Ion-exchange Centrifugal Partition Chromatography Research Articles

Purification of dirucotide, a synthetic 17-aminoacid peptide, by ion exchange centrifugal partition chromatography

Dirucotide is a synthetic drug candidate for the treatment of multiple sclerosis. This 17-aminoacid peptide was successfully purified by ion exchange centrifugal partition chromatography. The optimized conditions involved the biphasic methyl tert-butyl ether/acetonitrile/n-butanol/water (2:1:2:5, v/v) solvent system in the descending mode, the di(2-ethylhexyl)phosphoric acid cation-exchanger with an exchanger (di(2-ethylhexyl)phosphoric acid)/dirucotide mole ratio of 100 and Ca2+ ions in aqueous solution as displacer. Critical impurities were efficiently eliminated and dirucotide was recovered in high yield and purity (69% and 98%, respectively) and with a productivity of 2.29g per liter of stationary phase per hour.

Centrifugal partition chromatography - A review of recent applications and some classic references.

Countercurrent chromatography, based on liquid-liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid-liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one-axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two-phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual-mode or elution-extrusion procedure, pH-zone-refining centrifugal partition chromatography, ion-exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).

One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography

Aristolochic acids are biologically active and highly toxic secondary metabolites that recently became an important pharmacological tool for in vivo models of acute kidney disorders. Only aristolochic acid I and a mixture of aristolochic acids I and II are currently commercially available. A method based on the strong-ion exchange displacement mode in centrifugal partition chromatography was developed to extract and purify arictolochic acids, naturally present in Aristolochia bracteolata Lam. (Aristolochiaceae). The extraction and purification of aristolochic acids were performed at a flow rate of 2 mL min−1 in the descending mode by using the biphasic solvent system methyl-ter-butylether, acetone, methanol and water (3:1:1:3, v/v/v/v). Trioctylmethylammonium chloride (Aliquat 336®) was used as the anion exchanger in the organic stationary phase and sodium iodide as the displacer in the aqueous mobile phase. From 5 g of crude extract of A. bracteolata, 45.5 mg, 77.2 mg and 35.1 mg of aristolochic acids I, II and IIIa were obtained respectively with purities greater than 95%.

One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography

Aristolochic acids are biologically active and highly toxic secondary metabolites that recently became an important pharmacological tool for in vivo models of acute kidney disorders. Only aristolochic acid I and a mixture of aristolochic acids I and II are currently commercially available. A method based on the strong-ion exchange displacement mode in centrifugal partition chromatography was developed to extract and purify arictolochic acids, naturally present in Aristolochia bracteolata Lam. (Aristolochiaceae). The extraction and purification of aristolochic acids were performed at a flow rate of 2 mL min−1 in the descending mode by using the biphasic solvent system methyl-ter-butylether, acetone, methanol and water (3:1:1:3, v/v/v/v). Trioctylmethylammonium chloride (Aliquat 336®) was used as the anion exchanger in the organic stationary phase and sodium iodide as the displacer in the aqueous mobile phase. From 5 g of crude extract of A. bracteolata, 45.5 mg, 77.2 mg and 35.1 mg of aristolochic acids I, II and IIIa were obtained respectively with purities greater than 95%.

Concentration and selective fractionation of an antihypertensive peptide from an alfalfa white proteins hydrolysate by mixed ion-exchange centrifugal partition chromatography

This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a l-valyl-l-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.

Ion exchange centrifugal partition chromatography: An innovative tool for bioactive peptide capture in complex protein hydrolyzate

Ion exchange centrifugal partition chromatography: An innovative tool for bioactive peptide capture in complex protein hydrolyzate

Ion-exchange centrifugal partition chromatography: A methodological approach for peptide separation

This article presents the scope and optimization strategies employed in ion-exchange centrifugal partition chromatography (IXCPC). Both the weak and the strong modes were used to separate the constituents of a model mixture of dipeptides. Thus, the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger, and of two displacers: calcium chloride and hydrochloric acid, has proven to be efficient for the preparative separation of the model mixture of five dipeptides (GG, GY, AY, LV and LY, in the order they were collected). The separation was optimized by splitting the stationary phase into two sections that differed by their triethylamine concentration. Moreover, the chemical nature of the exchanger/analyte entities that were involved in the chromatographic process was determined by 31P and 1H DOSY NMR experiments.

Preparative isolation of glucosinolates from various edible plants by strong ion-exchange centrifugal partition chromatography

Glucosinolates (GSLs) are a class of phytochemicals found in all cruciferous plants (Brassicaceae) family as well as in the whole order of Brassicales (syn. Capparales). GSLs and their hydrolysis products ( e.g. isothiocyanates) are known to play a defensive role by protecting the plant against exterior aggressions and may be potent antitumor, anticancer, antioxidant and antibiotic agents. In order to obtain pure GSLs standards for developing research, an efficient two-step method was developed to preparatively isolate and separate GSLs from papaya ( Carica papaya), upland cress ( Barbarea verna), cauliflower ( Brassica oleracea L. var. botrytis L.), and broccoletti seeds ( Brassica rapa ruvo). In this process, solvent extraction was followed by a strong anion-exchange centrifugal partition chromatography protocol. TLC, HPLC-PDA, ESI-MS and NMR analysis of the collected fractions, demonstrated that the GSLs from papaya and upland cress seeds ( i.e. glucotropaeolin and gluconasturtiin, respectively) as well as all the GSLs from cauliflower and broccoletti seeds ( i.e. sinigrin, glucoiberin, glucoiberverin, gluconapin, glucobrassicanapin and gluconasturtiin) can be purified in a single chromatographic step. Gram amounts of pure reference standards were obtained. These results will facilitate investigation of the biological activities and the isolation of such compounds in other crucifers.

Pilot‐scale ion‐exchange centrifugal partition chromatography: Purification of sinalbin from white mustard seeds

The purification of p-hydroxybenzylglucosinolate (sinalbin) on a multigram scale from a crude aqueous extract of white mustard seeds (Sinapis alba var. concerta) was successfully achieved by scaling up a strong ion-exchange centrifugal partition chromatography (SIXCPC) laboratory procedure. Thus, the one-step sinalbin purification was performed with 2.35 g of crude extract in approximately 170 min (830 mg/h) up to 70.3 g in approximately 160 min (26.3 g/h) by switching from a 200 mL laboratory scale column to a 5.7 L pilot-scale column. The required biphasic solvent system contained ethyl acetate, n-butanol, and water in 3:2:5 v/v/v proportions, Aliquat 336 (trioctylmethyl ammonium chloride) was added to the organic stationary phase (80 mM) and acted as ion-exchanger. Potassium iodide in the aqueous mobile phase (80 mM) was used as sinalbin displacer. The 28.5 mass scale factor arose from the increase in mobile phase flow-rate (from 2 to 50 mL/min), from the higher mass of injected white mustard seed extract (from 12 to 350 g), and from the calculated productivity (from 830 mg to 26.3 g). These results demonstrate that industry scale production of glucosinolates is easily performed by SIXCPC, thus providing pure reference standards for pharmacology studies.

Large-scale purification of glucosinolates by strong ion-exchange centrifugal partition chromatography. From lab-scale CPC to 5L-pilot-scale FCPC

The glucosinolates (GSLs) constitute a large class of hydrosoluble plant secondary metabolites acting as phytoanticipins. They are mainly present in the Brassicaceae, Capparidaceae families in the Capparales order, such as broccoli, mustard, rapeseed, cauliflower, cress, caper and papaya [1]. Naturally occurring GSLs are anionic, β-D-thioglucoside-(Z)-N-hydroxyiminosulfates that contain an amino acid-derived side chain. GSLs and their hydrolysis products are of particular interest owing to their potential benefit to health: they have been described as chemopreventive, chemoprotective, antioxidant and antibiotic substances [2]. However, metabolite fate and bioavailability studies as well as clinical evaluation require large amounts of pure GSLs that are often not commercially available at all. Indeed, due to their particular physicochemical properties, the separation and isolation of glucosinolates from crude plant extracts is an extremely difficult task and only few reference samples can be bought. Therefore, new chromatographic methods have recently been developed in order to obtain the required amounts of pure GSLs (3). Previously, the gram-scale purification of pure sinalbin and glucoraphanin from white mustard and broccoli seed extracts was successfully achieved using strong ion-exchange displacement centrifugal partition chromatography (SIX-CPC) [4]. In this study, the same protocol was successfully applied to the purification of glucotropaeolin from papaya, gluconastrutiin from cress, glucoiberin, glucoiberverin and sinigrin from cauliflower and glucoiberin, sinigrin, gluconapin, glucobrassicanapin and gluconasturtiin from brokoletti seeds. Finally, our protocol was easily scaled up from a 200 mL lab scale CPC column to a 5L FCPC apparatus, in order to prove that our method can be applied to industry-scale GSLs production. In a single run that lasted for 3 hours, 310g of a crude white mustard seeds extract were processed to yield 64g of highly pure sinalbin.

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 2.6 g of glucoraphanin were obtained in one step in 2.5 and 3.5 h respectively, starting from 12 and 25 g of mustard and broccoli seed aqueous extracts, using a laboratory scale CPC column (200 mL inner volume).