Polymer fractionation by continuous chromatography

A review is given of general chromatographic theory, the factors affecting the performance of chromatographi c columns, and aspects of scale-up of the chromatographic process. The theory of gel permeation chromatography (g. p. c.) is received, and the results of an experimental study to optimize the performance of an analytical g.p.c. system are reported. The design and construction of a novel sequential continuous chromatographic refining unit (SCCR3), for continuous liquid-liquid chromatography applications, is described. Counter-current operation is simulated by sequencing a system of inlet and outlet port functions around a connected series of fixed, 5.1 cm internal diameter x 70 cm long, glass columns. The number of columns may be varied, and, during this research, a series of either twenty or ten columns was used. Operation of the unit for continuous fractionation of a dextran polymer (M. W. - 30,000) by g.p.c. is reported using 200-400 µm diameter porous silica beads (Spherosil XOB07S) as packing, and distilled water for the mobile phase. The effects of feed concentration, feed flow rate, and mobile and stationary phase flow rates have been investigated, by means of both product, and on-column, concentrations and molecular weight distributions. The ability to operate the unit successfully at on-column concentrations as high as 20% w/v dextran has been demonstrated, and removal of both high and low molecular weight ends of a polymer feed distribution, to produce products meeting commercial specifications, has been achieved. Equivalent throughputs have been as high as 2.8 tonnes per annum for ten columns, based on continuous operation for 8000 hours per annum. A concentration dependence of the equilibrium distribution coefficient, KD observed during continuous fractionation studies, is related to evidence in the literature and experimental results obtained on a small-scale batch column. Theoretical treatments of the counter-current chromatographic process are outlined, and a preliminary computer simulation of the SCCR3 unit t is presented.

Effect of the Hofmeister anions upon the swelling of a self-assembled pH-responsive hydrogel

We report the effect of a range of monovalent sodium salts on the molecular equilibrium swelling of a simple synthetic microphase separated poly(methyl methacrylate)-block-poly(2-(diethylamino)ethyl methacrylate)-block-poly(methyl methacrylate) (PMMA88-b-PDEA223-b-PMMA88) pH-responsive hydrogel. Sodium acetate, sodium chloride, sodium bromide, sodium iodide, sodium nitrate and sodium thiocyanate were selected for study at controlled ionic strength and pH; all salts are taken from the Hofmeister series (HS). The influence of the anions on the expansion of the hydrogel was found to follow the reverse order of the classical HS. The expansion ratio of the gel measured in solutions containing the simple sodium halide salts (NaCl, NaBr, and NaI) was found to be strongly related to parameters which describe the interaction of the ion with water; surface charge density, viscosity coefficient, and entropy of hydration. A global study which also included nonspherical ions (NaAce, NaNO3 and NaSCN) showed the strongest correlation with the viscosity coefficient. Our results are interpreted in terms of the Collins model,(1) where larger ions have more mobile water in the first hydration cage immediately surrounding the gel, therefore making them more adhesive to the surface of the stationary phase of the gel and ultimately reducing the level of expansion.

Bioreaction and separation in preparative batch chromatographic columns : the hydrology of lactose to yield glucose,galactose and oligosaccharides

The initial aim of this project was to improve the performance of a chromatographic bioreactor-separator (CBRS). In such a system, a dilute enzyme solution is pumped continuously through a preparative chromatographic column, while pulses of substrate are periodically injected on to the column. Enzymic reaction and separation are therefore performed in a single unit operation. The chromatographic columns used were jacketed glass columns ranging from 1 to 2 metres long with an internal diameter of 1.5 cm. Linking these columns allowed 1, 2, 3 and 4 metre long CBRS systems to be constructed. The hydrolysis of lactose in the presence of β~galactosidase was the reaction of study. From previous work at Aston University, there appeared to be no difficulties in achieving complete lactose hydrolysis in a CBRS. There did, however, appear to be scope for improving the separative performance, so this was adopted as an initial goal. Reducing the particle size of the stationary phase was identified as a way of achieving this improvement. A cation exchange resin was selected which had an average particle size of around half that previously used when studying this reaction. A CBRS system was developed which overcame the operational problems (such as high pressure drop development) associated with use of such a particle size. A significant improvement in separative power was achieved. This was shown by an increase in the number of theoretical plates (N) from about 500 to about 3000 for a 2 metre long CBRS, coupled with higher resolution. A simple experiment with the 1 metre column showed that combined bioreaction and separation was achievable in this system. Having improved the separative performance of the system, the factors affecting enzymic reaction in a CBRS were investigated; including pulse volume and the degree of mixing between enzyme and substrate. The progress of reaction in a CBRS was then studied. This information was related to the interaction of reaction and separation over the reaction zone. The effect of injecting a pulse over a length of time as in CBRS operation was simulated by fed batch experiments. These experiments were performed in parallel with normal batch experiments where the substrate is mixed almost instantly with the enzyme. The batch experiments enabled samples to be taken every minute and revealed that reaction is very rapid. The hydrodynamic characteristics of the two injector configurations used in CBRS construction were studied using Magnetic Resonance Imaging, combined with hydrodynamic calculations. During the optimisation studies, galactooligosaccharides (GOS) were detected as intermediates in the hydrolysis process. GOS are valuable products with potential and existing applications in food manufacture (as nutraceuticals), medicine and drug targeting. The focus of the research was therefore turned to GOS production. A means of controlling reaction to arrest break down of GOS was required. Raising temperature was identified as a possible means of achieving this within a CBRS. Studies were undertaken to optimise the yield of oligosaccharides, culminating in the design, construction and evaluation of a Dithermal Chromatographic Bioreactor-separator.

Combined bioreaction and separation in a simulated counter-current chromatographic bioreactor-separator system

The objective of this work has been to investigate the principle of combined bioreaction and separation in a simulated counter-current chromatographic bioreactor-separator system (SCCR-S). The SCCR-S system consisted of twelve 5.4cm i.d x 75cm long columns packed with calcium charged cross-linked polystyrene resin. Three bioreactions, namely the saccharification of modified starch to maltose and dextrin using the enzyme maltogenase, the hydrolysis of lactose to galactose and glucose in the presence of the enzyme lactase and the biosynthesis of dextran from sucrose using the enzyme dextransucrase. Combined bioreaction and separation has been successfully carried out in the SCCR-S system for the saccharification of modified starch to maltose and dextrin. The effects of the operating parameters (switch time, eluent flowrate, feed concentration and enzyme activity) on the performance of the SCCR-S system were investigated. By using an eluent of dilute enzyme solution, starch conversions of up to 60% were achieved using lower amounts of enzyme than the theoretical amount required by a conventional bioreactor to produce the same amount of maltose over the same time period. Comparing the SCCR-S system to a continuous annular chromatograph (CRAC) for the saccharification of modified starch showed that the SCCR-S system required only 34.6-47.3% of the amount of enzyme required by the CRAC. The SCCR-S system was operated in the batch and continuous modes as a bioreactor-separator for the hydrolysis of lactose to galactose and glucose. By operating the system in the continuous mode, the operating parameters were further investigated. During these experiments the eluent was deionised water and the enzyme was introduced into the system through the same port as the feed. The galactose produced was retarded and moved with the stationary phase to be purge as the galactose rich product (GalRP) while the glucose moved with the mobile phase and was collected as the glucose rich product (GRP). By operating at up to 30%w/v lactose feed concentrations, complete conversions were achieved using only 48% of the theoretical amount of enzyme required by a conventional bioreactor to hydrolyse the same amount of glucose over the same time period. The main operating parameters affecting the performance of the SCCR-S system operating in the batch mode were investigated and the results compared to those of the continuous operation of the SCCR-S system. . During the biosynthesis of dextran in the SCCR-S system, a method of on-line regeneration of the resin was required to operate the system continuously. Complete conversion was achieved at sucrose feed concentrations of 5%w/v with fructose rich. products (FRP) of up to 100% obtained. The dextran rich products were contaninated by small amounts of glucose and levan formed during the bioreaction. Mathematical modelling and computer simulation of the SCCR-S. system operating in the continuous mode for the hydrolysis of lactose has been carried out. .

Bioreaction and separation in batch chromatographic columns

The objective of this work has been to study the behaviour and performance of a batch chromatographic column under simultaneous bioreaction and separation conditions for several carbohydrate feedstocks. Four bioreactions were chosen, namely the hydrolysis of sucrose to glucose and fructose using the enzyme invertase, the hydrolysis of inulin to fructose and glucose using inulinase, the hydrolysis of lactose to glucose and galactose using lactase and the isomerization of glucose to fructose using glucose isomerase. The chromatographic columns employed were jacketed glass columns ranging from 1 m to 2 m long and the internal diameter ranging from 0.97 cm to 1.97 cm. The stationary phase used was a cation exchange resin (PUROLITE PCR-833) in the Ca2+ form for the hydrolysis and the Mg2+ form for the isomerization reactions. The mobile phase used was a diluted enzyme solution which was continuously pumped through the chromatographic bed. The substrate was injected at the top of the bed as a pulse. The effect of the parameters pulse size, the amount of substrate solution introduced into the system corresponding to a percentage of the total empty column volume (% TECV), pulse concentration, eluent flowrate and the enzyme activity of the eluent were investigated. For the system sucrose-invertase complete conversions of substrate were achieved for pulse sizes and pulse concentrations of up to 20% TECV and 60% w/v, respectively. Products with purity above 90% were obtained. The enzyme consumption was 45% of the amount theoretically required to produce the same amount of product as in a conventional batch reactor. A value of 27 kg sucrose/m3 resin/h for the throughput of the system was achieved. The systematic investigation of the factors affecting the performance of the batch chromatographic bioreactor-separator was carried out by employing a factorial experimental procedure. The main factors affecting the performance of the system were the flowrate and enzyme activity. For the system inulin-inulinase total conversions were also obtained for pulses sizes of up to 20 % TECV and a pulse concentration of 10 % w/v. Fructose rich fractions with 100 % purity and representing up to 99.4 % of the total fructose generated were obtained with an enzyme consumption of 32 % of the amount theoretically required to produce the same amount of product in a conventional batch reactor. The hydrolysis of lactose by lactase was studied in the glass columns and also in an SCCR-S unit adapted for batch operation, in co-operation with Dr. Shieh, a fellow researcher in the Chemical Engineering and Applied Chemistry Department at Aston University. By operating at up to 30 % w/v lactose feed concentrations complete conversions were obtained and the purities of the products generated were above 90%. An enzyme consumption of 48 % of the amount theoretically required to produce the same amount of product in a conventional batch reactor was achieved. On working with the system glucose-glucose isomerase, which is a reversible reaction, the separation obtained with the stationary phase conditioned in the magnesium form was very poor although the conversion obtained was compatible with those for conventional batch reactors. By working with a mixed pulse of enzyme and substrate, up to 82.5 % of the fructose generated with a purity of 100 % was obtained. The mathematical modelling and computer simulation of the batch chromatographic bioreaction-separation has been performed on a personal computer. A finite difference method was used to solve the partial differential equations and the simulation results showed good agreement with the experimental results.

Biosynthesis and separation of dextran fructose mixtures in a chromatographic reactor

A literature review of work carried out on batch and continuous chromatographic biochemical reactor-separators has been made. The major part of this work has involved the development of a batch chromatographic reactor-separator for the production of dextran and fructose by the enzymatic action of the enzyme dextransucrase on sucrose. In this reactor, simultaneous reaction and separation occurs thus reducing downstream processing and isolation of products as compared to the existing industrial process. The chromatographic reactor consisted of a glass column packed with a stationary phase consisting of cross linked polysytrene resin in the calcium form. The mobile phase consisted of diluted dextransucrase in deionised water. Initial experiments were carried out on a reactor separtor which had an internal diameter of 0.97cm and length of 1.5m. To study the effect of scale up the reactor diameter was doubled to 1.94cm and length increased to 1.75m. The results have shown that the chromatographic reactor uses more enzyme than a conventional batch reactor for a given conversion of sucrose and that an increase in void volume results in higher conversions of sucrose. A comparison of the molecular weight distribution of dextran produced by the chromatographic reactor was made with that from a conventional batch reactor. The results have shown that the chromatographic reactor produces 30% more dextran of molecular weight greater than 150,000 daltons at 20% w/v sucrose concentration than conventional reactors. This is because some of the fructose molecules are prevented as acting as acceptors in the chromatographic reactor due to their removal from the reaction zone. In the conventional reactor this is not possible and therefore a greater proportion of low molecular weight dextran is produced which does not have much clinical use. A theoretical model was developed to describe the behaviour of the reactor separator and this model was simulated using a computer. The simulation predictions showed good agreement with experimental results at high eluent flowrates and low conversions.

Studies on survival of Pseudomonas aeruginosa 6750

The growth of Pseudomonas aeruginosa 6750 as a biofilm was investigated using a novel system based on that of Gilbert et al (1989). The aim was to test the effect of controlled growth of the organism on antibiotic susceptibility and examine the survival of the organism as a biofilm. During the investigations it became clear that, because of the increasing growth of P.aeruginosa and production of exopolysaccharide, a growth rate controlled monolayer could not be achieved and so the method was not used further. The data, however, showed that there was an increase in the smooth colony type of the organism during growth. Investigations were focused on the survival of P.aeruginosa in batch and chemostat studies. Survival or percentage culturability, as measured by total and colony count ratio, was found to decrease both in extended batch culture and for chemostat cells with decreasing growth rate. Extended batch culture, however, did not exhibit further increases in resistance to ciprofloxacin and polymyxin B. Survival was also measured using other parameters namely the direct viable count, vital staining, effect of temperature downshift and measurement of lag. In batch culture, the most notable change was a decrease in cell size along the growth curve. This was accompanied by an increase in the cellular protein content. Protein per volume was calculated from the data which showed a marked increase in batch culture, which was not demonstrated for chemostat cells with decreasing growth rate. Outer membrane protein profiles were obtained for batch and chemostat cells. An LPS profile of batch culture cells was also demonstrated. In general, there was little difference in the outer membrane protein profiles of cells from early and late stationary phases.The result of the LPS profile showed that there appeared to be an increase in the B-band of the region of the LPS in the older stationary phase cultures.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components

Flow cytometry and confocal microscopy were used to quantify and visualize FITC-lectin binding to cell-surface carbohydrate ligands of log and stationary phase acapsular and capsular Cryptococcus neoformans strains. Cell populations demonstrated marked avidity for terminal a-linked mannose and glucose specific FITC-Con A, mannose specific FITC-GNL, as well as N-acetylglucosamine specific FITC-WGA. Exposure to other FITC-lectins specific for mannose, fucose and N-acetylgalactosamine resulted in little cell-surface fluorescence. The nature of cell-surface carbohydrates was investigated further by measurement of the fluorescence from surfaces of log and stationary phase cell populations after exposing them to increasing concentrations of FITC-Con A and FITC-WGA. Cell fluorescence increased significantly with small increases in FITC-Con A and FITC-WGA concentrations attaining reproducible maxima. Measurements of this nature supported calculation of the lectin binding determinants EC 50, Hn, Fmax and relative Bmax values. EC50 values indicated that the yeast-cell surfaces had greatest affinity for FITC-WGA, however, relative Bmax values indicated that greater numbers of Con A binding sites were present on these same cell surfaces. Hn values suggested a co-operative lectin-carbohydrate ligand interaction. Imaging of FITC-Con A and FITC-WGA cell-surface fluorescence by confocal microscopy demonstrated marked localization of both lectins to cell surfaces associated with cell division and maturation, indicative of dynamic carbohydrate ligand exposure and masking. Some fluorescence was associated with entrapment of FITC-Con A by capsular components, but FITC-Con A and FITC-WGA readily penetrated the capsule matrix to bind to the same cell surfaces labelled in acapsular cells.

Investigating the structure of biomass-derived non-graphitizing mesoporous carbons by electron energy loss spectroscopy in the transmission electron microscope and X-ray photoelectron spectroscopy

We have investigated the microstructure and bonding of two biomass-based porous carbon chromatographic stationary phase materials (alginic acid-derived Starbon® and calcium alginate-derived mesoporous carbon spheres (AMCS) and a commercial porous graphitic carbon (PGC), using high resolution transmission electron microscopy, electron energy loss spectroscopy (EELS), N2 porosimetry and X-ray photoelectron spectroscopy (XPS). The planar carbon sp -content of all three material types is similar to that of traditional nongraphitizing carbon although, both biomass-based carbon types contain a greater percentage of fullerene character (i.e. curved graphene sheets) than a non-graphitizing carbon pyrolyzed at the same temperature. This is thought to arise during the pyrolytic breakdown of hexauronic acid residues into C5 intermediates. Energy dispersive X-ray and XPS analysis reveals a homogeneous distribution of calcium in the AMCS and a calcium catalysis mechanism is discussed. That both Starbon® and AMCS, with high-fullerene character, show chromatographic properties similar to those of a commercial PGC material with extended graphitic stacks, suggests that, for separations at the molecular level, curved fullerene- like and planar graphitic sheets are equivalent in PGC chromatography. In addition, variation in the number of graphitic layers suggests that stack depth has minimal effect on the retention mechanism in PGC chromatography. © 2013 Elsevier Ltd. All rights reserved.

Molecular phase space transport in water:non-stationary random walk model

Molecular transport in phase space is crucial for chemical reactions because it defines how pre-reactive molecular configurations are found during the time evolution of the system. Using Molecular Dynamics (MD) simulated atomistic trajectories we test the assumption of the normal diffusion in the phase space for bulk water at ambient conditions by checking the equivalence of the transport to the random walk model. Contrary to common expectations we have found that some statistical features of the transport in the phase space differ from those of the normal diffusion models. This implies a non-random character of the path search process by the reacting complexes in water solutions. Our further numerical experiments show that a significant long period of non-stationarity in the transition probabilities of the segments of molecular trajectories can account for the observed non-uniform filling of the phase space. Surprisingly, the characteristic periods in the model non-stationarity constitute hundreds of nanoseconds, that is much longer time scales compared to typical lifetime of known liquid water molecular structures (several picoseconds).

GLM-beamformer method demonstrates stationary field, alpha ERD and gamma ERS co-localisation with fMRI BOLD response in visual cortex

Recently, we introduced a new 'GLM-beamformer' technique for MEG analysis that enables accurate localisation of both phase-locked and non-phase-locked neuromagnetic effects, and their representation as statistical parametric maps (SPMs). This provides a useful framework for comparison of the full range of MEG responses with fMRI BOLD results. This paper reports a 'proof of principle' study using a simple visual paradigm (static checkerboard). The five subjects each underwent both MEG and fMRI paradigms. We demonstrate, for the first time, the presence of a sustained (DC) field in the visual cortex, and its co-localisation with the visual BOLD response. The GLM-beamformer analysis method is also used to investigate the main non-phase-locked oscillatory effects: an event-related desynchronisation (ERD) in the alpha band (8-13 Hz) and an event-related synchronisation (ERS) in the gamma band (55-70 Hz). We show, using SPMs and virtual electrode traces, the spatio-temporal covariance of these effects with the visual BOLD response. Comparisons between MEG and fMRI data sets generally focus on the relationship between the BOLD response and the transient evoked response. Here, we show that the stationary field and changes in oscillatory power are also important contributors to the BOLD response, and should be included in future studies on the relationship between neuronal activation and the haemodynamic response. © 2005 Elsevier Inc. All rights reserved.

Thin film bearings with phase change of lubricant

The possible evaporation of lubricant in fluid film bearings has been investigated theoretically and by experiment using a radial flow hydrostatic bearing supplied with liquid refrigerant R114. Good correlation between measured and theoretical values was obtained using a bespoke computational fluid dynamic model in which the flow was assumed to be laminar and adiabatic. The effects of viscous dissipation and vapour generation within the fluid film are fully accounted for by applying a fourth order Runge-Kutta routine to satisfy the radial and filmwise transverse constraints of momentum, energy and mass conservation. The results indicate that the radial velocity profile remains parabolic while the flow remains in the liquid phase and that the radial rate of enthalpy generation is then constant across the film at a given radius. The results also show that evaporation will commence at a radial location determined by geometry and flow conditions and in fluid layers adjacent to the solid boundaries. Evaporation is shown to progress in the radial direction and the load carrying capacity of such a bearing is reduced significantly. Expressions for the viscosity of the liquid/vapour mixture found in the literature survey have not been tested against experimental data. A new formulation is proposed in which the suitable choice of a characteristic constant yields close representation to any of these expressions. Operating constraints imposed by the design of the experimental apparatus limited the extent of the surface over which evaporation could be obtained, and prevented clear identification of the most suitable relationship for the viscosity of the liquid/vapour mixture. The theoretical model was extended to examine the development of two phase flow in a rotating shaft face seal of uniform thickness. Previous theoretical analyses have been based on the assumption that the radial velocity profile of the flow is always parabolic, and that the tangential component of velocity varies linearly from the value at the rotating surface, to zero at the stationary surface. The computational fluid dynamic analysis shows that viscous shear and dissipation in the fluid adjacent to the rotating surface leads to developing evaporation with a consequent reduction in tangential shear forces. The tangential velocity profile is predicted to decay rapidly through the film, exhibiting a profile entirely different to that assumed by previous investigators. Progressive evaporation takes place close to the moving wall and does not occur completely at a single radial location, as has been claimed in earlier work.