Cross-axis Coil Planet Centrifuge Research Articles

Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns.

Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of partition efficiency and shortening theseparation time. The locular multilayer coils modified with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl (MU) sugar derivatives using the small-scale cross-axis coil planet centrifuge. Proteins were well separated from each other and the separation was improved at a low flow rate of the mobile phase. On the other hand, 4-MU sugar derivatives were sufficiently resolved with short separation time at a highflow rate of the mobile phase under satisfactory stationary phase retention. Effective separations were achieved using the locular multilayer coil for proteins with aqueous-aqueous polymer phase systems and for 4-MU sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule.

Counter-Current Chromatographic Separation of Nucleic Acid Constituents with a Polar Volatile Organic-Aqueous Two-Phase Solvent Systems with ELSD Detection

Highly hydrophilic volatile organic/aqueous two-phase solvent systems containing an organic salt such as, acetonitrile/800 mM and 1200 mM ammonium acetate (1 : 1, v/v) were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate hydrophilic compounds. The retention of the upper and the lower stationary phases in the column of the cross-axis coil planet centrifuge (CCC instrument) was studied by changing the flow-rate of the mobile phase (1.0–3.0 ml/min). Using the acetonitrile/800 mM ammonium acetate two-phase solvent system, the stationary phase was retained at 46.3% relative to the total column capacity of 65 ml by the reversed-phase elution mode at a flow-rate of 1.0 ml/min. The best retention of the stationary upper phase of 51.5% was obtained by the solvent system of the acetonitrile/1200 mM ammonium acetate at the above flow-rate. With the acetonitrile/800 mM ammonium acetate system the base line separation of adenine and adenosine monophosphate (AMP) detected by evaporative light scattering detector (ELSD) and UV was achieved with lower phase mobile at a flow-rate of 1.0 ml/min within 70 min.

PARTITION EFFICIENCY OF HIGH-PITCH LOCULAR MULTILAYER COIL FOR COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF PROTEINS USING SMALL-SCALE CROSS-AXIS COIL PLANET CENTRIFUGE AND APPLICATION TO PURIFICATION OF VARIOUS COLLAGENASES WITH AQUEOUS-AQUEOUS POLYMER PHASE SYSTEMS

Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially higher stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, IV, V, and X derived from Clostridium histolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample. Collagenase from C. histolyticum well retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities.

Performance comparison of three types of high-speed counter-current chromatographs for the separation of components of hydrophilic and hydrophobic color additives

The performance of three types of high-speed counter-current chromatography (HSCCC) instruments was assessed for their use in separating components in hydrophilic and hydrophobic dye mixtures. The HSCCC instruments compared were: (i) a J-type coil planet centrifuge (CPC) system with a conventional multilayer-coil column, (ii) a J-type CPC system with a spiral-tube assembly-coil column, and (iii) a cross-axis CPC system with a multilayer-coil column. The hydrophilic dye mixture consisted of a sample of FD&C Blue No. 2 that contained mainly two isomeric components, 5,5′- and 5,7′-disulfonated indigo, in the ratio of ∼7:1. The hydrophobic dye mixture consisted of a sample of D&C Red No. 17 (mainly Sudan III) and Sudan II in the ratio of ∼4:1. The two-phase solvent systems used for these separations were 1-butanol/1.3M HCl and hexane/acetonitrile. Each of the three instruments was used in two experiments for the hydrophilic dye mixture and two for the hydrophobic dye mixture, for a total of 12 experiments. In one set of experiments, the lower phase was used as the mobile phase, and in the second set of experiments, the upper phase was used as the mobile phase. The results suggest that: (a) use of a J-type instrument with either a multilayer-coil column or a spiral-tube assembly column, applying the lower phase as the mobile phase, is preferable for separating the hydrophilic components of FD&C Blue No. 2; and (b) use of a J-type instrument with multilayer-coil column, while applying either the upper phase or the lower phase as the mobile phase, is preferable for separating the hydrophobic dye mixture of D&C Red No. 17 and Sudan II.

Partition Efficiency of Newly Designed Locular Multilayer Coil for Countercurrent Chromatographic Separation of Proteins Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems

Countercurrent chromatographic performance of the locular multilayer coil separation column newly designed in our laboratory, was evaluated in terms of theoretical plate number, peak resolution, and retention of the stationary phase in protein separation with an aqueous polymer phase system, using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The locular column was made from 1.0 mm I.D., 2.0 mm O.D., or 1.5 mm I.D., 2.5 mm O.D. PTFE tubing, compressed with a pair of hemostat at 2 or 4 cm intervals. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system under 1000 rpm of column revolution. The 1.5 mm I.D., 2.5 mm O.D. locular tubing compressed at 2 cm intervals yielded better partition efficiencies than the non-clamped tubing, using both lower and upper mobile phases with satisfactory retention of the stationary phase. The overall results suggest that the newly designed locular multilayer coil is useful to the preparative separation of proteins with aqueous-aqueous polymer phase system using our small scale X-axis CPC.

Countercurrent chromatographic separation and purification of various ribonucleases using a small-scale cross-axis coil planet centrifuge with aqueous–aqueous polymer phase systems

Countercurrent chromatographic (CCC) separation and purification of various ribonucleases (RNases) was performed using the small-scale cross-axis coil planet centrifuge (X-axis CPC) with aqueous–aqueous polymer phase systems. RNases B and A were well resolved from each other with an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate (pH 9.2) as the mobile lower phase. The commercial RNase A samples obtained from three different companies were also highly purified using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6) using the upper phase as the mobile phase. Recombinant RNase Po 1, an RNase T 1 family enzyme, was further successfully separated from the crude extract using the same solvent system with the lower phase used as the mobile phase. The RNase activities were well preserved during the CCC separation. The overall results demonstrate that the small-scale X-axis CPC is useful for a simple and rapid purification of various RNases in a preparative-scale.

Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge.

This review describes protein purifications from cell culture medium or cell-lysate by high speed counter-current chromatography using the cross-axis coil planet centrifuge. Purifications were performed using aqueous two phase systems composed of polyethylene glycols and dextrans.

New small-scale cross-axis coil planet centrifuge: Partition efficiency and application to purification of bullfrog ribonuclease

The new small-scale cross-axis coil planet centrifuge (X-axis CPC) previously designed and fabricated in our laboratory has a distinctive feature such that four separation columns of similar weight are mounted symmetrically around the rotary frame to achieve stable balancing of the centrifuge under a high revolution speed. In this column layout, neighboring columns must be rotated in the opposite direction if viewed from the center of the centrifuge to avoid twisting the interconnecting flow tubes. The effect of rotational direction of the columns on the partition efficiency was evaluated with separation of a set of test samples such as cytochrome c, myoglobin, and lysozyme using an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate under 1000 rpm of column revolution. A series of experiments was performed using a set of two diagonally located columns (connected in series) each consisting of five coiled layers of 1 mm I.D. with a total capacity of 27.0 mL. Both right- and left-handed coils were tested each under the optimized conditions for choice of mobile phase and direction of the column rotation so that the satisfactory volume of the mobile phase was retained in the column by the aid of Archimedean screw effect. The results of these studies showed that one particular combination of handedness of the coil and direction of the rotation yielded the best peak resolution for each mobile phase. In order to demonstrate the capability of the apparatus, the purification of ribonuclease (RNase) from the extract of bullfrog egg, sialic acid binding lectin (cSBL), was carried out using both organic–aqueous and aqueous–aqueous polymer phase systems. When using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system, cSBL was successfully separated from other proteins present in the extract while commercial RNase A was eluted at near the solvent front by the lower phase mobile. The cSBL retained its native RNase activity. The overall results demonstrated that the present new small-scale X-axis CPC is useful for the purification of bioactive compounds without loss of their native activities.

New small-scale cross-axis coil planet centrifuge: The design of the apparatus and its application to counter-current chromatographic separation of proteins with aqueous–aqueous polymer phase systems

The cross-axis coil planet centrifuge ( X-axis CPC) is useful for partitioning macromolecules with aqueous–aqueous polymer phase systems. The floor model we have built with a pair of separation columns had some shortcomings such as requirement of large space, short life of the flow tubes, and difficulty in installing columns. In order to improve the partition efficiency and the utility of counter-current chromatography (CCC), a new small-scale X-axis CPC was designed and fabricated in our laboratory. The down-sizing of the apparatus was done by reducing the scale to about 1/2 of our original model of X-1.5 L type with several improvements. Performance of the apparatus was evaluated on protein separation using an aqueous–aqueous polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate with four multilayer coiled columns. A series of experiments revealed that the combination of right- and left-handed coils produced the best partition efficiencies for both lower and upper mobile phases by selecting the revolution direction. The overall results indicate that the head–tail elution mode substantially affects to the peak resolution and stationary phase retention. This new X-axis CPC would be useful for the separation of various kinds of biologically active compounds.

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000–6% (w/w) dextran T500 containing 10 mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0 ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500 mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.

Purification of alcohol dehydrogenase from bovine liver crude extract by dye–ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye–ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge ( x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000–7.0% dextran T500–0.1 M potassium phosphate buffers and 16% PEG 1000–12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000–potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6 h with minimum loss of its enzymatic activity.

PROTEIN SEPARATION BY CROSS-AXIS COIL PLANET CENTRIFUGE WITH SPIRAL COLUMN ASSEMBLIES

ABSTRACT A newly fabricated spiral column assembly was first applied to the countercurrent chromatographic separation of proteins using the cross-axis coil planet centrifuge (cross-axis CPC). The separation was performed by a set of stable proteins such as cytochrome C, myoglobin, and lysozyme with an aqueous–aqueous polymer phase solvent system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate. Three sets of left-handed single-layer spiral columns with different IDs (1.0, 1.5, and 2.0 mm) were employed to investigate the effect of column ID on partition efficiency at four different elution modes. The elution modes consisted of the combinations of the direction of revolution (PI=counterclock-wise; PII=clockwise), the head–tail elution mode (H=head to tail; T=tail to head), and the inward–outward elution mode (I=inward; O=outward). Among these experiments, the best separation of proteins was attained using a 1.5 mm-ID column assembly with the PI-H-O elution mode of the lower phase mobile. The resolution between cytochrome C and myoglobin peaks was 0.9 and between myoglobin and lysozyme peaks was 1.0, while the retention of the stationary phase was 32.7%. In order to improve the partition efficiency, the four-layer spiral column assembly with 1.0 mm-ID column tubing was applied to the protein separation. The resolution between these peaks was improved to 1.1 and 1.2, respectively, while the stationary phase retention decreased to 20.9%. Further studies using a single-layer spiral coiled column also revealed the effective peak resolution of proteins, but with lower stationary phase retention. The overall results demonstrated the spiral column assembly was useful for the protein separation using the cross-axis CPC with the aqueous-aqueous polymer phase system.

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540–15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540–ammonium sulfate polymer phase system by adding a retainer (10 m M acetic acid) to the upper stationary phase and an eluter (100 m M sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.

COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF WATER-SOLUBLE VITAMINS BY CROSS-AXIS COIL PLANET CENTRIFUGE USING AN ION-PAIR REAGENT WITH POLAR TWO-PHASE SOLVENT SYSTEM

Countercurrent chromatographic separation of water-soluble vitamins, such as riboflavin sodium phosphate, cyanocobalamin, riboflavin, pyridoxine hydrochloride, and thiamine nitrate was performed using the cross-axis coil planet centrifuge equipped with a pair of eccentric coil assemblies at off-center position. The solvent system used in the present studies was prepared by adding 1.5% of 1-octanesulfonic acid sodium salt, as an ion-pair reagent, to the polar two-phase solvent system composed of 1-butanol and aqueous 0.15 M monobasic potassium phosphate. The resolution was remarkably improved as compared with that obtained in our previous studies. Overall experimental results revealed that the cross-axis coil planet centrifuge is useful for the separation of extremely hydrophilic and ionic compounds, such as water-soluble vitamins, by adding a suitable ion-pair reagent to the polar two-phase solvent system.

COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF BIOTIC DICARBOXYLIC ACIDS WITH POLAR TWO-PHASE SOLVENT SYSTEMS USING CROSS-AXIS COIL PLANET CENTRIFUGE

Various aliphatic dicarboxylic acids were separated by countercurrent chromatography using cross-axis coil planet centrifuge (cross-axis CPC) equipped with a pair of eccentric coil assemblies mounted in an off-center position. Partition coefficients of these samples were optimized by adjusting the volume ratio of a polar two-phase solvent system composed of methyl t-butyl ether/1-butanol/acetonitrile/aqueous 0.1% trifluoroacetic acid. Maleic acid and fumaric acid were separated at a volume ratio of 5:0:0:5 by the lower aqueous phase mobile, while tartaric acid, succinic acid, and fumaric acid were well resolved by a more polar solvent system at a volume ratio of 2.9:0:2.9:4.3. Using the most polar binary solvent system composed of 1-butanol/water, oxalic acid, malonic acid, and succinic acid were resolved from each other by the lower aqueous phase mobile. The overall results of the present studies indicate that the cross-axis CPC is useful for the separation of various polar organic acids.

SEPARATION OF CHICKEN EGG WHITE PROTEINS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

Chicken egg white proteins were fractionated by high-speed counter-current chromatography (HSCCC) using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% polyethylene glycol (PEG) 1000 and 12.5% potassium phosphate buffer at different pH of 6.8, 8.0, and 9.2. From 20 g of the crude egg white solution, the best separation was achieved with a polymer phase system at pH 8.0 by eluting the lower phase at 1.0 mL/min. After the sequential elution of ovotransferrin, ovalbumin, and lysozyme from the column, ovomucin still retained in the column was eluted in the reverse direction with upper PEG-rich phase. All four proteins in the CCC fractions were identified by SDS gel electrophoresis with Coomassie brilliant blue staining.

ONE-STEP PURIFICATION OF CHOLINESTERASE FROM HUMAN SERUM BY CCC

The type XL cross-axis coil planet centrifuge (CPC) was successfully utilized for purification of cholinesterase (ChE) from human serum by countercurrent chromatography (CCC). Aqueous-aqueous polymer phase systems composed of polyethylene glycol-potassium phosphate buffers were tested at various pHs, ranging from 6.8 to 9.2 for the distribution ratios (D) of ChE, human serum albumin, α- and γ-globulins. The CCC separation was performed with optimized polymer phase systems in two different coiled columns. The best purification was achieved with 16.0% PEG 600–12.5% potassium phosphate at pH 9.2, by eluting the lower phase at 0.5 mL/min through the small capacity column in which ChE was purified directly from human serum within 2h. The ChE fractions confirmed by the enzymatic activity were free of serum proteins by SDS PAGE analysis.

Counter-current chromatographic separation of glycoprotein components from Morchella esculenta (L.) with a polymer phase system by a cross-axis coil planet centrifuge

Using a cross-axis coil planet centrifuge, glycoproteins were separated from fermentation media of Morchella esculenta (L.) by high-speed counter-current chromatography. The performance of the apparatus was optimized with four standard proteins including pepsin, lysozyme, ovalbumin and hemoglobin and a polymer phase system composed of 12.5% (w/w) polyethylene glycol 8000 and 25% (w/w) potassium phosphate in distilled water at various pH values. Separations were performed by eluting the lower phosphate-rich phase at a flow-rate of 1.0 ml/min. Under the optimized conditions three glycoprotein components in Morchella esculenta (L.) were resolved within 6 h.

COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF VITAMINS BY CROSS-AXIS COIL PLANET CENTRIFUGE WITH ECCENTRIC COIL ASSEMBLIES

Vitamins were resolved by high-speed countercurrent chromatography using a cross-axis coil planet centrifuge equipped with eccentric coil assemblies. A two-phase solvent system composed of 1-butanol/ethanol/aqueous 0.15 M monobasic potassium phosphate (8:3:8) was used for the separation of water-soluble vitamins such as thiamine nitrate, pyridoxine hydrochloride, and nicotinamide, while 2,2,4-trimethyl pentane (isooctane)/methanol (1:1) was used for the separation of fat-soluble vitamins such as calciferol, vitamin A acetate, and (±)-α-tocopherol acetate. Overall results of experiments revealed that the cross-axis coil planet centrifuge is useful for the separation of vitamins by selecting suitable two-phase solvent systems.