Isolation and purification of chemical constituents from the pericarp of Sophora japonica L. by chromatography on a 12% cross-linked agarose gel

A chromatographic method using 12% cross-linked agarose gel Superose 12 as the separation medium was developed for isolation and purification of the chemical constituents from the pericarp of Sophora japonica L. The mobile phase used for the separation was 2% acetic acid and 7% acetic acid in gradient elution. As a result, eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained in a one-step separation. A straightforward explanation of the separation mechanism of flavonoids and isoflavonoids on Superose 12 is also given. The flavonoids and isoflavonoids are retained on Superose 12 by a combination of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of aglycone and the residues of the cross-linking reagents used in the manufacture of Superose 12.     

Isolation and purification of flavonoid and isoflavonoid compounds from the pericarp of Sophora japonica L. by adsorption chromatography on 12% cross-linked agarose gel media

A method for isolation and purification of flavonoid and isoflavonoid compounds in extracts of the pericarp of Sophora japonica L. was established by adsorption chromatography on the 12% cross-linked agarose gel Superose 12. The crude extracts were pre-separated to two parts, sample A and sample B, on a D-101 macroporous resin column by elution with 20% ethanol and 40% ethanol, respectively. Samples A and B were then separated by adsorption chromatography on Superose 12 with 40% methanol as the mobile phase. Eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained by the proposed method. The adsorption mechanisms of flavonoids and isoflavonoids on Superose 12 were also discussed.     

Purification of hydroxyanthraquinones and cinnamic acid from the roots of Rheum officinale Baill

A chromatographic method for isolation and purification of chemical constituents from the well-known traditional Chinese drug Da-huang (roots of Rheum officinale Baill.) was established by using 12% cross-linked agarose gel, Superose 12, as the separation media. A two-step separation procedure is employed. Sixty five percent methanol was used as the eluent for separation of cinnamic acid, rhein, physcion and emodin form Da-huang crude extract. The fraction containing aloe-emodin and chrysophanol was then separated by using 55% methanol containing 0.5% acetic acid as the eluent. As a result, cinnamic acid and five kinds of hydroxyanthraquinones including rhein, aloe-emodin, chrysophanol, physcion and emodin were obtained. The retention behavior of hydroxyanthraquinones on Superose 12 was also studied. The retention of hydroxyanthraquinones on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the hydroxyanthraquinones and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.     

One-step separation and purification of hydrolysable tannins from Geranium wilfordii Maxim by adsorption chromatography on cross-linked 12% agarose gel

The hydrolysable tannins corilagin and geraniin, the major active components of the traditional Chinese medicine Geranium wilfordii Maxim, have been separated and purified from crude extracts in one step by adsorption chromatography on cross‐linked 12% agarose gel (Superose 12 10/300 GL). The separation was achieved by gradient elution using mobile phase A composed of 5% ethanol and 5% acetic acid and mobile phase B composed of 30% ethanol and 30% acetic acid. The gradients were composed as follows: 0–240 mL, 0–25% B; 240–480 mL, 25–40% B; after 480 mL, 100% B. The purities of the collected corilagin and geraniin were 92.4 and 87.2%, and the corresponding yields were 88.0 and 76.8%, respectively.     

Investigation of the adsorption behavior of glycine peptides on 12% cross-linked agarose gel media

The highly cross-linked 12% agarose gel Superose 12 10/300 GL causes retardation of glycine peptides when mobile phases containing varying concentrations of acetonitrile in water are used. An investigation has been made into the retention mechanism behind this retardation using the glycine dipeptide (GG) and tripeptide (GGG) as models. The dependence of retention times of analytical-size peaks under different experimental conditions was interpreted such that the adsorption most probably was caused by the formation of hydrogen bonds but that electrostatic interactions cannot be ruled out. Thereafter, a nonlinear adsorption study was undertaken at different acetonitrile content in the eluent, using the elution by characteristic points (ECPs) method on strongly overloaded GG and GGG peaks. With a new evaluation tool, the adsorption energy distribution (AED) could be calculated prior to the model selection. These calculations revealed that when the acetonitrile content in the eluent was varied from 0% to 20% the interactions turned from (i) being homogenous (GG) or mildly heterogeneous (GGG), (ii) via a more or less stronger degree of heterogeneity around one site to (iii) finally a typical bimodal energy interaction comprising of two sites (GG at 20% and GGG at 10% and 20%). The Langmuir, Tóth and bi-Langmuir models described these interesting adsorption trends excellently. Thus, the retardation observed for these glycine peptides is interpreted as being of mixed-mode character composed of electrostatic bonds and hydrogen bonds.     

Cascade-mode multiaffinity chromatography. Fractionation of human serum proteins.

The group-resolving power of cascade-mode multiaffinity column chromatography (CASMAC), was demonstrated with human serum as a model mixture. More than 99% of the serum proteins were adsorbed in the same high salt-containing buffer on a tandem column consisting of (1) immobilized Zn2+ on triscarboxymethyl diamine gel followed by (2) thiophilic (T) gel, (3) Zn2+ bound to the new tridentate chelating adsorbent dipicolylamine (DPA) agarose, (4) hexyl-thioether C6-S agarose and (5) Ni(2+)-DPA agarose. After the adsorption step the immobilized metal ion affinity gels were attached to the top of tandem columns of other adsorbents (T gel, Sephadex G-25 for desalting and Mono-Q) and the elution conditions were selected such that further group separation was achieved. High resolution, high recovery, easy manipulation and high capacity are characteristic features of the cascade process with these adsorbents. The advantage of CASMAC is particularly striking when, with a given number of adsorbents, the overall number of operations involving adsorption, desorption, washing, buffer change and substance concentration can be effectively minimized.     

Synthesis of new hydrophobic adsorbents based on homologous series of uncharged alkyl sulphide agarose derivatives

A homologous series of uncharged thioalkyl derivatives of agarose were prepared by a simplified synthetic route and their adsorption behaviour towards human serum proteins was evaluated and compared with that of a commercially available alkyl ether derivative of agarose. The influence of the spacer arm length on the adsorption efficiency was also investigated. The degree of substitution of the derivatives can be estimated conveniently by sulphur analysis. The four different types of thiolkyl derivatives (C6, C8, C12 and C14) investigated here behave in all respects like hydrophobic adsorbents. The coupling yield obtained is high (75% or more) and is better than that obtained by alternative synthetic routes reported so far. The adsorption capacity towards serum proteins of the various derivatives increases with increasing alkyl chain length and degree of substitution. Desorption is achieved by a progressive decrease in the polarity of the eluent and the recovery of the applied material is in the range 80-90%. The role played by the thioether as a possible modulator of the observed hydrophobic adsorption is discussed. For the group separation of serum proteins the optimum adsorbent, as regards capacity combined with ease of elution of adsorbed material, should be substituted with chains of six or eight carbon atoms and have a ligand concentration in the range 80-120 mumole g-1 dry gel.     

The Separation of Polyphenols by Isocratic Hydrogen Bond Adsorption Chromatography on a Cross-Linked 12% Agarose Gel

The highly cross-linked 12% agarose gel, Superose™ 12 HR 10/30 is shown to possess hydrogen bond acceptor properties suitable for the separation of polyphenolic solutes such as phenolic acids, flavonols and flavonoids. The separation is achieved isocratically in the presence of solvent mixtures of acetic acid and ethanol. The extent of hydrogen bond adsorption is reviewed based on data obtained from the elution behaviour of a variety of simple polyphenolic solutes including dihydroxybenzoic acids.     

One-Step Purification of Resveratrol and Polydatin from Polygonum cuspidatum (Sieb. & Zucc.) by Isocratic Hydrogen Bond Adsorption Chromatography on Cross-Linked 12% Agarose

Resveratrol and polydatin (piceid), the major active components of the traditional Chinese medicinal herb Polygonum cuspidatum (Sieb. & Zucc.), have been separated and purified from crude root extracts in one step by isocratic hydrogen bond adsorption chromatography on cross-linked 12% agarose (Superose? 12 HR 10/30). Separation was achieved by step-wise elution with mobile phases composed of mixtures of ethanol and acetic acid: 0–75 mL, 10% ethanol, 10% acetic acid; 75–150 mL, 20% ethanol, 20% acetic acid; 150–250 mL, 30% ethanol, 30% acetic acid. At a sample load of 40 mg crude extract dissolved in 0.5 mL mobile phase (corresponding to a load of 1.7 mg mL^?1 gel) a resveratrol purity of about 96% with a recovery of 61% was obtained by proper peak cutting.     

One-step purification of epigallocatechin gallate from crude green tea extracts by mixed-mode adsorption chromatography on highly cross-linked agarose media

(-)-Epigallocatechin gallate (EGCG) was purified in one step from a green tea polyphenol (GTP) crude extract by adsorption chromatography on a Superose 12 HR 10/30 column. The mobile phase used was a mixture of acetonitrile and water with an optimum mobile phase compositions regarding purity, recovery and separation time of 78/22 (v/v). Maximum practical sample loading was 100 mg GTP per run (corresponding to 4.2 mg/ml Superose). An EGCG purity of 99% with recoveries in the range 60-65% was achieved in one step directly from the crude GTP extract. Full column regeneration was obtained using solvents in the following order: 0.5 M NaOH, distilled water and 30% acetic acid.     

One-step purification of 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde from Salvia miltiorrhiza Bunge by isocratic stepwise hydrogen bond adsorption chromatography on cross-linked 12% agarose

Three major active components of the traditional Chinese medicinal herb Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde, are separated and purified from a crude water extract in one step by isocratic hydrogen bond adsorption chromatography on cross-linked 12% agarose (Superose 12 HR 10/30). Separation is achieved by stepwise elution with mobile phases composed of mixtures of ethanol and acetic acid: 0-50 mL, 5% ethanol, 5% acetic acid; 50-100 mL, 20% ethanol, 20% acetic acid; and 100-200 mL, 30% ethanol, 30% acetic acid. The 3,4-dihydroxyphenyllactic acid is obtained with a purity of 97.3% and with a recovery of 88.1%. The corresponding figures for protocatechualdehyde are a purity of 99.4% with a recovery of 90.7%, and for salvianolic acid B a purity of 90.4% with a recovery of 50.3%, respectively. At a sample load of 40 mg crude extract dissolved in 0.5 mL mobile phase (corresponding to a load of 1.6 mg/mL gel), a 3,4-dihydroxyphenyllactic acid purity of approximately 94% with a recovery of 80.2% is obtained.     

Mixed-mode retention mechanism for (-)-epigallocatechin gallate on a 12% cross-linked agarose gel media

The adsorption behaviour of (-)-epigallocatechin gallate (EGCG), the major polyphenolic substance in green tea extracts, on the cross-linked agarose gel Superose 12 HR 10/30, has been studied using a variety of solvent systems and shown to be based on a mixture of hydrogen bonding and hydrophobic interaction. The hydrogen bonding was studied in acetonitrile in the presence of different co-solvents possessing varying hydrogen bond donor (HBD) and/or hydrogen bond acceptor (HBA) characteristics. The HBA-value of the co-solvent had the highest effect whereas the HBD-value played a subordinate role. Retention due to hydrophobic interaction could be demonstrated when mobile phases containing high water content were applied. The retention of EGCG, and its analogues (-)-epigallocatechin (EGC) and (-)-catechin (C) were thus shown to be dependent on the polarity of the organic modifiers added. However, the elution order of EGC and C, was inversed to that observed in reversed phase chromatography, indicating that some hydrogen bonding was still in effect. The retardation of EGCG in the presence of a wide concentration range of acetonitrile in water confirmed the interpretation that the retention mechanism is of mixed-mode character based on both hydrogen bonding and hydrophobic interaction.     

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents: Hydrophobic, thiophilic and electron donor–acceptor adsorbents

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol–divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene–divinyl sulfone agarose (DVS–TCP gel), tricyanoamino-propene–bisoxirane agarose (bisoxirane–TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor–acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor–acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for ‘hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor–acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor–acceptor ligand devoid of sulfur atoms.     

Boronate-containing copolymers: polyelectrolyte properties and sugar-specific interaction with agarose gel

Copolymers of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with acryamide (AA), N,N-dimethylacrylamide (DMAA), and N-isopropylacrylamide (NIPAM) were found to adsorb on cross-linked agarose gel (Sepharose CL-6B) in the pH range from 7.5-9.2, due to specific boronate-sugar interactions. The molar percentages of phenylboronic acid (PBA) groups in the boronate-containing copolymers (BCCs), as estimated by 1H NMR spectroscopy, were 13, 10, and 16%, respectively, whereas the apparent ionization constants, the pKa values, of the copolymers were similar and equal to 9.0 +/- 0.2 at 20 degrees C. The copolymers adsorption capacities were in the range of 15-30 mg x ml(-1) gel (14-36 micromol pendant PBA ml(-1) gel) at pH 9.2 and decreased with decreasing pH value. The interaction of monomeric NAAPBA with Sepharose CL-6B was characterized by an equilibrium association constant of 53 +/- 17 M(-1), the chromatographic capacity factor k' = 1.8, and a total content of binding sites of 27 +/- 10 micromol x ml(-1) gel at pH 9.2. The weak reversible binding of monomeric NAAPBA and almost irreversible binding of NAAPBA copolymers to the gel at pH 9.2 suggested a multivalent character of the copolymer adsorption. At pH 7.5, the maximal adsorption capacity was displayed by the AA-NAAPBA copolymer (15 mg x ml(-1) gel). All the BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH values from 7.5-9.2. The strong adsorption of AA-NAAPBA on agarose gel probably relates to the conformation of the copolymer in aqueous solution and provides opportunities for biomedical applications of the copolymer under physiological conditions. Multivalent, weak-affinity adsorption of BCCs to the agarose gel seems to be a tentative model for the copolymers' binding to oligo- and polysaccharides of cell membranes and mucosal surfaces.     

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.     

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

Phenylboronate-chitosan resins for adsorption of β-amylase from soybean extracts

Isolation and purification of bioproducts from crude extracts can be obtained by affinity methods based on reversible binding of a specific molecule to ligand immobilized in a porous matrix. In the present work, nicrospheres based on chitosan matrix, which incorporated aminophenylboronic acid as a derivative, were prepared and characterized, aimed at developing a β-amylase adsorption process. Kinetic curves and adsorption isotheriru of the crude extracts as well as the breakthrough curves for a frontal chromatographic separation method of a commercial sample of β-amylase from soybean are presented. These results were compared to similar data obtained with a comercial microspheres gel based-on agarose.     

Precise control of agarose media pore structure by regulating cooling rate

A porous structure is the key factor to successful chromatography separation. Agarose gel as one of the most popular porous media has been extensively used in chromatography separation. As the cooling process in the agarose gelation procedure can directly influence the pore structure, ten kinds of 4% agarose media with different cooling rates from 0.132 to 16.7°C/min were synthesized, and the pore structure was determined accurately by using low‐field NMR spectroscopy. The curves of pore structure and cooling rate can be divided into two stages with the boundary of 6°C/min. In stage I, the pore structure met a power equation with the decrease of the cooling rate, and in stage II, the process reached a plateau. Confirmatory experiments proved that, by adjusting the cooling rate, a precise control of the pore structure of agarose media can be realized, furthermore, cooling rate optimization was an effective way to control the pore size of agarose media and can further tailor the pore structure for more effective separation of different proteins.