Linda L. Lloyd

Carbohydrate polymers as wound management aids

Wound management aids have, in recent years, seen a transition from being simple natural coverings which give a wound protection from the external environment during the formation of the scab to specialized high technology materials which are produced from both synthetic and natural polymers. Much of the development has resulted from a greater understanding of the processes involved in wound healing coupled with advances in technology to produce biocompatible materials with the necessary physical and chemical characteristics for enhancement of the healing process. Polysaccharides, being naturally occurring biomolecules, were an obvious choice for investigation as potential wound management aids. In recent years it was recognised that not only can polysaccharides be produced with the required physical characteristics for a wound management product but that the actual polysaccharide or polysaccharide derivative may itself actively participate in the process of wound healing. This paper sets out to review the various types of polysaccharides which were used as wound management aids, the physical forms in which they are used and also the biological properties of polysaccharides which enable them to participate actively in the wound healing process.

Rigid macroporous copolymers as stationary phases in high-performance liquid chromatography

Abstract Rigid macroporous copolymers of styrene and divinylbenzene have been developed which have the high physical/mechanical stability necessary to operate under high-performance liquid chromatographic conditions of pressure and flow-rate. The matrices can be produced in a range of porosities, 100, 300, 1000 and 4000 A each with the controlled uniform pore geometry and pore size distribution essential for high efficiency separations. The small pore—high surface area macroporous poly(styrene—divinylbenzene) copolymer, PLRP-S 100 A, can be used in the unmodified form for reversed-phase separations of small molecules or by increasing the pore size, PLRP-S 300 A or greater, for the analysis of biological macromolecules. The surface characteristics of the hydrophobic poly(styrene-divinylbenzene) matrix can be altered by derivatization or coating to produce ion exchange functionalities or a hydrophilic surface for gel filtration chromatography. The hydrophilic surface can then be further derivatized to be utilised as a base matrix for affinity chromatography.

Preparative high-performance liquid chromatography on a unique high-speed macroporous resin

Abstract The use of high-performance liquid chromatography (HPLC) for the analysis and purification of biological macromolecules has increased with the commercial availability of packing materials spefically designed for large-molecule analysis. However, the speed of analysis is limited as the efficiency of the HPLC columns for macromolecules has been significantly lower that that acheived for small molecule separations. One possible solution to the speed/capacity/resolution triangle experienced with large biological molecules is to increase the pore size of the HPLC matrix. A rigid macroporous polymer with a 4000 A pore size has, therefore, been developed for evaluation as a high performance matrix for macromolecule HPLC separations. The mechanical stability of this polymer enables the material to be packed in conventional columns, 4.6 mm I.D., and operated under HPLC conditions of flow rate and pressure. The high speed, fast flow matrices evaluated are for reversed-phase HPLC, PLRP-S 4000 A, and for anion-exchange HPLC, PL-SAX 4000 A. The matrices/columns have been assessed for stability, permeability, efficiency, selectivity and capacity with macromolecules and their suitability for analytical and preparative applications evaluated.

l-Ascorbic acid stability in aseptically processed orange juice in TetraBrik cartons and the effect of oxygen

Abstract The rate of degradation of l -ascorbic acid in commercial single-strength orange juice aseptically processed in TetraBrik cartons was evaluated at different storage temperatures. In this type of sample, the level of dissolved oxygen present in the sample after packaging significantly affected the l -ascorbic acid content, the effect being directly related to temperature. Likewise, the rate of consumption of dissolved oxygen is directly dependent on the concentration of l -ascorbic acid. Both aerobic and anaerobic degradation of l -ascorbic acid occurs in the same system. The aerobic process predominates and the anaerobic process takes place when the level of dissolved oxygen has reached equilibrium.

A cellulosic exopolysaccharide produced from sugarcane molasses by a Zoogloea sp.

Abstract An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri- O -methylhexitol (74.7%) and 2,3,-di- O -methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β- d -glucopyranosyl residues.

The identification and quantitation of the hydroxycinnamic acid substituents of a polysaccharide extracted from maize bran

The composition and carbohydrate structure of hemicelluloses namely branan ferulate isolated from maize bran has been determined. This novel polysaccharide material is the base polysaccharide of Sterigel®, a Seton Scholl preparation which is being used successfully as a wound management aid. The structure of the polysaccharide was determined by methylation followed by GC-MS analysis, and 1 H- and 13 C-NMR spectroscopic analysis. It was shown to be composed of a (1 → 4)-β-D-xylan backbone substituted with a variety of side-chain components, such as single α-D-glucuronic acid residues (via an α-D-G1cA-(1 → 2)-linkage to the xylan backbone), single α-L-arabinose residues (via α-L-Araf-(1 → 2)- and/or α-L-Araf-(1 → 3)-linkages to the xylan backbone) and longer chains of 2-3 monosaccharide residues containing arabinose, xylose and galactose (in the form α-D-Galp-(1 → 4)-β-D-Xylp-(1 → 2))-α-L-Araf, linked to the xylan backbone via α-L-Araf-(1 → 2)- and/or α-L-Araf-(1 → 3)-linkages). In addition to the carbohydrate, non-carbohydrate moieties such as hydroxycinnamic acids and some proteins, detected using scanning UV spectroscopy (190-450 nm), are present in the branan ferulate. The hydroxycinnamic acids appear to be bound to the polysaccharide chains via ester linkages between their carboxylic acid groups and the C-5 hydroxyl group of a proportion of the α-L-Araf residues, the location of which are detailed above. Predominantly trans-ferulic acid (0.38% w/w), and small amounts of cis-ferulic acid (0.06% w/w) and trans-p-coumaric acid (0.02% w/w) were extracted with ethyl acetate after hydrolysis using 2M NaOH and identified using GC and GC-MS. Hydrolysis with an aryl ester hydrolase (EC 3.1.1.2) was carried out to confirm the presence of the ferulate ester linkages. HPLC results confirmed that the ferulic acid is released by the enzyme (0.25% w/w). GPC analysis using UV (280 nm) and RI detection confirmed that the ferulic acid was bound to the polysaccharide chain since the distributions directly overlapped, indicating the presence of little or no free ferulic acids. Indeed, free ferulic acid contents of <0.02% w/w were determined in extracts of solid branan ferulate and an aqueous solution of branan ferulate, by HPLC.

Quantitative analysis of vitamin C (l-ascorbic acid) by ion suppression reversed phase chromatography

Abstract Ion suppression reversed phase chromatography of vitamin C ( l -ascorbic acid) is described using a pH-stable, polymeric adsorbent (PLRP-S), a low pH sodium dihydrogen phosphate buffer as the mobile phase, and spectrophotometric detection. The optimization of the method was performed with reference to the separation of l -ascorbic acid and d -erythorbic acid ( d -isoascorbic acid). The quantitative assay is demonstrated by the determination of vitamin C in fruit juice.

Reversed-phase poly(styrene-divinylbenzene) materials optimised for large scale preparative and process purification of synthetic peptides and recombinant proteins.

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.

Determination of the molecular weight distribution of hydroxyethylcellulose by gel filtration chromatography

Abstract Due to the importance of the knowledge of the molecular weight distribution of hydroxyethylcellulose, (HEC), in predicting its performance, a study on the use of gel filtration chromatography for the determination of this information has been carried out. Results presented demonstrate that it is possible to use gel filtration columns with 100% aqueous eluents for the size separation and gel filtration analysis of a representative selection of water-soluble HEC samples. Data obtained correlate with the viscosity grading supplied with the samples by the manufacturer.

Rigid polymerics: the future of oligonucleotide analysis and purification

A family of rigid macroporous HPLC materials, reversed phase and anion exchange, has been evaluated for the analysis and purification of a range of de-protected, dimethoxytrityl-off, oligonucleotides. A 25-base pair (bp) double-stranded DNA ladder was used to determine the resolving range for the four pore sizes of reversed-phase media. The 100 A pore size resolves up to 50-75 bp, the 300 A up to 250-300 bp, the 1000 A up to 400-450 bp and the 4000 A pore size is capable of resolving in excess of 500 bp. The dynamic capacity of these four pore sizes was also determined using a synthetic oligonucleotide with two ion-pairing agents at ambient and 60 degrees C. The dynamic capacity was shown to decrease with increasing pore size and that with the triethylammonium acetate ion-pairing agent there was negligible temperature dependency. The dynamic capacity was higher when tetrabutylammonium bromide was used at elevated temperature. A strong anion-exchange functionality on a pH-stable polymeric particle was used to investigate the selectivity and resolution of the technique. Using a poly-T-oligonucleotide size standard, resolution of full length oligonucleotide (n) from the truncated species due to coupling failure (n-1, n-2, etc.) was demonstrated up to at least the 30mer. Resolution of a phospho diester contaminant from a phospho thioate oligonucleotide and a truncated sequence was demonstrated using anion-exchange HPLC at high pH.