J. Čoupek

Chromatography of biopolymers and their fragments on ion-exchange derivatives of the hydrophilic macroporous synthetic gel spheron.

New ion-exchange materials have been developed from the synthetic glycol methacrylate gel Spheron: carboxymethyl-Spheron (weakly acidic); phosphonyl-Spheron (medium acidic); sulphonyl-Spheron (strongly acidic); diethylaminoethyl-Spheron (Type 1, weakly basic; Type 2, medium to strongly basic). Information is presented on the chromatographic characteristics of these new, macroporous, hydrophilic and rigid ion-exchangers as determined by experiments on the separation of mixtures of proteins, peptides, amino acids, nucleic acids, oligonucleotides, and nucleotides. The possibility is discussed of the use of ion-exchangers of this type in high-performance liquid chromatography of biopolymers.

Affinity chromatography on hydroxyalkyl methacrylate gels. I. Preparation of immobilized chymotrypsin and its use in the isolation of proteolytic inhibitors.

Abstract Heterogeneous suspension copolymerization of hydrophilic methacrylate monomers with dimethacrylates was used to obtain neutral hydrophilic gels with a defined specific surface and pore distribution. Such macroporous carriers can be activated with bromocyanogen for binding biologically active compounds and applied in affinity chromatography. Chymotrypsin was used to investigate the dependence of the amount of a covalently bound enzyme and of its esterase and proteolytic activity on the specific surface and pore size of the carrier. In a similar manner to chymotrypsin-Sepharose, there was a shift of the pH optimum of esterase activity toward the alkaline region compared with the pH optimum of free chymotrypsin, while the pH optimum of the proteolytic activity of free and immobilized chymotrypsin remained unchanged. Affinity chromatography with covalently bound chymotrypsin was used to isolate a chymotryptic inhibitor from potatoes, and a trypsin inhibitor from lung. If the trypsin inhibitor from lung was bound to a hydroxyalkyl methacrylate carrier, the affinity chromatography of a commercial chymotrypsin sample raised its activity by one order of magnitude. The new hydrophilic carriers are characterized by an excellent mechanical stability which gives them outstanding flow properties.

Accelerated reversed-phase chromatography of carbohydrate oligomers

Abstract Two different ways of accelerating analyses of glucose oligomers by reversed-phase chromatography and of improving their sensitivity are presented. A specially developed glass cartridge packed with a C 18 bonded silica of the Separon type was used for high speed separations of maltodextrins up to a degree of polymerization of 8 at ambient temperature in 5 min using water as eluent. The effect of temperature upon the separation of carbohydrates on a conventional reversed-phase column in terms of heats of transfer, capacity factors, selectivity and column efficiency was also considered. The capacity factors decrease markedly with increasing column temperature owing to relatively high values of the heat of transfer. The non-linear increase in the heat of transfer with the degree of polymerization leads also to some reduction in the column selectivity at increased temperatures. An increase in temperature accelerates the rate of interconversion between the α- and β-anomers of saccharides, thus eliminating unwanted double peaks. The combined effects of elevated temperature result in a considerable acceleration in the separation of maltose (10 min for up to a degree of polymerization of 10) and cellobiose oligomers (10 min for a degree of polymerization of 6 at 70-80°C), which means a 2-20 fold acceleration of their analyses with a substantial gain in sensitivity.

Hydroxyalkyl methacrylate gels derivatized with epichlorohydrin as supports for large-scale and high-performance affinity chromatography

Abstract The preparation of hydroxyalkyl methacrylate copolymers derivatized with epichlorohydrin (epoxide group content 140–1770 μmol/g) has been accomplished. The coupling of various amino derivatives on to derivatized carriers has been investigated as a function of pH, time of coupling, concentration of the compound bound, degree of epoxidation of the carrier and specific structural features of amino compounds. The unreacted epoxide groups were eliminated using hydrolysis with 0.1 M perchloric acid. For the preparation of a specific sorbent of carboxylic proteinases, a procedure for the synthesis of ϵ-aminocaproyl- L Phe- D Phe-OCH 3 and for its binding on to an epoxide carrier has been devised. The sorbents prepared were used in affinity chromatography of a raw sample of pepsin and proteinases from Aspergillus oryzae . The same efficiency was achieved with pepsin solutions, the enzyme concentration differing within the range of two orders of magnitude. The mechanical stability of the support made possible an analytical application of the high-performance liquid affinity chromatography of pepsin, by which the pepsin concentration in solution can be determined within 30 min with high sensitivity.

Ion-exchange derivatives of spheron : I. characterization of polymeric supports

Abstract In order to characterize matrices suitable for the preparationof ion exchangers, three commercially available types of glycol methacrylate macroreticular gels, SpheronTM P-100, P-300 and P-1000 (particle size, 20–40 μm), have been characterized by their bulk rate, degree of swelling, working volume and inner surface area. Prior to modification, the gels were extracted with dilute acid, dilute alkali. 8 M urea, pyridine and hot organic solvents. Extracted and dried Spherons have been characterized by elemental analysis, and by the determination of the inner surface area, exclusion limit, specific pore volume, the most frequent pore diameter, specific unpenetrable volume, number of unpolymerized double bonds and capacity for small ions. The particle-size distribution of Spheron P-300 has also been determined. Porosimetric data obtained by nitrogen sorption and desorption measurements are compared with those obtined by mercury porosimetry. The inner structure of Spheron is discussed in relation to the experimentl results (electron microphotography and the course of thermal vacuum depolymerization, ion-exchange capacity after the highest attainable ionogenic substitution, unpenetrable volume and particle porosity). Extracted Spheron P-300 exhibits an advantageous, approximately level dependence of the height equivalent to a theoretical plate on the flow-rate within the range 25–280 ml/h. The suitability of a Spheron matrix for the preparation of ion exchangers is demonstrated.

Ion-exchange derivatives of spheron

Abstract The preparation of diethylaminoethyl derivatives of Spheron (DEAE-Spheron 300) having different capacities for small ions (nominal capacities 0.11, 0.26, 0.60, 1,23, 1.63, 2.05 and 2.20 mequiv./g) is described. Routes leading to a high degree of ionogenic substitution are discussed. The ion exchangers were characterized analytically and by titration curves, which indicate that the ion exchangers are homoionic. The chromatographic properties of the DEAE-Spherons were tested in the chromatography of serum albumin, of an artificial mixture of proteins (lysozyme, chymotrypsinogen and serum albumin), blood plasma, a mixture of peptides and of glutamic, aspartic and cysteic acids using an amino acid analyzer and also in the chromatography of adenosine phosphates. In all cases the best separations were obtained with anion exchangers having the highest nominal capacities. The effect of degree of ionogenic substitution on the decrease of the partial hydrophobicity of the Spheron matrix is discussed. It is shown that at a nominal capacity exceeding 1.2 mequiv./g the effect of hydrophilic iogenic groups balances the partial hydrophobicity of the Spheron matrix, so that no hydrophobic sorption of proteins takes place.

Antioxidants and stabilizers. XXXIII. Analysis of stabilizers of isotactic polypropylene: application of gel permeation chromatography

Abstract Mixtures of stabilizers, consisting of an antioxidant, light-absorber and synergist, designed to protect isotactic polypropylene against atmospheric ageing, were analysed by means of gel permeation chromatography. An acetone extract of the polymer was chromatographed on styrene—divinylbenzene gel having a molecular weight exclusion limit of 1000; the eluent was tetrahydrofuran. The values of the relative zone velocity, R, of some important stabilizers are listed and examples of quantitative analyses are discussed.

Determination of acetylcholine and choline by flow-injection with immobilized enzymes and fluorometric or luminometric detection

A method for determination of picomolar quantities of acetylcholine and choline in solutions and tissue extracts is described. The analytes are injected into a continuous stream of a simple medium flowing through a sequence of enzyme reactors containing acetylcholinesterase, choline oxidase, and peroxidase. Additional reactors with choline oxidase and catalase are used to remove endogenous choline from the tissue extracts in which the content of acetylcholine is to be measured. Reaction products are detected fluorometrically or luminometrically. The limits of sensitivity are about 10 pmol/sample with luminometric and 0.2 pmol/sample with fluorometric detection.

Modified hydroxyethyl methacrylate copolymers as sorbents for ion chromatographya : I. Synthesis and properties of sorbents

Abstract The preparation and properties of ion chromatography sorbents based on Separon hydroxyethyl methacrylate copolymers are described. Sorbents with amino, ethylamino, diethylamino, dicyclohexylamino, diethanolamino and triethylamino functional groups were prepared by reaction of the appropriate amine with a precursor gel containing epoxy groups. Sorbents with the required ion-exchange capacity and additional cross-linking on column performance were studied. Examples of applications include the determination of nitrate in milk, separation of polythionates, etc.

Hydroxyethyl methacrylate-based sorbents for high-performance liquid chromatography of proteins

Abstract TESSEK Separon HEMA sorbents are based on a copolymer of ethylene dimethacrylate and hydroxyethyl methacrylate, whose biocompatibility has been demonstrated by its widespread use in soft contact lenses. The copolymer has a high resistance to hydrolysis and microbial attack, high mechanical strength and a high surface hydroxyl group content. In addition to size-exclusion chromatography, a wide range of derivatives have been prepared for ion-exchange, hydrophobic interaction and affinity chromatography. New modifications for reversed-phase, immobilized metal affinity and protein A chromatography have increased the number of possibilities for separating proteins.