Handbook of Size Exclusion Chromatography and Related ...

less steep and still linear at higher molecular weights. Furthermore, although the
slopeofthecurvesathighmolecularweightwereindependentofsaltconcentration,
below 15,000 dalton the slope became steeper with decreasing phosphate
concentration. No marked effect of SDS concentration was detected for
polypeptides 10,000 dalton or higher. For polypeptides with molecular weight
less than 10,000 dalton, the plot in 1% SDS lost linearity and became steeper.
If SEC is being performed for preparative purification or desalting, such
volatile buffers as ammonium bicarbonate or acetate may be preferred because
theyarereadilyremovedbyfreezedrying.Organicmodifiers,suchasacetonitrile,
arevolatilebutmayleadtoproteinaggregation.Triethylamineformate,pH 3.0,is
also avolatile denaturing agent. Reference 119 lists more volatile buffer systems.
As shown in Fig. 3, mobile phase flow rate has astrong influence on
resolution. For larger molecules (polynucleotides and proteins), the mass transfer
term is much larger and the flow rate must be correspondingly decreased. Typical
flow rates are 0.5–1.0 mL/min for 7.5-mm ID columns, and although better
resolution can be obtained at much lower flow rates (see Fig. 1), these rates
represent the best compromise between separation efficiency and time.
5.4 Temperature
Although most SEC applications are run at room temperature, increased
temperature may be utilized to improve the resolution of difficult separations or to
decrease the viscosity. As long as the macromolecule is well dissolved, the
influence of temperature on the slope and position of a molecular weight
calibration curve is relatively minor (8). Some high molecular weight polyolefins
and polyamides require temperatures of 100–1358C because the samples are not
soluble at lower temperatures (45). With low molecular weight molecules,
increasing the temperature may decrease adsorption. The extent and rate of
formation of aggregates was investigated by Watson and Kenney using SEC at
elevated temperatures (125). They found that the formation of aggregated species
was the main reason for loss of monomer for interleukin-2 analog and g-interferon.
6 REFERENCES
1. J Porath, P Flodin. Nature 183:1657, 1959.
2. J Moore. J Polym Sci, Part A 2:835, 1964.
3. J Giddings. Anal Chem 35:2215, 1963.
4. J Kirkland. J Chromatogr 125:231, 1976.
5. F Regnier, R Noel. J Chromatogr Sci 14:316, 1976.
6. K Fukano, K Komiya, H Sasaki, T Hashimoto. J Chromatogr 166:47, 1978.
7. Y Kato, Y Yamasaki, H Moriyama, K Tokunaga, T Hashimoto. J Chromatogr
404:33, 1987.
© 2004 by Marcel Dekker, Inc.