Counter-current Chromatography Separation Research Articles

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.

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.

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.

High-speed countercurrent chromatography for separation of americium from lanthanoids

The separation of americium from lighter lanthanoids has been performedby high-speed countercurrent chromatograph (CCC) equipped with a small coiledcolumn. An extractant having higher affinity for americium, 5,8-diethyl-7-hydroxydodecan-6-oneoxime (LIX 63) was employed as a stationary phase. Americium was chromatographicallyfractionated by gradient elution from large amounts of lighter lanthanoidsthrough a coiled column.

High-speed counter-current chromatography separation and purification of resveratrol and piceid from Polygonum cuspidatum

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of resveratrol and piceid from the dried roots (20.0 g) of Polygonum cuspidatium. The EtOAc extracts were separated with chloroform–methanol–water (4:3:2, v/v). Resveratrol was identified in fraction 5. The water extracts were separated first with EtOAc–EtOH–water (10:1:10, v/v) and then with the same solvent system at the modified volume ratio of 70:1:70. Yields of resveratrol and piceid obtained were 2.18% and 1.07%. Chemical structures of the purified resveratrol and piceid were confirmed by electrospray ionization MS and 1H nuclear magnetic resonance spectroscopy.

COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF HIPPURIC ACID AND RELATED COMPOUNDS USING CROSS-AXIS COIL PLANET CENTRIFUGE WITH ECCENTRIC COIL ASSEMBLIES

Countercurrent chromatographic separation of hippuric acid (HA) and related compounds was performed using the cross-axis coil planet centrifuge equipped with a pair of eccentric coil assemblies mounted in an off-center position. Partition coefficients of HA and a set of related compounds were determined on a polar two-phase solvent system composed of methyl t-butyl ether, 1-butanol, acetonitrile and aqueous 0.1% trifluoroacetic acid at various volume ratios. The optimal volume ratio of 1 : 0 : 0 : 1 was successfully used to resolve p-amino HA, HA, p-methyl HA, and (±)-mandelic acid by the lower aqueous phase mobile, and benzoic acid, p-methyl HA and HA by the upper organic phase mobile.

Analysis of simulated moving-bed reactors

The simulated moving-bed reactor (SMBR) combines continuous countercurrent chromatographic separation with chemical reaction. This is a hybrid process, not energy-intensive and is competitive with traditional processes in which reaction and separation are carried out in different devices. The ideal application of this technology is to reactions where the once-through conversion is limited by chemical equilibrium. In this case SMBRs can achieve simultaneously the goals of complete conversion and separation of the products. Examples are esterifications, transesterifications, etherifications, acetilations, some isomerisations, hydrogenations, some enzyme reactions and others. A complete analysis of the behaviour of SMBs without chemical reaction is available. SMBR units are more complex than SMBs and more properties have to be accounted for, such as the reaction rate and the equilibrium conversion. The purpose of this work is to illustrate the behaviour and develop a design strategy for SMBRs, based on our understanding of the corresponding non-reactive units.

HPCPC Separation of Proteins using Polyethylene Glycol-Potassium Phosphate Aqueous Two-Phase

ABSTRACT High Performance Centrifugal Partition Chromatography (HPCPC) is a practical and suitable method, particularly on the preparative scale, for the separation of biomolecules such as proteins, enzymes, etc. Aqueous two-phase system is also very attractive for the isolation of biomolecules. Aqueous polymer phase system composed of polyethylene glycol 6000-potassium phosphate has been used for the countercurrent chromatographic separation of bovine serum albumin (BSA) and lysozyme using HPCPC. The separation of BSA and lysozyme under various conditions such as various flow rates, rotational speeds, pH of the solvent, and the retention of stationary phase has been studied in the present investigation. The baseline separation of BSA and lysozyme has been also observed. The results of this study demonstrate that HPCPC is useful for separation of proteins with aqueous two-phase systems.

Countercurrent Chromatographic Separation of Proteins by Cross-Axis Coil Planet Centrifuge: Choice of Polymer Phase Systems and Revolution Speed

Countercurrent chromatographic separation of proteins by the cross-axis coil planet centrifuge was maximized by selecting the suitable polymer phase system and revolution speed. Polymer phase systems composed of polyethylene glycol (PEG) 1000 and several inorganic salts were examined to determine partition coefficient values (K) for various proteins. The overall results indicated that the polymer phase system composed of 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate yielded suitable K values for most proteins except for cytochrome C and apo-transferrin which may be separable with a solvent system composed of 12.5% (w/w) PEG 1000 and 24% (w/w) potassium citrate. A series of experiments with the PEG 1000 - potassium phosphate system under various revolution speeds revealed that the best separation was achieved at 850 rpm. The above optimized conditions may be applied to separations of other protein samples.

Recycling High-Speed Countercurrent Chromatography for Separation of Taxol and Cephalomannine

A mixture of taxol and cephalomannine was subjected to recycling countercurrent chromatography (CCC) which allows the use of the same column for repetitive separation of the peak fractions to improve the peak resolution. When the total amount of 50 mg of the two components was recycled twice, peak resolution (Rs) increased from 0.7 to 1.27. The results also showed that Rs value increases according to the formula: Rs-n = n1/2Rs-1 where n is the number of CCC runs, and Rs-1 and Rs-n, peak resolutions after the first and the nth run, respectively.

Polyethylene Glycol-Potassium Phosphate Aqueous Two-Phase Systems for Countercurrent Chromatography of Proteins

Aqueous-aqueous polymer phase systems composed of polyethylene glycol 1000-potassium phosphate were used for the countercurrent chromatographic separation of proteins using the type XL cross-axis coil planet centrifuge. It was found that the peak resolution of proteins is highly dependant on the pH of the solvent system. The best separation of cytochrome c, myoglobin and human serum albumin was achieved with 16.0% PEG 1000-12.5% potassium phosphate buffer at pH 9.2. The resolution and separation time were improved by pH-peak-focusing countercurrent chromatography.

Toroidal Coil Countercurrent Chromatography Separation of Staphylococcus aureus Enterotoxin A in Food

Countercurrent Chromatography (CCC) has the potential to play a major role in food analysis because it permits analysis of crude and complex samples. CCC was evaluated for its ability to separate S. aureus enterotoxin A (SEA), which is a common cause of food poisoning. Mushrooms containing native- or heat-denatured SEA were separated by Toroidal Coil CCC and the fractions containing the toxin were analyzed by Western immunoblotting. Both native SEA, used to spike mushroom samples, and heat-denatured SEA, resulting from S. aureus contamination of canned mushrooms, were detectable using CCC. Ths method increases the level of sensitivity of Western immunoblottmg by at least an order of magnitude by concentrating the sample. Our results suggest that CCC, in combination with a suitable detection method. is a potentially useful method for toxin analysis in food.

Continuous chromatographic separation process: simulated moving bed allowing simultaneous withdrawal of three fractions

Continuous counter-current chromatographic separation has been carried out in a simulated moving bed system (SMB). We have worked with a SMB pilot plant (8 columns, 4.4 litres of resin each) which allows the continuous withdrawal of two different fractions. A mixture of glucose-fructose has been separated. To calculate the concentration profile within the separator an axial dispersed plug flow model and an equilibrium stage model have been employed; software has been created to simulate the behaviour of the separator. The necessary parameters of the mode: the adsorption equilibrium constant, the height equivalent to a theoretical plate and the bed voidage, have been acquired experimentally from elution chromatography measurements. The results calculated by simulation give a good representation of the experimental concentration profiles; other separations like xylitol-arabitol have been simulated. The influence of some factors like desorbent flow-rate, feed flow-rate and the bed voidage have been studied using the software. Once the system has worked in a two withdrawal way, an extension of the pilot plant has been constructed so as to obtain a third one. The necessary parameters of the three withdrawal model will be studied.

Modelling studies on the transient and steady state behaviour of a simulated counter-current chromatographic system

A simulated counter-current chromatographic (SCC) separation process is simulated using a dispersed plug flow model with a direct approach. The solution of the governing partial differential equations is solved with a numerical technique based on the method of orthogonal collocation. Mass transfer resistances and axial dispersion are accounted for in the model. This model is used to simulate the separation of praziquantel enantiomers in the SCC system. The model accounts fully the sequential operation of the system. The transient and steady state behaviour of the separation of praziquantel enantiomers by this chromatographic system can be described adequately by this model.

Counter-current chromatographic separation of polyunsaturated fatty acids

Hexadecatrienoic acid (C16:3), octadecatetraenoic acid (C18:4), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) were purified in two steps by counter-current chromatography (CCC). The selected liquid phases were heptane-acetonitrile-water for the first step and heptane-methanol-water for the second. The upper non-polar phase was kept stationary in the coil of the instrument by a rotational force field. The separation was followed by UV detection at 210 nm and by analysing the eluted fractions by gas chromatography after esterification. The purity of the fatty acids therefore purified was checked by capillary gas chromatograpy. CCC may be an alternative to HPLC for the preparative-scale purification of such compounds.

High-speed counter-current chromatographic separation of biflavanoids from Garcinia kola seeds

Garcinia kola Heckel (Guttiferae) known in commerce as “bitter kola” is used extensively in Nigeria and Ghana for the treatment of coughs, mouth infection, liver disorders and antidote for arrow poisons. In our studies extracts of seed displayed appreciable activity against several selected microorganisms. To identify the antimicrobial compounds and study other biological effects of G. kola and its constituents, we have separated the constituents of the ethyl acetate extract of defatted seeds by high-speed counter-current chromatography. Using this technique with the solvent system n-hexane-ethyl acetate-methanol-water (1:4:2.5:2.5, upper phase as the stationary phase and the lower phase as the mobile phase), the ethyl acetate extract provided seven products. Four of these have been characterized as 3–8 linked biflavanoids, kolaflavanone, GB-1, GB-1a and GB-2 by spectral data.

Discrepancy Between the Theoretical Plate Number (N) and Peak Resolution (Rs) for Optimizing the Flow Rate in Countercurrent Chromatography

Abstract This paper deals with a problem in optimizing the flow rate of the mobile phase in countercurrent chromatography (CCC), A set of data including theoretical plate number (N), resolution factor (Rs), % retention, etc. are obtained from the separation of three indole auxins in a two-phase solvent system of n-hexane / ethyl acetate / methanol / water (1:1:1:1) by applying the flow rates of the mobile phase at 0.5- 8.0ml/min. The experimental results revealed a disagreement between the efficiencies expressed in parameters N and Rs at the high flow rates of the mobile phase in the CCC separation. Namely, the partition efficiency expressed in terms of Rs decreases with an increased flow rate of the mobile phase, while the N rises with increasing the flow rate. Since Rs represents the actual separation of the solute peaks, the results indicate that the N used in high performance liquid chromatography is not always a reliable measure in CCC separation. However, the application of a high flow rate yields b...

High-Speed Countercurrent Chromatography Separation of Taxol and Related Diterpenoids fromTaxus Baccata

Abstract Taxol and related taxane diterpenoids were isolated from pre-purified extracts of stem-barks of Taxus bacccata by HSCCC. The described method allowed to obtain a mixture of taxol and cephalomannine without any other interfering constituent and furthermore a partial separation of taxol (40 % of the total recovery) from cephalomannine. In addition, the HPLC and TLC separation conditions of these compounds for the semi-preparative and analytical purposes were improved. The proposed HSCCC method could be adapted to the scaling-up separation of taxol from vew material.

Purification of Some Water Soluble Azo Dyes by High-Speed Countercurrent Chromatography

Abstract High-speed countercurrent chromatography (CCC) has been used to prepare preparative amounts of pure magenta azo dyes by a method involving the continuous extraction of an aqueous solution of the dyes by a suitable mobile phase. This slight variation in the normal way of running CCC separations permits both the removal of the impurities from the desired color component and an increase in the quantity of dye that can be purified in a single experiment.