Comparison and optimization of different peak integration methods to determine the variance of unretained and extra-column peaks.

ABSTRACT

Different automatic peak integration methods have been reviewed and compared for their ability to accurately determine the variance of the very narrow and very fast eluting peaks encountered when measuring the instrument band broadening of today's low dispersion liquid chromatography instruments. Using fully maximized injection concentrations to work at the highest possible signal-to-noise ratio's (SNR), the best results were obtained with the so-called variance profile analysis method. This is an extension (supplemented with a user-independent read-out algorithm) of a recently proposed method which calculates the peak variance value for any possible value of the peak end time, providing a curve containing all the possible variance values and theoretically levelling off to the (best possible estimate of the) true variance. Despite the use of maximal injection concentrations (leading to SNRs over 10,000), the peak variance errors were of the order of some 10-20%, mostly depending on the peak tail characteristics. The accuracy could however be significantly increased (to an error level below 0.5-2%) by averaging over 10-15 subsequent measurements, or by first adding the peak profiles of 10-15 subsequent runs and then analyzing this summed peak. There also appears to be an optimal detector intermediate frequency, with the higher frequencies suffering from their poorer signal-to-noise-ratio and with the smaller detector frequencies suffering from a limited number of data points. When the SNR drops below 1000, an accurate determination of the true variance of extra-column peaks of modern instruments no longer seems to be possible.