We are resuming the Technical Tip of the Month feature for 2010.
This year's topics will deal mostly with data interpretation.

TODAY'S LESSON: USING ENVIROQUANT (Part 3)

What to do it the system cannot find and properly integrate a peak and how to create a custom integration file for a specific compound

1. Use INT\Ion chromatogram to extract the quant ion
2. Use INT\Load integration parameters to ensure that RTEINT.P is loaded.
3. Make the necessary changes RTEINT.P based on what you learned in previous tech tips.
4. Use INT\integrate until it integrates properly
5. SAVE as "_______.P"
6. Enter this newly created file on Page 3 of INIT CAL\EDIT COMPOUNDS
7. Go to the apex and double right click- a spectrum appears
8. Type "NORM 100" [RETURN]
9. Type TAB [RETURN]
10. Write down which qualifier ions you wish to use and their percentage and enter that information on Page 1.

TRACE MODE QUANT

Another helpful feature can be found under Quant\Trace Mode Quant. When you click this it brings up a command box asking you to enter the compound number to quantitate. Trace Mode Quant lets you quant for a SINGLE compound only. It is very useful when doing method development or troubleshooting a system and you can't get a few compounds to be detected. The Trace Mode Quant gives you detailed information about the compound of interest including the following:

Peaks Found in integration list
Quant Ions and Qualifier Ions-expected Ion ratios and actual obtained
The acceptable Retention Time Range as specified in your calibration table
Actual Retention time, Expected Retention time and the Difference between them
Quantitation Results (if any): peak area and calculated concentration.

This information is helpful when a peak is not showing up in your quant report or is constantly being misidentified. You may need to make changes in the retention times, ion qualifiers or integration routine in order to make the Enviroquant software detect the peak automatically so Trace Mode Quant enables you to troubleshoot the difficult compounds by providing all the above information on the one compound without having to quant the entire list. Once you figure out the problem with the information provided by Trace Mode Quant, you can then Calculate and generate a report for the entire compound list.

THE CON CAL MENU

The only point I wish to make here is that EACH TIME you run a CCC/SPCC Cal check, you should update reference spectra. It takes but a minute and is accessed by:
Con Cal\Update Reference Spectra

QUANTITATING AND REVIEWING DATA

Next let's consider the finer points of the Quant menu bar. I'm sure everyone is familiar with "Calculate and Generate Report" and "Generate Report". But let's look at "Edit Quant Report Options".  Look on the bottom right corner of the screen and you will see the following:

Omit Target Compounds that are missed
have qualifiers out of range
If the "are missed" box is checked off, the quant report will omit entirely the names of compounds that were not found. If the "are missed" is unchecked- it will list the compound name and the term "N.D." for not detected on the quant report.
If the "have qualifiers out of range" box is checked off, the quant report will omit entirely the names of compounds that were found but with the ion qualifiers out of range. If the "have qualifiers out of range" is unchecked- it will list the compound name and then flag such compounds with qualifiers out of range on the quant report with a "#" sign.

Here's how I use these boxes. For standards, I expect every compound to be in, so I leave both boxes UNCHECKED. That way if a compound or is missed I can easily tell by scanning the quant report for an "N.D." flag. If a compound has ion qualifiers out the quant report flags it with the "#" sign so I can correct the problem and requant until ALL compounds are found with NO "#" signs. For samples, I CHECK both boxes because if the compound is not found or has outlying qualifiers, then I want those compounds left off the quant report entirely. Some analysts like to CHECK the "are missed" box and UNCHECK the "have qualifiers out of range" because if the software finds the quant ion of a compound but has qualifiers out, they like to look carefully at the peak in QEDIT and decide for themselves if it is a hit or not. Whatever you choose to do is your own prerogative, I am merely pointing out the options available.

I just mentioned QEDIT in the last paragraph. Let's discuss this feature in detail. You will probably spend more time using QEDIT in the data reduction part of your analysis than any other part of Enviroquant so it is important to know how to use it efficiently. After you have calculated and generated a report, you may QEDIT the data. Click Quant\Qedit Quant Result to load the QEDIT menus.

At this point Quick QEdit should load automatically. It will be a box containing a list of target compounds and should be visible at the top right-center of the screen. It puts an "x" mark next to the target compounds that were found. If Quick QEdit does not automatically load upon entering QEDIT, you can load it manually by clicking QEDIT\Restart Quick QEdit.

Here's a tip that may very well save you from getting carpal tunnel syndrome. Let's say you run a standard and want to scroll through every compound for a few seconds ensuring proper identification of isomers and complete integration (a good idea to do especially when changing flows, columns or anything else that might cause retention times to shift or peak shape to change). You could double right click the bottom right hand box (the one that lists the compound, RT, area and ions) and go from compound to compound that way. Of course, that can get very tedious. An easier way would be to click the CONFIGURE box at the top of Quick Qedit. That opens a dialog box that allows you to input the number of seconds that will elapse in between each compound as the system scrolls though each one for you. You can stop the automatic timed scrolling anytime by clicking STOP and then resume it by clicking START. I love this feature especially when doing method development. The Quick QEdit also allows you to QDEL (delete) a compound with a click of the mouse. Once done, click EXIT- the software will confirm that you want to SAVE the changes you made in QEDIT (e.g. re-integrations are re-identifications) and then exit back into the main Enviroquant menu.

If you make changes and then decide you want to go back to the original data, click Qedit\Abort Changes and Exit.

If you want to display the reference spectra in the bottom left hand box, click Spectrum\Display Reference Spectra. The reference spectrum for each compound as obtained when you clicked Con Cal\Update Reference Spectra (mentioned previously) will appear underneath the sample spectrum at the bottom left.


LIBRARY SEARCHES

Next let's consider the finer points of the LSC and Lib menu bars. I'm sure everyone is familiar with the basic procedure to perform library searches but there are a few key points I wish to make. The first is that to improve the quality of your library searches you should do the following under Click Lib\Edit Strategy:
The parameters in the Search Strategy dialog box have been carefully chosen by the Agilent software engineers to give the best search results in most cases. The recommended default values and possible changes are:

U + A = 2. The range is from 1-9. The default is 2. If you are having difficulty obtaining matches increase this setting. It will slow the search but sometimes returns better matches.

Tilting = on. This features allows the searching algorithm to scale the reference spectrum to fit the unknown spectrum. I suggest leaving it ON as it is in the default mode.

Flag Threshold = 3. The range is 0-99. This is the Threshold setting so to speak of the library search. For example, if it is set to 3, then ions below 3% of the base peak won't be taken into consideration. I suggest leaving it at the default of 3%.

Cross-correlation sort = off (box unchecked). By setting this to off, the search algorithm is what's known as PBM match quality (probability based matching) which is the industry standard for EPA methods. I suggest leaving this parameter with default off setting.

Minimum Estimated Purity = 50%. The range is 20-80. The default is 50%. The way this works is as follows: the search algorithm first identifies the base peak in the library match. If that base peak ion is also present at or above the specified % relative to the base peak in the unknown, then the routine reports that match as a possible hit. Note: if the base peak in the library match is totally absent from the unknown hit, this feature is deactivated. This is good because if this weren't the case, the disparity caused by differences in scan ranges between what environmental labs use and what the contributors of the NIST/NBS/Wiley Library used would make certain matches impossible.


Example:

Unknown spectrum has ion 59 at 100%, ion 45 at 80% and ion 74 at 60%.
Possible library search hit has ion 74 at 100%, ion 45 at 30%, ion 59 at 5% and ion 41 at 95%
The base peak in the Possible library search hit is ion 74. It now checks to see if ion 74 is present in the unknown. It is. It now checks the Minimum Estimated Purity setting. In this case it is set to 50%. Since ion 74 is present at 60% in the unknown, then it passes this test and shows up on the list of possible hits. If the Minimum Estimated Purity was set to 70%, then ion 74's 60% abundance would be BELOW the specified setting and this hit would be rules out and hence not show up on the list of possible hits.
This may all seem complicated, and indeed it is, so I think that it's best to use the default setting of 50% which is right in the middle of the possible range anyway.

None of the following parameters have any effect on the search itself or on the speed of the search. They all prune entries from the results list after the search is completed.

Low report MW (molecular weight) = 0
High report MW (molecular weight) = 9999
By setting the MW window from 0-9999 you will not exclude any compounds by virtue of their molecular weight. Realistically, you could use 0-800 and obtain essentially the same results.

Max hits = 10. This is the number of hits that will be reported. 10 is a good number.
Remove duplicate CAS numbers = off

The only one I disagree with is "Remove duplicate CAS numbers" which I always set to ON. The reason for this is that for many compounds there are numerous entries of its spectra in the NIST/NBS/Wiley Mass Spectral Library. By setting "Remove duplicate CAS numbers" = off then if an unknown peak is identified in your search, the top 3 or 4 matches will all be the same compound. Well, I don't see any value in that, so by setting it to "Remove duplicate CAS numbers" = on (i.e. checking the box) then the search only returns the best hit of that compound and then uses the other spots to search for other potential matches. My experience reducing data has been that "Remove duplicate CAS numbers" = on works better.

The next technical tip will be in August.

If you have any questions about this application please
feel free to e-mail Mark Ferry by clicking here:

|| ECS/MDL Main Page ||