Is the published paper integration correct ??

[Newchromatographer]

I know the answer is correct because it was published , but honestly if have done this work according to my knowledge i will repeat it many times because i feel it is not seperated the preaks from each other !

to follow my question please see the following :

1- download the picture taken from one paper from ( http://www.mediafire.com/?momtm3y0hht) , it is trutable web don't worry
2-in picture ( c) do u thing peak 3 well seperated from peak 4 ?
3- do u guess how can he integrate the peaks , is it forexample , from the beginig of the peak and he draws to basline or what ??

i really want to know more about integration like from John Or Tom

please help me with this issue

thanks

[bisnettrj2]

I had some extra time on my hands, so I'm posting more information regading Newchromatographer's post...

Journal of Chromatography A
Volume 1216, Issue 2, 9 January 2009, Pages 211-216

Determination of carbamate pesticides using micro-solid-phase extraction combined with high-performance liquid chromatography - Chanbasha Basheera, Anass Ali Alnedharyb, B.S. Madhava Raob and Hian Kee Leea



Fig. 5. Liquid chromatograms of spiked soil extracts after μ-SPE: (a) Soil sample spiked at 2 ng g−1 of each carbamate; (b) soil sample spiked at 10 ng g−1 of each carbamate and (c) soil sample spiked at 40 ng g−1 of each carbamate. Peaks: (1) carbaryl, (2) propham, (3) methiocarb, (4) promecarb, (5) chlorpropham and (6) barban. (text added in (c) by Newchromatographer)


My opinion in regard to the questions:

1. No, the peaks aren't well separated, that much is obvious.

2. Using sophisticated software known as 'Microsoft Paint', I calculated a peak resolution using the equation Rs = 2(tRB – tRA)/(wB + wA) for peaks 3 and 4 at 0.64.

3. The integration I would perform would be a baseline drop to the perceived baseline from before peak 2 and after peak 4, unless there was some regular baseline interference somwhere in the elution times of the peaks. I would also quant using peak height instead of area due to the lack of resolution between peaks.

[tom jupille]

I think the "resolution" value is misleading in this case because the peaks in question show significant shoulders on the back side. Our usual judgments concerning resolution values (e.g., Rs=1.5 is "99% baseline resolution") assume approximately Gaussian peaks, which these are not*! All the peaks (well, almost all, per my comment at the end) have shoulders. I expanded bisnettrj2's graphic and stretched out the x-axis go give this (ugly) view; you can see that the shoulder on peak 3 creates a "plateau" between peaks 3 and 4:


In general, when resolution is incomplete, choosing an integration algorithm is little more than educated guesswork. Check my post near the bottom of this thread for a more detailed comment: viewtopic.php?t=11946 . I agree that a perpendicular drop from the valley to the baseline would be the "best", but there is a lot of ambiguity as to where the minimum of the valley occurs.

The bottom line, though, is that if the standards and the samples behave the same way and are treated the same way, integration errors may cancel out. The proof would be in the validation of the method to demonstrate that it is good enough for the purpose.

Now, as to why the peaks have shoulders. I'd be tempted to guess a physical problem with the column (either a headspace or a partial inlet blockage) because almost all the peaks show the same problem -- which suggests that it occurred while the peaks were still together. It's hard to tell for sure about peak 1, but in the first chromatogram, it shows the same type of shoulder.

[bisnettrj2]

The peak shape error may also be sample-diluent based - I think they are injecting 100% methanol in a 60:40 water:methanol (maybe 40:60?) mobile phase, if I remember the article correctly from this morning.

[danko]

Hi Bisnettrj2,

Your suggestion as to what causes the tailing doesn’t fit with the actual observation.
Strong solvent effect would result in fronting, rather than tailing. Also the fastest eluting peaks (shortest retention times) would suffer the most.
My personal guess is the same as Tom’s. And if the utilized column were performing adequately I would’ve expected baseline separation between peak 3 and 4.

Best Regards

[bisnettrj2]

In any case, we've strayed from the original question. I think that the purpose of the paper was demonstrating the effectiveness of a micro-extraction scheme, rather than a robust analytical method, so perhaps a little more method development could have been undertaken (or maybe they could have used a column other than a Phenomenex? )

[danko]

Agree.

Best Regards

[Peter Apps]

There are some pretty dramatic retention shifts as well

Peter