Peak area Integration (Chemstation)

[letsdoit]

Last week the integration of peaks using changed to some other way.
I am using Chemstation
I dont know what happened. But due to that I have to now manually integrate the peaks myself everytime.
I am unable to understand whats going on???


Could someone please explain why did this happen?

[jaj]

This is most likely due to a retention time shift. Some event( air leak, new column, etc.) has happened with your system that is causing the peaks to elute sooner or later than the retention times you have specified in your Chemstation method.

[letsdoit]

Thanks for the reply.
I changed the gradient flow. But then how do I make the chemstation integrate the peaks the same way as it did earlier??

This is most likely due to a retention time shift. Some event( air leak, new column, etc.) has happened with your system that is causing the peaks to elute sooner or later than the retention times you have specified in your Chemstation method.

[jaj]

Hi!

I guess that you are running an LC. I run GC/MS, so our systems maybe quite different. On my chemstation, you can enter the retention times in the data analysis method where your calibration information is stored.
I am sure that you know that recalibration is a good idea if your retention times have shifted.


Good Luck,
JAJ

[Tim]

What do you mean when you say the integration changed? Are peaks that were being picked up now not picked up or additional peaks being detected. Is the way the baseline is being drawn not acceptable?

One potential thing to try is ensure that the "Advanced Baseline" option is turned on for the method, since this gives better baseline tracking than the standard integration (but you MUST recalibrate if you do activate it).

You could also try using the "AutoIntegrate" function in ChemStation. That tries to give an optimum set of parameters for a chromatogram. You may need to modify the values it gives to work for every chromatograms, but it is usually a good starting point.

[letsdoit]

Thanks for your reply.
It integrates with a baseline that is not correct.
Actuaaly it takes the lowest point on the chromatogram (where the mobile phase actually elutes)
So may be I should try to use the advanced Baseline.

Where do I find that option??

Also I would Recalibrate it for sure

What do you mean when you say the integration changed? Are peaks that were being picked up now not picked up or additional peaks being detected. Is the way the baseline is being drawn not acceptable?

One potential thing to try is ensure that the "Advanced Baseline" option is turned on for the method, since this gives better baseline tracking than the standard integration (but you MUST recalibrate if you do activate it).

You could also try using the "AutoIntegrate" function in ChemStation. That tries to give an optimum set of parameters for a chromatogram. You may need to modify the values it gives to work for every chromatograms, but it is usually a good starting point.

[Tim]

From what you say I am guessing that the integrated baseline doesn't follow the signal, but instead stays below it as a straight line to which ChemStation drops vertical lines at the start and end of each peak. If that is the case, you should also use the "Valley at Baseline" integration function.

To get both of these, you need to have the Integration Events panel selected on the Data Analysis view. Advanced Baseline is an option box on the left side of the screen above the integration events list.

To turn on Valley at Baseline, select it from the drop-down box on the right side of the window above the chromatogram, then click on the chromatogram where you want it to be turned on (usually right at the start of the chromatogram). Check that the event has been added to the timed events on the integration table and that it has a value of "ON".

Make sure you save and exit from the integration events window before recalibrating, especially if you are using Agilent ChemStore alongside.

Hope this helps

[Consumer Products Guy]

I actually think I might know what's going on here (amazing to think that I am somewhat knowledgeable about any software). I think you previously had been using the ChemStation "standard" integrator and that you/someone clicked "OK" on the monitor message "Update to the Enhanced Integrator?" which can appear. Unfortunately, if one does that, it's like the roach motel of software (once a roach goes in, it never comes out), you cannot return to standard integrator for THAT METHOD.M . You'd have to load up a previously-saved file of that METHOD directory, or load up one of your other methods that have been saved using the standard integrator, or maybe the software default method (I'm not in the lab now, don't remember if it's lc_xxx.M or def_lcx.M, something like that). After loading such, you can make your other modifications than save back to the original Method.M file. What version of Chemstation are you using? I believe once one gets to A.10 that ONLY the enhanced integrator is used, and we use enhanced on our version A.09 HPLC-MS. Otherwise, I really and honestly prefer the "standard" integrator tons better, and that's what we use. We caution our operators at the production facilities to NEVER update to the enhanced integrator should that question appear. If you can't get there, leave your E-mail address and I can E-mail a "default Method.M" in your Chemstation revision level.

[letsdoit]

I guess you kinda exactly understand the problem.
It does mention that at the end of run that the results were obtained using an enahnced integrator.
So could you please email me the a "default Method.M" for my Chemstation .
My email ID is rithrr@yahoo.com
I have the version 8.03 and we are planning to get the version 8.10
Waiting for your reply.
thanks

I actually think I might know what's going on here (amazing to think that I am somewhat knowledgeable about any software). I think you previously had been using the ChemStation "standard" integrator and that you/someone clicked "OK" on the monitor message "Update to the Enhanced Integrator?" which can appear. Unfortunately, if one does that, it's like the roach motel of software (once a roach goes in, it never comes out), you cannot return to standard integrator for THAT METHOD.M . You'd have to load up a previously-saved file of that METHOD directory, or load up one of your other methods that have been saved using the standard integrator, or maybe the software default method (I'm not in the lab now, don't remember if it's lc_xxx.M or def_lcx.M, something like that). After loading such, you can make your other modifications than save back to the original Method.M file. What version of Chemstation are you using? I believe once one gets to A.10 that ONLY the enhanced integrator is used, and we use enhanced on our version A.09 HPLC-MS. Otherwise, I really and honestly prefer the "standard" integrator tons better, and that's what we use. We caution our operators at the production facilities to NEVER update to the enhanced integrator should that question appear. If you can't get there, leave your E-mail address and I can E-mail a "default Method.M" in your Chemstation revision level.

[Consumer Products Guy]

I have E-mailed to you. You didn't state whether it was HPLC or GC Chemstation though, I'll send both if I don't hear back.

[letsdoit]

I am so sorry that I forgot to mention that.
It is a LC
Thanks

I have E-mailed to you. You didn't state whether it was HPLC or GC Chemstation though, I'll send both if I don't hear back.

[letsdoit]

How can I upload an image so that its easy to explain what problems I am having with the peak area integration
I mean I still am confused about some things....(this makes it more difficult to understand the differences in integration events)
Please respond to this

[Tim]

Tom Jupille posted information how to do this at:

http://www.sepsci.com/chromforum/viewtopic.php?t=2617

If the above link doesn't work, it's a "sticky" message within the HPLC section of the forum. There may be other ways too, but not tried, so someone else will have to speakup if there are.

[letsdoit]

I guess you guys can now understand this better.
In the chromatogram attached you can see that the chemstation drops these pink lines on the baseline it considers so I have to manually integrate the peak by changing the baseline it sees.
It did that automatically for me first but now it has changed due to some reason I do not know.
Any suggestions would be greatly appreciated...

Tom Jupille posted information how to do this at:

http://www.sepsci.com/chromforum/viewtopic.php?t=2617

If the above link doesn't work, it's a "sticky" message within the HPLC section of the forum. There may be other ways too, but not tried, so someone else will have to speakup if there are.

[tom jupille]

What you need to do is to "lock out" integration around that big negative dip near the beginning of the chromatogram.