, irrespective of the type of support material, the amount of stationary phase in an LC column is primarily determined by its surface area. In addition, the amount of available stationary phase on a bonded phase can be modified by adjusting the molecular size (chain length) of the bonded material. The chain length of the bonded material can be critical when separating proteins as dispersive interactions between the alkane chains and the dispersive (hydrophobic) groups of the protein can be strong enough to cause structural deconformation; (i.e., the protein becomes denatured). Reducing the chain length of the bonded material, the dispersive forces can be reduced significantly and the deconformation diminished. In practice, carbon chains only two or four carbon atoms long are among those most commonly used for separating labile proteins. Stationary Phase Limitation by Chiral Selectivity. The extent to which an enantiomer can interact with the

Bayer and his coworkers (19), synthesized a stationary phase that consisted of a chiral agent attached by an amide linkage to a carboxyl group of a polymer matrix of dimethylsiloxane or (2-carboxypropyl)-methylsiloxane. This combined the chiral selectivity of L-valine-t-butylamide with the high thermal stability and low volatility of the polysiloxanes. This stationary phase could be used over the temperature range of 30˚C to 230˚C. and would allow the separation of all the racemic protein amino acids to be separated in one chromatogram taking only about 30 min. An example of the separation of the N-(O,S)-pentafluoro-propanoyl-isopropylesters of the amino acids, employing this phase system, is shown in figure 32. It is seen that the column bleed is quite manageable at 185˚C and that a clean separation of all the enantiomers is achieved. Chiral polysiloxanes can also be prepared in a number of ways. For example, the cyano groups of OV-225 can be  hydrolyzed and the

to provide adequate peak capacity (see Plate Theory and Extensions). The particle diameter of the packing was 6 mm and thus, at the optimum velocity, an efficiency of about 25000 theoretical plates should be produced. It is seen in figure 61 that the dead volume time is about 19 minutes and a flow-rate of 1.0 ml/min this would be equivalent to a dead volume of 19.5 ml. A more typical application of micro-reticulate resins in exclusion chromatography is shown by the separation of a standard protein mixture depicted in figure 62.   Courtesy of Asahipak Inc. Figure 62. The Separation of a Standard Protein Mixture by Exclusion  Chromatography on a Vinyl Alcohol-Styrene Co-Polymer Hard Gel

Due to their being stable to extremes of pH,  their use for the separation of peptide and proteins at both high and low pH has been well established. An example of a macro-reticulated resin phase used as an exclusion medium in the separation of a crude protein extract is shown in figure 39. The column, 9TSKgel QC-PAK GFC 399GL0, was 15 cm long and 8 mm in diameter and the separation was carried out at a flow rate of 1 ml/min. The mobile phase consisted of 0.5M NaCl in 0.05M sodium phosphate buffer at pH 7.0.  The sample was 5ml of a rat liver extract. An excellent separation based on molecular size is obtained from which the molecular weight range of the mixture and even that of the individual components could be estimated. LC Mobile

with almost any desired pore size, ranging from 20Å to 5,000Å. Underivatized, they exhibit strong dispersive type interactions with solvents and solutes together with some induced polarizability arising from the aromatic nuclei in the polymer if strongly polar solutes are being separated. Consequently, the untreated resin has found some use as an alternative to the C8 and C18 reverse phase columns based on silica.  Courtesy of TOSOHAAS Inc. Figure 39. The Separation of a Crude Protein Extract by Exclusion on a Micro-Reticulated Resin Column

gradient will compensate for the strongly concave form of the unassociated methanol concentration profile shown in figure 47 which will be the strongest eluting component of the mobile phase. The strong association of methanol with water could also account for the fact that proteins can tolerate a significant amount of methanol in the mobile phase before they become denatured. It is clear that this is because there is virtually no unassociated methanol present in the mixture which could cause protein denaturation since all the methanol is in a deactivated state by association with water. Katz, Lochmüller and Scott also examined acetonitrile/water, and tetrahydrofuran(THF)/water mixtures in the same way and showed that there was significant association between the water and both solvents but not to the same extent as methanol/water. At the point of maximum association for methanol, the solvent mixture contained nearly sixty percent of the methanol/water associate. In contrast