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A novel on-line comprehensive two-dimensional liquid chromatography (2D-LC) method by coupling mixed-mode gel liquid chromatography (MMG-LC) with reversed phase liquid chromatography (RPLC) was developed. A mixture of 17 reference compounds was used to study the separation mechanism. A crude water extract of Flos Carthami was applied to evaluate the performance of the novel 2D-LC system. In the first dimension, the extract was eluted with a gradient of water/methanol over a cross-linked dextran gel Sephadex LH-20 column. Meanwhile, the advantages of size exclusion, reversed phase partition and adsorption separation mechanism were exploited before further on-line reversed phase purification on the second dimension. This novel on-line mixed-mode Sephadex LH-20 × RPLC method provided higher peak resolution, sample processing ability (2.5 mg) and better orthogonality (72.9%) versus RPLC × RPLC and hydrophilic interaction liquid chromatography (HILIC) × RPLC. To the best of our knowledge, this is the first report of a mixed-mode Sephadex LH-20 × RPLC separation method with successful applications in on-line mode, which might be beneficial for harvesting targets from complicated medicinal plants.

The presence of Ostreopsis cf. ovata on the Mediterranean coast represents a serious concern to human health due to production of toxins–putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d, -e, -f and -g). However, purified ovatoxins are not widely available and their toxicities are still unknown. In the present study, we report on HR LC-MS/MS analysis of a French Ostreopsis cf. ovata strain (IFR-OST-0.3 V) collected at Villefranche-sur-Mer (France) during a bloom in 2011. Investigation of this strain of Ostreopsis cf. ovata cultivated in our laboratory by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS) confirmed the production of ovatoxins -a to–e and revealed the presence of a new ovatoxin analogue, named ovatoxin-h. Ostreopsis cf. ovata extracts were pre-purified by Sephadex LH-20 to obtain a concentrated fraction of ovatoxins (OVTXs). This method provided a recovery of about 85% of OVTXs and a cleanup efficiency of 93%. Different stationary phases were tested with this fraction of interest to elucidate the structure of the new OVTX congener and to obtain purified ovatoxins. Eight reversed phase sorbents were evaluated for their capacity to separate and purify ovatoxins. Among them Kinetex C18, Kinetex PFP and Uptisphere C18-TF allowed for best separations almost achieving baseline resolution. Kinetex C18 is able to sufficiently separate these toxins, allowing us to identify the toxins present in the extract purified by Sephadex LH-20, and to partly elucidate the structure of the new ovatoxin congener. This toxin possesses one oxygen atom less and two hydrogens more than ovatoxin-a. Investigations using liquid chromatography coupled to high resolution tandem mass spectrometry suggest that the part of the molecule where ovatoxin-h differs from ovatoxin-a is situated between C42 and C49. Uptisphere C18-TF was proposed as a first step preparative chromatography as it is able to separate a higher number of ovatoxins (especially ovatoxin-d and ovatoxin-e) and because it separates ovatoxins from unknown compounds, identified using full scan single quadrupole mass spectrometry. After pre-purification with Sephadex LH-20, purification and separation of individual ovatoxins was attempted using an Uptisphere C18-TF column. During recovery of purified toxins, problems of stability of OVTXs were observed, leading us to investigate experimental conditions responsible for this degradation.

Research on the isolation and preparation of anthocyanins has intensified in recent years because of the requirements of quantitative and bioactive analyses. However, simple and effective methods for the scale purification of pure anthocyanins from natural products are rarely reported. In this study, high-purity anthocyanin mixtures and monomers were successfully isolated from wild blueberries using a combination of column chromatography and semi-preparative HPLC. We established an effective elution system to separate high-purity anthocyanin mixtures with aqueous ethanol containing 0.01% HCl first in an Amberlite XAD-7HP column (ethanol/H2O=35:65) and then in a Sephadex LH-20 column (ethanol/H2O=25:75). Crude anthocyanin extracts were isolated using the Amberlite column, and a purity of 32% was obtained based on UV-vis analysis. Three fractions of anthocyanin mixtures were isolated from the crude extracts using the Sephadex column with purities ranging from 59% to 68%. Three pure monomeric anthocyanins of malvidin-3-O-glucoside, petunidin-3-O-glucoside, and delphinidin-3-O-glucoside were also isolated by semi-preparative HPLC and identified by HPLC-DAD-ESI-MS/MS. The purities of these anthocyanins were determined by analytical HPLC and estimated to be 97.7%, 99.3%, and 95.4%, respectively. The results of this study may help promote the purification of anthocyanins from most blueberry varieties as well as from other plant materials.

The interaction of a series of dodecaborate cluster compounds B(12)X(12)(2-) and B(12)X(11)Y(2-) (X=H, Cl, Br, I and Y=SH, OH, NR(3)) with hydrophilic column materials (Superdex 200, Sepharose 4B, Sephadex G-50, Sephadex G-100, alumina, silica gel and anion exchange material) was studied. Almost all the dodecaborate cluster compounds were retained strongly on Superdex 200. The halogenated cluster compounds interacted with Sepharose 4B, Sephadex G-50, Sephadex G-100 and alumina; on alumina, also the non-halogenated clusters were retained. Silica gel showed the least interaction with all compounds. The thermodynamic parameters were investigated for a selection of compounds on Superdex 200 and Sephadex G-100. Values for ΔH° were found to be negative on both gels. As the change in entropy ΔS° was also negative, it compensated ΔH° to a large extent. The clusters interacted also strongly with anion exchange material in ion chromatography; the interaction decreased with increasing acetonitrile concentration, implying a large contribution from solvent effects.

Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v). Salfredin B(11) (22.1mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9mg, HPLC purity 97.3%) and crude sample 2 (555mg) were separated from 600mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12mg) and 3-O-[beta-d-xylopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-arabinopyranosyl]-hederagenin (45mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12mg of hederagenin and 45mg of 3-O-[beta-d-xylopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, (1)H NMR and (13)C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant.

A glycoconjugate constituted by the Streptococcus pneumoniae serotype 14 capsular polysaccharide (CPS14) and bovine serum albumin (BSA) was prepared, and the unique properties of Sephadex LH-20 were used to separate the conjugate from the unconjugated material. The strength of this approach consists in its capacity to produce pure polysaccharide-protein conjugate in good yield and free from unconjugated material, a common residual contaminant of this type of immunobiologicals. The CPS14-BSA conjugate prepared via an improved 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)-activation technique was characterized chemically and its immunogenicity was evaluated in mice. The purified conjugate, unlike the corresponding polysaccharide, produced a T-cell-dependent response in this species.

A novel high yield isolation procedure for lipophilic cyclic peptide derivatives is presented. Destruxin (dtx) A, B, D, E, and E-diol retrieval from Metarhizium anisopliae culture broth was achieved with a three-step purification protocol. After liquid-liquid extraction column chromatography over Sephadex LH-20 served as enrichment step. High-speed counter-current chromatography (HSCCC) was used for the final purification. Within the first chromatographic step dtx D and dtx E-diol were separated in purities exceeding 90%. The separation of dtx A, B, and E was achieved from an enriched Sephadex LH-20 fraction by a HSCCC protocol using light petroleum-ethyl acetate-methanol-water = 2:5:2:5 (v/v) as eluent system. These derivatives were obtained in purities above 98% and total yields exceeding 40%.

kappa-Carrageenan is a polysaccharide from red seaweed which gets precipitated by K+ ions and dissolves again in water. This smart, K(+)-responsive polymer was found to selectively bind pullulanase activity from Bacillus acidopullulyticus. Gel filtration on Sephadex G-200 showed the formation of the polymer-pullulanase complex at the pre-precipitation stage. On the other hand, phospholipase D, an enzyme which did not co-precipitate with kappa-carrageenan, did not form any complex with the polymer. Thus, K+ ions could be used to selectively precipitate the pullulanase activity. Then, 92% enzyme activity could be eluted with 1 M maltose solution. The single step protocol resulted in 50-fold purification, with a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Peptides are difficult to isolate from wine because they are present in a complex mixture together with non-peptidic compounds. A method for the isolation, separation and purity assessing of small peptides is proposed. Small peptides (Mr

A sample solution containing sodium or potassium dihydrogenphosphate or disodium or dipotassium hydrogenphosphate was eluted from a Sephadex G-15 column with either sodium or potassium chloride solution in various sample-eluent systems, and every one of the four kinds of ions employed was determined in the eluate. Then, the elution profiles showed the phenomenon that a negative peak of chloride ion coexisted in the fractions of a positive peak of phosphate ion, in all sample-eluent systems employed. This phenomenon was assumed to occur by the coupled reaction of change-partner and ion-exchange reactions including ion exclusion.

The benzodiazepine receptor from calf brain was solubilized with sodium deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inhibitors, and bound flunitrazepam with an equilibrium dissociation constant (K-d) of 2.7+/-1.2 nM and with 0.40+/-0.04 pmol binding sites per mg protein (B-max). Up to 60% of the benzodiazepine binding sites (average 25%) could be reconstituted in lipid vesicles, upon size-exclusion chromatography of protein-detergent-lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the reconstituted receptor varied with the lipid composition (K-d 1.4-4 nM), Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, doubled the number of operative flunitrazepam binding sites. When the proteoliposomes were stored at -20 degrees C or -80 degrees C or in lyophilized state, the receptor retained its benzodiazepine binding affinity and B-max over a period of 2 months. (C) 1997 Elsevier Science B.V.

Fiber optic sensors show promise for real-time monitoring of local conditions inside chromatography columns. Experiments on a Sephadex G-50 column, with blue dextran as the test analyte, showed that insertion of a single fiber or a lattice of fibers did not interfere with column fluidics, provided the fiber diameter was within the same order of magnitude as the diameters of the particles composing the column packing. Experiments on a Sephadex G-50 column, with fluorescein as the test analyte, showed that column packing particles do not interfere with the sensor's ability to accurately reflect differences in fluorescence produced by fluorescein under different conditions (e.g., concentration, pH). Experiments with fluorescein on a G-50 column also showed that fiber optic sensors can be used to monitor local analyte concentrations within a column and to diagnose column problems which lead to reduced column efficiency.

The successive isothiocyanato complexes of chromium(III) including the geometric isomers, [Cr(NCS) n (OH 2 ) 6− n 3− n + ( n = 0–6), were completely separated for the first time by using gel chromatography. Together with a cation exchanger, Toyopearl HW-40 was mainly employed to separate the n = 0–3 complexes and Sephadex G-25 for the n = 3–6 complexes. The adsorption mechanism of the complexes on the gels was discussed on the basis of the thermodynamic functions obtained from the temperature dependence of the K av values of the complexes. The adsorption could be interpreted in terms of the difference in the degree of the water structure breaking by the hydrophobic ions in the bulk and gel phases, and the hydrogen bonding between the inert H 2 O ligand of the complexes and hydrophilic groups in the gel matrix.

We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

The molecular mass distribution of peptides in isoelectric soluble soybean protein hydrolysates and their hydrophobic peptide fractions was determined by gel permeation HPLC on a Zorbax Bio Series GF-250 column. The hydrophobic bitter peptide fraction of isoelectric soluble soybean protein hydrolysate with degree of hydrolysis 15% was fractionated on a Sephadex G-25 Fine column. The most bitter low-molecular-mass fraction of the peptides was separated by reversed-phase HPLC on a Spherisorb ODS-2 column. Fourteen low-molecular-mass hydrophobic bitter peptides containing three to six amino acid residues were isolated. They are predominantly composed of hydrophobic amino acids and have leucine, valine or tyrosine at their C-terminal position

Soluble polysaccharides have been isolated from sugar cane by size-exclusion chromatography using two successive columns of Sephadex G-10 and G-50. Two main fractions of different molecular mass were separated and their homogeneity was tested by HPLC using a Zorbax GF-450 and a Zorbax GF-250 column in series. Whereas the high-molecular-mass soluble polysaccharide (SP) fraction seems to be composed of two different polymers with retention times of about 16.5 and 24.5 min, the mid-molecular-mass carbohydrate (MMMC) fraction is resolved as only one peak with a retention time of about 2.45 min. This implies that Sephadex G-50 is not able to discriminate between the two categories of macromolecules, as SP eluted from Sephadex G-50 is clearly contaminated by MMMC. Fractionation of clarified juice with 80% (v/v) ethanol separates SP as an insoluble fraction and MMMC as an ethanol-soluble macromolecule. The natural fluorescence of both polymers reveals that they are glycoproteins and, in addition, they can clearly be recognized by the degree of tryptophan exposure to its chemical microenvironment.

Stokes radii (R) of sodium hyaluronate fractions were estimated from their available volumes (K) by gel chromatography on a column of Sephacryl S-1000 instead of the Sepharose series previously examined. The relationships between R and K already established for the Sephadex and Sepharose series were also applicable to Sephacryl S-500 and S-1000 gels. R values were determined with the use of bovine serum albumin as a standard of known size and the average radius (5.8 nm) of fibres constructing Sephacryl gels. The molecular masses (M) of the glycosaminoglycan fractions, calculated from their intrinsic viscosities according to Mark-Houwink equations, were in the range 3.9 · 104−8.8 · 105. Their Stokes radii were in the range 5.9–19.6 nm. The plots of R and M12 for these fractions gave R = 0.030 M12 and R = 0.016 M12 + 4.3 with M below and above 105, respectively. Although an upper limit of M for hyaluronic acid on Sepharose 2B appeared to be about 4 · 105, the use of Sephacryl S-1000 seems to extend this limit to about 1.5 · 106 under the experimental conditions

A self-contained sample preparation chromatographic device for the facile and rapid desalting, separation and purification of enzymes and other proteins is described. The method, dynamic column solid-phase extraction (DC-SPE), was applied to the extraction and separation of peroxidase isozymes from crude extracts of fresh leaf tissue of three maize lines and of cucumber leaf extracts, in screening plant resistance properties. When packed with Sephadex G-25(M), the DC-SPE device also gave highly satisfactory desalting of the plant protein solutions in a much shorter time (10–15 min) than that required to obtain similar results with Pharmacia DP-10 columns and/or by dialysis overnight.

The ability of a two-trap tandem system, one containing 300 mg of graphitized carbon black (GCB) and the other filled with 50 mg of Sephadex QAE A-25 strong anion exchanger (SAX), to extract trace amounts of phenols from environmental waters and isolate them from base-neutral species was evaluated. After the water sample had passed through the GCB cartridge, the latter was connected to the SAX cartridge and base—neutral species were removed from the GCB surface by a neutral eluent. Co-eluted very weakly acidic phenols were selectively readsorbed on the SAX surface. Still maintaining the two cartridges in series, an acidified eluent was allowed to flow through the two cartridges to recover the most acidic phenols from the GCB cartridge and the least acidic phenols from the SAX cartridge. After partial removal of the solvent, the final extract was submitted to reversed-phase high-performance liquid chromatography with UV detection. Recoveries of seventeen phenols of environmental concern added to 21 of drinking water at levels between 0.2 and 2 μg/1 were higher than 90%. The effect of the presence in water of fulvic acids on the efficiency of the extraction device was assessed. In term of recovery, the two-trap tandem system was compared with other two single extraction cartridges, one containing a chemically bonded siliceous material (C18) and the other SAX material. The limits of detection of the analytes considered were well below 0.1 μg/1.

Stokes radii (R) of sodium hyaluronate fractions were estimated from their available volumes (K) by gel chromatography on a column of Sephacryl S-1000 instead of the Sepharose series previously examined. The relationships between R and K already established for the Sephadex and Sepharose series were also applicable to Sephacryl S-500 and S-1000 gels. R values were determined with the use of bovine serum albumin as a standard of known size and the average radius (5.8 nm) of fibres constructing Sephacryl gels. The molecular masses (M) of the glycosaminoglycan fractions, calculated from their intrinsic viscosities according to Mark-Houwink equations, were in the range 3.9 · 104−8.8 · 105. Their Stokes radii were in the range 5.9–19.6 nm. The plots of R and M 1 2 for these fractions gave R = 0.030 M 1 2 and R = 0.016 M 1 2 + 4.3 with M below and above 105, respectively. Although an upper limit of M for hyaluronic acid on Sepharose 2B appeared to be about 4 · 105, the use of Sephacryl S-1000 seems to extend this limit to about 1.5 · 106 under the experimental conditions

The purification of multiple forms of cytochrome P-450 (P450) from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated marmosets using fast protein liquid chromatography (FPLC) is described. The main aim was to achieve a better separation of certain closely related P450 sub-forms from each other than that previously obtained using conventional chromatography. An 8-aminooctyl-Sepharose fraction of cholate-solubilized microsomes was obtained first and, after fast desalting on Sephadex G-25, loaded on to a preparative Mono Q column. Five of the six gradient peaks contained P450 and were each rechromatographed on an analytical Mono Q column. The pass-through peak was fractionated further using a Mono S column. Other HPLC-quality anion- and cation-exchange gels were compared. For removal of excess of non-ionic detergent, five types of hydroxyapatite gels were compared. Seven purified forms of P450 and cytochrome b5 and P420 were isolated and characterized according to PHAST sodium dodecylsulphate-polyacrylamide gel electrophoretic apparent molecular masses, catalytic, spectral and magnetic properties and also TCDD-binding capacity (molar ratio of [14C]TCDD to P450). There are at least two sub-forms which appear to be TCDD inducible, one showing a substantial ethoxyresorufin-O-deethylase activity and the other having a high TCDD-binding molar ratio. Two other forms appear to be constitutive, as deduced from comparisons with forms purified from untreated animals.

When a sample solution containing sodium-22-labelled sodium chloride and carrier-free phosphorus-32-labelled phosphoric acid was eluted from a Sephadex G-15 column with either 0.025 M sodium or potassium phosphate buffer (pH 7.0), the labelled phosphate ion was eluted earlier than the sodium-22. The presence of cold 0.72 M sodium chloride with the sodium-22-labelled sodium chloride in the sample did not affect the elution sequence. When 1 M monosodium phosphate was eluted with distilled water from fresh and phosphate-treated Sephadex columns, the sodium and phosphate ions were eluted together in approximately the same fractions in both instances. From these observations, it is concluded that sodium ion repeatedly exchanges its partner phosphate ion with that in the eluent during its elution from Sephadex.

Three geometrical isomers of electrically neutral [Co(EBAA)(aa)] [EBAA = ethylenebis(aminoacidate); aa = an amino acidate] complexes have been synthesized and characterized. They have been resolved into the enantiomers, except for symmetrical cis-mer-[Co (EBAA)(aa)] isomers, by gel permeation chromatography on a Sephadex G-10 column with water as the eluent, and their absolute configurations have been determined based on their circular dichroism spectra. It has been revealed that, except for [Co(EBG)(β-ala)] [EBG = ethylenebis(glycinate)], the Δ enantiomers have a substantially greater optical purity.

The use of capillary isotachophoresis (ITP) for the characterization of humic substances was studied. ITP separations of these complex mixtures were considerably improved on the addition of polyvinyl-pyrrolidone (PVP) to the leading electrolyte. It was preferred to work in the spike mode to improve the interpretability of the isotachopherograms obtained using a photometric detector (405 nm). Interaction of PVP with humic substances was found to differentiate humic and fulvic acids. ITP was useful also for characterizing differences in the molecular weights and hydrophobicities of humic substances fractionated by chromatography on Sephadex G-50 and on Phenyl-Sepharose CL-4B.

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.

A recently described vesicular chromatographic packing material (VP), consisting of purified plant cell walls with vesicular and cellular morphology, was characterized with respect to the minimum Stokes′ diameter necessary for complete exclusion (size limit of exclusion = SLE) and the maximum Stokes′ diameter permitting permeation into the whole stationary liquid volume (size limit of permeation = SLP). Using vesicle chromatography, the size fractionation of a polydisperse dextran preparation with a defined size distribution was carried out to determine the percentages of completely excluded ( P ex ) and completely permeable ( P perm ) dextran molecules. SLE may be derived from P ex and SLP from P perm taking into account the molecular size range of the fractionated polydisperse dextran sample. Values determined for the size limits of the vesicle membrane with the help of dextran 35 (calibrated on Sephadex G-200) were nearly equal to those determined with the help of dextran 15 (calibrated on Sephadex G-75). The SLP of the standard VP is 5.6 nm. However, negatively charged proteins with a Stokes′ diameter slightly below the SLP (pepsin and ovalbumin) are excluded from the VP. The method may be applied in controlling the separation limits of VPs with altered ultrafiltration properties.

The combination of a divinylbenzene-based reversed-phase (RP) column and acetic acid gradients in water as mobile phase described in the accompanying paper was used for characterizing the extractable polypeptides from the normal and the diabetic human pancreas. The pancreas was lyophilized, minced and extracted three times in 3 M acetic acid. After mechanical clarification, the raw extracts were applied directly to the RP column. Alternatively, the extracts were lyophilized and subjected to size-exclusion chromatography on Sephadex G-50 in 3 M acetic acid. Two fractions with mol. wt. > 6000 dalton (Peak I) or with mol. wt. ⩽ 6000 dalton (Peak II) were obtained. The Sephadex G-50 size-exclusion chromatography and the RP-high-performance liquid chromatographic (HPLC) analyses of the crude extracts from a normal pancreas clearly demonstrated the weight distribution and differences between the exocrine pancreas (containing primarily the major digestive enzymes) and the endocrine pancreas (containing insulin, glucagon, etc.). RP-HPLC analyses of crude extracts from various normal pancreatic glands resulted in very similar UV profiles, whereas those from a number of individual diabetic glands differed. Chromatograms of acetic acid extracts from normal pancreata were similar when analysed before or after lyophilization, whereas lyophilization of acetic acid extracts of diabetic glands resulted in severely obscurred chromatograms. RP-HPLC analyses clearly demonstrated several differences between the diabetic and the normal pancreas. In the crude extracts, the extractable proteins from the diabetic pancreas were shifted towards lower molecular weight and/or hydrophobicity. Further, a peak co-eluting with authentic, human insulin could be demonstrated in the raw extract and in the peak II material from the normal pancreas, whereas virtually no mass signal was seen in the UV-profiles of similar materials from the diabetic gland. This finding was further verified by insulin radioimmunoassay (RIA) performed on the isolated fractions after RP-HPLC of a crude extract from a normal and a diabetic pancreas. The insulin content in the diabetic pancreas was found to be ca. 1% of that in the normal pancreas. When authentic glucagon was added to crude extracts from a diabetic pancreas, a single component was found after immediate analysis, but after several hours at room temperature the glucagon was found to be degraded. Added insulin was stable under these conditions. Similar RP analyses were performed on a silica C4 column eluted with an acetonitrile gradient in trifuoroacetic acid. Although the chromatograms were more complex than those obtained with acetic acid as mobile phase, they were comparable in outline, and the differences between the normal and the diabetic glands could again be demonstrated. One of the reasons for the differences between the normal and the diabetic pancreas may be that the normal glands were removed and frozen immediately after death, whereas the diabetic glands were obtained after an ischaemia time of 6 h or more, leaving considerable time for post mortem enzymatic degradation. The RP-HPLC system is very suitable for these analyses. The acetic acid extracts can be applied directly to the RP column and, after lyophilization, the isolated fractions may be re-analysed [RIA or enzyme-linked immunosorbent assay (ELISA)], or subjected to a second (alternative) RP-HPLC. After such double RP-HPLC purification, almost all major individual components in the peak I and II material from a normal pancreas were sufficiently pure for microsequencing, and identification (based on 30 steps in the gas-phase sequence and comparing the sequence information obtained with databases) again reflected the distribution of the principal components from the exocrine and the endocrine pancreas in the peak I and II materials.

Two novel gel filtration media, Superdex 75 prep grade and Superdex 200 prep grade are described. The new media have high selectivities similar to those of Sephadex G-75 and G-200, respectively, but permit the use of high flow-rates as with cross-linked agarose hbeads. They consist of cross-linked agarose beads modified with dextran to obtain the desired selectivity. The mean particle size is ca. 33 μm (30–36 μm) and a reduced plate height of ca. 2.0 is regularly obtained in packed columns. A flow-rate of 50cm/h can be used even in large columns. The media are chemically stable at pH 3–14 and can be used with a wide range of eluents. The performance of the media with respect to flow-rate, sample load and sample concentration for given model systems is described. Superdex 75 prep grade was used to separate monomeric forms of a recombinant fusion protein (ZZ—brain insulin-like growth molecular weight 22 000 dalton) from dimers and oligomers. Superdex 200 prep grade was used to obtain separation of a monoclonal antibody from antibody dimer and contaminating transferrin.

A procedure is described for the determination of α-methyldopa (MD) [ l -3-(3,4-dihydroxyphenyl)-2-methylalanine], its metabolite and catecholamines in the urine and plasma of patients undergoing MD therapy, by high-performance liquid chromatography with dual working electrode coulometric detection. An efficient sample preparation procedure is presented for the isolation of endogenous MD, its metabolite and catecholamines from plasma or urine. After deproteinization of a plasma sample with methanol containing 2% of 0.5 M perchloric acid and dilution of a urine sample (1:200), MD, dihydroxyphenylacetic acid (DOPAC), 3-O-methylmethyldopa (3-OMMD) and homovanillic acid (HVA) were separated with a Supelcosil LC-18 column. Catecholamines were extracted from the supernatant of deproteinized plasma or from urine by ion exchange on a Sephadex CM-25 column and subsequent adsorption on alumina. The use of the same mobile phase for the concurrent assay of MD, its metabolite and catecholamines increased considerably the efficiency of sample separation. Recoveries were close to 100% for MD, DOPAC, 3-OMMD and HVA and 70% for catecholamines. The effects of variuos experimental parameters related to mobile phase composition on chromatographic performance are reported. The purity of the eluted compounds was tested by recording both the first detector response (oxidation current) and the second detector response (reduction current). The ratio of the detector responses yielded a chemical reversibility ratio for the detected compound. A number of applications such as monitoring data from patients under MD therapy are presented.

Monoiodinated tyrosine methyl ester, a derivative of 5-hydroxy-6,8,11,14-eicosatetraenic acid containing 125I in the phenolic orhto position was prepared with high specific radioactivity and separated by column chromatography on a Sephadex LH-20 gel. The adsorption behaviour of the labelled product was studied by absorption chromatography suing a Sephadex LH-20 adsorbent with ethanol—water as a binary eluent and by reversed-phase high-performance liquid chromatograhy using C18-silica as a stationary phase with aqueous binary containing ethanol, methanol or acetonitrile. In both separation systems, a linear relationship was found between the logarithmic capacity factor or distribution coefficient and the logarithmic concentration of the organic solvent.

For synthetic purposes mannobiose is isolated from the polysaccharide mannan of ivory nut flower. The chain length of mannan A is determined to approximately 15 mannose units on average with a definite distribution from 5 to 25 mannose units. Acetolysis is optimized and yields 41% mannobiose. The separation on Sephadex G-15 yields pure mannobiose, and in addition its higher homologues in gram amounts. Detection of the eluates is performed using a differential refractometer. Correspondingly laminaribiose can be obtained from the polysaccharides laminaran (brown algae Laminaria hyperborea) and pachyman (fungus Poria cocos Wolf, Bukuryo) by selectively optimized acetolysis in 30% yield. Depending on the starting material separations are favourably performed on silica gel or on Sephadex G-15. Laminaribiose and its higher homologues are obtained in gram amounts.

Evaluation of the elution behaviou of some classes of pesticides in gel chromatography The elution behavious of insecticides and herbicides of the organophosphorus, carbamate, phenylurea, triazine, chlorophenoxy acid and ester types has been studied on gel such as Sephadex, Bio-beads, and Merckogel. By comparing the elution volumes of the compounds within each class it was found that in most cases the order of elution does not follow the pure gel permeation principle, but is modified by several infuences of substituents, π-systems and the overall polarities of the compounds chromatographed. In triazine herbicides some linear relationships within several subgroups have been evaluated. The effects described may be used for differentiation and identification of pesticides.

Partition chromatography (Sephadex LH-20 or C 18 -partition) followed by high-performance liquid chromatography facilitated the organic and mutagenic characterization of synfuel samples. The mutagens in oil shale retort waters were polar, whereas those in a shale oil ranged from moderately polar to polar. In contrast, the mutagens in an solvent refined coal-II (SRC-II) distillate blend were primarily moderately polar. The mutagens in an SRC-I process solvent were both less polar and less heterogeneous than those of an SRC-I solid product. Known mutagens (primary aromatic amines and aza-polynuclear aromatic hydrocarbons) were identified in the SRC-I and SRC-II samples but not in the shale oil and retort waters.

Methods of preparing whole-fiber gel-filtration columns are described. Reproducible results were obtained with columns prepared by packing with randomized fiber mat type discs (such as cut from absorbent cotton mat rolls). Results from these columns compared favorably with Sephadex columns. The columns had a useful life exceeding one year. Techniques for reducing void volume and increasing the accessible pore volume are described.

A protein has been isolated from the venom of the western diamondback rattlesnake (Crotalus atrox) which induces acute myocardial depression when administered to experimental animals. Purification was achieved by gel filtration on Sephadex G-100, DEAE- and CM-cellulose ion-exchange chromatography, ultrafiltration, and adsorption chromatography on hydroxyapatite. Amino acid analysis of the highly purified protein indicated N-terminal isoleucine and C-terminal tyrosine residues, and the absence of free sulfhydryl groups. Rabbits were immunized against the myocardial depressor protein (MDP) and a highly specific antiserum prepared which made it possible to study other snake venoms for the presence or absence of MDP. All of the North American Crotalid species of snakes contain MDP in varying degrees of concentration, but none of the Asiatic snake venoms tested reacted with the antiserum to the myocardial depressor protein. Intravenous administration of MDP to experimental animals (dogs, cats) produces an immediate and profound decrease in the cardiac output, the left ventricular systolic and mean pressures, the velocity of shortening of the contractile element, the systemic arterial pressure and an elevation in the left ventricular end-diastolic and pulmonary wedge pressures. These hemodynamic changes indicate that MDP administration induces an acute myocardial failure which is dose dependent. The potential use of this protein for the reproducible causation of left ventricular failure, obviating the need for the more commonly used surgical ligation of the coronary arteries, warrants a full investigation into its structure, active site and its mechanism of action on the myocardial cell.

Culture medium conditioned by human mononuclear leukocytes (MNL) stimulated with formalin fixed heat-killed Staphylococcus aureus induces a small respiratory burst in human neutrophils, and dramatically increases the response of neutrophils to stimuli such as N-formyl- l -methionyl- l -leucyl- l -phenylalanine. The data presented show that the activity is not unique to Staphylococcus aureus. Similar neutrophil modulating activities were produced by medium conditioned by MNL cultured in the presence of Streptococcus pneumonia, and Group B streptococcus. The activity was relatively resistant to heating: significant reduction of activity was observed only when 80°C was reached. Neutrophil stimulating activity production by stimulated MNL was dependent on protein and RNA synthesis and the activity appeared to be released by the non-adherent fraction of the MNL, suggesting that it is not of macrophage origin. The activity was not sensitive to soya bean trypsin inhibitor, but was sensitive to trypsin and was not removed when stimulated conditioned medium was depleted of immunoglobulin and albumin by affinity chromatography. Purification by gel filtration on Sephadex G-100 and high-performance liquid chromatography with Bio-Sil TSK250 columns showed that the major activity had an apparent molecular weight of 35 000–43 000 under conditions in which ionic interactions and association with albumin were reduced; by using polyethylene glycol or high salt (0.46 M Na+) in the elution buffer.

The gel chromatographic abehaviour of tetraaklkylammonium (TAA) ions on Sephadex G-10 was studied chiefly by a thermodynamic and an extra-thermodynamic approach. The pH dependence of the distribution coefficients of TAA ions was observed, and compared with those of alkali metal and halide ions. The extra-thermodynamic approach showed that no enthalpy-compensation was operative in the partitioning of TAA ions between the mobile and the gel phase. Examination of the effects of eluent concentration, organic eluent and temperature suggested that hydrophobic interaction and sieving effects contribute to various extents to the separation mechanism of individual TAA ions in the temperature range studied.

Separation of N-heteropolycyclic aromatic hydrocarbons from polycyclic, aromatic hydrocarbons. Separation by ion-exchange chromatography and fractionation of cigarette smoke condensate Chromatography on strong acidic or strong basic ion exchangers of Sephadex gels enables separation of a mixture into N-heteropolycyclic aromatic hydrocarbons aza-PAH (acridine type), aza-PAH (carbazole type) and PAH. Compounds sorbed on the ion exchangers can be eluted by addition of acids and bases to the solvent. By liqid-liquid distribution procedures a concentrate of aza-PAH and PAH is produced from cigarette smoke condensate. This concentrate is separated by column chromatography on Sephadex ion exchangers into three fractions which should contain aza-PAH (carbazole type), aza-PAH (acridine type) and PAH.

Elution volumes of NaI and Na 125 I appear to differ significantly when chromatographed on Sephadex G-15 or G-10. Using distilled water as the eluent, radioiodide was eluted in the void volume of the column while NaI was found eluted after NaCl. However, in most other eluent systems studied, radioiodide was retarded in comparison with NaI. This retardation was greatest with organic acid eluents and was more pronounced on Sephadex G-10 than on G-15. The difference in the elution volumes between NaI and Na 125 I could be decreased by elution with 0.1 M NaOH or by addition of NaI or KI to the loading sample. Sephadex G-25 did not appear to discriminate between NaI and Na 125 I under the conditions studied.

The spatial distributions of individual components of carrier ampholyte mixtures obtained by isoelectric focusing in cylindrical (6 mm × 75 mm) polyacrylamide gels (PAG) and in a thin-layer Sephadex gel (TLSG) were determined by ion-exchange chromatography of the eluates of gel slices. A ca. 1-mm slice of PAG separating the A and B forms of β-lactoglobulin (isoelectric points of 5.21 and 5.34, respectively) was found to contain at least 12–14 ampholyte components out of a total of at least sixty in the ampholyte mixture (LKB Ampholine, pH 3.5–10, Lot No. 17). Fifteen to twenty components were found in each 2-mm slice of a sequence of six slices of PAG subsequent to isoelectric focusing of sperm whale myoglobin with the same ampholyte mixture. An ampholyte mixture prepared by the copolymerization of triethylenetetramine with acrylic acid was focused in TLSG on 20 cm × 5 cm plates and the eluates obtained from ca. 5-mm slices were analyzed by ion-exchange chromatography. The results were compared with the caramelization pattern obtained by the Felgenhauer technique. The spatial distributions of individual ampholyte components were broad with half-band widths of ca. 1 cm. The effects of sample load, ampholyte concentration, and duration of focusing on spatial distribution were investigated in the PAG focusing of [ 14 C]histidine. The narrowest and most symmetrical distributions were obtained with small sample load and high ampholyte concentration.

Metabolites of methoxyphenamine in urine obtaied after oral administration of the drug to rats have been isolated by Sephadex LH-20 column chromatography and identified as the O-, N,O- and N-demethylated derivatives of methoxyphenamine by thin-layer chromatography, spectrometry, and comparisons with synthesized compounds. The unchanged drug and the metabolites excreted in urine have been extracted and determined by gas chromatography after treatment with trifluoroacetic anhydride.

A method is described for the separation and isolation of the naturally-occuring cytokinins from plant extracts. The method also separates any geometric isomers that occur. The procedure involves tissue extraction with ethanol and the partitioning of the cytokinins into 1-butanol at pH 9.0. Preliminary separation of the cytokinins into well defined groups is achived by chromatography on a Sephadex LH-20 column. Final resolution of the individual cytokinins and their isomers is achieved by high-performance liquid chromatography on a column packed with LiChrosorb RP-8, 10 μm mean particle size.

The behaviour of 23 steroid oestrogens on Sephadex LH-20 using six solvent systems was investigated. The fractions eluted by gel column chromatography were analysed by thin-layer chromatography and spectrometry. The method was used for the analysis of the radiochemical purity of labelled compounds by isotopic dilution.

A “mixed solute exclusion” method for teh porous structure analysis of gel substances has been developed. A mixed solution of polymer and oligomers covering a sufficiently wide molecular weight range is brought into contract with a gel sample and the resulting differential dilution of the solute fractions is determined as a function of the molecular size by means of gel permeation chromatography (GPC). This method and two other versions of the solute exclusion technique, the single-point method (equilibrium partitioing) and the column method (inverse GPC), were applied to a cross-linked dextran gel (Sephadex G-100) using dextran and/or poly(ethylene oxide) fractions as the probe polymer. The results of the three methods were consistent with each other and demostrated the efficacy of the mixed solute exclusion method for the rapid analysis of gel structure. An important limitation, however, is the resolution of solute exclusion methods in general, as shown by examining the assumptions in interpreting a solute exclusion curve as a pore size distribution curve.

Rapid preparative purification and fragmentation of a cytokinin complex from tomato root pressure exudate is achieved using Bondapak C18/Porasil B, a preparative reversed-phase high-pressure liquid chromatography column packing material. Bondapak C18/Porasil B achieved the same resolution of the cytokinin complex from tomato root pressure exudate as Sephadex LH-20 in approximately one-thirtieth of the separation time. The tomato root pressure exudate was found to contain at least eight cytokinin-like components. The total cytokinin activity of the root pressure exudate was estimated to be between 17.0 and 26.0 μg of kinetin equivalents per liter of exudate.