Continous annular chromatography for the separation of carbohydrate mixtures

The continuous separation of beet molasses resulting in a sucrose rich product and a non-sugar waste product was carried out using a rotating annular chromatograph. The annulus was 12 mm wide and 1.4 m long and was packed with a sodium charged 5.5% cross-linked polystyrene ion exchange resin. Separation was achieved by the simultaneous mechanisms of ion exclusion, size exclusion and partition chromatography. The entire packed bed was slowly rotated while beet molasses was fed continuously through a stationary feed nozzle to the top of the bed. Each molasses constituent having a different relative affinity for the packing and the deionised water mobile phase describes a characteristic helical path as it progresses from the stationary feed point to the bottom of the rotating bed. Each solute then elutes from the annulus at a different angular distance from the feed and separation of the multicomponent mixture is thereby achieved. When a 35% w/w sucrose beet molasses feed was used the throughput achievable was 45.1 kg sucrose m~3 resin h"1. In addition to beet molasses separation other carbohydrate mixtures were separated. In particular the separation of glucose and fructose by Ligand exchange chromatography on a calcium charged ion exchange bed was carried out. The effects of flowrates, concentration, rotation rate, temperature and particle size on resolution and dilution of constituents in the mixtures to be separated were studied. A small test rig was designed and built to determine the cause of liquid maldistribution around the annulus. The problem was caused by the porous bed support media becoming clogged with fines being introduced by eluent flows and off the resin. An outer ring was constructed to house the bed support which could be quickly replaced with the onset of maldistribution. The computer simulation of the operation of the rotating annular chromatograph has been carried out successfully.

Radioimmunoassay of insulin and glucagon with studies on the obese hyperglycaemic syndrome in mice

Sensitive and precise radioimmunoassays for insulin and glucagon have been established. Although it was possible to employ similar precepts to the development of both hormone assays, the establishment of a reliable glucagon radioimmunoassay was complicated by the poor immunogenicity and instability of the peptide. Thus, unlike insulin antisera which were prepared by monthly injection of guinea pigs with crystalline insulin emulsified in adjuvant, the successful production of glucagon antisera was accomplished by immunisation of rabbits and guinea pigs with glucagon covalently linked to bovine plasma albumin. The conventional chloramine-T iodination with purification by gel chromatography was only suitable for the production of labelled insulin. Quality tracer for use in the glucagon radioimmunoassay was prepared by trace iodination, with subsequent purification of monoiodinated glucagon by anion exchange chromatography. Separation of free and antibody bound moieties by coated charcoal was applicable to both hormone assays, and a computerised data processing system, relying on logit-log transformation, was used to analyse all assay results. The assays were employed to evaluate the regulation of endocrine pancreatic function and the role of insulin and glucagon in the pathogenesis of the obese hyperglycaemic syndrome in mice. In the homozygous (ob/ob) condition, mice of the Birmingham strain were characterised by numerous abnormalities of glucose homeostasis, several of which were detected in heterozygous (ob/+) mice. Obese mice exhibited pancreatic alpha cell dysfunction and hyperglucagonaemia. Investigation of this defect revealed a marked insensitivity of an insulin dependent glucose sensing mechanism that inhibited glucagon secretion. Although circulating glucagon was of minor importance in the maintenance of hyperinsulinaemia, lack of suppression of alpha cell function by glucose and insulin contributed significantly to both the insulin insensitivity and the hyperglycaemia of obese mice.

Efficient purification of chromatin architectural proteins:histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells. This journal is © 2012 The Royal Society of Chemistry.