Chromatography Forum

It recently is becoming popular again to use the term peak capacity, in gradient elution.

I have two questions
1. Gradient run time. Is it the time from start to the time where the last peak enters the detector or form start until the end of the gradient program?
3. At what height do you need to calculate the peak width?
2. How dangerous is it to compare Pc of separations with different gradient slopes?

Depends on what you are doing and what you want to measure. If you got a generic chromatogram, say a run that you will always use for an analysis of a protein hydrolysate or for a combichem sample, you definitely use the gradient run time as the measure. For comparing the quality of a separation for a specific sample, say an impurity profile run under different circumstances, you are probably better off to use something close to the retention time of the last peak (just beyond the last peak).

The standard way to estimate the peak capacity is to use the 4-sigma peak width, which can be conveniently obtained from the software.

You will get different peak capacities when you vary the gradient parameters, such as the gradient slope. Peak capacity is not a parameter of the column, but a parameter of the separation. You change the separation conditions, you affect the peak capacity.

Here is a reference: U. D. Neue, The Theory of Peak Capacity in Gradient Elution, J. Chromatogr. A 1079 (2005), 153-161

Thanks for the comments! I already read the article.
But would it not make sense to come up with a standard calculation for this?

I think that it makes more sense, if you have a very complex mixture, to measure from the first identified compound of interest until the last identified of interest.

In this way you define really your separation window without "cheating" either in the early or end of the chromatogram.

For example, for a peptide mixture, I could go from 0-5% ACN for one hour followed by a very steep gradient to 90% ACN. This will report very high peak capacities which are not true...

For the late eluters, with a let's say normal gradient, I could use polymers eluted very late as actual peaks and then again report very high peak capacities...

My suggestion would be... the separation window is from the first identified peak of interest to the last identified peak of interst. Then you use the same sample to characterize different variations of your gradient or even different columns (of the same nature)...

Kostas