Handbook of Size Exclusion Chromatography and Related ...

volume. It is clear, however, that Spherisorb and Superlcosil have narrower pore
size distributions than Zorbax and, particularly,LiChrospher.
The application of the silicas shown in Fig. 5in SEC is demonstrated in
Fig. 6, in which six narrow molecular weight polystyrene standards ranging from
4,480,000 to 890 dalton are separated on 15 cm 4.6 mm ID columns packed
with 5-mm LiChrosorb Si-100, Spherisorb S5W,Supelcosil LC-Si, and Zorbax
BP-SIL,respectively (R.Eksteen, unpublishedresults, 1986). Toluene isincluded
in the mix to mark the total inclusionvolume. The calibration curves for the four
silicas, as well as for Nucleosil 120-5 and YMC-GEL SIL 120A S5, are shown
in Fig. 7. To simplify the comparison of the different packing materials,
normalized retention volume (VE=Vi) 1, is plotted on the x-axis instead of
elution volume. The normalized retention volume, which is zero for a totally
excluded solute, is a direct measure of the retention of a compound beyond the
interstitial volume.
It is evident from the chromatograms in Fig. 6 that of all the columns,
LiChrospher provides the best separation for polystyrenes above 17,500 dalton
molecular weight, followed by Supelcosil. LiChrospher is also the best choice for
separations below 17,500 dalton molecular weight, followed closely by Zorbax.
This last result is expected based on the large number of small pores that were
measured for LiChrospher and Zorbax in Fig. 5. In support of the data shown in
Fig. 5, the calibration curve for LiChrospher Si-100 in Fig. 7a also confirms
the presence of pores much larger than 100 A ˚ . In terms of the available pore
volume, both the LiChrospher and the YMC silicas are considerably more porous
than the other silicas shown in Fig. 7. Although this property is particularly
attractive for their use in SEC, silicas with large pore volumes are more fragile, as
shown later in this section. It is interesting to note that the interparticle porosity for
both high pore volume silicas was only 34% of the empty column volume, but that
of the other siicas was 40%. A low interparticle porosity can result when a silica
has a broad particle size distribution such that the smallest particles can occupy
the interparticle space between the larger particles. It is also possible that some
particle fracturing took place during column packing. The backpressure for the
LiChrospher column was about 25% higher than that for the more robust
Spherisorb, Supelcosil, Nucleosil, and Zorbax columns, and the backpressure for
the YMC column was twice as high. In comparison with the stronger silicas, the
efficiency for the 15-cm LiChrosorb and YMC columns was about 7000 vs.
10,000 theoretical plates and the peak asymmetry factor was 0.6 vs. 0.9,
respectively. Despite these lower values for the column performance parameters, it
is clear from Fig. 6 that good overall peak shape and resolution were obtained for
the polystyrene test mixture on the more fragile LiChrospher silica. Note also that
all silicas shown in Figs 5 to 7 were primarily developed for analysing small
molecular weight compounds. Although, as shown in Fig. 7, even small solutes
are partially excluded from entering all pores, silicas with pores in the range
© 2004 by Marcel Dekker, Inc.