Possibly obvious questions from a desperate access student

[Lowryn]

Hi, I discovered this site when searching for information on chromatography for my chemistry assignment.

I have to answer some questions on chromatograms although I have not been taught anymore than the basics. If I explained my problem, could you give me some pointers?

I have been asked to analyse the following chromatograms





What could I say about them? I understand how the different peaks correspond to the difference substances, but that won't give me 20 points!

What does the 135mV mean?

Thanks in advance
Nikki

[tom jupille]

Sorry, we won't do your homework for you . . .

. . . but, if you post the homework questions and what you think the answers are, we'll let you know if you're right, (and if not, we'll point you in the right direction).

For starters, what questions were asked about those chromatograms?

[Lowryn]

There are no questions, I have been asked to analyse the chromatograms.

I don't understand why the coffee has 135mV next to it and the black tea has 26mV next to it, is this relevant?

Is the one marked "standard" similar to a control? I mentioned that it was being used as a control and the known substances put through it to see where the peaks would occur - am I way off the mark?

So far I have this:
The peaks shown on the chromatogram indicate the different substances separated by the mixture. The substances that are heaviest or that have greatest affinity for the mobile phase will emerge first. The time taken for the components to emerge is recorded, as is the quantity. The total quantity of the component can be calculated by looking at the area of the peak. In Fig 1:3 the chromatograms show the amounts of three particular substances in a mixture, Theobromine, Theophylline and Caffeine. The first chromatogram is the control in which the three substances are tested to see how where they appear on the graph. The other two graphs show the analysis of coffee and black tea. From the graph it is clear that all three substances are present in each mixture, caffeine being in a significantly higher quantity than the other two substances.

I don't know how else to interpret them

Thanks

[PJ8]

mV stands for microvolts, which is simply a measure of the strength of the response of each analyte. So the peak for caffeine in coffee is bigger than the peak for caffeine in tea (though you can't say there's more caffeine without knowing the concentrations of the sample.)

[Lowryn]

Thank you for respondind, is there anything else I ought to mention?
Does my write up so far make any sense?

[PJ8]

Mostly... the standard is mainly so you can identify the peaks in the samples (providing you run the same conditions and have no co-elution), also you can quantify peaks provided you know the concentrations of your sample and standard and the areas of your peaks.

Try reading the FAQ here and an HPLC text book, that should give you the basics you need.

[tom jupille]

Actually, very well done.

In your write-up, I would mention that the identity of an analyte is established by it's retention time, and the quantity is established by the size (either height or area) of the peak.

You should also know that chromatography is never an absolute technique; everything is measured by comparison to standards. We establish the identity of a peak by matching it's retention time to that of a known standard. We establish the quantity of a compound by comparing the size of the peak to the sizes of known standards of that compound.

The one place you were wrong was in stating that the caffeine is present in higher concentration than the theobromine and/or theopylline. Quantitation can only be established by comparison with known standards of the same compound. Different compounds have different "response factors", so it is quite possible for a large peak to result from a small amount of analyte.

That said, don't feel bad, a lot of experienced scientists have made the same error!

[Lowryn]

Thank you, that is so helpful. I guess I should just say that the peak is talller, not make assumptions that this means that there is greater quantity!

[Dan]

Lowryn,

You are getting it, good work.

Let me add one correction:

substances that are heaviest or that have greatest affinity for the mobile phase will emerge first.

I suggest that you remove the part about "heaviest" as this may not be a factor for the determination of elution order. Just leave in the part for "greatest affinity". Things such as size, shape, polarity come into play for the "affinity" issue but, in general, not the molecular weight.

Regards,
Dan