ABSTRACT
In order to take full advantage of the sensitivity of modern protein characterization techniques, such as amino acid composition and sequence analysis, fairly pure samples of a single polypeptide are usually required. Although HPLC has been used extensively for isolation and purification of peptides and proteins at levels above one microgram, problems with sensitivity, resolution and recovery are often encountered when isolating nanogram (low picomole) levels of a polypeptide from a complex sample. This paper explores the requirements for nanopreparative purification of polypeptides by HPLC and details the design of a microbore HPLC system which allows reproducible, high sensitivity, high resolution separations of peptides and proteins at the low picomole level. The application of this microbore HPLC system to the isolation and characterization of a trace variant from a partially purified recombinant protein sample is also detailed.
Subject(s)
Chromatography, High Pressure Liquid/methods , Peptides/isolation & purification , Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Chromatography, High Pressure Liquid/instrumentation , Sensitivity and SpecificityABSTRACT
In the process of developing a new analytical technology (the chromatophoresis process) which couples reversed-phase high-performance liquid chromatography (HPLC) to sodium dodecyl sulfate polyacrylamide gel electrophoresis in a real-time automated system, it was apparent that improvements in resolving power for the first-dimension (HPLC) separation were necessary. The present paper describes the optimization of the column for our initial work on reversed-phase HPLC separations. Polymeric (polystyrene) packings having particle diameters of 5 micron and pore diameters of 300 A were generally superior in terms of resolution, sample recovery and minimization of "ghosting". Optimum column dimensions were 50 x 1.0 mm I.D. for the flow-rates required in our system (10-100 microliter/min).
Subject(s)
Chromatography, High Pressure Liquid/instrumentation , Proteins/isolation & purification , Indicators and Reagents , Molecular Weight , Particle Size , Silicon DioxideABSTRACT
The effects of separation variables such as temperature, pH and composition of the mobile phase (including additives such as chaotropes, ion-pairing agents and surfactants), sample size and sample pretreatment for reversed-phase high-performance liquid chromatography (RP-HPLC) of proteins is examined. Experimental optimization of these parameters using the preferred instrumental and column conditions described previously lead to well behaved chromatographic performance for most proteins. This allowed us to achieve the required level of performance for the first dimension (RP-HPLC) separation of most protein samples by the chromatophoresis process.
Subject(s)
Chromatography, High Pressure Liquid/instrumentation , Proteins/isolation & purification , Bacterial Proteins/analysis , Buffers , Electrophoresis, Polyacrylamide Gel , Escherichia coli/analysis , Hydrogen-Ion Concentration , Indicators and Reagents , Protein Conformation , Solvents , TemperatureABSTRACT
It has been shown that various amino acids will polymerize under plausible prebiotic conditions on mineral surfaces, such as clays and soluble salts, to form varying amounts of oligomers (n = 2-6). The investigations of these surface reactions required a quantitative method for the separation and detection of these amino acid oligomers at the picomole level in the presence of nanomole levels of the parent amino acid. In initial high-performance liquid chromatography (HPLC) studies using a classical postcolumn o-phthalaldehyde (OPA) derivatization ion-exchange HPLC procedure with fluorescence detection, problems encountered included lengthy analysis time, inadequate separation and large relative differences in sensitivity for the separated species, expressed as a variable fluorescent yield, which contributed to poor quantitation. We have compared a simple, automated, pre-column OPA derivatization and reversed-phase HPLC method with the classical post-column OPA derivatization and ion-exchange HPLC procedure. A comparison of UV and fluorescent detection of the amino acid oligomers is also presented. The conclusion reached is that the pre-column OPA derivatization, reversed-phase HPLC and UV detection produces enhanced separation, improved sensitivity and faster analysis than post-column OPA derivatization, ion-exchange HPLC and fluorescence detection.
Subject(s)
Amino Acids/analysis , Chromatography, High Pressure Liquid/methods , Glycine/analysis , Oligopeptides/chemistry , Chromatography, High Pressure Liquid/standards , Evaluation Studies as Topic , Fluorescence , Indicators and Reagents , Minerals/analysis , Time Factors , Ultraviolet Rays , o-PhthalaldehydeABSTRACT
Column chromatography of technical toxaphene on activated alumina reproducibly yields eight major fractions from which 2,2,5-endo,6-exo,8,9,10-heptachlorobornane and 2,5,6-exo,8,8,9,10-heptachlorodihydrocamphene may be readily isolated by preparative gas--liquid chromatogrpahy. These and some other reported toxaphene components were characterized by gas--liquid chromatographic retentions relative to aldrin on packed columns of OV-101, OV-17, and QF-1, and on a capillary open-tubular column of OV-101. Structural assignments were revised for some components on the basis of retentions. The fractionation and isolation procedure may have general utility for separating toxaphene components, and the retention data form a convenient basis for component comparison.
