Violation of basic principles of HPLC seperation

[najmulhasan]

How could it be possible that two different molecules of different physical and chemical properties are eluted on same retention time on same analytical parameters for HPLC. (According to my studies this phenomenon is against the principles of HPLC) I need expert comments plz....

[DR]

It'a all about the partition coefficients.

[tom jupille]

To elaborate a bit on DR's response: all that is required is for the two molecules to have approximately the same partition (distribution) coefficient between the mobile and stationary phase. That is more probable than you might imagine because liquid chromatography has only a limited peak capacity: for a full-range gradient in reversed-phase with a 10,000 plate column, the peak capacity at Rs = 1 is approximately 200. That's directly proportional to the desired resolution, so if you look for Rs = 0.1 (at which point the naked eye can't see the difference), the peak capacity is still only 2,000.

To make a gross oversimplification, if you assume that there are a couple of million possible organic compounds, that means any arbitrary compound you choose has at least 1,000 other compounds that would have close to identical retention.

[lmh]

and to elaborate still further: if you have chemicals eluting randomly with a uniform distribution from one end of a chromatogram to the other, and your chromatogram is long enough to fit 200 peaks, then if you want to be 90% sure that the peaks are pure, you cannot have more than 6 peaks. Since retention times won't generally have a uniform distribution, the situation is actually much, much worse.

[DR]

... which is why we typically go right for the alpha portion of the resolution equation (by trying a different stationary phase) when a major change is needed to separate a pair of co-eluters.

No violations here, play on.

[prepcolumn]

Because of the possibility of giving same retention time of different compounds, ICH Q6A states that;

b) Identification: Identification testing should establish the identity of the new drug substance(s) in the new drug product and should be able to discriminate between compounds of closely related structure which are likely to be present. Identity tests should be specific for the new drug substance, e.g., infrared spectroscopy. Identification solely by a single chromatographic retention time, for example, is not regarded as being specific. However, the use of two chromatographic procedures, where the separation is based on different principles, or combination of tests into a single procedure, such as HPLC/UV diode array, HPLC/MS, or GC/MS, is generally acceptable.