Chemistry - High performance liquid chromatography (HPLC)
High performance liquid chromatography (HPLC)
HPLC is an integral part of modern chemical analysis. It is more
sophisticated than paper or thin-layer chromatography, allowing for
quantification as well as separation and identification.
A schematic diagram of an HPLC is shown opposite.
In HPLC analysis, the sample is injected into the mobile phase and carried
onto a column containing the stationary phase. For effective separation,
the components must interact with both the stationary and mobile phases as
they pass through the column.
The retention time of a component is the time it takes to pass from the
injection point through the column to the detector.
The components that interact least with the stationary phase leave the
column first and have the lowest retention times.
In separating the components of mixtures using HPLC, the mobile phase is
always a liquid and the stationary phase can be either a liquid or a solid.
The separation principle is the same as in paper or thin-layer
chromatography - relative attraction to the mobile and stationary phases.
The output (chromatogram) from an HPLC can provide us with not only the
identity of the components in the mixture but also the relative amounts of
Just like_ R_f values in paper chromatography, retention times are the key
identifying to components of the mixture. The area under the peaks is the
key to calculating the relative amount of each component.
As with all instruments, some form of calibration is required. This can
take the form of a library of retention times for substances passed through
a particular column at a particular flow rate for a particular mobile
phase. Substances separated under these conditions can then be identified
by comparing their retention times with previously recorded retention
Alternatively, the suspected identity of an element or compound can be
verified by spiking a sample with that element or compound and checking
which peak on the chromatogram is affected.
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