Handbook of Size Exclusion Chromatography and Related ...

where ais the separation factor, Nis the number of theoretical plates, and k 0 is
the capacity factor. In SEC, k 0 equals 1. Therefore, the calculation of resolution
can be simplified to:
1
RS ¼= 8(a 1)N 1=2
In order to improve resolution in SEC, aand Nshould be maximized. As
in HPLC, Ncan be increased by increasing the column length or by reducing
the particle size of the gels. Toincrease the total column length, one can simply
run several low MW columns in series. Regarding particle size, 5mm SEC gels
for low MW are currently very popular. Several SEC column manufacturers
also provide columns with 3mm gels. However, either method will induce
higher backpressure. Unfortunately, the small-pore gels are more fragile than
the SEC gels with larger pore sizes as mentioned previously. The pressure
fluctuation during sample injection, when the zero-pressure sample loop is
connected to the high-pressure flow line, will reduce the lifetime of the smallpore
gels. Placing a small guard column, which serves as a pulse damper,
before the analytical low MW columns will greatly reduce the pressure
fluctuation on the analytical column. Because of the low viscosity nature, the
concentration effect in SEC of high MW polymer samples is usually not taken
into account in the case of low MW material (4). Therefore, in order to reduce
the pressure fluctuation and to extend the lifetime of acolumn, injection of a
small sample volume with ahigh concentration is also preferred.
The ain SEC depends mainly on the slope of the calibration curve: the
flatter the slope the better resolution, as shown in Fig. 1. There are at least two
ways to reduce the slope of the calibration curve and to maximize a: first,
increasing Dtr by increasing the total pore volume of the gels, and secondly,
minimizing the MW separation range (DlogMW) by selecting columns with
minimum but adequate MW range.
In order to increase the total pore volume, one may simply connect more
columnsofthesameporesizeinanSEC(columnsetBinFig.1).Inthiscase,the
numberoftheoretical plateswillalsobeapproximatelydoubled.Theotherwayto
increase the pore volume is to increase the pore-to-solid-body ratio, because the
interstitial volume of an SEC column is inherently fixed at approximately 40% of
the total columnvolume. The OligoPore column, recently introduced by Polymer
Laboratories, is designed based onthis principle (5). The calibration curvefor the
OligoPore column compared to that of aregular low MW column is shown in
Fig. 2.However,larger totalporevolumewith thesame pore size means athinner
solid wall, and thus more fragile particles. This type of column should be used
with care. Yet another means to increase the total pore volume is to use a larger
internal diameter column. This also has the advantage of reducing backpressure.
© 2004 by Marcel Dekker, Inc.
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