Separation and purification of xylose oligomers using centrifugal partition chromatography

Xylose oligomers, which have a prebiotic effect, have been used as additives to human and animal food. These oligomers are also the primary intermediate in hemicellulose degradation during the pretreatment of biomass. Centrifugal partition chromatography (CPC) was used in this study to separate and purify xylan-derived oligomers from birchwood xylan. The xylan was partially hydrolyzed to achieve varying degrees of polymerization at 130°C using 0.98% aqueous sulfuric acid for 20 min with a 2.5% solid loading. The CPC solvent system consisting of dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and water in a 1:6:3 volumetric ratio was used because of its ability to dissolve xylose oligomers of different degrees of polymerization. The CPC was operated in the ascending mode with the water- and DMSO-rich bottom phase acting as the stationary phase, while the THF-rich top phase was the eluent. This paper delineates a method for the production and purification of xylose monomer and xylose oligomers (up to xylopentaose) using CPC. The amount and purity of compounds collected from the CPC fractionation based on 1 g of birchwood xylan were 25.26 mg of xylose at 91.86% purity, 10.71 mg of xylobiose at 85.07% purity, 4.15 mg of xylotriose at 54.71% purity, 5.03 mg of xylotetraose at 38.33% purity and 3.31 mg of xylopentaose at 30.43% purity.     

Preparative isolation and purification of geniposide from gardenia fruits by centrifugal partition chromatography

The iridoid glycoside, geniposide was purified by centrifugal partition chromatography (CPC) with a two-phase solvent system composed of ethyl acetate:isopropanol:water (3:2:5, v/v) from an 80% methanolic extract of fruits of Gardenia jasminoides. Preparative CPC yielded 56.2 mg of geniposide in a one-step separation of 500 mg of extract, with a purity of 95% as determined by HPLC. Isolated geniposide was identified from its 1H-NMR, 13C-NMR and MS spectra.     

Large scale purification of factor X by hydrophobic chromatography

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.     

Large scale purification of plasmid DNA

A simple and rapid procedure for large scale purification of plasmid DNA is described. The procedure consists of two main steps: 1. Alkaline extraction of plasmid DNA (by a slight modification of the method of Birnboim and Doly (1)) and 2. Purification of the crude extract by hydroxyapatite chromatography. The plasmids obtained are biologically active and can be used in gene manipulation experiments.     

Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS

The development of new antiplasmodial drugs is of primary importance due to the growing problem of multi-drug resistance of malaria parasites. Spilanthes acmella, a plant traditionally used for the treatment of toothache, was targeted as a lead for its potential antiplasmodial activity. A systematic approach for investigating a suitable centrifugal partition chromatography (CPC) solvent system for N-alkylamides separation was reported. The partition behavior of three N-alkylamides has been studied using several biphasic solvent mixtures in search of an adequate CPC solvent system for this class of compounds. Major N-alkylamides in S. acmella were isolated from a methanolic crude extract of flowers by CPC with the solvent system heptanes-ethyl acetate-methanol-water (3:2:3:2, v/v/v/v). Four N-alkylamides were purified and the structures were illustrated by electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), 1H nuclear magnetic resonance (1H NMR) and 13C nuclear magnetic resonance (13C NMR). The CPC fractions, which contained natural mixtures of phytochemicals, demonstrated significantly higher antiplasmodial activity compared to corresponding purified N-alkylamides, thus suggesting that interactions between these N-alkylamides may potentiate antiplasmodial bioactivity.     

Large scale purification of human fibroblast interferon

Human fibroblast interferon was partially purified, about 4,000-fold, on a chromatographic tandem of columns: concanavalin A-agarose leads to phenyl-agarose, to a specific activity of ca 4 x 10(7). The overall recovery of interferon activity was ca 60%.     

Large scale purification of rapeseed proteins (Brassica napus L.)

Rapeseed (Brassica napus L.) cruciferin (12S globulin), napin (2S albumin) and lipid transfer proteins (LTP) were purified at a multi-g scale. The procedure developed was simple, rather fast and resolutive; it permitted the recovery of these proteins with a good yield, such as 40% for cruciferin and 18% for napin. Nanofiltration eliminated the major phenolic compounds. The remaining protein fraction was fractionated by cation exchange chromatography (CEC) on a streamline SP-XL column in alkaline conditions. The unbound neutral cruciferin was polished by size exclusion chromatography. The alkaline napin isoforms and LTP, adsorbed on the beads, were eluted as a whole fraction and further separated by an other CEC step at acidic pH. Napins were polished by hydrophobic interaction chromatography (HIC). The fractions were characterized by reverse phase HPLC, electrophoresis, N-terminal sequencing and mass spectrometry. All the fractions contained less than 5% of impurities.     

Preliminary report on fractionation of fucans by ion-exchange displacement centrifugal partition chromatography

A new method combining ion-exchange displacement chromatography with centrifugal partition chromatography (CPC) was used for the fractionation of partially depolymerized fucans (polysulphated polysaccharides). The ion-exchanger was Amberlite LA2, a high-molecular-mass liquid secondary amine miscible with most common organic solvents and immiscible with aqueous solutions. Ion-exchange displacement centrifugal partition chromatography was performed with LA2 in methyl isobutyl ketone (MiBK) as the stationary phase, water as the mobile phase, Cl⁻ as the carrier and OH⁻ as the displacer. A complex mixture of partially depolymerized fucans was resolved into adjacent families characterized by their peak molecular mass and polydispersity. The Dubois test (sugar) and the azur A test (SO₃⁻) confirmed the displacement mode of the process, and size-exclusion chromatographic controls confirmed its efficiency.     

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.     

Pilot scale affinity chromatography: purification of beta-galactosidase

β-Galactosidase has been purified from an ammonium sulfate precipitate of E. coli strain ML308 by biospecific adsorption on a column of agarose gel substituted with p-aminophenyl-β-D -thiogalactopyranoside. The system described using a 1.8 liter column has a useful processing capacity of 3.8 × 10⁶ units of β-galactosidase per 2 hr cycle. This corresponds to about 5 g of pure enzyme. An electromechanical timing device operates a set of six solenoid valves and carries out a preset program consisting of sample application, washing, and elation operations.     

Preparation of electrophoretic variants of corticosteroid-binding globulin (CBG) using liquid liquid partition chromatography

Human corticosteroid-binding globulin (CBG) was purified to homogeneity by application of three different chromatographic methods. After fractionation of pregnancy serum with ammonium sulfate the 80%-pellet was used for affinity chromatography based on tresyl activated Sepharose (Pharmacia, Uppsala, Sweden). The affinity eluate was injected into a Mono Q anion exchange column (Pharmacia). Fractions containing CBG were finally purified by liquid liquid chromatography on LiParGel 750 (Merck, Darmstadt, F.R.G.). The purified protein was characterized by IEF and PAGE. This paper describes a method for the chromatographic separation of the two variants of CBG without a loss of binding activity towards steroids for each of the two characteristic bands of this protein.     

Large-scale separation of clavine alkaloids from Ipomoea muricata by pH-zone-refining centrifugal partition chromatography

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids, directly from a crude extract of Ipomoea muricata. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)–acetonitrile–water (4:1:5, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and trifluoroacetic acid (10 mM) to the aqueous mobile phase as an eluter. From 4 g of crude extract, 210 mg lysergol and 182 mg chanoclavine were obtained in 97% and 79.6% purities. Total yield recovery was >95%. Isolated alkaloids were characterized on the basis of their ¹H, ¹³C NMR and ESI-MS data.     

High-performance centrifugal partition chromatography (HPCPC) for efficient isolation of diphlorethohydroxycarmalol (DPHC) and screening of its antioxidant activity in a zebrafish model

High-performance centrifugal partition chromatography (HPCPC) can be used for the rapid isolation of biologically active metabolites from natural sources. The present study investigates the one-step isolation of diphlorethohydroxycarmalol (DPHC), an algal polyphenol, from the brown seaweed Ishige okamurae by HPCPC and its protective effect against 2,2’-azobis dihydrochloride (AAPH) induced oxidative stress in zebrafish embryos. HPCPC was found to be efficient and effective for the isolation of DPHC from Ishige okamurae under optimized solvent conditions, yielding a high purity product. The present purification method helps overcome compound wasting and possible degradation, which cause a low yield in conventional column separations. Further, zebrafish embryos exposed to AAPH were compared with and without DPHC treatment, two days after fertilization for ROS generation, cell death, lipid peroxidation, survival rate, and heartbeat rates. These evaluations revealed that DPHC treatment significantly enhanced protection against oxidative stress in zebrafish embryos. HPCPC was, therefore, established as an efficient DPHC isolation method and could be used for separating other complex phlorotannins from seaweeds.     

Fractionation of UV-B absorbing molecules and of free radical scavenging compounds from Solieria chordalis by using centrifugal partition chromatography

Liquid-liquid chromatography can lead to the isolation of compounds or enrichment of fractions. To investigate the structural characteristics of antioxidants and UV-B absorbing molecules, a preparative centrifugal partition chromatography (CPC) method was developed to prepare enriched bioactive fractions from the red seaweed Solieria chordalis (Gigartinales, Solieriaceae). After a methanolic extraction of dried Solieria chordalis and liquid-liquid separation using ethyl acetate/water (v/v), the ethyl acetate phase was submitted to CPC using Arizona Y of the quaternary solvent system composed of n-heptane/ethyl acetate/methanol/water (19/1/19/1, v/v) in ascending mode. Among the fifteen collected fractions, three fractions gave up to 23.50% of DPPH radical scavenging activity. The CPC fractionation was monitored by HPLC and, four compounds exhibited UV-B absorbing capacity regarding their absorption at 310 nm.     

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography

Introduction. Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. Objective. To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). Methodology. The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative‐scale separation of lancemasides by CPC using n‐hexane:n‐butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two‐phase solvent system. The upper phase (organic phase) of the two‐phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. Results. The demonstrated separation sequence, hot water extraction, DIAION HP‐20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. Conclusion. The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.     

A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography

Introduction – Eisenia bicyclis (Kjellman) Setchell (Laminariaceae) is a common brown alga that inhabits around the coast of Korea, Japan and China. It contains fucoxanthin, a major carotenoid of brown algae which shows a variety of pharmaceutical functions. Objective – The aim of this investigation was the quantification and preparative isolation of fucoxanthin from fresh E. bicyclis using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – The fucoxanthin fraction (Fuco fraction) was prepared by solvent partition method from the acetone extract of fresh E. bicyclis. Fuco fraction was used for CPC using a two‐phase solvent system of n‐hexane–ethyl acetate–ethanol–water (5:5:7:3, v/v/v/v). The flow rate of mobile phase was 2 mL/min with descending mode while rotating at 1000 rpm. The eluate was monitored at 410 nm. The content and structure of fucoxanthin in the CPC fraction were confirmed with HPLC, UV, APCI/MS and NMR spectra. Results – A preparative CPC yielded 20 mg of fucoxanthin (87% recovery from Fuco fraction) in a two‐step separation from 516 mg of Fuco fraction containing 4.59% fucoxanthin. The purity of the isolated fucoxanthin was about 81% in the first CPC step and over 98% in the second CPC step based on the calibration curve. The isolated fucoxanthin was identified as all‐trans‐fucoxanthin with APCI/MS (parent ion at m/z 641 [M + H ? H₂O]⁺) and ¹H, ¹³C and 2‐D NMR spectra. Conclusion – High‐purity fucoxanthin was successfully isolated from fresh E. bicyclis, suggesting further potential applications in the industrial use of this valuable carotenoid. Copyright © 2011 John Wiley & Sons, Ltd.