Handbook of Size Exclusion Chromatography and Related ...

Figure 15 Effect of sample volume on column efficiency. Column, TSKgel G3000SW,
10 mm, 60 cm 7.5 mm; mobile phase, 0.1 M phosphate and 0.2 M sodium chloride,
pH 7.0; flow rate, 1.0 mL/min; detection, 280 nm, UV. (Adapted from Ref. 83.)
TSKgel G3000SW column (83). For a 0.5-mg sample load, column efficiency
does not decline until the injection volume increases above 250 mL, or 2% of the
empty column volume, in reasonable agreement with the predicted value. Note
that mass overloading can be detrimental at much lower injection volumes. As
demonstrated, dilution of the sample actually improves efficiency beyond the
injection volume at which volume overload becomes apparent.
The construction of the detector cell and detector electronics can seriously
detract from the efficiency of the column. Although generally some capillary tubing
is contained in the detector, we assume that this can be neglected in comparison with
the amount of capillary tubing used to connect the column to the injector and
detector. This assumption is not valid when the column effluent is directed through a
large-volume heat exchanger before entering the detector cell, as in most refractive
index detectors. To minimize the band broadening of early peaks, the volume of the
cell should be less than one-tenth the volume of the peak of interest (8,45).
© 2004 by Marcel Dekker, Inc.