Chromatography of proteins on hydrophobic interaction and ion-exchange chromatographic matrices: mobile phase contributions to selectivity.

The effect of mobile phase pH on the retention characteristics of eleven proteins was examined in hydrophobic interaction chromatography (HIC) on a SynChropak propyl stationary phase. Selectivity was shown to change with eluent pH. The effect of the displacing salt on the separation of proteins on a weakly hydrophobic weak-anion-exchange chromatography (AEC) packing was examined. Some differences in selectivity were observed when sodium sulfate was used as the displacing salt, compared to that observed with sodium chloride in the eluent. It was demonstrated that these AEC packings exhibited both electrostatic and hydrophobic properties, depending upon the type and concentration of salt used in the mobile phase. The addition of 20% ethylene glycol to the mobile phase was shown to reduce the hydrophobic interactions. The application of weakly hydrophobic weak-cation-exchange packings to HIC of proteins was demonstrated. Elution of such columns with descending sodium sulfate gradients was found to provide a selectivity different from that observed with a propyl stationary phase. Manipulation of mobile phases was shown to provide useful selectivity as a result of the combination of electrostatic and hydrophobic contributions to the separation process.

Influence of pore and particle size on the frontal uptake of proteins. Implications for preparative anion-exchange chromatography.

Several silica-based anion-exchange packings were synthesized with nominal pore sizes of 250, 500 and 1000 A in 10-, 20- and 50-micron particles. The static ("equilibrium") adsorption capacities for bovene serum albumin (mol. wt. 69,000), alpha-lactalbumin (17,500) and ferritin (440,000) were first measured using bulk material. The media were then packed into columns for frontal uptake experiments to measure adsorption from a flowing mobile phase. In general, frontal uptake was inversely related to both flow-rate and particle size. However, the magnitude of these relationships was strongly dependent on the pore to protein diameter ratio. More specifically, the uptake of bovine serum albumin was significantly more sensitive to linear velocity and particle size than alpha-lactalbumin. A mathematical model of the chromatographic process was used to calculate radial adsorption profiles across the chromatographic particle during frontal uptake. It was shown that restricted intraparticle diffusion due to insufficient pore size causes incomplete utilization of internal surface area. Under such conditions, protein is only bound within a finite shell on the outermost side of the particle; therefore, the effective loadability of the packing is greatly reduced. These data suggest that a pore size of at least 500 A will be required for the preparative chromatography of proteins with a molecular weight higher than ca. 100,000. This observation is especially evident when using particle sizes greater than 10 micron.

Factors contributing to intrinsic loading capacity in silica-based packing materials for preparative anion-exchange protein chromatography.

Properties of the matrix and stationary phase which affect the intrinsic loading capacity of silica-based packing materials for preparative anion-exchange chromatography of proteins were investigated. Polyethyleneimine-coated controlled porosity glass beads ranging from 100 to 2000 A in pore diameter were used to evaluate the effects of pore diameter and surface area. Protein binding was found to depend on accessible, rather than total, support surface area. Consequently, wide-pore, high surface area media provide maximum intrinsic loading capacity. Increasing the number of positively charged sites on the stationary phase by increased coating or by quaternization of amines increases hemoglobin-binding capacity.

Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode.

Several high-performance stationary phases suitable for protein chromatography were synthesized. Columns packed with these materials could be operated independently in either the anion-exchange or hydrophobic-interaction mode. Two approaches were used to prepare these materials. In the first method, a polyamine was adsorbed on the surface of macroporous silica and then crosslinked with a multifunctional oxirane. The hydrophobicity of the crosslinking agent and the extent of interconnection were used to modulate the electrostatic and solvophobic interactions. The second approach also utilized a crosslinked polyamine stationary phase; however, the forces of interaction were attenuated through controlled acylation of surface amines with a small anhydride molecule. The resolving ability of these columns, functioning in either mode, was comparable to commercial high-performance liquid chromatographic columns, designed to operate by a single retention mechanism. Column selectivity for proteins was completely different in each mode. Protein fractions collected from a multimodal column, operated in the anion-exchange mode, could be further purified by rechromatographing them on the same column in the hydrophobic-interaction mode. Utility of the multimodal column was demonstrated with the fractionation of several cytochromes and ferredoxins from the cyanobacterium Microcystis aeruginosa.

Synthesis of cation-exchange stationary phases using an adsorbed polymeric coating.

We have prepared several silica-based cation-exchange materials that were suitable for the high-performance liquid chromatography of basic proteins. Two synthetic routes were examined. Central to both procedures was the adsorption of a low molecular weight polyamine. One method crosslinks the adsorbed polyamine with a multifunctional oxirane, which is then extensively derivatized with a monomeric cyclic anhydride. The second involves an adsorbed uncrosslinked polyethyleneimine layer which is reacted with polyacrylic anhydride, thereby crosslinking and imparting anionic character simultaneously. The resulting media prepared by either of these methods bound more than 40 mg of hemoglobin per gram of support depending on the reaction conditions. These cation-exchange stationary phases also exhibited good chromatographic performance, successfully resolving (horse heart) cytochrome c and lysozyme. Two of the more promising support materials were effectively used to isolate cytochrome c553 from a crude extract of cyanobacteria.

Synthesis of an adsorbed reversed-phase packing material for the separation of proteins and peptides.

We have described the preparation and chromatographic evaluation of an adsorbed hydrophobic stationary phase suitable for reversed-phase chromatography of proteins and peptides. The synthetic procedure involves three steps: the adsorption of a polyamine to the silica surface; crosslinking of the adsorbed polyamine layer with a bis-phenyl difunctional epoxide; and the benzoylation of the remaining accessible amino groups. Performance of this chromatographic material compared favorably with SynChropak RP-8 silica (SynChrom, Linden, IN, U.S.A.) and was stable to 40% formic acid. Good separations were obtained between the components of sample mixtures containing proteins or the cyanogen bromide fragments of sperm whale myoglobin. However, in both cases, the adsorbed hydrophobic stationary phase was less retentive. Furthermore, this medium exhibited slightly different selectivity. Whereas the heme which was present in the cyanogen bromide digest of myoglobin desorbed as the second peak from the RP-8 column, it eluted last from the adsorbed stationary phase. Comparable performance, acid stability and alternate selectivity suggest that this material is an interesting alternative to organosilane reversed-phase coatings.

Stationary phase contributions to retention in high-performance anion-exchange protein chromatography: ligand density and mixed mode effects.

Several anion-exchange stationary phases (based on polyethyleneimine-coated silica) were synthesized so as to vary in ligand density and hydrophobicity. These materials were first examined for hemoglobin-binding capacity and then evaluated chromatographically. Protein binding, retention and resolution increased concomitantly with ligand density. Ferritin (molecular weight 440,000) could not be eluted from the more highly-charged surfaces, but was desorbed from a low ligand density support. The above parameters also varied with the hydrophobic character of the stationary phase. Retention and resolution increased as more hydrophobic moieties were added. Data from a non-ionic hemoglobin-binding assay correlated reasonably well with anticipated matrix hydrophobicities. Possible explanations and applications of the observed phenomena are discussed.

Mobile phase selection for the high-performance ion-exchange chromatography of proteins.

Proper mobile phase selection significantly improved high-performance ion-exchange fractionations of proteins. The pH and salt content of the eluant affected chromatographic behavior on both strong and weak ion-exchange columns. The retention and resolution of a number of proteins was examined on strong and weak ion-exchange supports with regard to these mobile phase variables. The strong ion-exchange columns were found to be superior for the protein separations performed in this study. The selectivity of both weak and strong ion-exchange columns was pH dependent, however, strong ion-exchange columns were operable over a broader range of pH. Examination of the effect of salts demonstrated that their "displacing activity" could be divided into three categories: weak, intermediate, and strong with regard to the protein mixture utilized. Intermediate salts provided best resolution along with good recoveries. Strong displacing salts were useful for eluting strongly retained proteins. The selection of a mobile phase with respect to protein retention is discussed.

A system for coupled multiple-column separation of proteins.

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.