Kazufusa Shinomiya

Studies on a new cross-axis coil planet centrifuge for performing counter-current chromatography. I: Design of the apparatus, retention of the stationary phase, and efficiency in the separation of proteins with polymer phase systems

An improved model of the cross-axis synchronous flow-through coil planet centrifuge has been designed in light of previous studies. The apparatus has a versatile feature in that both analytical and preparative columns can be accommodated in both off-center and central positions. Each has merit in separations. Retention of stationary phase was examined with various two-phase solvent systems used for the separation of biopolymers. Both analytical and preparative columns showed satisfactory retention of the stationary phase under optimum conditions. The apparatus was evaluated in separation of a set of protein samples using a polyethylene glycol-potassium phosphate biphasic system. In both types of columns all proteins were resolved with partition efficiencies of 260 to 670 theoretical plates. Further studies indicated that the relatively low partition efficiency of proteins is mainly attributed to their high molecular mass or molecular heterogeneity within each species rather than due to the high viscosity of the polymer phase system.

Determination of human urinary hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharides by high-performance liquid chromatography

A method for the determination of hyaluronic acid (HA), chondroitin sulphate (CS) and dermatan sulphate (DS) was developed. HA, CS and DS were converted to the corresponding unsaturated disaccharides by digestion with chondroitinase ABC and/or chondroitinase AC-II and determined by high-performance liquid chromatography with fluorimetric detection using 2-cyanoacetamide as a post-column derivatization reagent. The calibration graphs for the unsaturated disaccharides were linear over the range 2 ng - 2 micrograms for each unsaturated disaccharide. This method was applied to the analysis of normal human urine.

Protein Separation by Improved Cross-Axis Coil Planet Centrifuge with Eccentric Coil Assemblies

The new prototype of the cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory provides various improvements over the original unit such as ambient temperature control, good visibility of the rotary frame and substantial reduction of the torque by a round transparent case, direct motor shaft coupling to the rotary frame to stabilize the system, and ease of belt tension adjustment using idler pulleys. The capability of the system was demonstrated in the separation of stable proteins with a polymer phase system using a pair of eccentric coil assembly separation columns. Cytochrome C, myoglobin and trypsinogen were well resolved and eluted in 5.5 h at a partition efficiency of 200 theoretical plates. The method provides a gentle environment for proteins without causing their deactivation or loss.

Countercurrent Chromatographic Separation of Biotic Compounds with Extremely Hydrophilic Organic‐Aqueous Two‐Phase Solvent Systems and Organic‐Aqueous Three‐Phase Solvent Systems

Abstract Highly polar organic‐aqueous two‐phase and three‐phase solvent systems were applied to the countercurrent chromatographic (CCC) separation of biotic compounds. The ethanol/2 M ammonium sulfate (3∶5) system was used for protein separation between human serum albumin and lysozyme, and also for separations of sugars including D‐(+)‐glucose and L‐(−)‐fucose, D‐(+)‐xylose and α‐L‐rhamnose, and α‐D‐galacturonic acid, mannuronic acid lactone and D‐(+)‐glucoronolactone. When using the acetonitrile/1 M sodium chloride (5∶4) system for applying water‐soluble carboxylic acids, 2‐naphthoic acid, p‐methyl hippuric acid and p‐amino hippuric acid were well separated with upper phase mobile, and maleic acid and fumaric acid were resolved with lower phase mobile. An organic‐aqueous three‐phase solvent system composed of n‐hexane/methyl t‐butyl ether/acetonitrile/water (5∶5∶7.5∶5) was also applied to the simultaneous CCC separation of fat‐soluble vitamins and water‐soluble vitamins. Using the middle phase as the stationary phase, the separation of thiamine hydrochloride and nicotinamide was achieved first with the lower mobile phase and then vitamin K1 and K3 were eluted after the mobile phase was switched to the upper phase reversing the direction of elution. The overall results demonstrated that both extremely hydrophilic organic‐aqueous two‐phase solvent systems and organic‐aqueous three‐phase solvent systems are useful for the CCC separations of polar compounds.

Protein separation by nonsynchronous coil planet centrifuge with aqueous–aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.

Determination of Phenolic Anti-Oxidants in Edible Oil by High-Performance Liquid Chromatography with Amperometric Detector

Abstract A high-performance liquid chromatography (HPLC) with amperometric detection was investigated for the analysis of 2-and 3-tert-butyl-4-hydroxyanisole (BHA), 3,5-di-tert−butyl-4-hydroxytoluene (BHT), and tert−butyl-hydroquinone (TBHQ) in edible oil. The reversed-phase system developed was combined with an amperometric detector, the working electrode of which was made of glassy carbon, in order to compare the sensitivity and selectivity of ultraviolet and fluorometric detection. For the amperometric detection of HPLC, cyclic voltammetry was used to monitor the electrochemical properties of the phenolic antioxidants. A simple isolation procedure, based on the continuous liquid-liquid partition technique, was examined for the extraction and clean up of the antioxidants from edible oil. The recovery rates of BHA, BHT, and TBHQ added salad oil were between 90.2-107.7% in the range of 1-50 ppm of the antioxidants. By the present method, BHA, BHT, and TBHQ were well separated, identified and quantitated w...

Countercurrent chromatographic analysis of ovalbumin obtained from various sources using the cross-axis coil planet centrifuge.

The present studies have been conducted to investigate the cause of an unusually broad peak of ovalbumin obtained by countercurrent chromatography (CCC) reported earlier [K. Shinomija et al., J. Chromatogr., 644 (1993) 215]. A series of CCC experiments using our prototyte of the cross-axis coil planet centrifuge revealed that commercial ovalbumin products were classified into two groups: group A formed two peaks of ovalbumin at pH 7.0 and 5.8, while group B showed a relatively sharp single peak in a broad range of pH. Electrophoresis indicated that the group A ovalbumin consisted of both natural and denatured products: the natural ovalbumin is a monomer (Mr 45 000) whereas the denatured products form dimers (Mr 90 000). The abnormally broad peak obtained from the group A ovalbumin at pH 9 is apparently caused by the heterogeneity of the sample protein.

Enantiomeric Separation of Commercial D,L-Kynurenine with an Aqueous Two-Phase Solvent System by Cross-Axis Coil Planet Centrifuge

Commercial D,L-kynurenine was resolved by high-speed countercurrent chromatography using a cross-axis coil planet centrifuge. The separation was performed with an aqueous-aqueous polymer phase system composed of 10% (w/w) polyethylene glycol 8000 and 5% (w/w) dibasic sodium phosphate containing 6% (w/w) bovine serum albumin as a chiral selector. The lower phosphate-rich mobile phase eluted the L-enantiomer first followed by the D-enantiomer. The peak resolution was 0.94 for 2.5 mg of the sample. The separation was completed within 3.5 h.

COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF SUGARS AND THEIR p-NITROPHENYL DERIVATIVES BY CROSS-AXIS COIL PLANET CENTRIFUGE

Sugars and their p-nitrophenyl (PNP) derivatives were separated by high-speed countercurrent chromatography using a cross-axis coil planet centrifuge (cross-axis CPC) equipped with a pair of eccentric coil assemblies. A polar two-phase solvent system composed of 1-butanol/acetic acid/water (4:1:5) was used for the separation of sucrose and fucose, and glucuronic acid (lactoid form) and galacturonic acid, while 1-butanol/ethanol/water (4:1:4) was used for the separation of free and lactoid forms of glucuronic acid. PNP-sugar derivatives such as neutral sugars, uronic acids, and amino sugars were separated with a less polar solvent system composed of n-hexane/ethyl acetate/1-butanol/methanol/water at various volume ratios. PNP-glucose derivatives were further separated according to the number of sugar chains, and five PNP-neutral sugars were resolved by adding 0.1 M sodium tetraborate in the two-phase solvent system. Overall results of experiments revealed that the cross-axis CPC is useful for the separatio...

Study of the measurement of chondroitin sulphates in rabbit plasma and serum

A highly sensitive high-performance liquid chromatography method, which was established by us for the determination of chondroitin sulphates in biological substances as their unsaturated disaccharides, was applied to elucidate the qualitative and quantitative differences in chondroitin sulphates in rabbit plasma and serum samples. In this work, it was found that rabbit plasma contains low-sulphated chondroitin 4-sulphate (approximately 40% sulphation at the 4-position of N-acetyl galactosamine), while serum contains the low-sulphated chondroitin 4-sulphate and fully sulphated chondroitin 4-sulphate (approximately 96% sulphation). The latter was released from platelets during coagulation of blood.