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How to Read a Chromatogram?

Over the years chromatography has gained an enviable position in analytical laboratories
involving separation and quantification of organic compound mixtures. However, a
chromatogram is not a display of results in concentration units but rather a graphical display in
real time of peaks generated as the separated components pass through the detector.
The chromatogram makes little sense to the layman as the peaks provide no information on the
identity of the mixture components nor any information on the amount present.
First of all it is necessary to understand what a chromatogram depicts. The chromatogram is a
two-dimensional plot with the ordinate axis giving concentration in terms of detector response
and the abscissa represents the time. The detector gives response as a peak whose height should
be ideally dependent on concentration of the particular component.

Retention Time (tr)


However, due to analysis conditions peaks may deviate from ideal shape and peak height can no
longer be a true measure of the concentration and instead the area under the peak is considered as
a measure of component concentration.
Each peak represents a component present in the sample. Retention time is time interval between
sample injection and the maximum of the peak. It is characteristic of the identity of the
component under the operating conditions. Identity of the component can be confirmed by
making injections of reference material under the same operational conditions. The matching of
retention time of reference material and the component peak confirms the identity of the
unknown sample component.

Now let us consider a sample which contains more than one sample component. Likewise each
component will be eluted at different retention times depending upon solute stationary phase
interactions and mobile phase flow characteristics.

Calculation of results
From the area measurements using simple arithmetic it is simple to calculate the concentration
of each component as a percent of the total.

%A =
Real Chromatogram
Let us now look at the actual chromatogram printout of HPLC separation of a mixture of
vitamins A and E in a food matrix and see what the chromatogram represents

Actual Chromatogram
The ordinate is in units of volts and abscissa in minutes. The signals are recorded at a wavelength
of 284 nm using a UV detector.
Retention time of each peak is marked above the peak and in the tabulated data below the
chromatogram details of the retention time, area (as digital units), peak area%, height and height
%. You can observe that due to non-ideal shape of peaks percentage area is different from
percentage height for each component so area measurement is a more reliable measure of
concentration.
You have been introduced to simple concepts on how to read a chromatogram. Please let us have
your comments on the article and if you found it useful.

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