Karen M. Gooding

High-performance liquid chromatography of proteins.

Proteins were separated on microparticulate bonded phase steric exclusion and anion-exchange chromatography supports. A post-column enzyme detector was developed which gives a specific and sensitive response for enzymes. The three iso-enzymes of creatine phosphokinase were separated and assayed in 4 min and the five isoenzymes of lactic dehydrogenase in 6 min.

Use of oxiranes in the preparation of bonded phase supports

Summary A variety of stationary phases were bonded to silaceous supports through an intermediate silane coupling agent, γ-glycidoxypropylsilane. Consequently, a series of ion-exchange, hydrophobic, and hydrophilic supports were prepared that are stable, withstand high mobile phase velocities, and have sufficient porosity to allow the partitioning of biopolymers. Liquid-liquid partition chromatography of proteins was achieved with phosphate buffers while the separation of a series of aromatic compounds was accomplished on the same columns with hexane. Anion- and cation-exchange supports were found to have hemoglobin ion- exchange capacities similar to classical cross-linked dextrans while allowing separation speeds 10–20 times those of carbohydrate gel supports.

Analysis of Proteins with new, mildly hydrophobic high-performance liquid chromatography packing materials

Abstract A series of packings that had alkyl and aryl groups incorporated into a hydrophilic polymeric matrix were developed for hydrophobic interaction chromatography. Using an inverse salt gradient of ammonium sulfate or sodium chloride, a series of proteins were purified and the activity of trypsin was monitored using post-column detection. Retention times of proteins generally increased in the following order of ligands: hydroxypropyl, propyl, benzyl, isopropyl, phenyl and pentyl.

Capillary hydrodynamic chromatography

Abstract Liquid chromatographic separation in capillary tubing of particles ranging from 0.5 to 30 μm is reported. Elution volumes are inversely related to particle diameter for materials of diverse composition such as latex, pollen, bacterial spores, silica, and whole cells. Relative elution times of particles are dependent on column diameter and both the velocity and viscosity of the mobile phase. Viscosity appears to affect the relative retention of small particles more than particles over 1 μm in diameter. Addition of ethyleen glycol to aqueous mobile phases diminishes peak trailing wiht latex particles. The practical limit on column length is approximately 300 ft.

A continuous-flow enzyme detector for liquid chromatography

A detection system has been developed for the selective and sensitive detection of enzymes eluting from a liquid chromatographic column. This system monitors a reaction that the enzyme catalyzes and provides a chemical amplification ranging from 10(4) to 10(5). The detection system consists of a reagent or substrate pump, a post-column reactor packed with non-porous spherical glass beads, and a photometric detector. A linear and selective response to a series of enzymes of clinical importance is demonstrated.

Ion selectivity in the high-performance cation-exchange chromatography of proteins

Abstract The effects of varying the ionic composition of some aqueous mobile phases were observed during the analysis of five proteins by gradient elution on a weak cation-exchange column, SynChropak CM 300. Changes in both selectivity and band broadening occurred when either cations or anions were varied, contrary to similar studies for small molecules. Cations generally follow the same order for retentive properties for proteins as they do for small molecules. However, anions affect the retention of proteins in an inverse order from the way they affect small molecules on anion-exchange.

Effect of mobile phase and ligand arm on protein retention in hydrophobic interaction chromatography

Abstract The retentive properties of a series of hydrophobic interactin chromatography packings with six different lignd arms (SynChropak Hydroxypropyl, Methyl, Propyl, Butyl, Pentyl, and Benzyl) were investigated with mobile phases of different ionic compositions and pH. Substitution of ammonium acetate for ammonium sulfate resulted in decreased retention for most combinations of proteins and ligands, although the retention of some proteins, such as lysozyme on the pentyl ligand, was unchanged by the salt substitution. Generally, lower pH resulted in reduced retention, but the elution of lysozyme was more affected by pH than that of ovalbumin.

Optimization of preparative hydrophobic interaction chromatographic purification methods

The chromatographic behavior of five proteins on hydrophobic interaction matrices having six different ligand arms was investigated using gradient elution with ammonium sulfate and ammonium acetate buffers at two pH values. The nature of the mobile phase and/or the ligand chain arm of the matrix was found to have substantial effect on the resolution, retention, and selectivity. Ovalbumin was moderately or highly retained with ammonium sulfate on all columns; however, with ammonium acetate, ovalbumin was not retained on SynChropak Hydroxypropyl and Propyl columns. Chromatographic conditions developed for analytical hydrophobic interaction chromatography columns containing 6.5-micron packings were adapted to preparative columns packed with 30-micron SynChroprep packings for the separation of serum components. Dynamic load capacities were 4-13 mg of ovalbumin per ml of column volume.

Comparison of weak strong high-performance anion-exchange chromatography

Abstract A comparison of the chromatographic characteristics of weak anion exchangers with those of strong anion exchangers was made for a series of proteins. On both SynChropak AX300 Q300 the resolution of bovine serum albumin from its dimer was better at pH 6 than at pH 8. Conversely, catalase components separated better at pH 8 than at pH 6. A pH effect which may be due to hydrophobicity was observed for ovalbumin lactate dehydrogenase on the weak anion exchangers. A protein with a molecular weight of 140 000 shows equivalent separations on both 300-A 1000-A column materials, whereas smaller proteins are fractionated better on the 300-A columns.

Purification of trypsin and other basic proteins by high-performance cation-exchange chromatography

Abstract Conditions for the purification of basic proteins by high-performance cation-exchange chromatography were examined on SynChropak CM 300, a carboxymethyl column packing material. The pH and salt composition of the mobile phase were varied to determine the effect on the retention of trypsin, chymotrypsin, lysozyme and cytochrome c. In the course of these studies, the resolution of trypsin and chymotrypsin was optimized so that purification of either enzyme could be effected. Enzyme activity was monitored during the analyses. The capacities of an analytical and a semi-preparative column were determined for chymotrypsin.