[Readers Insight] The Interplay between Column Properties and Peak Integration

Author: Chromatography Mound

Several weeks ago, I was asked a compelling question: Can you write an article about the relationship between columns and peak integration?

At first glance, I had no clue, as these two concepts seem unrelated: one is a piece of separation hardware, and the other a mathematical procedure performed on a chromatogram. Where should I start with?

But as I dig into it, a clear and practically important link emerged.

Integration is, in essence, the calculation of the chromatographic peak area: the detector signal above the baseline is traced and the enclosed area is computed by software. Since the area represents the total mass of the analyte passing through the detector, the integrated area should (in theory) remain invariant regardless of whether the peak is sharp and tall or broad and short, provided the concentration remains constant.

However, empirical observations often contradict this theoretical constancy. When comparing chromatograms of the same analyte across different retention conditions, discrepancies in integrated peak areas of peaks eluting at different retention time is frequently observed.

Consider a scenario where we hold the column, detector (DAD in this example), and analyte concentration constant, and only modify the mobile phase composition to delay retention. It is obvious that the peak will be broadened. Theoretically, since the concentration is unchanged, the peak area should remain constant and the peak height should fall. But in practice, the peak height can even be higher and the peak area becomes larger (see the chromatogram below). Similar phenomena also exist when only switching the columns used.

The mechanism underlying the above discrepancy resides in the velocity at which the analyte zone traverses the detector's flow cell.

A detector operates at a finite sampling frequency, imposing temporal constraints (detector response time, A/D sampling rate, time constants, and on-line filtering or smoothing). These parameters determine how faithfully a transient signal is recorded.

When an analyte elutes at a high linear velocity, the duration it spends within the flow cell is minimized, which can lead to a reduction in the number of data points collected across the peak. If the sampling rate is insufficient to capture the rapid flux of molecules at high velocities, the resulting integration may suffer from undersampling, leading to a slight loss in recorded area. 

Conversely, a column that provides greater retention causes the analyte to pass through the detector at a lower velocity. This increased residence time allows the detector to perform more sampling cycles for the same mass of analyte, potentially leading to a more comprehensive and accurate representation of the peak profile.

Much like a high-speed camera capturing a fast-moving object, the precision of the final image—or in this case, the integrated area—is intrinsically linked to the speed of the subject relative to the capture rate of the device.