Significance of peak resolution, Rs

[joelyue]

I recently came across the term peak resolution for chromatograms, Rs.
Can someone expain what it means and its significance?

[tom jupille]

It's the ratio of the center-to-center separation between two peaks to their average baseline width. If both peaks are symmetrical and equal in size, at Rs = 1.5, there will be just less than 1% overlap between the peaks.

[joelyue]

So what happens if peak overlap is >1%. What are its effects on the peak results?

[tom jupille]

What are its effects on the peak results?

If you think about it, quantitation in chromatography is based on relating the peak area to the amount of sample injected. For two adjacent peaks, the quality of the separation is directly related to how well you can allocate areas (i.e., tell how much area belongs to which peak). If the peaks overlap significantly, that allocation uses algorithms that involve assumptions about the exact shape of the peaks. Norman Dyson wrote the standard text on integration, and at the end of the chapter on integration algorithms, he says something like 'all of the preceding algorithms are capable of generating extremely precise and totally inaccurate values of peak areas' (I'm not at the office, so I'm writing that from memory). Translation: no matter which way you slice 'em, it's going to be wrong; the best you can hope for is to be wrong the same way all the time.

To be a bit more specific, if you have two approximately equal sized symmetrical peaks with 1% overlap, you are unlikely to be very far off, but that begs the question: if you know the peaks are equal, why do the chromatography? If one peak is small and the other large (say 99% with a 1% impurity), then that "1%" overlap will render the smaller peak virtually unquantifiable.

Most chromatographers can "eyeball" a chromatogram and make a fairly good judgement as to the quality of the separation. The problem is that it's hard to go to the FDA with a method that you characterize as "pretty good", or "not too bad", or "adequate". That's why we use a number like resolution.

[joelyue]

Thanks Tom for the explanation. i get the idea now.