Flow-through Coil Planet Centrifuge Research Articles

Purification of mast cells with an improved nonsynchronous flow-through coil planet centrifuge.

A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells.

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.

Cross-Axis Synchronous Flow-Through Coil Planet Centrifuge (Type XLL) III. Performance of Multilayer Coils in Preparative Separation of Dipeptides

Abstract Performance of the type-XLL cross-axis coil planet centrifuge with three different size of the multilayer coils was evaluated by separating dipeptide samples on a polar biphasic solvent composed of n-butanol/acetic acid/water (4:1:5, v/v/v). Best results were obtained from the large helical diameter coils with β values of 1.00-1.20 [1]| by eluting with the upper nonaqueous phase at 120 ml/h. The four dipeptides. L.-tryptophyl-L-tyrosine, L-leucyl-L-tyrosine, L-valyl-L-tyrosine and L-tyrosylglycine were completely rcsolved in 9 hours with high peak resolution (Rs > 1.5).

Studies on the peparative capability of the horizontal flow-through coil planet centrifuge and high-performance liquid chromatography in the separation of polar compounds from Oxytropis ochrocephala Bunge

Horizontal flow-through planet centrifuge (CPC) and high-performance liquid chromatography (HPLC) techniques were used for separation of polar compounds from a crude ethanol extract of Oxytropic ochrocephala Bunge, a poisonous legume plant widely distributed in northwestern China. The performance of these two chromatographic methods was compared in terms of column efficiency, peak resolution, separation time, sample loading capacity, etc. The results indicated that two polar compounds in the crude extract were equally well separated by these two methods. HPLC gave comparable peak resolution in shorter separation time while its sample loading capacity was limited to the mg range. The CPC method required a long separation time, but yielded a higher purity of fractions with a much greater capacity.

Cross-axis synchronous flow-through coil planet centrifuge (type XLL) : II. Speculation on the hydrodynamic mechanism in stationary phase retention

The hydrodynamic mechanism involved in the retention of stationary phase in the present x-axis coil planet centrifuge system is discussed. A statistical treatment of the retention data disclosed important clues such as the effect of the inward-outward elution mode, the close correlation between the planetary motion and the head—tail elution mode, and the superior retention capacity of the left-handed coils. The combined effects of the radial and lateral centrifugal force field derived from the mathematical analysis of acceleration acting on the coil provide an explanation for the phenomena.

Protein separation with aqueous—aqueous polymer systems by two types of counter-current chromatographs

Two different types of counter-current chromatographs, the cross-axis coil planet centrifuge ( X-axis CPC) and horizontal flow-through coil planet centrifuge (horizontal CPC), were evaluated for protein separation with an aqueous-aqueous two-phase polymer system. The sample solution, containing 10–200 mg each of cytochrome c, myoglobin, ovalbumin and hemoglobin in 2 ml of each phase was eluted with the lower phase. In both instruments, the effects of the flow-rate, revolution speed, and parameter β (helical diameters of the multilayer coil) on the protein separation were investigated. The best results were obtained from the X-axis CPC operated at 750 rpm and a flow-rate of 2.0 ml/min using a multilayer coil with a small helical diameter (β = 0.25–0.60). Four protein samples were well resolved in less than 5 h.

Cross-axis synchronous flow-through coil planet centrifuge (type XLL) : I. Design of the apparatus and studies on retention of stationary phase

the fourth prototype holds a pair of column holders in the lateral position at 15 cm from the center of the rotary shaft horizontally mounted on the rotary frame at 7.6 cm from the central axis of the apparatus. Using short coils of 2.6 mm I. D. PTFE (polytetrafluoroethylene) tubing with 7.6 cm and 24 cm helical diameters, retention of the stationary phase was measured in ten pairs of two-phase solvent systems under various experimental conditions. Satisfactory retention was obtained by choosing proper combinations of three factors, i. e., the direction of planetary motion, head—tail elution mode, and inward—outward elution mode. The polar butanol solvent systems showed excellent retention from 65 to 80% in the 7.6 cm helical diameter left-handed coil

Chromatography of peptides on a multi-coil counter-current chromatograph

A modified horizontal flow-through coil planet centrifuge has been constructed that has the potential for preparative choromatography. The multi-coil counter-current chromatograph, equipped with a set of four multi-layer coils, has have improved performance with polar solvents suitable for peptide elution. Separations of a group of dipeptides and purification of a cholecystokinin fragment and an undecapeptide were achieved with n-butanol—acetic acid—water at high flow-rates with good resolution.

Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography : I. Design of the apparatus and analysis of accelaration

A novel design of the cross-axis synchronous flow-through coil planet centrifuge is introduced. The apparatus holds a pair of large coil holders symmetrically, one on each side of the rotary frame, at a lateral position 12.5 cm from the center of the holder shaft held 10 cm from the centrifuge axis. Mathematical analysis of acceleration generated by the planetary motion of the apparatus revealed a unique centrfugal force field which promises high retention of the stationary phase in the multilayer coil to perform efficient preparative-scale counter-current chromatography.

Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography : II. Studies on retention of stationary phase in short coils and preparative separations in multilayer coils

Performance of the apparatus was evaluated in terms of stationary phase retention, partition efficiency and sample loading capacity. Preliminary studies with short coils revealed high retention of the stationary phase under a proper combination of the head—tail elution and planetary motion. Preparative capability of the apparatus was successfully demonstrated on efficient multigram separations of 2,4-dinitrophenyl amino acids, indole auxins, and bacitracin in a pair of large multilayer coils with a total capacity of 1.5 1.

Cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography : III. Performance of large-bore coils in slow planetary motion

A preparative capability of the present cross-axis synchronous flow-through coil planet centrifuge was demonstrated with 0.5 cm I.D. multilayer coils. Results of the model studies with short coils indicated that the optimal separations are obtained at low revolutional speeds of 100–200 rpm in both central and lateral coil positions. Preparative separations were successfully performed on 2.5–10 g quantities of test samples in a pair of multilayer coils connected in series with a total capacity of 2.5 l. The sample loading capacity will be scaled up in several folds by increasing the column width.

Cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography : II. Studies on partition efficiency in short coils and preparative separations with multilayer coils

A series of experiments was conducted to evaluate the partition efficiency of the cross-axis synchronous flow-through coil planet centrifuge. The preliminary studies indicated that short coils (5 m) mounted on 15- and 25-cm diameter holders in either a central or a lateral location produce a high partition efficiency of over 100 theoretical plates. Under the optimum operating conditions, the preparative capability of the countercurrent chromatograph was demonstrated on gram-scale separations of DNP-amino acids and dipeptides in a pair of multilayer coils connected in series with a total capacity of 1600 ml.

Large Scale Purification of Apocarotenoids from Cochlospermum Tinctorium by Countercurrent Chromatography

Abstract Carotenoids are well-known for their instability so as usual analytical methods such as HPLC or preparative TLC applied to the separation of the constituents of Cochlospermum tinctorium failed to the isolation of pure pigments for further chemical characterization. Thus high-speed CCC using a horizontal flow-through coil planet centrifuge was used to conveniently and efficiently achieve this separation for quantities up to 500mg of the crude plant extract.

Separation of Flavonoids in Crude Extract from Sea Buckthorn by Countercurrent Chromatography with Two Types of Coil Planet Centrifuge

Abstract Flavonoid constituents in a crude ethanol extract from dried fruits of sea buckthorn (Hippophae rhamnoides) were subjected to countercurrent chromatography with a two-phase solvent system composed of chloroform, methanol and water at a 4:3:2 volume ratio. Separations were performed with two different types of the coil planet centrifuge: One is called the multilayer coil planet centrifuge and the other, the horizontal flow-through coil planet centrifuge. Both instruments permit continuous elution through the rotating column without the use of rotary seals. Although the horizontal flow-through coil planet centrifuge produced efficient peak resolution for five flavonoid components, the multilayer coil planet centrifuge yielded much superior results in terms of partition efficiency, separation time and sample loading capacity.

Horizontal flow-through coil planet centrifuge equipped with a set of multilayer coils around the column holder: Counter-current chromatography of proteins with a polymer-phase system

Horizontal flow-through coil planet centrifuge equipped with a set of multilayer coils around the column holder: Counter-current chromatography of proteins with a polymer-phase system

Preliminary applications of cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography

The cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius and a total capacity of 1600 ml was successfully applied to preparative counter-current chromatography of various biological samples, which include sea buckthorn extract, steroid reaction mixture, indole plant hormones, and dinitrophenylamino acids. The present system offers advantages of stable balance of the centrifuge, a large column capacity, and high resolution.

Cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography : I. Apparatus and studies on stationary phase retention in short coils

Using a new cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius, the retention of the stationary phase for nine solvent systems was studied with short coils mounted at two different locations on three holders with 5-, 15- and 25-cm hub diameters. Coils mounted 10 cm to the left of the center of a holder produced a much improved retention of most of the solvent systems compared with the same coils mounted at the center of the holder. In the lateral coil position the retention was found to be affected by the direction of the planetary motion and the head-tail elution mode. This phenomenon may be attributed to the effect of the lateral force field acting asymmetrically between the upper and lower halves of the coil.

Cross-Axis Synchronous Flow-Through Coil Planet Centrifuge Free of Rotary Seals for Preparative Countercurrent Chromatography. Part II. Studies on Phase Distribution and Partition Efficiency in Coaxial Coils

Abstract Potential capability of the apparatus in performing countercurrent chroma-tography has been examined with three different types of coiled columns, all coaxially mounted around the holder. In single-layer coils, typical solvent systems display characteristic hydrodynamic distribution which ensures a stable retention of the stationary phase against heavy sample loading in preparative separations. Direct observation of the hydrodynamic motion in the rotating spiral column revealed vigorous mixing of the two solvent phases throughout the area which promises a high partition efficiency of the present method. Gram-quantity preparative separations were performed in the multilayer coil with two different sets of test samples: Isocratic elution of dinitrophenyl amino acids yielded high partition efficiency of 1600 theoretical plates while the versatility of the method was demonstrated on gradient elution of dipeptides including two pairs of sequential isomers.

Cross-Axis Synchronous Flow-Through Coil Planet Centrifuge Free of Rotary Seals for Preparative Countercurrent Chromatography. Part I. Apparatus and Analysis of Acceleration

Abstract A novel type of the coil planet centrifuge is introduced. The apparatus holds a coil holder in such a way that the axis of the holder is positioned perpendicular to and at a fixed distance away from the centrifuge axis. In maintaining the above orientation the holder undergoes synchronous planetary motion, i.e., revolution around the central axis-of the centrifuge and rotation about its own axis at the same angular velocity. Mathematical analysis of acceleration generated by this planetary motion revealed a unique distribution pattern of centrifugal force vectors which promises a high performance of the present scheme in counter-current chromatography.

Counter-current chromatographic purification of [ dala 1]peptide t amide

A recently proposed octapeptide, [ dAla 1]peptide T amide ( d-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH 2), has been shown to bind to the CD4 receptor on human T-cell lymphocytes and block human immunodeficiency viral infectivity. This peptide may not only be itself a promising therapeutic for the disease of AIDS (acquired immune deficiency syndrome), but might open a new avenue of research towards more efficacious drugs. Accordingly, a counter-current chromatography system is here presented, utilizing the Ito flow-through coil planet centrifuge (solvent system, 1% trifluoroacetic acid— n-butanol (1:1), upper phase mobile) which allows facile preparative purification of [ d-Ala 1]peptide T amide. The method may be applicable to purifying analogues of [ d-Ala 1]peptide T amide as they are developed.