Handbook of Size Exclusion Chromatography and Related ...

discussed in Table 8(33). Based on the data in Table 8, it was expected that the
TSKgel G3000SW and SynChropak GPC 100 columns would show similar
behavior, but larger KD values were expected for the remaining columns. Instead,
lysozyme shows similar retention on the TSKgel and the LiChrosorb columns and
much longer retention on SynChropak and Waters columns.
The importance of hydrophobic interactions as another secondary retention
mechanism is also illustrated in Table 8, in which the distribution coefficient for
phenylethanol is listed as a function of ionic strength for the same set of
commercial GFC columns (33). Indicative of hydrophobic interaction, KD values
increase with increasing ionic strength for this uncharged solute. Thus, a balance
must be stuck between the need to increase ionic strength to reduce ionic
interactions and to decrease ionic strength to limit hydrophobic interaction. In
practise, hydrophobic interaction is not a strong component of protein retention in
size exclusion chromatography because the hydrophobic side chains of the amino
acids are predominantly located in the interior of the protein. The addition of
5–20% of a nondenaturing solvent, such as ethylene glycol, to a high ionic strength
mobile phase was shown to eliminate the hydrophobic interaction of globular
proteins on a diol bonded phase column (98). In contrast to proteins, hydrophobic
interaction can be significant in SEC of peptides, some of which may require high
concentrations of organic solvents to obtain retention dominated by size exclusion
(101,102). Mant et al. (103) demonstrated the effectiveness of 0.1% trifluoroacetic
acid or addition of organic solvents to overcome hydrophobic interactions.
Additionally, the advantageous use of nonideal SEC behavior is detailed.
Kato and co-workers recommend the use of 0.05 M sodium phosphate buffer
(pH 7.0) containing 0.3 M NaCl to obtain true size exclusion behavior for most
proteins on 5-mm TSK-GEL SW XL columns (7). Not surprisingly, Mori and Kato
(104) recommend a very similar mobile phase, 0.1 M phosphate and 0.1 M NaCl at
pH 7.0, for size exclusion on diol bonded porpous glass columns. Okazaki and
Hara (105) recommend 0.15 M NaCl with lipoproteins, but various aqueous
buffers with salts are satisfactory as long as the pH is less than 8.5. Salt
contcentration, buffering, and pH all may alter the lipoprotein separation and
improve resolution. Increasing the buffering substance or salt concentration leads
to peak broadening, indicating a salting-out effect.
5 PRACTICAL CONSIDERATIONS
5.1 Extracolumn Effects
Since the advent of high-performance liquid chromatography, it has been
emphasized that the analyst be aware of the influence of the HPLC system
components on column efficiency. In a chromatographic system, the observed
column efficiency is caused not only by dispersion processes in the column.
© 2004 by Marcel Dekker, Inc.