Agilent 3395/3396 Integrator
Contents
1. TIMETABLE STOP The 0 456 peak contains a letter N in the peak type column to show that this is a negative peak inverted for integration It is a very well shaped peak The distortion apparent in the earlier runs was due to clipping when it reached the mechanical limit of the plotter Optimizing the Baseline 4 15 Analyzing Data from Other Sources Make your first run without using the integration functions This will probably do a fair to good job of integrating your peaks Then use the integration func tions where necessary to optimize your integration The User s Manual contains a complete discussion of integration and the inte gration functions Note Do not disable the default chromatogram or change any method parameters at this time You will be using them in the next section 4 16 Optimizing the Baseline Archiving Run Data When you store run data in the Memory Disk M it is saved in a file named SIGNAL An appended extension identifies the type of data stored in the file E M SIGNAL RAW Raw data is unprocessed integrator signal input E M SIGNAL BNC Bunched signal data is raw data that has been processed by the peak width filter It contains much less noise than raw data and can be stored in much less memory Unless your application requires raw data store run data in bunched files Bunched data requires less memory and takes less time to reintegrate than raw data2. Archiving Files by Renaming Them Unfortunately the SIGNAL file is overwritten with new data after each run destroying the results of the previous analysis To prevent run data from being overwritten you can archive the Signal file by renaming it after each run RENAME M SIGNAL BNC M RUN11 BNC Automatically Renaming Result Files You can run the AUTONAME program to assign unique and meaningful names automatically to the Signal Data Processed Peak and Report files This pre vents the files from being overwritten and archives them automatically Optimizing the Baseline 4 17 Processed Peak Files Processed Peak files are not overwritten after each run Each Processed Peak file is automatically assigned a unique name based on the integrator clock time The first character is always Q for example E M QABCDEF1 PRO Processed peak file from raw or bunched data E M QABCDEF1 PRA Processed peak file from an ANALYZE com mand E M OABCDEF1 RPT Report file 4 18 Optimizing the Baseline Saving Your Method RUN PARAMETERS ZERO 0 TIMETABLE EVENTS 0 000 INTG 8 0 000 INTG 13 0 380 INTG 11 0 480 INTO 11 1 300 STOP CALIBRATION NO CALIB TBL INTEORATION PLOT TYPE In this chapter m Listing Your Method uva 5 2 m Saving Your Method for Future Recall 5 3 m Ending the Tutorial Lesson 5 4 Saving Your Metho
3. M SIGNAL BNC PEAK FILE M Q1EB8512 PRO IBP 1 149 AREAS RT AREA TYPE WIDTH 082 370294 BV 039 3173 79248 VB 043 TIMETABLE STOP 499 36383 BV 031 574 209960 VB 044 832 506592 BB 053 1 149 3502 I BP 052 TOTAL AREA 1205979 MUL FACTOR 1 0000E 00 This baseline looks much better Note the IF mark where the clamping started The baseline penetration has vanished and the third and fourth peaks now have reasonable baselines Optimizing the Baseline 4 11 However look over the chromatogram and report of run 9 not its replot with baseline m There are not as many tick marks as in run 8 and some of them have changed type W The areas reported for the last two peaks have decreased The 1 149 peak does not have a tick mark showing when it ends its type in the report is now I BP incomplete baseline point to penetration point IF 12 the clamp is the cause The clamping function occurs at 0 38 minute and the baseline remains unchanged for the remainder of the run The true baseline drifts very slightly but the clamp prevents the software from compen sating for the drift The solution to this drift dilemma is simple make another timetable entry to release the clamp as soon as the baseline penetration has passed The time selected must be after the end of the baseline penetration but before the re tention time of the next peak An event time of 0 48 minute seems reasonable here 5 Press
4. Peak Type Baseline Construction 0 082 BV Starts at a baseline point It ends at a dropline from a valley point 0 173 VB Begins at a valley dropline and ends at a baseline point 0 499 PV Begins at a penetration point and ends at a dropline from a valley point 0 574 VB Begins at a valley dropline and ends at a baseline point 0 832 PB Begins at a penetration point and ends at a dropline from a valley The dropline is too short to be visible Optimizing the Baseline Obviously the third and fourth peaks are measured from a very odd baseline but what is the correct baseline That depends on the type of data and to some extent on the information you want to extract In this case it s quite clear that you must optimize the baseline construction so that the baseline penetration doesn t cause mismeasurement of the 0 499 and 0 574 peaks In the runs that follow only the replotted chromatogram and baseline per the Baseline program are shown The report for each run is superimposed for your reference Optimizing the Baseline 4 7 Using the Integration Functions to Optimize the Baseline The tools for correcting or customizing the baseline construction are the inte gration functions all controlled by the INTG key These functions must be time programmed The 15 Integration Functions 4 8 Function No Resulting Action Optimizing the Baseline 0 D IN t A N e MP e e me e WO N
5. goal of optimizing your analysis When you are finished you will save the integrator parameters in a method Once you start this manual try to finish it in one sitting if possible it should take no more than two hours E Before you start disconnect any external devices from the integrator and verify that it is in good working order W Keep the Quick Reference Card handy You will find it useful as a refer ence as you perform the tasks in this tutorial Introduction 1 3 Getting Out of Trouble 1 4 Introduction During the course of this tutorial you may make a mistake If you do don t panic You are bound to make a few mistakes when learning a new product Some of the common problems that a new user may encounter are listed below Stopping the Tutorial To stop the demo chromatogram and end the tutorial simply press and hold down both the CTRL and DEL keys This resets the integrator to its default condition Correcting a Typing Error Correct a typing error in one of the following ways W Use the BKSP key to erase the error and repeat your entry The printhead does not back up as a typewriter does A block char acter is printed instead and the character to the left of the block is erased from integrator memory W Press ESC to clear the entire entry and start again W Press BREAK to cancel the entire command and start over When You Hear a Beep You will hear a beep when m You at
6. 4 7 B Archiving Run Data at sante 4 16 Optimizing the Baseline 4 1 Running the Baseline Program In section 3 you improved the chromatogram by adjusting the main integration controls PK WD and THRSH In this section you will use the Baseline program to examine how the baseline is drawn and locate any peak or baseline situations that require special atten tion The Baseline program is a basic program that can be scheduled to execute af ter each run When the Baseline program executes it replots the chromatogram obtained during the analysis and draws in the baseline showing exactly how the integration was performed Storing Run Data The Baseline program requires that you store the signal and processed peak files 1 Press OPO 2 ENTER to select Option 2 and respond to the dia log as shown below RUN DATA STORAGE Store signal data Y N Y ENTER Device M ENTER Bunched or raw data B R B ENTER Store processed peaks Y N Y ENTER Device M ENTER 4 2 Optimizing the Baseline Setting Up the Baseline Program Assigning the Baseline program to Key 0 will make it execute after each run as a postrun program 2 Type the command string ASSIGN 0 E BASELINE BAS and press ENTER to assign the Baseline program to Key 0 ASSIGN 0 E BASELINE BAS When each run completes the Baseline program executes replotting the original chromatogram and drawing in the baseli
7. TIME 48 INTGO 12 ENTER to release the clamp TIME 48 INTG 12 8e 6 Press LIST TIME ENTER to display the list of your timetable entries LIST TIME 0 000 INTG 8 0 000 INTG 13 0 380 INTG 12 0 480 INTG 12 1 300 STOP In the next run the baseline penetration will be inverted and we can tell if it is a chromatographic peak 4 12 Optimizing the Baseline 7 Press START to begin run 10 The baseline and droplines indicate the peak area allocations PROCESSING RUN 10 BV ee eee Bes 0 082 VB De m IF IF 0 499 BV VB 0 574 PB 0 832 RUN 10 JUN 1 1990 02 02 23 SIGNAL FILE M SIGNAL BNC PEAK FILE M Q1EB8632 PRO AREAS RT AREA TYPE WIDTH BV 1 149 082 370294 BV 039 173 79248 VB 043 499 37210 BV 032 574 211909 VB 044 TIMETABLE STOP 832 511110 PB 053 1 149 6638 BV 070 TOTAL AREA 1216409 MUL FACTOR 1 0000E 00 Notice that the last two peaks now have the areas they had before we experimented with the clamp This means that the drift compensation is work ing again and that the clamp has been effectively canceled The IF mark on the leading edge of the 0 499 peak shows that the time chosen for the event was appropriate Optimizing the Baseline 4 13 Analyzing Liquid Chromatographic Data With some HPLC sample and detector combinations negative peaks are per fectly valid data The baseline penetration does no
8. TIMETABLE STOP For illustration purposes marks have been added to the chromatograms to indicate baseline points The baseline points are labeled with the letter B and a number to reference them Valley points are labeled with the letter V Optimizing the Baseline 4 5 4 6 The baseline starts at the first data point in the chromatogram and then goes from baseline point to baseline point in this instance from B1 to B6 unless some special situation arises There is a baseline penetration between points B2 and B3 This section of the baseline is allocated as a rubber band line anchored at the penetration point and B3 However because the negative going peak is clipped the baseline cannot be drawn as allocated It has to be drawn horizontally while inside the clipped region The baseline is drawn normally once it is outside the clipped region Negative peaks should be inverted or clamped to make the baseline allocation obvious This is especially true when using the Baseline program At the end of the run the last baseline point is B6 The line is drawn vertically down from that point until the run ends and passes below point V3 How do you know If the line went through V3 it would be another baseline point not a valley point If it went above it V3 would become a penetration point and lose the V label The table below shows the peak types that appear in the printed report and the baseline constructions they represent
9. access the demo chromatogram 1 Hold down the CTRL SHIFT and BREAK keys simultane ously until the instrument starts printing If you release any of them too soon the diagnostics will not start 2 The integrator should display the following message SELF TEST Press M key for more help Il V If the self test message does not appear you probably released one of the keys too soon Wait for the instrument to finish whatever it s doing and then try again 3 TypeM for menu to list the menu of integrator self tests Making Your First Run 2 5 gt M Press the keys for the tests you want to perform If you select no tests you will return to the system software After you have selected the tests you want press ENTER The tests will run continuously unless an error halts them Clear all tests ROM cre and bank select test Quick RAM test Extended RAM test 10 min 8051 ROM and RAM test 8051 interface test RS232 port test HP IL port test HP IL bus test Remote control and sample input test A D Noise test P P test Keyboard test High Speed Printer Test Run all tests Enable Demo Chromatogram Print error messages Suppress error messages Continue testing if error occurs Halt testing if error occurs press SPACE to continue TONVDHPRARPDTOMIDNUARWNERO Enabling the Demo Chromatogram The demo chromatogram is item T in the menu listing 1 Type T to enable the demo chromatogram The integ
10. change the data they just change the plot appearance If you find differences between the re ports the most likely reason is that you ended a run too soon and missed the last peak What you have accomplished so far is to improve the appearance of the chromatogram to make the integration and baseline marks easier to observe This will be important when you attempt to optimize the baseline Optimizing the Chromatogram Setting a Timed Stop So far you ve been using the STOP key to end the runs This is satisfactory for single runs however it is usually more convenient to have the instrument stop the runs automatically Note that the retention time of the last peak is 1 150 minutes Stopping the run at 1 3 minutes should not interfere with that peak 1 Press TIME 1 3 STOP to program the run to stop automatically 1 3 minutes after the run is started TIME 1 3 STOP Optimizing the Chromatogram 3 7 Run 4 1 Press START to begin a new run with a time programmed Stop command RUN 4 JUN 1 1990 00 31 15 START Um 0 082 0 173 0 574 0 832 1 150 TIMETABLE STOP RUN 4 JUN 1 1990 00 31 15 AREAS RT AREA TYPE WIDTH AREAS 082 369702 BV 039 28 85754 173 79010 VB 042 6 16722 499 67789 PV 040 5 29135 574 246119 VB 048 19 21111 832 511335 BP 053 39 91288 1 150 7173 VP 072 55990 TOTAL AREA 1281128 MUL FACTOR 1 0000E 00 2 This time the run ends automatic
11. e O Set baseline now Set baseline at next valley Set baseline at all valleys Tangent skim from next peak Turn off tangent skim Draw horizontal baseline Measure and update threshold Turn off retention time labeling Turn on start stop marks Turn off integration Increment threshold Invert negative peaks Clamp negative peaks Show functions 11 and 12 Start peak sum window Choosing the Integration Functions When a baseline penetration goes well below the expected baseline use integration function 11 invert negative peaks and integration function 12 clamp negative peaks The penetration may actually be a peak you want to quantify Function No Resulting Action 11 Sets a baseline level and inverts any signal changes that go below that level This turns negative peaks into positive peaks before integration 12 Clamps the signal at the baseline level any signal that goes below baseline is ignored 13 Does not change the signal treatment it just causes the chart to display the result of functions 11 or 12 if they are used The inversion or clamping occurs regardless of whether or not you make it visible with function 13 Consider the Source Before you program the integration functions consider the data source You may be collecting any one of the following types of data W Gas Chromatographic Data W Liquid Chromatographic Data W Data from Some Other Source Each source of chromatographic data has differen
12. number date and time W The chromatogram appears next in this case it looks terrible The peaks are jammed together and they run off the right edge of the chart W The report of the analysis follows the chromatogram Until you have improved the chromatogram you cannot accurately correlate much in the chromatogram with the report Your next step is Optimizing the Chromatogram 2 8 Making Your First Run Go Optimizing the Chromatogram 0 082 0 173 In this chapter m Optimizing Your Analysis 3 2 m Setting the Chart Parameters 243 saut ace eere eas 3 3 BY Setting Timed StODosas re ea Se RO e nee Ra a 3 7 m Adding Integration Event Marks to the Chart 3 9 m Evaluating the Integration 3 12 m Changing the Integration Parameters 3 14 m Evaluating the Improved Integration 3 16 Optimizing The Chromatogram 3 1 Optimizing Your Analysis To obtain the most reliable data you should perform your analysis under the most favorable conditions by optimizing the analysis Each analysis has three components to be optimized W the chromatography E the chromatogram E the baseline The Chromatography You optimize the chromatography by varying instrument temperatures carrier gas flow and the column selection This is an important first step A good analysis always starts with good chromatography In this tutor
13. 82 369702 BV 039 28 85754 173 79010 VB 042 6 16722 499 67789 PV 040 5 29135 574 246119 VB 048 19 21111 832 511335 BP 053 39 91288 1 150 7173 VP 072 55990 TOTAL AREA 1281128 MUL FACTOR 1 0000E 00 2 Use STOP to end the run after the peak at 1 150 is finished This chromatogram is much improved Individual peaks are now sepa rated and appear on scale You can make further improvements by increasing the chart speed and decreasing the attenuation This will make some of the details at the base of the peaks become more apparent 3 Press ATT 21 4 ENTER to decrease the attenuation to 4 ATT 2 4 4 Press CHT SP 4 ENTER to double the chart speed CHT SP 4 Optimizing the Chromatogram 3 5 Run 3 3 6 RUN 3 JUN 1 1990 START pore ne STOP RUN AREAS RT 082 1 73 499 574 832 1 150 1 Press START to begin a new run using the new chart parameters 2 Press STOP to end the run after the peak at 1 150 is finished 00 24 05 150 3 JUN AREA TYPE 369702 BV 79010 VB 67789 PV 246119 VB 511335 BP 7173 VP TOTAL AREA 1281128 MUL FACTOR 1 00001 E 00 1 1990 WIDTH 039 042 040 048 053 072 00 24 05 28 39 AREAS 85754 16722 29135 19 91288 55990 21111 e 0 082 Now compare the three reports that you ve generated The reports should be identical since changes in ATT 2 and CHT SP do not
14. 90 00 07 11 MUL FACTOR 1 0000E 00 1 To review the default integrator values press LIST LIST LIST LIST PEAK CAPACITY ZERO ATT 2 CHT SP A T 1244 Optimizing the Chromatogram 3 3 Attenuation ATT 2 ATT 2 attenuation sets the scale for the chart width Low numbers expand the chromatogram s width large values compress it The valid range is from 8 to 36 Currently the value is 0 and the peaks go off scale a larger value is needed to keep them on the paper 2 Press ATT 21 5 ENTER to increase the attenuation to 5 ATT2 5 Since each increase of 1 divides the peak heights by 2 going from 0 to 5 will scale them down by a factor of 32 Chart Speed CHT SP CHT SP is the chart or paper speed Low numbers cause peaks to be squeezed together into groups of narrow peaks A faster chart speed spreads out the peaks The current setting of 1 cm min is too slow Try doubling it and then check the results 3 Press CHT SP 2 ENTER to double the chart speed CHTSP2 Notice that each key press is printed on the chart so you can keep track of the changes The ENTER key is acknowledged with an symbol 3 4 Optimizing the Chromatogram Run 2 1 Press START to begin a new run using new chart parameters RUN 2 JUN 1 1990 00 16 58 START TUUM nn 0 082 0 173 0 574 0 832 1 150 STOP RUN 2 JUN 1 1990 00 16 58 AREAS RT AREA TYPE WIDTH AREAS 0
15. Agilent 3395 3396 Integrator Tutorial X Agilent Technologies Agilent 3395 3396 Integrators e e E e E Manuals These manuals may contain references to HP or Hewlett Packard Please note that Hewlett Packard s former test and measurement semiconductor products and chemicals analysis businesses are now part of Agilent Technologies The HP 3395 3396 Integrator referred to throughout this document is now the Agilent 3395 3396 Integrator E Agilent Technologies HP 3395 3396 Integrator Tutorial LU presets Manual Part No Edition 1 May 1992 03395 90235 Printed in USA Printing History The information contained in this document may be revised without notice Hewlett Packard makes no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Hewlett Packard shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material No part of this document may be photocopied reproduced or translated to another program language without the prior written consent of Hewlett Packard Company First edition May 1992 Printed in USA Copyright 1992 by Hewlett Packard Company All Rights Reserved Safety Information The HP 3395 3396 is an IEC International Electrotechnical Commission Safety Class 1 instrument This
16. ally Note that the notation at the end of the chromatogram has changed from STOP to TIMETABLE STOP 3 8 Optimizing the Chromatogram Adding Integration Event Marks to the Chart By adding integration event marks you can see exactly how each peak was in tegrated Then you can correct poor integrations by programming the appropriate integration function 3 Press TIME 0 INTG 8 ENTER to program integration function 8 to begin at the start of the run TIME 0 INTG 8 Programming integration function 8 will add integration event marks to the chart A list of all the integration functions appears in section 4 4 Press LIST TIME ENTER to verify that the timetable entry has been made 0 000 INTG 4 8 1 300 STOP 5 Press LIST LIST to list the current values for all the control pa rameters ZERO ATT 2 CHT SP AR REJ THRSH PK WD 0 002 oooPr PrOoO Optimizing the Chromatogram 3 9 Run 5 1 Press START to make a run with integration event marks RUN 5 JUN 1 1990 00 54 45 START IF TT UU 0 082 0 173 0 499 0 574 0 832 1 150 TIMETABLE STOP RUNS 5 JUN 1 1990 00 54 45 AREAS RT AREA TYPE WIDTH AREAS 082 369702 BV 039 28 85754 173 79010 VB 042 6 16722 499 67789 PV 040 5 29135 574 246119 VB 048 19 21111 832 511335 BP 053 39 91288 1 150 7173 VP 072 55990 TOTAL AREA 1281128 MUL FACTOR 1 0000E 00 3 10 Optimizing the Chromatog
17. are finished the chart will look similar to this 1 8 amp 0123456789 lt gt ABCDEFGHIJKLMNOPORSTUVWXYZ _ abcde fghijklmnopqrstuvwxyz 9 lt gt ABCDEFGHIJKLMNOPQRSTUVWXYZ _ abcde co Performing self test unit will accept commands when KEYBD led is ON A model number and revision code will be displayed If your integrator fails to perform the self tests put the tutorial aside and call your Hewlett Packard service representative 2 2 Making Your First Run The Default Integrator Parameters When you first turn the integrator power on the integrator parameters are automatically set to their default values To list the default parameters listed below press LIST METH RUN PARAMETERS ZERO 0 ATT 2 0 CHT SP 1 AR REJ 0 THRSH 0 PK WD 0 TIMETABLE EVENTS EMPTY CALIBRATION NO CALIB TBL INTEGRATION PLOT TYPE FILTERED RUN DATA STORAGE Store signal data NO Store processed peaks NO REPORT OPTIONS Suppress local report NO HEIGHTS report NO Report uncalibrated peaks NO Extended report NO POST RUN LIST OPTIONS Large font ice x d YES Store post run report NO External post run report NO List run parameters NO List timetable NO List calibration table NO List remote method NO Form feed before repor
18. d 5 1 Listing Your Method 1 To list the method you have created by following this tutorial en ter the following key commands LIST METH ENTER RUN PARAMETERS ZERO 0 ATT 2 4 CHT SP 12 0 AR REJ 0 THRSH 2 PK WD 0 04 TIMETABLE EVENTS 0 000 INTG 8 0 000 INTG 13 0 380 INTG 11 0 480 INTG 4 11 1 300 STOP CALIBRATION NO CALIB TBL INTEGRATION PLOT TYPE FILTERED Presentation plot NO RUN DATA STORAGE Store signal data YES D yice anal M Bunched or raw data BUNCHED Store processed peaks YES DEV TCE scat DC M n ERE M REPORT OPTIONS Suppress local report NO HEIGHTS report NO Report uncalibrated peaks NO Extended report NO PRINT amp POST RUN LIST OPTIONS Large front Qe sese ide sa YES Store post run report NO External post run report NO List run parameters NO List timetable u 05000883 NO List calibration table NO List remote method NO Form feed before report NO Form feed after report NO Skip perforations in report NO Skip perforations in plot NO 5 2 Saving Your Method Saving Your Method for Future Recall You may wish to save the current method After you have created or edited a method Before you switch off the integrator clearing the memory and era
19. et 1 2 m Introducing the Tutorial Manual 1 3 m Getting Out of Trouble 1 4 Introduction 1 1 The Integrator s Manual Set 1 2 Introduction Installation Maintenance and Service Manual This is a three part manual It is the first manual you should open when your instrument arrives because it contains installation information It also contains user maintenance troubleshooting and servicing informa tion Tutorial This is the manual you are using now User s Manual The User s Manual contains procedures and facts needed during everyday operation Using Application Programs This manual provides operating information for the application programs installed in your integrator Using these programs you can schedule postrun programs autoname data files manage your files reprocess data files automate runs plot peak calibration curves and plot chromatographic baselines Quick Reference Card The Quick Reference Card illustrates many operating tasks of the integra tor Introducing the Tutorial Manual Welcome to the Hewlett Packard Integrator If you are ready to use your integrator for the first time you have come to the right place This Tutorial Manual introduces you to many of the major integrator functions by having you analyze a demo chromatogram supplied by Hewlett Packard You will edit several integrator parameters with the
20. ey marker instead of an up tick It doesn t look like a valley because there s no following peak visible 1 150 Starts and ends at valley markers but where are the valleys 3 12 Optimizing the Chromatogram A word of warning the signal makes a strong negative downscale move just before the 0 499 peak What is this negative object There are three major possibilities m This is data from a gas chromatograph Since GC peaks are usually positive the negative object is probably a signal disturbance caused by a valve operation or some other happening that is not related to the data Thus you should probably ignore the object m This is data from a liquid chromatograph LC data may go in either di rection from the baseline depending on the detector and sample The object may be perfectly valid data and you must be prepared to handle it m This is data from some other instrument You must use your knowledge of that instrument to decide how to process the object The integrator cannot know how to process everything that may happen some times the decisions depend on information that is not part of the signal In such cases you must provide some assistance For the moment ignore the 0 499 peak and examine the other two questionable ones The problem for the 0 832 and 1 150 peaks is that you are integrating with a noisy signal You can prove this to yourself by making a run with very low at tenuation Th
21. g Hiermit wird bescheinigt daf das Gerat System HP 3395 3396 in bereinstimmung mit den Bestimmungen von Postverfugung 1046 84 funkentstort ist Der Deutschen Bundespost wurde das Inverkehrbringen dieses Gerates Systems angezeigt und die Berechtigung zur Uberprufung der Serie auf Einhaltung der Bestimmungen eingeraumt Contents Chapter 1 Introduction This chapter introduces you to the Integrator s manuals It then describes the Tutorial Manual and how to best use it It also contains information for getting out of trouble Chapter 2 Making Your First Run This chapter teaches the basics of making a run You can analyze the demo chromatogram using the default an integrator conditions Chapter 3 Optimizing the Chromatogram In this chapter you optimize the chromatogram by aosd making several runs changing the chart and integration ae parameters after each run Chapter 4 Optimizing the Baseline In chapter 4 you eliminate deficiencies in the default baseline by programming integration functions The inte grator reallocates the baseline using your instructions to create an optimized baseline Chapter 5 Saving Your Method In this chapter you see how the integrator parameters can be saved in a method file Review all the method entries you made in the course of this tutorial and save them in a method file for future recall Introduction In this chapter m The Integrator s Manual S
22. ial you are using a demo chromatogram so you can assume that the chromatography is good The Chromatogram Once the chromatography is under control you take steps to optimize the chromatogram First you adjust the chart parameters attenuation and chart speed to improve the appearance of the chromatogram This is important when optimizing the integration where you must be able to see the integration marks clearly Next you select the appropriate peak width and threshold parameters to con trol how the peaks are recognized and integrated The Baseline Finally you optimize the baseline construction using the integration functions Your goal here is to get the best baseline fit for each peak in the chromatogram 3 2 Optimizing the Chromatogram Setting the Chart Parameters Run 1 RUN START 1 150 STOP RUN AREAS RT 082 173 499 574 832 1 150 TOTAL AREA 1281128 The chart parameters are ATT 2 and CHT SP and ZERO In Run 1 the peaks run off scale and are too crowded to be seen properly You have to adjust the ATT 2 and CHT SP values to improve the appearance of the chromatogram The default integrator parameters are not suitable for the data supplied by the demo chromatogram T JUN 1 1990 00 07 11 AREA TYPE WIDTH AREAS 369702 BV 039 28 85754 79010 VB 042 6 16722 67789 PV 040 5 29135 246119 VB 048 19 21111 511335 BP 053 39 91288 1173 VP 072 55990 JUN 1 19
23. is will expand the area near the baseline and show you how noisy this signal is The integrator is much more sensitive than your eyes and it is finding valley points between noise spikes and real peaks Optimizing the Chromatogram 3 13 Changing the Integration Parameters So far you have simply improved the chromatogram s appearance The changes you ve made have not affected the peak areas In this part of the tutorial the objective is to find a combination of integration parameters that will find peaks where they exist and mark them with start and stop tick marks m The start of a peak is marked by a long down tick m The end ofa peak is marked by an up tick m Ifthe signal does not reach the baseline between peaks a short down tick marks the end of one peak and the start of the next The two main integration control parameters are peak width and threshold Peak width controls the peak finding process threshold controls integrator sensitivity Changing the Peak Width Value PK WD peak width controls the ability to find peaks Compare the present value 0 04 with the measured peak widths in the WIDTH column of the re port 2 PK WD should be Greater than WIDTH 4 and Less than 2 WIDTH In this case it is for all peaks so the current value is satisfactory Changing the Threshold Value THRSH threshold controls sensitivity Increasing the threshold value makes the integrator less responsive to noise i
24. n the signal The present value 0 is very low the range is from 6 to 28 3 Press THRSH 2 ENTER to increase the threshold to 2 THRSH 2 amp Since THRSH uses the same kind of scale as ATT 2 that is a factor of 4 in sensitivity reduction 3 14 Optimizing the Chromatogram Run 6 Press START to make a run with an increased threshold value from 0 to 2 RUN 6 JUN 1 1990 01 08 45 START II S Se a 0 173 0 499 0 574 0 832 1 149 TIMETABLE STOP RUN 6 JUN 1 1990 01 08 45 AREAS RT AREA TYPE WIDTH AREAS 082 370294 BV 039 28 89322 173 79248 VB 043 6 18355 499 67827 PV 040 5 29239 574 246478 VB 048 19 23213 832 511110 PB 053 39 88078 1 149 6638 BV 070 51 795 TOTAL AREA 1281595 MUL FACTOR 1 0000E 00 Optimizing the Chromatogram 3 15 Evaluating the Improved Integration This integration has almost twice as many tick marks showing that the data integration works better with this value of THRSH than with the original one m The first four peaks have the same tick marks as before m The 0 574 peak now ends with a definite end of peak mark rather than the noise induced valley mark m The 0 832 peak also ends with an end mark m The last peak now begins with a start mark but still ends with a valley mark Its retention time has changed from 1 150 to 1 149 something that can occur with very small peaks and a lot of noise You might want to ex
25. ne according to the integration marks As you continue with this exercise compare the baselines that you obtain with those in the illustrations Optimizing the Baseline 4 3 Examining the Current Baseline Increase the chart speed to 12 to spread out the chromatogram making the baseline easier to see 3 Press CHT SP 12 ENTER to increase the chart speed to 12 CHT SP 12 4 Press START to begin your examination of the default baseline RUN 8 JUN 1 1990 01 17 53 START SS 0 082 0 173 0 499 0 574 7 0 832 RUN JUN 1 1990 01 17 53 SIGNAL FILE M SIGNAL BNC PEAK FILE M Q1EB8423 PRO 1 149 AREA RT AREA TYPE WIDTH 082 370294 BV 039 TIMETABLE STOP 173 79248 VB 043 499 67827 PV 040 574 246478 VB 048 Closing Signal File M SIGNAL BNC 832 511110 PB 053 Storing processed peaks to M Q1EB8423 PRO 4 4 Optimizing the Baseline 1 149 6638 BV 070 TOTAL AREA 1281595 MUL FACTOR 1 0000E 00 Examining the Baseline Construction First note that the replotted chromatogram has fewer integration event marks than the original run This is because any false start of peak integration marks that appear on the original chromatogram are not saved in the processed peak file When the Baseline program replots the chromatogram it includes only those integration event marks stored in the processed peak file PROCESSING 0 082 B6 1 1 149 v3
26. periment with other values for THRSH but 2 is about optimal for this data Lower values get back into a lot of valley marks and higher values starting at 4 reduce sensitivity so much that the last peak is not found A Suggested Strategy for Setting the Integration Parameters The strategy in selecting a certain peak width or threshold value is not to get the most or least possible number of tick marks Instead you try to find a combination of values that produces the right kind of tick marks for each peak Ideally each peak begins with a long down tick and ends with an up tick If peaks are merged the valleys are marked with short down ticks There will often be some excess marks usually with more down ticks than up ticks This happens when the instrument believes that a peak is starting and writes a down tick but for some reason the data that follows does not con tinue to look like a peak Thus the down tick is a false alarm but it has already been printed and the integrator cannot back up and erase it It could suppress the extra marks by reducing sensitivity but this would almost certainly lose some real data Your next step is Optimizing the Baseline 3 16 Optimizing the Chromatogram Optimizing the Baseline In this chapter m Running the Baseline Program 4 2 m Examining the Current Baseline 4 4 Using Integration Functions to Optimize the Baseline
27. ram Integration Event Marks and Baseline Points There are three kinds of integration event marks 1 Long to the left This marks the start of a peak It is often called a long down tick It is a baseline point 2 Short to the left This marks the valley between two peaks that are not completely separated It may be a baseline point 3 Long to the right This marks the end of a peak It is often called an up tick It is a baseline point When the baseline is drawn it passes through all the baseline points A baseline point is marked by a long down tick or an up tick Optimizing the Chromatogram 3 11 Evaluating the Integration Notice the IF mark on the chart right at the beginning of the plot This tells you that an integration function is executed at that time The particular integrator function is not identified you must consult the LIST TIME printout for that Peak R T Where integration begins and ends 0 082 Begins with a long down tick Ends at the valley between it and the second peak which is marked with a short down tick 0 173 Begins at the valley between it and the previous one ends at the up tick mark 0 499 This ends at the valley between it and the 0 574 peak but where did it start There is no long down tick mark 0 574 This is very similar to the 0 173 peak It begins at a marked valley and ends at the up tick 0 832 Begins at the long down tick which is reasonable but is followed by a vall
28. rator prints the following message gt Demo Chromatogram Enabled BREAK While running the demo chromatogram the integrator cannot process an external analog signal 2 To terminate this demonstration simultaneously press both the CTRL and DEL keys to reset the default conditions and ready it for normal operation Otherwise continue to Making a Run on the next page 2 6 Making Your First Run Making a Run RT 082 173 499 574 832 1 150 TOTAL AREA 1281128 To initiate a run using the default method 1 Press START to run the Demo chromatogram 2 After the last peak labeled 1 150 appears stop the run by press 1 JUN AREA TYPE 369702 BV 79010 VB 67789 PV 246119 VB 511335 BP 7173 VP ing STOP JUN MUL FACTOR 1 0000E 00 1 1990 00 07 11 1 1990 WIDTH 039 042 040 048 053 072 00 07 11 AREAS 85754 16722 29135 2 11 91288 55990 Making Your First Run 2 7 Reviewing the Run Data The run data is displayed below RUN 1 JUN 1 1990 00 07 11 STOP RUN 1 JUN 1 1901 00 07 11 AREAS RT AREA TYPE WIDTH AREAS 082 369702 BV 039 28 85754 173 79010 VB 042 6 16722 499 67789 PV 040 5 29135 574 246119 VB 048 19 21T11 832 511335 BP 053 39 91288 1 150 7173 VP 072 55990 TOTAL AREA 1281128 MUL FACTOR 1 0000E 00 To review the run data W The first line identifies the run by supplying the run
29. sing the current method Before you load a new method overwriting the current method To save your method the current method with the name DEMO enter the following keystrokes STORE METH M DEMO MET ENTER Saving Your Method 5 3 Ending the Tutorial Lesson When you are finished with the tutorial you should return the integrator to its normal operation To return the instrument to normal nondiagnostic operation 1 Note Hold down the CTRL SHIFT and BREAK keys simultane ously until the instrument starts printing If you release any of them too soon the diagnostics will not start The integrator should display the following message SELF TEST Press M key for more help gt If the self test message does not appear you probably released one of the keys too soon Wait for the instrument to finish whatever it s doing and then try again Press 0 zero to return the integrator to its normal operation gt 0 BREAK You can also return the instrument to normal operation by press ing the CTRL and DEL keys at the same time Note that this action clears the integrator memory including the current method Congratulations You have completed the tutorial Take a break 5 4 Saving Your Method UE Agilent Technologies Agilent Technologies Inc 05 92 Part Number 03395 90235
30. t NO Form feed after report NO Skip perforations in report NO Skip perforations in plot NO The integrator parameters are stored in a file called a method Making Your First Run 2 3 Integrator Parameters and the Method When you edit any integrator parameter you are editing a method A method is simply a file that contains all the parameters required by the integrator to perform an analysis run parameters data storage options and report options A method will also contain a calibration table when a calibrated report is specified And for 3396 not 3395 it will contain INET instrument set points when INET instruments are connected to the integrator The Current Method Throughout this tutorial you will be editing the current active method The current active method is the method currently controlling the analysis The current active method has the following characteristics W There is always a method in memory the current active method E You create a new method by modifying the current active method and saving it W When you start a run all aspects of the analysis are controlled by the current active method Methods can be edited stored loaded and listed 2 4 Making Your First Run Accessing the Demo Chromatogram To ensure a common starting point you will have to set the default conditions The demonstration chromatogram is a set of data stored as part of the instrument diagnostics To
31. t characteristics which in turn require different treatment using the integration functions Optimizing the Baseline 4 9 Analyzing Gas Chromatographic Data 4 10 Assume that you are analyzing gas chromatographic data All the peaks should be positive The baseline penetration is almost certainly a baseline upset To prevent inaccurate areas for peaks nearby it should be ignored Use function 12 clamp negative peaks to do this and function 13 to display IF for integra tor function on the chart to see when it occurs Function 12 must be programmed to start before the baseline goes negative and after the end of the preceding 0 173 peak An event time of 0 38 minute seems reasonable 1 Press TIME 38 INTGQ 12 ENTER to clamp the baseline penetration TIME 38 INTG 12 2 Press TIME 0 INTGO 13 ENTER to display IF on the chart to see when an integrator function occurs TIME 0 INTG 13 Function 13 can be activated for the entire run it takes no action until function 12 occurs 3 Press LIST TIME ENTER to display the list of your timetable entries LIST TIME 8 0 000 INTG 8 0 000 INTG 13 0 380 INTG 12 1 300 STOP 4 Press START to make another run The baseline is drawn in and the peak area allocations are indicated with droplines Optimizing the Baseline PROCESSING D 0 082 0 832 RUN JUN 1 1990 01 51 32 SIGNAL FILE
32. t look much like a peak but we ll treat it as such for this experiment To do this you must replace the function 12 entries which clamp negative peaks with function 11 entries which will invert them To invert the baseline penetration instead of clamping it 1 Press DEL TIME 38 ENTER to delete the function 12 entry at 38 minute DELETE TIME 38 2 Press DEL TIME 48 ENTER to delete the function 12 entry at 48 minute DELETE TIME 48 3 Press TIME 38 INTGO 11 ENTER to invert the negative peak TIME 38 INTG 11 4 Press TIME 48 INTGO 11 ENTER to turn off function 11 TIME 48 INTG 11 8e If you left function 11 on all peaks after 38 minute would be inverted 5 Press LIST TIME ENTER to display the list of your timetable entries 0 000 INTG 8 0 000 INTG 13 0 380 INTG 11 0 480 INTG 11 1 300 STOP 4 14 Optimizing the Baseline 6 Press START to begin run 11 The baseline and droplines indicate the peak area allocations PROCESSING RUN 11 0 082 IF 0 499 0 574 0 832 RUN T1 JUN 1 1990 02 14 10 SIGNAL FILE M SIGNAL BNC PEAK FILE M Q1EB8752 PRO AREAS RT AREA TYPE WIDTH 082 370294 039 173 79248 043 456 18769 N 030 499 36099 031 574 210565 044 832 511110 053 1 149 6638 070 TOTAL AREA 1232723 MUL FACTOR 1 0000E 00
33. tempt to enter a command during plotting integrating or reintegrating Wait for the operation to end and try again W You type faster than the integrator can accept input Try typing a little slower W You attempt to enter an invalid key If You Get a in Response to Your Entry The numeric value is out of range Enter a correct value after the If You Get an Invalid System Command Message The command does not exist Enter the correct command after the Introduction 1 5 1 6 Introduction Making Your First Run In this chapter m Setting the Default Conditions m The Default Integrator Parameters m Integrator Parameters and the Method m Accessing the Demo Chromatogram m Making a Run m Reviewing the Run Data 0 082 0 173 Making Your First Run INO 2 1 Setting the Default Conditions Before starting this tutorial disconnect any external devices from your integrator The integrator performs a number of internal checks called self tests whenever the CTRL and DEL keys are simultaneously pressed and held down together When these tests are complete the integrator s operating conditions are automatically set to their default values This process will erase whatever may currently be in the instrument memory Be sure this is permissible before continuing To run the self tests simply press the CTRL and DEL keys at the same time When the tests
34. unit has been designed and tested in accordance with recognized safety standards Whenever the safety protection of the HP 3395 3396 has been compromised disconnect the unit from all power sources and secure the unit against unin tended operation WARNING A WARNING CALLS ATTENTION TO A CONDITION OR POSSIBLE SITUATION THAT COULD CAUSE YOU OR OTHERS INJURY CAUTION A Caution calls attention to a condition or possible situation that could damage or destroy the product or your work Important User Information for In Vitro Diagnostic Applications This is a multipurpose product that may be used for qualitative or quantitative analyses in many applications If used in conjunction with proven procedures methodology by a qualified operator one of these applications may be in vitro diagnostic procedures General instrument performance characteristics and instructions are included in this manual Specific in vitro diagnostic procedures and methodology remain the choice and the responsibility of the user and are not included RFI Certification for the Federal Republic of Germany Manufacturer s Declaration This is to certify that the equipment HP 3395 3396 is in accordance with the Radio Interference Requirements of Directive FTZ 1046 1984 The German Bundespost was notified that this equipment was put into circulation and the right to check the series for compliance with the requirements was granted Herstellerbescheinigun
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