Handbook of Size Exclusion Chromatography and Related ...

The detector time constant can distort column efficiency when the peak
width (in time units) becomes of the same order of magnitude as the response time.
High-efficiency columns produce very sharp peaks, and detectors with response
times greater than 0.5 s can contribute significantly to band broadening. Electronic
filtering can increase response time and cause measurable broadening of sharp
peaks. Refer to Ref. 108 for an exhaustive discussion of extracolumn effects in
detector systems.
Capillary tubing should be kept as narrow and short as possible, while
remaining practical. The length of tubing for a maximum band width increase of
5% can be calculated from Eq. (24), taken from Ref. 45:
L ¼ 40V 2 E Dm
pFNd 4 ct
(24)
in which Dm is the solute diffusion coefficient in cm 2 /s, F is the flow rate in mL/s,
dct is the ID of the capillary in cm, N is the plate number, and the retention volume
(VE) was earlier given by Eq. (19). Equation (24) can also be used to calculate the
dimensions of a detector cell for the ideal situation in which no mixing occurs in
the cell, that is, the plug flow model. Bending, coiling, or deforming the tubing
permits longer lengths with the same degree of band broadening as shorter lengths
of straight tubing (111).
5.2 Sample
As discussed, there is a limit to how much can be injected into an HPLC column in
terms of sample mass and volume at which the resolution deteriorates beyond
acceptable levels. SEC has the lowest loading capacity (g sample/g packing
material) for high-performance HPLC techniques because the separation is
performed under isocratic mobile-phase conditions and because the separation
takes place within the interstitial pore volume, that is, in the absence of a stationary
phase. In general, samples are injected as a large volume of a dilute solution. As
the increasing concentration overloads the inlet, asymmetrical and broad peaks are
seen and resolution decreases. Gooding et al. (112) derived Eq. (25) to calculate
the theoretical protein load in milligrams for a 25 cm long column:
C ’ r2
(25)
4:4
where C is the loading capacity and r is the column radius in mm. Thus, for a
column ID of 7.5 mm, the protein loading capacity is v3.2 mg/injection.
Kirkland and Antle (113) determined that 0.1 mg of a 4800 dalton polystyrene
polymer could be injected per gram packing material in GPC on 47 A˚ silanized
silica. Roumeliotis and Unger (99) found that 0.1 mg protein can be loaded per
gram LiChrosorb Diol material. They demonstrated that load is proportional to
© 2004 by Marcel Dekker, Inc.