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 tR. 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 tR with that of injected reference standards (Fig. 1b) in the same mobile and
stationary phase a chromatographer may be able to identify each compound.
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).
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