Getting Started in HPLC

Section 4A. Qualitative Analysis by HPLC

Fortunately, we usually know what compounds to expect in a particular sample. For example, if we are analyzing tablets for aspirin, we assume that aspirin will be present in each tablet. Or if we are trying to measure the purity of some compound "X" by determining its concentration in the sample, we assume that compound X is present. When we want to measure the concentration of a compound or compounds in a sample by means of HPLC, we first determine the retention time of the pure compound as sample. If we want to measure compound A, we inject pure A into our LC system, using the same conditions that we use to measure A in different samples. In this case we might obtain the chromatogram at the left.

Chromatogram of pure standard drug "A"

We see that compound A has a retention time of 4.25 minutes. Now if we want to find out if some mixture or sample contains this compound, we inject the sample and obtain the chromatogram at the right. In this case we see that the first peak also has a retention time of 4.25 minutes, and we therefore assume that peak #1 in the sample is compound A. We might also want to measure compounds B and C in the sample, so we inject them too: By matching up the various retention times, we see that peak #2 in our sample is compound B (retention time of 4.64 minutes) and peak #3 is compound C (retention time of 5.1 minutes). If our retention times don't match closely for the pure compound and for the sample, then probably we have made a mistake - the compound is not present in the sample.

Chromatogram of sample mixture. The first peak has the same retention time as that of the drug "A" standard (above). The second and third peaks have the same retention times as standards of drugs "B" and "C" (not shown)

When we run different samples containing compounds A, B, and C as above, we generally find that the size of the different peaks changes from run to run. This means that there are different amounts of A, B, and C present. However as long as A, B, or C is contained in the sample, its retention time should not change. That is, the retention time of a compound is normally not affected by peak size or the amount of the compound present in the sample. If compound A (retention time equals 4.25 minutes) is absent from a given sample, then no peak will be found at 4.25 minutes - we can then report that the concentration of A in the sample is zero.

In other cases, we may find extra peaks in the chromatogram besides those due to compounds we are analyzing for. This means that there are additional compounds in the sample, compounds we may not have expected to be present. The presence of unexpected bands in the chromatogram should be pointed out to the person for whom the LC analysis is intended - sometimes this affects the use that will be made of the sample analysis.

Often we find that retention times for different compounds remain fairly constant from day to day. That is, compound A gives the same retention time (4.25 minutes) in every LC run. In other cases we may see small variations in retention time from one day to the next: This may be normal, and it usually is when the changes in retention time are only a few hundredths of a minute - and when retention changes in the same direction for all bands. For larger changes in retention time, as shown below at the right, the pure compounds should be reinjected to confirm that the sample bands are the same compounds we are analyzing for. In any case, pure compounds should be injected at least once a day to confirm sample bands. Never rely on yesterday's injection of a pure compound to identify today's sample peak. Also keep in mind that changes in retention time can be a symptom of some problem with the analysis procedure. This is especially true when just one band shows a change in retention time, while other bands do not change as we see here.

Here peak #1 (compound A) shows changed retention time of 4.1 minutes in the second run. This could be the result of some interfering compound that is overlapping peak #1, which could cause serious error in the reported value of compound A. In this case pure compound A should be injected to confirm its retention time.

If we find that something is wrong with this run, the concentration of compound A in this sample should not be reported.

It is also possible to see changes in peak shape within the chromatogram. Here peak #2 has developed a tail and is no longer a symmetrical peak. This suggests that some interfering compound is overlapping peak #2 in the chromatogram, and again, the concentration of compound B in this sample should not be reported.



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Last revised: April 06, 2001.