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9.11 Injection volumes and peak broadening
Introduction
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.
Sample volume effects
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 chro-
matography (IEX), and hydrophobic interaction chromatography (HIC) if the retention
factor k is high.
Broadening from UV monitor to
fraction collection
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 chro-
matogram. This "hidden" effect is more pronounced for smaller peak volumes.
504 ÄKTA avant User Manual 29035184 AE
9 Reference information
9.11 Injection volumes and peak broadening