In this article we learn about how to determine the retention time of a chromatographic peak.
Relevance #
The retention time is arguably one of the most basic characteristics of a peak. Its most elementary application is the qualitative identification of a known compound by means of reference information. The retention time is also of importance for fundamental research including (i) the prediction of retention times in method development, (ii) retention modeling to characterize selectivity and (iii) assessing the performance of chromatographic systems.
Definitions #
We will investigate the computational methods to obtain the retention time below. However, before we do so it is first important to understand what we exactly mean with the term retention time.
Presentations of the retention time
it is important to realize that the recorded chromatogram reflects the time at which the analytes are observed at the detector (t_{R,i,obs}), and not at the exit of the column. For fundamental studies of retention it is thus important to subtract the extra-column time (t_{ec}) from the observed retention time. However, for most practical applications its value is so small that it may reasonably be ignored. In some cases, the net retention time (t'_{R,i}) is used where the dead time (t_0 or t_m) is subtracted.
Identity of the retention time
From the moment that we inject our analytes, various band broadening processes occur as the analytes migrate through the chromatographic system. This results in our chromatographic peak, which never represents a singular discrete retention time, but always a distribution of retention times. This is depicted in the chromatogram shown in Figure 2, where panel B highlights one of the peaks.
The most convenient method to determine the retention time is by simply regarding the apex (i.e. maximum) of the peak (Fig. 2-B, Point I). This is especially correct when the peak is symmetrical. An arguably more appropriate method to determine the retention time is by computing the mean of the chromatographic distribution (Fig. 2-B, Point II), which represents its center of gravity. Finally, a rare determination of the retention time considers the start or frontal slope of the peak (Fig. 2-B, Point III). While this is never useful for calculation of the retention factor, it can sometimes be useful for example in case of preparative separations, when the column is intentionally overloaded.
Determination #
We can now focus on extracting the retention time from a peak. This depends highly on the the identity of the retention time, as explained above. We will assume here that the peak has already been found and isolated. Later tutorials will address signal processing methods that aid in peak detection and background correction.
Peak apex
The peak apex is literally the highest point of a peak distribution. This can be literally the time associated with the point of the peak with the highest y-value and is typically identified by functions that seek datapoints that are larger than (multiple of) its neighbors.
Statistical moment
Peaks in chromatography are – especially in case of LC – rarely symmetrical. It is thus more appropriate to determine the first normalized moment of the peak, which is equal to.
