PubMed · 3841185
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L C Davis, T J Socolofsky, G A Radke. 1985. Computer-controlled scanning gel chromatography.. https://doi.org/10.1016/s0076-6879(85)17011-4
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Rigid macroporous copolymers of styrene and divinylbenzene have been designed for large-scale preparative and process-scale purification of synthetic peptides and recombinant proteins. The polymeric particles are mechanically stable and hence able to operate at the required high linear velocities. The pore size and pore morphology has been optimised to enable unhindered solute diffusion whilst providing maximum available surface area to enhance loading capacity. A 100 A pore size has been developed for synthetic peptides and a 300 A pore size for recombinant proteins. Precise control of particle size, within the range 10 to 20 microm, is possible which together with the very narrow particle size distribution enables maximum resolution/loading to be obtained within the pressure limits of the instrumentation being used. The chemical stability of the polymer enables cleaning in place with 1 M sodium hydroxide without particle dissolution or a deterioration in selectivity. These materials can be packed into compression hardware and are manufactured as single lots up to 100 kg (300 l) batch size.
This work describes the hydrophobic interaction chromatography of homo-deoxyoligonucleotides polyA, polyT and polyU with sizes up to 30 bases on a Sepharose gel derivatized with the 1,4-butanediol diglycidyl ether. The oligonucleotides interacted differently with the column according to the molecular mass, the hydrophobic character of the individual bases, the secondary structure of the molecule and the concentration of ammonium sulphate in the eluent. The retention factor, k', was determined from the chromatographic profiles at different concentrations of ammonium sulphate. A linear relationship between log k' and the concentration of ammonium sulphate in the eluent was found for all oligonucleotides at the higher concentrations (> 1.0 M) of ammonium sulphate. The slope of these plots, termed the hydrophobic interaction parameter, was found to be an increasing function of the number of nucleotides. The same plots reveal that polyA molecules with high molecular mass have lower retention factors when compared with polyT, an observation that was not expected since the hydrophobicity of adenine is higher than that of thymine. This behaviour was due to the existence of secondary structures in polyA, which decrease the exposed hydrophobic area of the molecule.
In a previous study, we reported a novel method for the separation and quantification of a strong negatively charged material, dextran sulfate sodium (DSS), using fluorometric labeling with 2-aminopyridine and size-exclusion high-performance liquid chromatography. In the present study, we developed a method for the separation of pyridylamino-DSS (PA-DSS) using reversed-phase high-performance liquid chromatography (RPLC). In vitro enzymatic degradation of the PA-DSS was carried out using alpha-amylase. In RPLC, depolymerized PA-DSS was eluted on the basis of molecular mass (in the order pentamer, trimer, dimer, and monomer of PA-DSS) and separations were more sharply than in size-exclusion chromatography. The combination of RPLC and size-exclusion chromatography also separated depolymerized PA-DSS as effectively as RPLC alone.