Identifying Compounds by LC/HPLC - Qualitative Analysis by LC / HPLC

Liquid chromatography is a powerful separation technique. Using this technique, it is also possible to identify, quantitate and collect the compound. The analyst, however, is often faced with the problem of positive identification of one or more of the peaks emerging from the chromatographic column.

The volume of liquid required to elute a compound from the liquid chromatography column is called the retention volume. The associated time is called the retention time t_R. Both these measures are commonly used to identify peaks in a chromatogram.

The retention time and the retention volume are characteristic of the compound, column and other conditions.

Therefore, retention times or retention volumes may be used to identify the compound by comparison with knowns.

With modern instruments, the retention time, or retention volume is highly reproducible. Typical relative standard deviations of retention time are less than 2%. This is well within the precision required for assigning the identity of the peaks.

In Fig. 1a five compounds are represented by five peaks separated in time in the chromatogram. Each elutes at a specific location, measured by the time elapsed between the moment of injection (time zero) and the time when the peak maximum elutes. By comparing each peak’s retention time t_R with that of injected reference  standards (Fig. 1b) in the same mobile and stationary phase a chromatographer may be able to identify each compound.

Fig 1. a) Chromatogram of an unknown mixture containing five compounds as shown by five peaks  1b) Chromatogram of a standard that contains caffeine (1), aspirin (2) and salicylamide (5). By comparing the two chromatograms and the retention times of peaks 1, 2 and 5 the peaks of the unknown mixture can be identified. 

In the chromatogram shown in Fig.1b, the chromatographer knew that under these chromatographic conditions compounds 1 (caffeine), 2 (aspirin) and 5 (salicylamide) elute at the specific retention times shown. By comparing the chromatogram of the unknown mixture in Fig. 1a compounds 1(caffeine), 2 (aspirin) and 5 (salicylamide) could be identified.

However, it should be made clear that is possible for different compounds to have identical retention times. In order to avoid these coincidental errors, confirmation of the identification should be made by mass spectrometry, infrared or nuclear magnetic resonance or some other independent method.

Once identity of the peaks is established, the next peace of important information is how much of each compound was present in the sample (quantitative analysis).