Liquid-liquid Partition Chromatography Research Articles

Gradient Elution in Countercurrent Chromatography.

Countercurrent chromatography is a form of liquid-liquid partition chromatography in which the stationary liquid phase is retained in the apparatus without the use of a solid support. Gradient elution in countercurrent chromatography can be used in many different ways, such as linear gradients, stepwise elution gradients, pH gradients, etc. The main goal of using the gradient approach is to shorten the duration of the separation and improve resolution, especially when the retention range of the sample to be purified is broadening and, thus, the compounds cannot be purified by only one solvent system. The principle is based on modifying the mobile phase to increase the elution strength with no or minimum changes in the stationary phase, which can be a difficult task since both phases are in intimate contact all the time. The most common ways to perform gradients in countercurrent chromatography are changing the mobile's phase polarity, flow rate, and pH, what is called linear or step gradient, flow rate gradient, pH gradient, respectively.

Scaled up countercurrent chromatography separation of secondary metabolites from Schinus therebinthifolius Raddi

Schinus therebinthifolius Raddi (Anacardiaceae) is a tree of medium size native to South and Central America, whose fruits known as pink pepper, are widely used for cooking. In folk medicine, it has been used to treat ulcers, arthritis, as well as in leprosy therapy. Previous phytochemical investigations have described the isolation of polyphenols, fatty and terpenoid acids1. Countercurrent chromatography (CCC) is a liquid-liquid partition chromatography in which the stationary liquid phase is retained in the apparatus without the use of a solid support, while the mobile phase is pumped through the coil. Scaled up CCC was performed to isolate metabolites from a CH2Cl2 extract of S. therebinthifolius berries.

Isolation, Synthesis, and SAR Study of Antiplasmodial Anthraquinones from Kniphofia caulescens

During our ongoing research as part of a collaboration established between Virginia Tech and the Natural Products Discovery Institute (NPDI), a CH2Cl2 extract of Kniphofia caulescens was found to exhibit antiplasmodial activity against a chloroquine resistant strain of Plasmodium falciparum, with an IC50 value of 6 µg/mL. Liquid-liquid partition, LH-20 and silica gel open column chromatography, and C-18 reverse phase HPLC were used to fractionate the crude extract to obtain seven known anthraquinones (1 – 2, 5 – 9) and two new anthraquinones (3 – 4), which displayed modest antiplasmodial activity, with IC50 values of 25.6 ± 3.6 and 8.9 ± 1.2µM, respectively. The structures of the two new compounds were elucidated based on analysis of their 1D and 2D NMR spectra, and mass spectrometric data. Various analogs of 3 and 4 were prepared to evaluate structure-activity relationships, and one analogue of compound 4, a caffeic acid derivative of aloe emodin, showed good activity, with an IC50 value of 1.3 ± 0.2µM against P. falciparum.

Liquid Chromatographic Determination of Etofenprox Residues in Foods with Mass-Spectrometric Confirmation

BACKGROUND: An official analytical method was developed to determine etofenprox residues in agricultural commodities using high-performance liquid chromatography (HPLC). METHODS AND RESULTS: The etofenprox residue was extracted with acetone from representative samples of five raw products which comprised rice grain, apple, mandarin, cabbage, and soybean. The extract was then serially purified by liquid-liquid partition and Florisil column chromatography. For rice and soybean samples, acetonitrile/n-hexane partition was additionally coupled to remove nonpolar lipids. Reversed phase HPLC using an octadecylsilyl column was successfully applied to separate etofenprox from co-extractives. Intact etofenprox was sensitively detected by ultraviolet absorption at 225 nm. Recovery experiment at the quantitation limit validated that the proposed method could apparently determine the etofenprox residue at 0.02 mg/kg. Mean recoveries from five crop samples fortified at three levels in triplicate were in the range of 93.6∼106.4%. Relative standard deviations of the analytical method were all less than 10%, irrespective of crop types. A selected-ion monitoring LC/mass spectrometry with positive atmospheric-pressure chemical ionization was also provided to confirm the suspected residue.

Separation of alkaloids from herbs using high‐speed counter‐current chromatography

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.

Isolation of four high‐purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Centrifugal partition chromatography (CPC), as a continuous liquid-liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak-chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. To separate and isolate high-purity saponins from extract of Panax notoginseng using CPC-ELSD with a simple and low toxicity solvent system. Samples were preparaed by extracting the root material with acetone, treated with n-butanol and then freeze-dried. CPC-ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate-n-butanol-water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high-performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI-MS(n) ) in the negative and positive ion modes with the authentic standards. A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n-butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC-ELSD. CPC-ELSD was proved to be a very fast and efficient tool for separation of high-purity dammarane saponins.

Counter-Current Chromatography for High Throughput Analysis of Natural Products

Counter-current chromatography (CCC) is a unique support-free liquid-liquid partition chromatography winning wide applications in the separation of various components from natural or synthetic mixtures. It has been one of the prime methods for isolating compounds from Traditional Chinese Medicines (TCM) and other comprehensive natural products. Although early CCC models produced a long-standing false image that CCC is a time-consuming technique, rapid and high-performance CCC devices and methods for high-throughput analysis of natural mixtures have been advanced. For instances, multi-channel CCC, dual CCC, elution-extrusion CCC, and solvent simplification protocols can provide high-throughput CCC analysis and produce high purity of compounds or large natural product libraries for drug discovery. This review summarizes the recent advancements of CCC in the high-throughput analysis of natural product with an emphasis on the developments of instruments and methods.

Determination of Buprofezin Residues in Rice and Fruits Using HPLC with LC/MS Confirmation

A high-performance liquid chromatographic (HPLC) method was developed to determine buprofezin residues in hulled rice and fruits. The buprofezin residue was extracted with acetone and the extract was stepwise purified by liquid-liquid partition and Florisil column chromatography. For rice samples, acetonitrile/n-hexane partition was additionally employed to remove nonpolar lipids. Reversed phase HPLC using an octadecylsilyl column was successfully applied to separate buprofezin from sample co-extractives, as detected by ultraviolet absorption at 250 nm. Recovery experiment at the limit of quantitation validated that the proposed method could evidently determine the buprofezin residue at the level of 0.02 mg/kg. Mean recoveries from hulled rice, apple, pear, and persimmon samples fortified at three tenfold levels were in the range of 80.8~85.2%, 89.1~98.4%, 88.8~95.7% and 90.8~96.2%, respectively. Relative standard deviations of the analytical method were all less than 5%, irrespective of sample types. A selected-ion monitoring LC/mass spectrometry with positive electrospray ionization was also provided to sensitively confirm the suspected residue.

Qualitative and Quantitative Analysis of Mycotoxins.

Mycotoxin toxicity occurs at very low concentrations, therefore sensitive and reliable methods for their detection are required. Consequently, sampling and analysis of mycotoxins is of critical importance because failure to achieve a suitable verified analysis can lead to unacceptable consignments being accepted or satisfactory shipments unnecessarily rejected. The general mycotoxin analyses carried out in laboratories are still based on physicochemical methods, which are continually improved. Further research in mycotoxin analysis has been established in such techniques as screening methods with TLC, GC, HPLC, and LC-MS. In some areas of mycotoxin method development, immunoaffinity columns and multifunctional columns are good choices as cleanup methods. They are appropriate to displace conventional liquid-liquid partitioning or column chromatography cleanup. On the other hand, the need for rapid yes/no decisions for exported or imported products has led to a number of new screening methods, mainly, rapid and easy-to-use test kits based on immuno-analytical principles. In view of the fact that analytical methods for detecting mycotoxins have become more prevalent, sensitive, and specific, surveillance of foods for mycotoxin contamination has become more commonplace. Reliability of methods and well-defined performance characteristics are essential for method validation. This article covers some of the latest activities and progress in qualitative and quantitative mycotoxin analysis.

Characterization of bergenin in Endopleura uchi bark and its anti-inflammatory activity

Endopleura uchi (Huber) Cuatrec. is an Amazon species traditionally used for the treatment of inflammations and female disorders. Pure bergenin was isolated from the methanolic extract of bark of E. uchi, firstly by using liquid-liquid partition chromatography followed by column chromatography over Sephadex LH-20 and then silica gel 60 flash chromatography. The structure of bergenin was identified on the basis of its NMR spectra. The in vitro anti-inflammatory activity was determined by the measurement of the inhibitory concentration (IC) of bergenin against three key enzymes: COX-1, COX-2 (cyclooxygenases) and phospholipase A2 (PLA2). These enzymes were selected because they are important targets for the discovery of new anti-inflammatory drugs associated with the biosynthesis of prostaglandins. The IC50 of bergenin for phopholipase A2 was determined as 156.6 µmol L-1 and bergenin was not considered active as compared to the positive control, tioetheramide PC. Bergenin did not inhibit COX-1 as well (IC50 = 107.2 µmol L-1). However, bergenin selectively inhibited COX-2 (IC50 = 1.2 µmol L-1). Because of the use of E. uchi in traditional medicine, bergenin was quantified in teas prepared as prescribed in traditional medicine by RP-HPLC as being 3% in the bark of E. uchi. The inhibitory activity towards COX-2 is important, since selective inhibitors of COX-2 have been clinically validated as anti-inflammatory therapeutics due to their enhanced gastrointestinal safety.

Purification of gangliosides by liquid-liquid partition chromatography

A liquid-liquid partition chromatographic technique was applied to separate amphiphilic glycolipids. A two-phase solvent system composed of n-butanol-t-butyl methyl ether-acetonitrile-water at a volume ratio of 3:1:1:5 was found to be suitable for separating the gangliosides from total lipids extracted from rat brain by liquid-liquid partition chromatographic systems, namely centrifugal partition chromatography (CPC) and high-speed counter-current chromatography. GM1 could be separated rapidly by using the upper phase as stationary phase for both systems. Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) could be separated individually by using the lower phase as stationary phase by CPC. The sample can be recovered without loss by these systems.

α-Eleostearic Acid and Its Dihydroxy Derivative Are Major Apoptosis-Inducing Components of Bitter Gourd

Bitter gourd ( Momordica charantia L.) pericarp, placenta, and seed extracts were previously shown to induce apoptosis in HL60 human leukemia cells. To determine the active component that induces apoptosis in cancer cells, bitter gourd ethanol extract was fractionated by liquid-liquid partition and silica gel column chromatography. Several fractions obtained by silica gel column chromatography inhibited growth and induced apoptosis in HL60 cells. Among them, fraction 7 had the strongest activity in inhibiting growth and inducing apoptosis in HL60 cells. A component that induced apoptosis in HL60 cells was then isolated from fraction 7 by another silica gel column chromatography and high-performance liquid chromatography (HPLC) using a C18 column and was identified as (9Z,11E,13E)-15,16-dihydroxy-9,11,13-octadecatrienoic acid (15,16-dihydroxy alpha-eleostearic acid). 15,16-Dihydroxy alpha-eleostearic acid induced apoptosis in HL60 cells within 5 h at a concentration of 160 microM (50 microg/mL). (9Z,11E,13E)-9,11,13-Octadecatrienoic acid (alpha-eleostearic acid) is known to be the major conjugated linolenic acid in bitter gourd seeds. Therefore, the effect of alpha-eleostearic acid on the growth of some cancer and normal cell lines was examined. alpha-Eleostearic acid strongly inhibited the growth of some cancer and fibroblast cell lines, including those of HL60 leukemia and HT29 colon carcinoma. alpha-Eleostearic acid induced apoptosis in HL60 cells after a 24 h incubation at a concentration of 5 microM. Thus, alpha-eleostearic acid and the dihydroxy derivative from bitter gourd were suggested to be the major inducers of apoptosis in HL60 cells.

<b>Lindane and propuxur residues in the top soils of some cocoa growing areas in five districts of the Central Region of Ghana</b>

Lindane and propoxur residues in some topsoil samples from five cocoa growing districts in the Central Region of Ghana have been determined. Ten soil samples were taken randomly at a depth of 0-10 cm from pre-selected farms in each district. Some physical and chemical properties of the soils were determined. The pesticide residues were extracted from 50 g of topsoil by continuous Soxhlet extraction using a (1:1 v/v) acetone-chloroform mixture for six hours. Clean up of the extract was done by liquid-liquid partitioning and column chromatography using alumina and silica gel adsorbents. The pesticide residues were finally analysed by gas- chromatography using 63 Ni electron capture detector (ECD) and flame ionization detector (FID) for lindane and propoxur, respectively. Lindane and propoxur pesticide residues were found to be present in all the soils to which these pesticides had already been applied. Generally, lindane residues occurred in greater amounts than propoxur. The concentrations of lindane ranged from 2.1 to 15.4 mg kg -1 and propoxur from 1.71 to 7.95 mg kg -1 . Both the extracted lindane and propoxur residues correlated negatively with pH, cation exchange capacity, moisture content, and organic carbon. Propoxur correlated positively with the amount of clay but there was no significant correlation between the amount of clay and the extracted lindane concentration. KEY WORDS : Lindane, Propoxur, Soil, Pesticide monitoring Bull. Chem. Soc. Ethiop. 2006 , 20(2), 193-199.

Preparative isolation of isoflavones from soy and red clover

Numerous studies have demonstrated the potential of isoflavones occurring in soy (Glycine max L.) and red clover (Trifolium pratense L.) to alleviate climacteric complaints. They have also shown beneficial effects on the cardiovascular system and in the prevention of osteoporosis. As a result, several companies offer nutraceuticals based on soy or red clover extracts. The aim of the present study was the isolation of pure isoflavones on a preparative scale, in order to obtain standards for biological tests and for the quantification of isoflavones in nutraceuticals and foods. High-speed countercurrent chromatography, a special type of liquid-liquid partition chromatography, was applied to the preparative isolation of isoflavones. By using this technique the major monoglucosylated and acetylated isoflavones from soy extracts were obtained after a cleaning-up step on Amberlite XAD-7 material. Furthermore, it was possible to isolate isoflavone aglycones as well as glycosides from a red clover extract. Purity and identity of the isolated isoflavones were confirmed by HPLC with DAD, HPLC-ESI multiple-MS, and NMR spectroscopy.

The Liquid-liquid InterfaceUnder Planetary Rotation

Countercurrent chromatography is a liquid-liquid partition chromatography method allowing high-resolution purifications. It utilises the hydrodynamic behaviour of two immiscible liquid phases in coil columns undergoing a planetary motion generated by a synchronous coil planet centrifuge. In this study planetary rotation is applied to liquid-liquid flows in two types of ring geometries. The influence of the epicyclic force field is studied by applying a variety of different operating conditions, namely rotational speed and the ratio of the planet and sun radii known as the beta value. The results show that variation of the beta value has far greater influence on phase distribution than variation of the rotational speed.

Practical high-performance liquid chromatography

Theoretical principles pumps preparation of equipment up to sample injection detectors columns and stationary phases column packing and regeneration HPLC packing and regeneration HPLC column tests adsorption chromatography reversed-phase chromatography liquid-liquid partition chromatography chromatography with chemically bonded phases ion-exchange chromatography ion-pair chromatography ion chromatography size exclusion chromatography affinity chromatography choice of method analytical HPLC preparative HPLC separation of enantiomers new prospects review papers on the separation of individual classes of compounds commercially available stationary phases for HPLC.

Isolation of secondary metabolites from roots ofPlumbago auriculata Lam. by countercurrent chromatography

Centrifugal liquid-liquid partition chromatography presents significant advantages for the separation and purification of plant metabolites owing to the short operational time of the process and the elimination of possible irreversible adsorption of compounds. The crude chloroform extract from roots of Plumbago auriculata was analysed by countercurrent chromatography using hexane:ethyl acetate:methanol:water (40:10:10:2, v/v) as solvent system. The isolation of the naphthoquinones plumbagin and epi-isoshinanolone, the steroids sitosterol and 3-O-glucosylsitosterol, plumbagic and palmitic acids was easily achieved. Naphthoquinones are typical components of Plumbago species and they show interesting biological activities.

Tubelactomicin A, a novel 16-membered lactone antibiotic, from Nocardia sp. I. Taxonomy, production, isolation and biological properties.

A novel 16-membered lactone antibiotic named tubelactomicin A was isolated from the culture broth of an actinomycete strain. The producing organism, designated MK703-102F1, was identified as a member of Nocardia. Tubelactomicin A was isolated from the culture broth by Diaion HP20 absorption, ethyl acetate extraction, silica gel and Sephadex LH-20 column chromatographies and centrifugal liquid-liquid partition chromatography (CPC). Tubelactomicin A showed strong activity against acid-fast bacteria including the drug-resistant strains.

Separation of Antibodies by Liquid-Liquid Aqueous Partition and by Liquid-Liquid Partition Chromatography

In this review, we describe the use of liquid-liquid aqueous partition as a method for the separation of antibodies. Water-based two-phase systems made up of polyethylene glycol and dextran have, by far, been the most frequently used systems. The distribution of a molecule in these systems depends on its exposed surface properties and is described by its partition coefficient. The separation may be performed in a single step in a batch experiment or in several steps using various forms of automated counter-current extraction methods, referred to in this review as liquid-liquid partition (LLP). The sensitivity and selectivity of the two-phase technique can be considerably improved by employing a column chromatographic approach, liquid-liquid partition chromatography (LLPC). In LLPC, the bottom-phase of the two-phase system is adsorbed onto a support and packed into a column which is eluted with the corresponding top-phase. In the first part of this review, the methodology behind UP and UPC is described and outlined in broader terms, before the properties and prestanda of the two approaches are compared. In the second part, the results obtained by LLP and LLPC on antibodies are described in more detail. This review shows, that liquid-liquid aqueous partition is a powerful tool for antibody analysis, that is for purification and fractionation, detection and separation of conformational isomeric forms, examination of surface properties related to antigen specificities and for providing new interesting information about the events upon antigen-antibody complexation and about possible ligand-induced conformational changes.

Surface properties of antigen-antibody complexes.

In this paper, the authors show that liquid-liquid partition chromatography in an aqueous two-phase system offers unique possibilities of comparing the overall surface properties of intact antibodies in solution before and after binding of antigen. The authors demonstrate that the surface properties of antigen-antibody complexes are dependent on the variable regions of the antibodies, the nature of the antigen and/or possible conformational changes induced by antigen binding. Thus, each antigen-IgG antibody pair formed one type of complex with respect to the exposed dominant surface. The antigen-binding sites of IgG antibodies were exposed and dominant even after binding of hapten or hapten-carrier. In contrast, the antibody-combining sites were concealed upon protein binding and the exposed surfaces of the protein-antibody complexes were related mainly to those of the antigen. IgA1, IgA2, IgE and IgM formed, in comparison to the IgG, hapten-antibody complexes which exhibited surface properties that could be related to both the antigen-binding sites and Fc parts of the antibodies. Moreover, the results indicated that antigen-induced conformational changes occurred in either IgA1, IgA2, IgE, or IgM, but not in IgG1, -2, -3 and -4, making the surfaces of their heavy chain constant regions more similar.