Scale-up of protein purifications using aqueous two-phase systems: comparing multilayer toroidal coil chromatography with centrifugal partition chromatography

Two different laboratory scale liquid-liquid extraction processes using aqueous two-phase systems (ATPS) are compared: centrifugal partition chromatography (CPC) and multilayer toroidal coil chromatography (MTCC). Both use the same phase system, 12.5% (w/w) PEG-1000:12.5% (w/w) K(2)HPO(4), the same flow rate of 10 mL/min and a similar mean acceleration field of between 220 × g and 240 × g. The main performance difference between the two processes is that there is a continuous loss of stationary phase with CPC, while for MTCC there is not - even when sample loading is increased. Comparable separation efficiency is demonstrated using a mixture of lysozyme and myoglobin. A throughput of 0.14 g/h is possible with CPC despite having to refill the system with stationary phase before each injection. A higher throughput of 0.67 g/h is demonstrated with MTCC mainly due to its ability to tolerate serial sample injections which significantly reduces its cycle time. While CPC has already demonstrated that it can be scaled to pilot scale, MTCC has still to achieve this goal.     

Fractionation of low-molecular-mass heparin by centrifugal partition chromatography in the ion-exchange displacement mode

Centrifugal partition chromatography in the ion-exchange displacement mode allowed a preparative and efficient fractionation of low-molecular-mass heparins from enoxaparin sodium. Amberlite LA2--a lipophilic liquid secondary amine--was chosen as a weak anion exchanger. The biphasic system methyl isobutyl ketone-water was selected. Protonated LA2 (10%, v/v) was added to the organic stationary phase. Hydroxide (Na+, OH-) was chosen as a displacer in the aqueous mobile phase. The observed pH and concentration profiles are typical of displacement chromatography, as supported by numerical simulation. The Dubois test for the analysis of sugar content and an analysis of sulfur content (and consequently sulfatation rate) were carried out to monitor the effectiveness of the procedure. Moreover, the fractions were analyzed by high-performance size-exclusion chromatography and the 1H NMR spectra confirmed the fractionation of the sample of enoxaparin sodium.     

Rapid linear scale-up of a protein separation by centrifugal partition chromatography

The scaling up of the separation of two proteins with an aqueous two-phase system (ATPS) from 176 mg with a 500 ml laboratory scale centrifugal partition chromatography (CPC) column to 2.2g with a 6.25 litre pilot-scale column is presented. A model sample system of a mixture of lysozyme and myoglobin was chosen for this study using an ATPS system comprising 12.5% (w/w) PEG-1000:12.5% (w/w) K2HPO4. It was found that the maximum sample concentration possible without precipitation was 2.2mg/ml for each constituent. The optimisation of rotor speed, mobile phase flow rate and sample loading was performed on a laboratory-scale device. It was found that a centrifuge speed of 2000 rpm (224 'g'), 10 ml/min mobile phase flow rate with a 43 ml (10% of active column volume) sample volume gave optimum operating conditions. This was linearly scaled up to pilot scale by increasing mobile phase flow rate, fraction size and sample loading in the ratio of the system capacities (i.e. 12.5:1). Flow rate was therefore increased from 10 ml/min to 125 ml/min, fraction size from 10 ml to 125 ml and sample loading from 43 ml to 500 ml. Rotor speed however was reduced from 2000 rpm on the laboratory device to 1293 rpm on the pilot-scale device to maintain the same 224 'g' field in each chamber, as the pilot-scale CPC unit has a larger rotor radius than the laboratory one. Resolution increased from Rs=1.28 on the 500 ml rotor to Rs=1.88 on the 6.25 litre rotor, giving potential throughputs in batch mode of over 40 g/day.     

A convenient separation strategy for fungal anthraquinones by centrifugal partition chromatography

As recently shown, some fungal pigments exhibit significant photoactivity turning them into promising agents for the photodynamic treatment of microbial infections or malignant diseases. In the present study, a separation strategy for fungal anthraquinones was developed based on centrifugal partition chromatography. A suitable method was explored employing a methanolic extract of the fruiting bodies of Cortinarius sanguineus (Agaricales, Basidiomycota). An excellent fractionation was achieved using a biphasic solvent system comprising chloroform/ethyl acetate/methanol/water/acetic acid (3:1:3:2:1, v/v/v/v/v) operating in ascending mode. Experiments on an analytical scale with extracts of closely related Cortinarius species exhibited broad applicability of the devised system. Up to six pigments could be purified directly from the crude extract. Preparative-scale fractionation of the methanol extracts of C. malicorius and C. sanguineus demonstrated that up-scaling was possible without compromising selectivity.     

Separation of xanthones and a biphenyl fromKielmeyera coriacea by centrifugal partition chromatography

Summary Eight xanthones and a biphenyl have been isolated from the dichloromethane extract ofKielmeyera coriacea (Guttiferae) stem bark by a combination of centrifugal partition chromatography and gel filtration on Sephadex LH-20, with HPLC-UV monitoring of fractions. The structures of the isolated compounds (Figure 1) were established by spectroscopic methods (UV, EI-MS, D/CI-MS,¹H NMR,¹³C NMR).     

Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode.

Centrifugal partition chromatography (CPC) in the pH-zone refining mode allowed a preparative and efficient isolation of vindoline, vindolinine, catharanthine and vincaleukoblastine from a crude mixture of Catharanthus roseus alkaloids. The separation protocol was tested with a synthetic mixture of vindoline, catharanthine and vincaleukoblastine. The fraction content was analyzed and the results compared with theoretical chromatograms obtained by numerical simulation. The increase in injected sample mass results in an improvement of the purity of the isolated compounds. This observation, confirmed by theory, is of prime importance for the development of preparative pH-zone refining CPC as a preparative separation method.     

Optimisation of the derivatization in cellulose-type chiral selectors for enantioseparation by centrifugal partition chromatography

Cellulose was chemically modified with hydrophobic dodecanoyl groups followed by 3,5-dimethylphenylcarbamoyl substituents forming mixed ester/carbamate derivatives in order to improve the solubility in lipophilic solvents compared to the corresponding homosubstituted cellulose tris(3,5-dimethylphenylcarbamate). Two mixed derivatives of different degree of substitution were prepared, tested as chiral selectors (CSs) in counter-current chromatography (CCC) and compared to the homo-substituted derivative. Alternatively, cellulose tris(3,5-dichlorophenylcarbamate), was synthesised and tested with the same purpose. The racemic drugs pindolol and warfarin were used as test compounds. Biphasic organic/aqueous solvent systems, composed of methyl isobutyl ketone, t-butyl methyl ether or ethyl acetate and aqueous ammonium acetate or sodium phosphate buffer, were used. Centrifugal partition chromatography, a variety of CCC, in the classical elution mode and the pH-zone-refining displacement mode was applied. The enantioseparation of pindolol and warfarin was achieved in the latter conditions. The presence of dodecanoyl chains on the CS increased solubility in the organic solvents used. The selectivity of the mixed dodecanoyl/3,5-dimethylphenylcarbamoyl cellulose derivative with low degree of dodecanoyl groups (degree of substitution 0.3/degree of substitution 2.6, respectively) was similar to that of the homo-substituted derivative. Furthermore, the loading capacity for pindolol was increased by a factor of three compared to cellulose tris(3,5-dimethylphenylcarbamate). Nevertheless, the increasing degree of substitution with dodecanoyl groups on the CS, although improved solubility in the stationary phase, contributed negatively to the enantioselectivity, where warfarin was more affected than pindolol.     

Strong ion-exchange centrifugal partition chromatography as an efficient method for the large-scale purification of glucosinolates

The glucosinolates sinalbin and glucoraphanin were purified by strong ion-exchange displacement centrifugal partition chromatography (SIXCPC). The optimized conditions involved the biphasic solvent system ethyl acetate/n-butanol/water (3:2:5, v/v), the lipophilic anion-exchanger Aliquat 336 (trioctylmethylammonium chloride, 160 and 408 mM) and a sodium iodide solution (80 and 272 mM) as displacer. Amounts as high as 2.4 g of sinalbin and 2.6g of glucoraphanin were obtained in one step in 2.5 and 3.5h respectively, starting from 12 and 25 g of mustard and broccoli seed aqueous extracts, using a laboratory scale CPC column (200 mL inner volume).     

Purification by centrifugal partition chromatography of amphiphilic compounds, glycolipids and pseudo-glycolipids synthesized by using cells

Centrifugal partition chromatography (CPC) was applied to separate amphiphilic glycolipids and pseudo-glycolipids synthesized by using cells. Neutral and acidic lipid fractions were isolated by CPC under suitable conditions respectively. Separation of neutral lipid, Gb3-type and Gb4-type oligosaccharide synthesized by using cells, was performed with a two-phase solvent system composed of chloroform-methanol-water at a volume ratio of 5:6:4. On the other hand, separation of acidic lipid, GM3-type oligosaccharide synthesized by using cells, and ganglioside extracted from rat brain were performed with a two-phase solvent system composed of butanol-ethanol-1% acetic acid at a volume ratio of 4:1:5. 8.3mg of Gb3 analogue, 5.1mg of Gb4 analogue, and 19.5mg of GM3 analogue were purified from 3.2l of culture medium obtained by incubation of African green-monkey kidney (Vero) cells with 50 microM n-dodecyl beta-lactoside using CPC.     

Separation of protoberberine quaternary alkaloids from a crude extract of Enantia chlorantha by centrifugal partition chromatography

High-performance centrifugal partition chromatography (HPCPC) has been successfully applied to the separation of four protoberberine quaternary alkaloids, namely palmatine, jatrorrhizine, columbamine and pseudocolumbamine, from a methanolic extract (M1, 1.47 g) of Enantia chlorantha Oliver stem bark. For their isolation, two successive biphasic solvent systems composed of dichloromethane-methanol-water (48:16:36, v/v) were selected. The aqueous-rich phase was the stationary phase and the organic-rich phase was the mobile phase. The first system, containing potassium perchlorate, allowed to isolate 600 mg of palmatine, and to obtain 146 mg of a mixture (M2) containing only jatrorrhizine, columbamine and pseudocolumbamine. The second biphasic system, prepared with water alkalinized with sodium hydroxide, was employed to isolate the M2 components. This system applied to the purification of 70 mg of M2 allowed to obtain 16 mg ofjatrorrhizine and 13 mg of columbamine. To obtain pseudocolumbamine (16 mg), the elution mode was reversed, the aqueous-rich phase becoming the mobile phase, and the organic-rich phase becoming the stationary one. Analytical reversed-phase high-performance liquid chromatography, NMR, high-resolution mass spectrometry and UV spectrometry were used to verify the identity and the purity of the isolated compounds.     

Salting-out gradients in centrifugal partition chromatography for the isolation of chlorogenic acids from green coffee beans

In addition to sample solubility constraints, the use of polarity gradients in normal-phase centrifugal partition chromatography (CPC) for the purification of complex mixtures is also limited by the instability of biphasic systems as a consequence of dramatic changes in the settling times along the gradient, leading in many cases to column bleeding when working under maximum efficiency conditions. In this paper an electrostriction approach is proposed as a strategy in reversed-phase CPC to fractionate intermediate polarity extracts in a single run by bringing its components into the “sweet spot” in a controlled fashion through a stepwise reduction of salt concentration in the aqueous mobile phase. The salting-out gradient method was successfully tested with the separation of the major chlorogenic acids (CGAs, hydroxycinnamoylquinic acids) present in green coffee beans (5-caffeoylquinic acid (5-CQA), 5-feruloylquinic acid (5-FQA) and 3,5-dicaffeoylquinic acid (3,5-diCQA)) using ethyl acetate-hexane as the stationary phase and an ionic gradient of LiCl (5.0, 2.5 and 0.1 M) as the mobile phase in one case and (NH₄)₂SO₄/KNO₃ (3.0 and 1.5 M/1.5 M) in another. Regioisomers of each chlorogenic acid obtained by base-catalyzed isomerisation were also separated by CPC using isocratic elution. The best resolution for both FQAs and diCQAs was achieved with a chloroform–n-butanol–0.01 M pH 2.5 phosphate buffer (84:16:100; v/v) system, while CQAs were best isolated using chloroform–n-butanol–0.01 M pH 2.5 phosphate buffer/5.0 M LiCl (82:18:100; v/v).     

Gradient elution method in centrifugal partition chromatography for the separation of a complex sophorolipid mixture obtained from Candida bombicola yeasts

Sophorolipids represent an important class of natural surfactants with a variety of environmental, cosmetic, and pharmaceutical applications. Despite their promising physicochemical and biological properties, the use of sophorolipids is hampered by the lack of information regarding their individual structure‐activity relationships. The major difficulty in isolating pure sophorolipids arises from the high complexity of crude fermentation media composition and from their strong structural similarities. In this work, a centrifugal partition chromatography method was developed in an original gradient elution mode for the separation of sophorolipids produced by the yeast Candida bombicola. Experiments were realized by using three sets of solvent systems composed of n‐heptane, ethyl acetate, n‐butanol, methanol, and water in different proportions. The separation was performed at 5 mL/min in the ascending mode by increasing progressively the polarity of the organic mobile phase. In these conditions, more than 80% of the sophorolipids present in the initial crude fermentation extract were eluted successively from the most hydrophobic lactone forms to the most hydrophilic acid forms. The structures of the isolated sophorolipids were further elucidated by HPLC and NMR analyses.     

Development of hybrid elution systems for efficient purification of stilbenoids using centrifugal partition chromatography coupled to mass spectrometry

The phytochemical study of the root extract of the stilbenoid-rich Vitis riparia × Vitis berlandieri grapevine was carried out by centrifugal partition chromatography (CPC). For this reason, we developed a new elution mode we named back-step, which allowed us to obtain cleaner fractions and a more efficient separation process when used in conjunction with a classical elution approach. Three hydroxystilbenes: (E)-resveratrol, (E)-?-viniferin and (E)-vitisin C, with greater than 90% purity were thus obtained through such process, with minimal sample handling and purification steps. Online coupling of CPC to ESI mass spectrometry was used for optimization of the separation parameters and to facilitate the characterization of the stilbenoids. This study details the first phytochemical investigation of stilbenoids from the hybrid species together with a new elution mode able to widen the range of ARIZONA biphasic systems.     

Multiple dual-mode countercurrent chromatography applied to chiral separations using a (S)-naproxen derivative as chiral selector

Countercurrent chromatography (CCC) is a liquid–liquid chromatographic technique without a solid support. Several alternative elution modes can be applied to take advantage of the special nature of the liquid stationary phase. Among these dual-mode (DM) and multiple dual-mode (MDM) consist of switching alternatively between Reversed and Normal Phase operation during the experiment (once for DM and several times for MDM). In this paper, MDM has been applied to the chiral CCC separations of two racemic mixtures, (±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide and N-(3,5-dinitrobenzoyl)-(±)-leucine, using (S)-naproxen N,N-diethylamide as chiral selector (CS). Although the behaviour of the two analytes differed, improved resolution factors were successfully obtained. Results are rationalized on the basis of the distinct partition behaviour of the CS/enantiomer complexes in the biphasic system.     

pH‐Zone‐refining centrifugal partition chromatography for preparative isolation and purification of steroidal glycoalkaloids from Solanum xanthocarpum

pH‐Zone‐refining centrifugal‐partition chromatography (CPC) was successfully applied in the separation of complex polar steroidal glycoalkaloids of close Rf values, directly from a crude extract of Solanum xanthocarpum. The experiment was performed with a two phase solvent system composed of ethyl acetate/butanol/water (1:4:5 by volume) where triethylamine (5 mM) was added to the upper organic mobile phase as an eluter and TFA (10 mM) to the aqueous stationary phase as a retainer. Separation of 1 g of crude extract over CPC resulted in two distinct pH‐zones. The fractions collected in pH‐zone i afforded 72 mg of solasonine while the fractions collected in pH‐zone ii were slightly impure, hence were purified over medium pressure LC, which afforded 30 mg of solasonine and further 15 mg of solamargine (SM). The steroidal glycoalkaloids, SM and solasonine were isolated in 93.3 and 91.6% purity, respectively. The isolated alkaloids were characterized on the basis of their ¹H, ¹³C‐NMR, and ESI‐MS data.     

Hyphenation of centrifugal partition chromatography with electrospray ionization mass spectrometry using an active flow‐splitter device for characterization of flavonol glycosides

Online coupling of centrifugal partition chromatography to electrospray ionization mass spectrometry (CPC/ESI‐MS) was investigated for the separation and characterization of flavonol glycosides. Structural identification and purification monitoring of analytes on milligram scale were demonstrated to be possible by using an active flow‐splitter device which transfers automatically and successively, at discrete frequencies, small aliquots of the chromatographic effluent to an independent auxiliary stream directed to an ESI quadrupole mass spectrometer. The CPC protocol used a biphasic solvent system composed of ethyl acetate/ethanol/water (4.5:1:4.5, v/v/v) in isocratic mode. During the separation process, continuous acquisition of mass spectral data of the isolated flavonols from the effluent was performed in the negative ion mode with an auxiliary stream composed of 50 mM ammonium acetate/ethanol (2:8, v/v) delivered by a secondary pump. To demonstrate the potential of this hyphenated technique, flavonol glycosides from an apple peel extract were identified, purified and quantitatively analyzed. Calibration curves and limits of detection are also detailed. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of centrifugal partition chromatography coupled with evaporative light scattering detection for the isolation of saikosaponins‐a and ‐c from Bupleurum falcatum roots

Centrifugal partition chromatography (CPC) coupled with evaporative light scattering detection (ELSD) was applied to separate saikosaponins‐a and ‐c preparatively from Bupleurum falcatum roots. The two‐phase solvent system composed of ethyl acetate/n‐butanol/methanol/water (15:1:3:15 by volume) was used to yield saikosaponins‐a (36.1 mg) and ‐c (28.7 mg) from 370 mg of saponin‐rich extract. The purities of isolated compounds were 96.6 and 97.3% for saikosaponins‐a and ‐c, respectively. Structure identification of these compounds was accomplished by comparison of spectroscopic data of ESI‐MS, ¹H NMR, and ¹³C NMR with those of previously reported values.     

New biphasic solvent system based on cyclopentyl methyl ether for the purification of a non‐polar synthetic peptide by pH‐zone refining centrifugal partition chromatography

A new type 1 ternary biphasic system composed of cyclopentyl methyl ether, dimethylformamide and water was developed, characterized and successfully used for the purification of a lipophilic, protected peptide by pH‐zone refining centrifugal partition chromatography. The protected peptide is an 8‐mer, key intermediate in bivalirudin (Angiomax®) synthesis and shows a very low solubility in the solvents usually used in liquid chromatography. All ionic groups, except the N‐terminal end of the peptide, are protected by a benzyl group. The purification of this peptide was achieved with a purity of about 99.04% and a recovery of 94% using the new ternary biphasic system cyclopentyl methyl ether/dimethylformamide/water (49:40:11, v/v) in the descending pH‐zone refining mode with triethylamine (28 mM) as the retainer and methanesulfonic acid (18 mM) as the eluter.