1 of 1 150] cpa 100] 6 = 70, Motes ~ 254:nm Band = 1 nm Channel A Results ~~ PDA Channel 1, 254 nm, 1 nm Band Peak 1 2 3 4 5 6 ; 8 9 10 i 42 13 Totals : ‘Time 1,19 2,35 2,93 4,46 5,60 6,82 8,57 9,81 11,34 13/13 14,27 15,86 17,93 Area Area 8 649427 1,128 8734920 15,175 1983387 3,446 11015106 19,137 3653258 6,347 2079587 3,613 8128079 14,123 346721 0, 602 6785649 11,789 11268552 19,577 2050302 3,562 680517 1,182 183591 0,319 57560092 100, 000 5Page 1 Page: 1 : 6:\class~vp\sequence\demo. seq Printed : Oct 6, 1997 18:56:49 Last Change Date : 6/ 5/1992 Level 1 Rep 1 Sample amt : 1 ISTD amt : 1 Mult Fact : 1 Sample ID : test,001 Method + multical.met Filename : multical,001 Vial 1 N/A Volume : N/A Pretreat : (None) Run 2 Type ‘Unknown Fail Act: Continue Level 2 Rep 1 Sample Amt : 1 ISTD Amt ; 1 Malt Fact : 1 Sample ID : test,002 Method multical-met Filename : multical,002 vial 2 N/R Volume : N/A pretreat (one) Run 3 Type Unknown, Fail Act: Continue Level, 3 Rep 1 Sample Amt : 1 IsTD Amt : 1 Mult Fact : 1 Sample ID : test,003 Method 1 multical-mat Filename : multical,003 vial PN/A Volume: N/A Pretreat : (None) Run 4 Type : Unknown Fail Act: Continue Level 4 Rep ia fample amt : 1 IsTD amt : 1 Mult Fact : 1 Sample ID : test,004 Method + multical.met Filename : multical 004 Vial, N/A Volume : N/A pretreat ‘Wene) Run 15 Type: _ Unknown Fail Act: Continue Level 5 Rep 1 Sample Amt : 1 ISTD amt : 1 Mult Fact : 1 Sample ID : test, 005 Method : multical.met Filename : multical 005 Vial 2 N/A Volume : N/A Pretreat : (None)Pippo e pluto in montagnaaen--war0z aon--e3302z Peak 10- Retention Time 6.10 Min em Eo ee 8 3 8 8 8 Up Stope (0.9000) Bog 8 Us Siepe «6 0000, 8 3 8 8 8 1007 eo] 8° Sigpe 10.0000See eee eee Peak 4- Retention Time 3.13 in 100,