Automated measurement of peak widths for the determination of peak capacity in complex chromatograms.

The peak capacity was measured for an ultrahigh-pressure gradient elution chromatogram of a fluorescently tagged tryptic digest of ovalbumin. The peak widths in the chromatogram were determined by measuring the peak height and the second derivative at the peak maximum. This approach for measuring peak widths was programmed into a computer, and the software accurately determined the general progression of peak widths by measuring 47 peaks throughout the chromatogram in under 10 s. Peak capacity was determined by taking the definite integral of the plot of reciprocal base peak width versus retention time. This calculation of peak capacity is a linear transformation with respect to separation space, so the method is more rigorously accurate than previous methods. The peak capacity for the chromatogram was calculated to be 316.