Handbook of Size Exclusion Chromatography and Related ...

Acetonitrile–trifluoroacetic acid eluants are attractive for reducing hydrophobic
interactions and because of the volatile nature and ultraviolet (UV) transparency of
this mobile phase. Protein resolution is dependent upon the acetonitrile
concentration and requires the low pH trifluoroacetic acid provides. However, a
severe limitation is the low solubility of proteins larger than 15,000 dalton in the
30–45% acetonitrile needed for optimum resolution. This low solubility leads to
severe protein aggregation and limits the use of this mobile phase to peptides and
low molecular weight proteins.
Detergents may be utilized to stop protein hydrophobic interactions with
silica matrices. Some detergents are mild and allow nondenaturing conditions (for
example, sodium deoxycholate, Triton, and Nonidet P40). Deoxycholate is the
most versatile detergent, with little absorbance at 280 nm. Triton and Nonidet P40
both exhibit strong absorbance in the UV range. The detergent binds to the
hydrophobic portion of the protein without forming large micelle structures (this is
controlled with the critical micelle concentration, CMC, of each detergent). Triton
and Nonidet form large micelles that decrease resolution. Typically, deoxycholate
can be used at 0.1%, pH 7.6–8.0, without forming large micelles (115).
Detergents, such as SDS, may cause multisubunit proteins to divide into individual
subunits, may change the protein quaternary structure from globular to elongated,
or, through adsorption, may increase the size of the protein. SDS is always used at
its CMC, and the amount of SDS bound is sensitive to the buffer concentration
within the range 0.1–0.4 M (123).
The use of denaturing mobile phases is particularly helpful in the analysis of
the composition of oligomeric structures (that is, cell organelles, viruses, and
multimeric enzymes), because they disrupt most noncovalent protein–protein
interactions. Most common denaturing conditions utilize 0.1% SDS or 6 M
guanidine hydrochloride. As mentioned earlier, denaturing conditions may be
advantagcous for molecular weight determination and lead to in increase in
resolution. The use of SDS provides much better resolution than phosphate–
guanidine hydrochloride systems because of the extended and uniform
conformations of proteins. Takagi et al. (123) and Konishi (124) report the effect
of salt concentration (phosphate) on complexes of SDS and polypeptides. Takagi
found good resolution within the phosphate concentration range 0.05–0.15 M,
although, in general, retention is a strong function of buffer concentration in SDS
systems. This effect can only partially be explained by the change in the effective
size of the complexes as a result of their polyelectrolyte-like nature. Ion exclusion
appears to be at play for the lower concentrations. The complexes were totally
excluded at lower buffer concentrations, repelled by the negative charges on residual
and accessible silanol groups. Konishi found a linear relationship between log MW
and KD for polypeptides ranging from 1000 to about 80,000 dalton when eluted in a
0.20 M phosphate buffer in the presence of 0.1% SDS (124). At lower phosphate
concentrations, the calibration curves were steep, but linear, up to 15,000 dalton and
© 2004 by Marcel Dekker, Inc.