Chromatographic Behavior of Proteins

In reality, many proteins demonstrate mixed mode interactions (e.g., additional hydrophobic or silanol interactions) with a column, or multiple structural conformations that differentially interact with the sorbent. These nonideal interactions may distribute a component over multiple gradient steps, or over a wide elution range with a linear gradient. These behaviors may be mitigated by the addition of mobile phase modifiers (e.g., organic solvent, surfactants, and denaturants), and optimization (temperature, salt, pH, sample load) of separation conditions. 

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The chromatographic behavior of proteins has become an important adjunct in recent years to the usual physical and chemical criteria used to evaluate the purity of a protein. The sensitivity of this technique is demonstrated by the studies of Palpus and Neilands (131) and of Margoliash (111) with cytochrome c, of Hirs, Moore, and Stein (82) with ribonuclease, of Porter et al. (6,132,133) with... 

Although the mechanism of SEC is in principle nonadsorptive, effects derived from different matrices can be observed. As a result, quite different chromatographic behaviors of one particular protein can be obtained depending on the type of sorbent used. 

A review on TLC and PLC of amino adds, peptides, and proteins is presented in the works by Bhushan [24,25]. Chromatographic behavior of 24 amino acids on silica gel layers impregnated tiraryl phosphate and tri-n-butylamine in a two-component mobile phase (propanol water) of varying ratios has been studied by Sharma and coworkers [26], The effect of impregnation, mobile phase composition, and the effect of solubility on hRf of amino acids were discussed. The mechanism of migration was explained in terms of adsorption on impregnated silica gel G and the polarity of the mobile phase used. 

Wu, S. -L., Benedek, K., and Karger, B. L., Thermal behavior of proteins in high-performance hydro-phobic-interaction chromatography. On-line spectroscopic and chromatographic characterization, /. Chromatogr., 359, 3, 1986. 

Proper protein folding is a concern in the production of a bioactive protein therapeutic, and it is of interest to verify that the conformation of a recombinant protein is the same as the wild-type molecule. Because retention in RPLC depends on the surface hydrophobic contact area, comparable chromatographic behavior of a recombinant protein with that of the wild-type molecule provides evidence of similar 3-D structure.17 38... 

Affinity complexation — Many proteins have affinities for other molecules that can be exploited to alter their retention characteristics in IEC. For example, some enzymes may be combined with synthetic substrates, cofactors, or products.1315 The same principle can be applied to other protein/receptor systems. One well-characterized example is the change in chromatographic behavior of fructose 1,6-diphosphatase in the presence of its negatively charged substrate... 

This influence of the valence and activity coefficients of the displacer salt on the retention behavior of polypeptides and proteins can be anticipated from theoretical treatments of the ion-exchange chromatographic separation of proteins. According to the nonmechanistic stoichiometric model of protein retention behavior in HP-IEX80,82-85 the influence of a divalent cation salt such as CaCl2 on the retention behavior of a protein in HP-IEC can be evaluated in terms of the following relationships ... 

Columns packed with silica-bound proteins have been devised bovine serum albumin-derived columns ( J.) are available from Macherey Nagel. These CSPs show a fairly extensive scope of action, although the nature of the chiral recognition processes employed is still vague. Owing to the low concentration of active sites, these columns require quite small samples (0.5 - 5 nmol). Mobile phase variation is possible (over a limited range) and alters the chromatographic behavior of the enantiomers markedly. 

The chromatographic behavior of low MW salts could be expected to be less complex than that of synthetic polyelectrolytes with variable macromolecular dimensions, and also simpler than that of proteins, for which hydrophobic interactions need to be considered. For this reason, and also on historical grounds, we will first review the literature on the elution of salts. Next we will consider the behavior of synthetic polyelectrolytes, treating polycations separately, because these compounds pose special difficulties. The role of electrostatic effects in the bEC of proteins will be the third topic. Lastly, we discuss models put forward to semi-quantitatively treat electrostatic effects. 

Fig. 2. Chromatographic behavior of apoHBD-phospholipid complexes on Sephadex G-100. (a) MPL (b) mixed micelles (c) egg lecithin. L, phospholipid P, protein...

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