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Saturday, 21 December 2013
Elution Modes in HPLC
Modes in HPLC
adsorption chromatography, solvent molecules compete with solute molecules for
sites on the stationary phase. Elution can be described as a displacement of
solute from the stationary phase by solvent.
Elution Modes in HPLC
elution–Performed with a single solvent or constant
solvent mixture (i.e The composition of the mobile phase kept constant throughout
elution–Continuous change of solvent composition to
increase eluent strength (i.e The composition of the mobile phase varied during elution).
The main purpose of gradient elution is to move strongly
retained components of the mixture faster, but having the least retained
component well resolved.
Starting with the low content of the organic component in the
eluent the least retained components are allowed to be separated. Strongly
retained components will sit on the adsorbent surface on the top of the column,
or will move very slowly.
When the amount of organic component increased then strongly
retained components will move faster and faster, because of the steady increase
of the competition for the adsorption sites.
Gradient elution also increase quasi-efficiency of the column.
In the isocratic elution, the longer a component is retained, the wider its
peak. In gradient elution, especially with the smooth gradient shape without a
flat regions, the tail of the peak is always under the influence of the
stronger mobile phase when compared to the peak front. Thus, molecules on the
tail of the chromatographic zone (peak) will move faster. This will tend to
compress zone and narrow the resultant peak.
Performance of the gradient elution is strongly dependent on the
instrumentation. Two main points the chromatographer needs to know about his
1.How large the volume between the component mixing point and
column inlet is. For the low-pressure gradient systems this volume usually
correspond to the pump volume, and about 2 - 3 ml.
2.How well does the system mix eluent components. If the system
does not mix components well then it will supply for the certain time one
component then another and so on. Chromatographic performance of such system
will be very low especially for the least retained components.