The General Elution Problem
Look at the chromatogram below in which six components are to be separated by an
elution process:
S
Retention Time
It is clear from the figure that the separation is optimized for the elution of the first
two components. However, the last two components have very long retention and
appear as broad peaks. Using a mobile phase composition that can optimize the
elution of the last two compounds will, unfortunately, result in bad resolution of the
earlier eluting compounds as shown in the figure below where the first two
components are coeluted while the resolution of the second two components becomes
too bad:
S
Retention Time
One can also optimize the separation of the middle too components by adjusting the
mobile phase composition. In this case, a chromatogram like the one below can be
obtained:
S
Retention Time
However, in chromatographic separations, we are interested in fully separating all
components in an acceptable resolution. Therefore, it is not acceptable to optimize the
separation for a single component while disregarding the others. The solution of this
problem can be achieved by consecutive optimization of individual components as the
separation proceeds. In this case, the mobile phase composition should be changed
during the separation process. First, a mobile phase composition suitable for the
separation of the first eluting component is selected, and then the mobile phase
composition is changed so that the second component is separated and so on. The
change in mobile phase composition can be linear, parabolic, step, or any other
formula. The chromatographic separation where the mobile phase composition is
changed during the elution process is called gradient elution. A separation like the one
below can be obtained:
S
Retention Time
Qualitative Analysis
Usually, the retention time of a solute is the qualitative indicator of a specific analyte.
The retention time of an analyte is thus compared to that of a standard. If both have
the same retention time, this may be a good indication that the identity of the analyte
is most probably that of the standard. However, there can be important uncertainties
since some different compounds have similar retention. In such cases, it is not wise to
use the retention time as a guaranteed marker of the identity of compound, except in
cases where the sample composition is known. Development in qualitative analysis in
chromatography involves use of detectors that can give structural details of solutes,
like diode array, Fourier transform infrared, mass spectrometers, etc. In such cases,
qualitative analysis with high degree of certainty can be accomplished. It should
therefore be clear that a similar retention time of a component and standard does not
imply a100% identification but rather a good possibility. However, if the retention
time of a compound in question does not match that of the standard, we are 100% sure
that the anticipated compound is either absent or present at a concentration below the
detection limit of the instrument.
Quantitative Analysis
Chromatographic separations provide very good and reliable information about
quantitative analysis of sample constituents. Either the peak height or peak area can
be used for quantitative analysis. Peak heights are easier and faster to use and usually
result in good precision, especially when reproducible sample injections are made.
However, late eluting peaks may have small peak heights but large width which may
cause large errors. Peak areas are better for quantitative analysis as the area under the
peak is integrated which is an accurate measure of concentration. However, this
process is slow and tedious especially when it is to be manually performed or when
the peak is very sharp. Generally, peak areas give better quantitative results.
The Internal Standard Method
Uncertainties in sample injection can be overcome by use of an internal standard. In
this method, a measured quantity of an internal standard is added to both standard and
sample, and the ratio of analyte signal to internal standard is recorded. Any
inconsistency in injection of the sample will affect both the analyte and internal
standard. Properties of the internal standard should include:
a. The retention times of internal standard and analyte should be different and the
two peaks must be well separated, R >1.25
b. The detector response factor for the analyte and the internal standard should be
the same.
Using internal standards can significantly improve precision to better than 1%.
The Area Normalization Method
This technique can also overcome the uncertainties associated with sample injections.
In this method, complete elution of all components is necessary where areas of all
eluted peaks are computed and calculated areas are corrected for detector response.
The concentration of the analyte is thus the ratio of its corrected peak area to total
corrected areas of all peaks. The method is not as versatile as the internal standard
method.