Low-pressure Column Chromatography Research Articles

Mathematical model of astaxanthin purification process using the low-pressure column chromatography method

Astaxanthin is an antioxidant compound that can be extracted from the microalgae Haematococcus pluvialis using the microwave-assisted extraction (MAE) method. Purification of the astaxanthin compound can be conducted using a low-pressure column chromatography (LPCC) method. However, the mathematical model illustrating the adsorption and desorption process using the LPCC method has not been built by the other authors yet. Therefore, the purposes of this study were (1) to build the mathematical model of the purification process using the LPCC method, and (2) to simulate the experimental data using the model to find the important kinetic constant values that can be used in the industrial scale-up in the future. The purification process was carried out with silica gel as a stationary phase and a mixture of n-hexane:acetone of 3:1 (v/v) as a mobile phase. The silica gel diameter was varied to 0.2–0.5 mm and 0.063–0.2 mm, while the eluent velocity was varied to 1.85 mL/min and 3 mL/min. The mathematical model having five kinetic constants of De1, De2, kc2, kc2.a, and H_A2 was successfully built. The De1 and De2 (m2/s) were the effective diffusivities of astaxanthin in the intraparticle direction of the adsorbent granule and in the axial direction of the column, respectively. The kc2 (m/s) was the mass transfer coefficient of astaxanthin from the solution to the adsorbent. The kc2.a (/s) was the volumetric mass transfer coefficient of astaxanthin from the solution to the adsorbent. The H_A2 (g silica gel/m3) was the Henry's constant for astaxanthin concentration at equilibrium at the liquid-solid interface. In the variation of silica gel diameter, the De2 value at the diameter of 0.2–0.5 mm was greater than that at 0.063–0.2 mm. However, the De1, kc2, kc2.a, and H_A2 values ​​were not affected by the different diameters. In the variation of eluent velocity, the kc2 and kc2.a values at 3 mL/min were greater than those at 1.85 mL/min. However, the De1, De2, and H_A2 values were not affected by the different eluent velocities.

Antimicrobial Testing of Schinus molle (L.) Leaf Extracts and Fractions Followed by GC-MS Investigation of Biological Active Fractions.

Schinus molle (L.) is a dioecious plant of the Anacardiaceae family, originating in South America and currently widespread in many regions throughout the world. In this work leaf extracts and derived low-pressure column chromatography (LPCC) fractions of S. molle L. male and female plants were investigated for the antimicrobial activity. Leaf extracts were tested on microbes Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Bacillus subtilis. Furthermore, the extracts showing antimicrobial activity were fractionated by LPCC and the obtained fractions tested on the same microorganism strains. Positive fractions were investigated by gas-chromatography/mass spectrometry (GC-MS) and were seen to be rich in sesquiterpenes, sesquiterpenoids and other terpens. The obtained effects highlighted the antimicrobial properties of S. molle (L.) leaf compounds and revealed their importance as a source of bioactive molecules of potential pharmaceutical interest. To our knowledge, this is the first paper reporting investigations on the chemical composition of the extracts and derived positive fractions from Schinus molle (L.) plants grown in central Italy

Features of the determination of amlodipine in a biological material

The purpose of the work is to determine the optimal conditions for isolating amlodipine, to purify it by the method of column chromatography and to develop a method for detecting it in biological material. TLC, GC-MS, low pressure column chromatography, and HPLC were used for analysis. We studied the comparative isolation of amlodipine from biological material using 13 isolating agents of organic nature, water, and aqueous solutions. The use of acetone as an insulating agent for the extraction of amlodipine from tissues of cadaver organs has been substantiated. The possibility of purification of the analyzed compound from the endogenous substances of the biomaterial is shown by the method of reversed phase chromatography in a column of the Silasorp S-18 sorbent of 30 μm. A technique has been developed for detecting amlodipine in the tissues of cadaveric organs (liver), which corresponds to the necessary parameters of linearity, selectivity, accuracy, precision and stability. The limits of detection and quantification of amlodipine by the proposed method are 0.25·10-6 and 4.0·10-6 g, respectively, in 1 g of the biomaterial.

Isolation of Methoxyfuranocoumarins From Ammi majus by Centrifugal Partition Chromatography.

Pure methoxyfuranocoumarins were isolated from Ammi majus L. by use of low-pressure column chromatography (LPCC) followed by centrifugal partition chromatography (CPC). The concentrated petroleum ether extract from fruits of A. majus was fractionated on a silica gel column using a gradient of ethyl acetate in dichloromethane (0-80%, v/v). Coumarin-rich fractions were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography with diode array detection (HPLC/DAD). Xanthotoxin (8-MOP) and isopimpinellin (isoP), structurally similar compounds, were isolated in one fraction (FR6). To avoid multistep and long-lasting TLC preparation, optimization of CPC conditions has been performed. In one run, an effective separation of 8-MOP and isoP was achieved. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10 : 8 : 10 : 9; v/v) in an ascending mode (the aqueous phase was a stationary phase, and the organic phase was a mobile phase), with flow rate 3 mL/min and rotation speed 1,600 r.p.m., was used. The identification and high purities of isolated 8-MOP (98.7%) and isoP (100%) were confirmed by HPLC/DAD assay, when compared with standards. The developed CPC method could be applied to the effective isolation of 8-MOP and isoP from plant extracts. The high purity of obtained compounds makes possible further exploitation of these components in biological studies.

The distribution of carbosulfan in the body of warm-blooded animals

The objective of the present work was to study the specific distribution patterns of carbosulfan in the body of warm-blooded animals by means of thin-layer chromatography, low-pressure column chromatography, electron spectrophotometry, and GCh/ MS. The carbosulfan distribution in the warm-blooded animals (rats) was investigated after the intragastric administration of this poisonous substance at a dose equivalent to three median lethal doses (LD50) in the form of an aqueous emulsion. The highest concentrations of carbosulfan were found in the contents of the stomach, small and large intestines, liver, and lungs. The same organs and spleen contained carbofuran, the main metabolite of carbosulfan.

The forensic chemical investigation of acetylsalicylic acid

The objective of the present study was to develop the universal approach to the quantitative determination of acetylsalicylic acid in biological tissues and fluids to be applied in the practice of forensic chemical expertise with the use of thin-layer chromatography, gas chromatography and mass spectrometry, low-pressure column chromatography, and spectrophotometry. A system of solvents consisting of acetone and ethyl acetate (7:3) was proposed as a universal agent for extracting acetylsalicylic acid from the cadaveric tissues and blood. It was shown that acetylsalicylic acid and its principal metabolite, salicylic acid, can be purified from the endogenous admixtures present in the biological materials by column chromatography on silica gel L 40/100 mcm. Salicylic acid in extracts from biological materials was identified and quantified with the use of thin-layer chromatography, gas chromatography/mass spectrometry, and electronic spectrophotometry. The method for forensic chemical investigation of acetylsalicylic acid has been developed and applied in the analysis of the material provided for expertise.

The chemico-toxicological determination of dilthiasem

The objective of the present study was to determine the optimal conditions for isolation of dilthiasem, its purification by the combination of the extraction and column chromatography techniques, and the development of the universal method for the detection of this compound in the biological material. Other research methods included thin layer chromatography (TLC), gas chromatography mass-spectrometry (GH-MS), extraction, low-pressure column chromatography, and spectrophotometry. The effectiveness of dilthiasem isolation from the biological material with the use of 12 organic substances, water, and aqueous solutions was compared. The use of acetone as the universal solvent for dilthiasem isolation from the tissues and biological fluids of the cadaveric organs was substantiated. It was shown that dilthiasem can be purified from endogenous substances contained in the biological materials by means of combined liquid-liquid extraction and chromatography on the 30 mcm Silasorb C-18 column. The new modifications of thin layer chromatography and gas chromatography mass-spectrometry (GH-MS) are proposed for the identification and quantitative determination of dilthiasem isolated from cadaveric blood and hepatic tissue.

Separation, Purification, and Identification of (3S,3′S)-trans-Astaxanthin from Haematococcus pluvialis

A simple, low-cost method of separation and purification for preparing (3S,3′S)-trans-astaxanthin from Haematococcus pluvialis was established in this study. Crude extracts were separated from dry algal cells by extraction. The extracts were then saponified at 4°C for 15 h and at 22°C for 3 h with 0.02 M NaOH in the reaction mixture with lower isomerization of trans-astaxanthin to cis isomers. The saponified pigment extracts were mixed with distilled water and n-hexane at 1:1:1 volume ratio. Low-pressure column chromatography of silica gel (300-400 mesh) with different eluents was used to prepare high-purity (> 80%) all-trans-astaxanthin. Supersaturated solution of astaxanthin in acetone was then crystallized for 72 h at 4°C, and the purity of all-trans-astaxanthin crystalline powder was confirmed to be > 96.5% by HPLC. And the 3S,3′S isomers were identified and characterized by HPLC-APCI-MS, 1H NMR, 13C NMR, and HPLC-UV.

Isolation and Purification of Glucoraphenin from Radish Seeds by Low-Pressure Column Chromatography and Nanofiltration

A new and low–cost method to isolate and purify glucoraphenin as a potassium salt from radish seeds was described in this article. Crude glucoraphenin was separated from radish seeds, and then used as raw material for further purification of glucoraphenin by low-pressure column chromatography. Compared with K2SO4 solution, KCl solution is a preferable eluent for increasing the purity and recovery of glucoraphenin, and reducing the usage of salt. In order to remove a large quantity of KCl, nanofiltration with NF270-400 membrane and precipitation with methanol were investigated. Consequently, compared with precipitation, the purity and recovery of glucoraphenin prepared by nanofiltration were both much higher. At the same time, nanofiltration also got better efficiency of desalination. The purity of glucoraphenin was 91.5%, the recovery of it was 89.0%, and the removal of KCl was 99.5%. The glucoraphenin product was identified by MS, 1H, and 13C NMR spectra.

Protein Purification

The science, art, and practice of protein purification have been with us for more than a century, yet, in many respects, the field is only now evolving past its adolescent roots. New methods are replacing old methods at such a dizzying pace, that even life-long experts in protein purification cannot keep up. In this article, we present many state-of-the-art protein purification techniques without totally ignoring the past. Our goal is to enable those relatively new to the field of protein purification to choose the best methods to solve their own purification problems. Each method we describe has been used and validated in our own research. We describe these methods, pointing out advantages, disadvantages, and limitations with practical examples rather than with complex, theoretical equations. This paper covers methods of extraction, clarification, batch purification, low pressure column chromatography, HPLC, and electrophoresis as applied to both genetically engineered, recombinant proteins and proteins isolated from natural sources. The relatively new methods of three-phase partitioning, hydrophobic charge induction chromatography, immobilized metal affinity chromatography, and perfusion chromatography are featured. Keywords: Protein purification, Extraction, Clarification, Chromatography, HPLC, Electrophoresis, Filtration

Rapid Preparation of Procyanidins B2 and C1 from Granny Smith Apples by Using Low Pressure Column Chromatography and Identification of Their Oligomeric Procyanidins

Research in the field of procyanidins is always hindered by the lack of procyanidin standards, and the preparation of procyanidins, especially in large scale, remains difficult and time-consuming. Commercial sources of procyanidin standards are scarce. In this study, a rapid preparation method of procyanidins by using low-pressure column chromatography was developed. Procyanidins in Granny Smith apples were extracted with boiled water and purified on an ADS-17 macroporous resin column to obtain a Granny Smith apple procyanidin extract (GSE). GSE was fractionated according to its degree of polymerization on a Toyopearl TSK HW-40s column. Procyanidins B2 (epicatechin-(4beta-8)-epicatechin) and C1 (epicatechin-(4beta-8)-epicatechin-(4beta-8)-epicatechin) were prepared without HPLC separation. Oligomeric procyanidins from Granny Smith apples were also identified by liquid chromatography-electrospray ionization-mass spectrometry.

Separation of Individual Tocopherols from Soybean Distillate by Low Pressure Column Chromatography

Abstractα‐Tocopherol, γ‐tocopherol and δ‐tocopherol were successfully separated and purified from soybean vitamin E concentrate (53% purity, from soybean oil deodorizer distillate) using a low‐pressure glass column (500 mm × 25 mm, I.D., packed with silica gel). The effects of eluent, flow‐rate and sample loading on separation efficiency were investigated using product purity and recovery as evaluation indices. On the basis of the single factor experiment results, an orthogonal test was designed to optimize the chromatographic separation conditions. The optimum conditions obtained by using analyses of extreme difference and variance are as follows: cyclohexane‐ethanol 99.7:0.3 (v/v), flow‐rate at 25 ml/min and loading amount being 2 ml (concentration 1 g/ml). Under these optimum conditions, purity of the α‐tocopherol, γ‐tocopherol and δ‐tocopherol were 92.35, 91.23, 89.95%, respectively; the recovery of those products were 35.21, 36.25, 61.25%, respectively. The advantage of this process is that high purity individual tocopherols can be obtained directly from soybean vitamin E concentrate at 53% purity, without additional purification steps.

Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography

Sulforaphane is an isothiocyanate that is present naturally in widely consumed Brassica oleracea vegetables and has been shown to block the formation of tumors. The contents of sulforaphane in five groups of B. oleracea seeds (broccoli, Brussels sprouts, cabbage, cauliflower and kale) were determined by RP-HPLC using linear gradient of acetonitrile in water. A new low-cost method to isolate and purify natural sulforaphane from B. oleracea seed meal was described in this work. Crude sulforaphane was first separated from B. oleracea seed meal by using immiscible solvent extraction with ethyl acetate, 10% ethanol and hexane, and the crude sulforaphane was used as raw materials to prepare high purity sulforaphane by low-pressure column chromatography of silica gel (200-300 mesh) with different eluents and elution modes. Compared with these different elution methods, the gradient elution was preferable to the isocratic elution for reducing the elution time and the eluent consumption and increasing the purity of sulforaphane product. The purity and recovery of sulforaphane were more than 90% in gradient elution.

Comparison of donor–acceptor and alumina columns for the clean-up of polycyclic aromatic hydrocarbons from edible oils

Two methods for clean-up and sample enrichment for the analysis of polycyclic aromatic hydrocarbons (PAHs) in edible oils are compared; a clean-up based on a donor–acceptor complex chromatography (DACC) column and a standardized method widely used in the food industry consisting in a low pressure column chromatography with alumina as stationary phase. Both methods are followed by a reversed-phase high-performance liquid chromatography with fluorescence detection for the separation and quantitation of each PAH. Certified materials were used in order to validate the methods. The limits of detection were lower than 1 ng/g and good selectivity was achieved in both cases. The DACC column clean-up is faster and better accuracies were obtained. The advantages and disadvantages of both methods are discussed.

Comparison of two sample clean‐up methodologies for the determination of polycyclic aromatic hydrocarbons in edible oils

AbstractAn off‐line high‐performance normal‐phase liquid chromatography procedure with a silica column followed by reversed‐phase high‐performance liquid chromatography (HPLC) with fluorescence detection for the determination of polycyclic aromatic hydrocarbons (PAHs) in edible oils is reported. The method was validated using certified reference materials and compared with a standardized method widely used in the food industry, consisting in low pressure column chromatography with alumina as stationary phase followed by reversed phase HPLC determination. The limits of detection were lower than 1 ng/g and good selectivity was achieved for both methods. There were no significant differences in accuracies and precisions obtained for each approach. The advantages and disadvantages of the two methods are discussed.

Isolation of a New Member of the Ecdysteroid Glycoside Family: 2-Deoxy-20-hydroxyecdysone 22-O- -D-Glucopyranoside

A new ecdysteroid glycoside, 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside, is isolated from the herb Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The compound is purified with multistep chromatography, such as classical column chromatography on alumina and droplet countercurrent distribution. Also, it is expanded using twice low-pressure reversed-phase liquid column chromatography. Chromatography in four steps results in the purified 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside. Two other ecdysteroids have also been separated, including the formerly identified integristerone A and 24(28)-dehydromakisterone A.

Isolation of 5 alpha- and 5 beta-dihydrorubrosterone from Silene otites L. (Wib).

5 alpha-Dihydrorubrosterone (2 beta, 3 beta, 14 alpha, 17 beta-tetrahydroxy-5 alpha-androst-7-ene-6-one), a new 19-carbon 5 alpha-ecdysteroid, was isolated together with its 5 beta counterpart from the aerial parts of Silene otites L. (Wib.) (Caryophyllaceae) by a combination of solvent partition, low-pressure column chromatography, thin-layer chromatography (normal-phase and reversed-phase) and finally HPLC. Mass spectrometry and nuclear magnetic resonance spectroscopic procedures were used for compound characterization.

Inhibition of calcium oxalate crystal growth and aggregation by prothrombin and its fragments in vitro: relationship between protein structure and inhibitory activity.

During blood coagulation, prothrombin (PT) is ultimately degraded to three fragments, thrombin, fragment 1 (F1) and fragment 2 (F2), which, collectively, contain all of the structural features of PT. One of these fragments, F1, is excreted in human urine and is the principal protein occluded into calcium oxalate (CaOx) crystals precipitated from it. This urinary form of F1, which we have named urinary prothrombin fragment 1 is present in calcium stones and is a potent inhibitor of CaOx crystallization in urine in vitro. The aim of this study was to determine whether PT itself and its other activation products, namely, thrombin, F1 and F2 also inhibit CaOx crystallization, by comparing their effects in a seeded, inorganic crystallization system. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. PT was isolated from a human blood concentrate rich in vitamin K-dependent proteins. Following initial cleavage by thrombin, the resulting fragments, F1 and F2, were purified by a combination of reversed phase HPLC and low pressure column chromatography. The purity of the proteins was confirmed by SDS/PAGE and their individual effects on CaOx crystallization were determined at the same concentration (16.13 nM) in a seeded, metastable solution of CaOx using a Coulter Counter. [14C]Oxalate was used to assess deposition of CaOx and crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that the proteins reduced the size of precipitated crystals in the order F1 > PT > F2 > thrombin. These findings were confirmed by scanning electron microscopy which showed that the reduction in particle size resulted from a decrease in the degree of crystal aggregation. [14C]Oxalate analysis demonstrated that all proteins inhibited mineral deposition, in the order F1 (44%) > PT (27.4%) > thrombin (10.2%) > F2 (6.5%). It was concluded that the gamma-carboxyglutamic acid domain of PT and F1, which is absent from thrombin and F2, is the region of the molecules which determines their potent inhibitory effects. The superior potency of F1, in comparison with PT, probably results from the molecule's greater charge to mass ratio.

Isolation of 9Z beta-carotene from Dunaliella bardawil and its stereoselective synthesis.

An isolation of 9Z beta-carotene from Dunaliella bardawil was accomplished by low-pressure column chromatography with Ca(OH)2 column. Furthermore, it was stereoselectively synthesized by Wittig reaction between the C10-phosphonium salt 2 and 9Z beta-apo-8'-carotenal 1, which was prepared by Emmons-Horner reaction of 9Z beta-ionylideneacetaldehyde 3 with phosphonates 6 and 7 or 8a and 9.

Autocrine/Paracrine Activator of Cell Proliferation: Purification of a 4–6 kD Compound with Growth-Factor-Like Effects in Tetrahymena thermophila

Extracellular medium from cultures of Tetrahymena thermophila was collected, concentrated and fractionated by ultrafiltration and low-pressure column chromatography. The effect on proliferation of various fractions was tested on cultures of T. thermophila in synthetic medium at low initial cell densities (<250 cells/ml). In unsupplemented cultures, cells failed to thrive and died before proliferation. Addition of the fraction isolated from the extracellular medium in the molecular weight range of 1–10 kD activated the cells to proliferate. The size of the main compound in this fraction, herein referred to as TPAF, was in the range of 4–6 kD as estimated by SDS-PAGE. Evidence of a dialysable factor produced by cells in the stationary phase inhibiting cell proliferation has also been found.