9.12 Injection volumes and peak broadening
The width of peaks at the fraction collector is influenced by the following:
•
the properties of the column,
•
the dimensions of the tubing,
•
the dimensions of the modules in the flow path, and
•
fluid dynamics.
Initial sample volume affects the peak width in gel filtration (GF) chromatography and other isocratic
techniques. A sample zone is broadened during passage through a GF column so that the sample is
diluted and the resolution decreases with increasing sample volume. Sample volume does not however
affect the resolution in adsorption chromatography techniques such as affinity chromatography (AC),
ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) if the retention
factor k is high.
The effect of peak broadening in the system from sample injection to peak detection (including dilution
on the column) is apparent in the chromatogram from the UV monitor, but broadening from the UV
monitor to fraction collection is not visible in the chromatogram. This "hidden" effect is more pronounced
for smaller peak volumes.
Narrow and short tubing reduces peak broadening. However, narrow tubing also increases the back
pressure. See for recommendations on tubing dimensions for prepacked columns.
ÄKTA pure User Manual 29119969 AB 493
9 Reference information
9.12 Injection volumes and peak broadening
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