Semi-preparative Column Research Articles

A modified Arg-Asp-Val (RDV) peptide derived during the synthesis of Arg-Glu-Asp-Val (REDV), a tetrapeptide derived from an alternatively spliced site in fibronectin, inhibits the binding of fibrinogen, fibronectin, von Willebrand factor and vitronectin to activated platelets

Characterization of a side-product obtained during the synthesis of Arg-Glu-Asp-Val (REDV) with inhibitory activity in thrombin-activated platelet aggregation was carried out. The semipreparative column fractionation of REDV peptide was rechromatographed on an analytical HPLC column and revealed two peaks which were re-tested for inhibitory activity. Using amino acid analysis with sequencing and fast atom bombardment mass spectrometry (FABMS), the first peak was determined to be REDV with molecular mass of 517 Da, and the second peak was determined to be a modified RDV with a mass of 608 Da. The modified RDV peptide inhibited thrombin-induced platelet aggregation with an IC50 of 200 μM, and complete inhibition occurred at 600 μM. However, the REDV peptide did not inhibit platelet aggregation up to 1 mM concentration. The modified RDV peptide eluted platelet glycoprotein IIb-IIIa complex that had been bound to GRGDSP-agarose. These studies show that the modified RDV peptide interacts with the platelet glycoprotein IIb-IIIa complex. Based on the collision-induced dissociation (CID) mass spectral data analysis, the modified RDV peptide has been characterized to contain an N-terminus blocking group on the Arg residue. The origin of this blocking group is presumed to have originated from decomposition products of the phenylacetamidomethyl (PAM) resin used in the solid-phase synthesis of the target peptide Arg-Glu-Asp-Val.

Fractionation and viscometric characterization of a (1→3), (1→4)-β- d-glucan from oat, and universal calibration of a high-performance size-exclusion chromatographic system by the use of fractionated β-glucans, alginates and pullulans

A polydisperse sample of mixed-linkage (1→3), (1→4)-β- D-glucans from oat aleurone was fractionated by conventional size exclusion chromatography on semipreparative columns of Sepharose CL 4B and 6B to produce fractions of low polydispersity. The fractions were characterized by their intrinsic viscosities and their number-average molecular weight. The HPSEC-chromatograms of the β-glucan fractions suggested very narrow molecular weight distributions in all fractionated samples. From knowledge of the relative weights of the fractions and by assuming that each fraction was strictly monodisperse, the weight and number-average molecular weight of the unfractionated sample of β-glucan were determined to be 71 900 and 49 000, respectively, in reasonable agreement with the number-average molecular weight determined by osmometry (55 000). The Mark-Houwink-Sakurada viscosity equation for monodisperse β-glucans was determined for samples having molecular weights from 26 × 10 3 to 137 × 10 3 g/mol as [ η ] = 6.7 × 10 − 2 M ¯ n 0.75 (in ml/g) (20°C, 0.1 mol/dm 3 NaCl). The β-glucan fractions were further characterized by high-performance size-exclusion chromatography, and compared with commercially prepared pullulan fractions of low polydispersity and alginate fractions obtained in another study in this laboratory. The validity of the universal calibration procedure was tested with the three different polysaccharides, showing the same linear relationship between hydrodynamic volume ([η] × M) and elution volume for all three polysaccharides. The commercial pullulan samples seemed well-suited for calibration purposes.

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30–70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant ( K M) and catalytic constant ( K cat)] and the ratio K cat/ K M against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH 2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 μg of cathepsin B and 50 μg each of cathepsins L and H are obtained in a single run.

Extraction and identification of a plasticizer, di-(2-ethylhexyl)phthalate, from a plastic bag containing contaminated corn

Residues of di-(2-ethylhexyl)phthalate (DEHP) were extracted along with deoxynivalenol (DON) from plastic bags containing corn contaminated byFusarium graminearum. The concentration/extraction of this residue involved a series of silica gel-400 chromatography columns followed by purification by semi-preparative column chromatography, using hexane/dichloromethane as the mobile phase. The purified product was identified by NMR (1H and13C) and mass spectrometry. Quantification of DEHP was also done by high performance liquid chromatography (HPLC).

Liquid fuel analyses using high-performance liquid chromatography and gas chromatography-mass spectroscopy

The potential of a coal- or shale-derived liquid fuel precursor to be upgraded to a fuel product can be assessed if the composition of the precursor is known. Herein is described a technique utilizing high-performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS) which provides sufficient compositional data to allow one to estimate fuel upgrading potential. Samples are first filtered and then subjected to a liquid-liquid extraction to remove solids and phenolics, which have deleterious effects on the HPLC separation. The liquids are then fractionated by using a semipreparative column into aliphatics and aromatics by ring size using HPLC and collected for off-line injection into the GC-MS, where further compositional information is obtained. Using this method, it is possible to quantitate the components present that are of most interest in assessing the potential of a fuel. These components include the various classes of aliphatics (such as straight chain, branched, cyclic, etc.) and the 1-, 2-, and 3-ring aromatics. A discussion of the experimental details along with data from several samples is presented

Versatility of Reversed Phase Chromatographic Techniques on the Separation and Purification of Complex Glutathione Conjugates Mixtures from Synthetic Origin

Abstract Reversed phase chromatographic techniques have shown to be versatile and convenient tools for separation and purification of the cis [c-GSH 1 (3S, 4R) and c-GSH 2 (3R, 4S)] and trans [t-GSH 1 (3R, 4R) and t-GSH 2 (3S, 4S), see Figure la] diastereomeric pairs of adducts derived from the reaction of glutathione with 3,4-epoxyprecocene II (1), a model of bioactive epoxide. Combination of analytical and semipreparative HPLC, Sep-Pack cartridges and open column flash chromatography, enabled the separation and eventual isolation of the above compounds at microgram, milligram, or gram scales. In addition, an open column procedure has also been developed for easy removal of salts and water from eluates in the above separations.

Improved high-speed counter-current chromatography with three multilayer coils connected in series : II. Separation of various biological samples with a semi-preparative column

The semipreparative capability of the newly developed high-speed counter-current chromatograph equipped with a set of three multilayer coils has been demonstrated in separations of a variety of biological samples including triterpenoic acids, indole auxins, bacitracin, flavonoids and tetracycline derivatives, each with a suitable two-phase solvent system. The sample quantities ranging from 50 to 500 mg were efficiently separated within a few hours. The separation of tetracycline derivatives was remarkably improved by adding ammonium acetate to the solvent system.

Separation schemes for coal tar analysis — a comparison of methods

A standard tar fractionation scheme used to separate coal-derived materials at CRE involves a solvent separation based on solubility in pentane and benzene. This method is operator-dependant. The solvent fractionation has been followed by open-column liquid chromatography on silica/alumina to generate saturate, aromatic and polar fractions. Our published work has shown that the aromatic fraction contains both saturate and polar material. Alternative methods have been investigated using three tars to compare performance. An initial separation into pentane solubles, asphaltenes and pre-asphaltenes by sequential elution of tar coated on beads, appears to be reproducible and not subject to operator variations. The subsequent fractionation of pentane soluble material into chemical class fractions has been investigated by three methods. They are open column chromatography on alumina, open column chromatography on OPN Porasil-C and HPLC on a semi-preparative PAC10 column. The alumina and OPN Porasil methods are more satisfactory than the silica column method, giving heterocyclic fractions which contained very little aromatic material. OPN Porasil-C is the more expensive packing, but it can be reused. The HPLC method is best for isolation of PAH ring size fractions free from polar compounds. A solvent gradient can be used to elute polar material free from the aromatics but large volumes of relatively expensive solvents are needed. It is cheaper to back-flush the column after elution of the aromatics and collect the polars with no separation. Mass spectrometry has been used to evaluate the various class fractions.

Semi-Preparative Reverse Phase HPLC Fractionation of Pesticides from Edible Fats and Oils

Abstract A semi-preparative (25 cm × 9.4 mm i.d.) HPLC ODS-bonded silica column was evaluated for the fractionation of pesticides from edible fats and oils of both animal and vegetable origin. Using acetonitrile as a mobile phase, organochlorine and organophosphorous pesticides with a wide range of polarity were eluted in the first 40 ml of eluate. Co-eluted lipid material ranged from 0 to 35 mg for 500 mg injections, depending on sample type. Excessively “dirty” samples (e.g., tallow) were further cleaned up on small Florisil columns. Recoveries of selected pesticides from fortified samples, which were determined by gas chromatography with electron capture and flame photometric detectors, ranged from 80 to 108%.

Rapid HPLC techniques for globin chain synthesis studies.

Globin chain synthesis and alpha/beta ratios were determined in a number of normal subjects, alpha-thalassemia-2 homozygotes, and beta-thalassemia trait using three different techniques. Cation exchange high performance liquid chromatography on a Pharmacia Mono-S, HR 5/5 and reversed phase high performance liquid chromatography on a semi-preparative Vydac C4 column were compared with the traditional carboxymethylcellulose chromatography. Both high performance liquid chromatography columns give excellent results when 2 mg of hemoglobin was chromatographed in each analysis. By modifying the protocols for column equilibration and gradient shape for preparative Vydac C4 columns, conditions were found yielding excellent resolutions of the labeled globin chains in less than an hour without the need for substantial increase of the flowrate. This method was found to be superior to other methods and may be a suitable alternative for the classical carboxymethylcellulose chromatography. Up to five specimens could easily be analyzed in a single day with this system.

Characterization of biotechnological processes and products using high‐performance liquid chromatography (HPLC) IV. SEC, HIC, and IEC separations of proteins

AbstractProteins are separated by means of size‐exclusion (SEC), hydrophobic‐interaction (HIC) and ion‐exchange chromatography (IEC). Analytical and semipreparative HPLC glass columns are the basis of the chromatographic analyses.Using a short (100 × 3.8 mm i.d.) column packed with Si 200 Polyol standard of different molecular weights (ovalbumin, MW = 43 000; chymotrypsinogen A, MW = 25 000; ribonuclease, MW = 13 700 and contrycal, MW = 6512) could be distinguished.Basic proteins (e.g., chymotrypsinogen A, cytochrome C) are separated on aluminium oxide (Li‐Chrosorb Alox T) by cation‐exchange chromatography. Correlations between the retention times of proteins and their isoelectric points or the buffer concentration of the mobile phase are investigated.Furthermore, two examples of liquid chromatographic purification procedures for enzymes of biotechnological interest are demonstrated. One enzyme extract (thermostable protease) is separated by hydrophobic‐interaction chromatography, another one (β‐galactosidase from a thermophilic microorganism) is purified on a weakly basic anion‐exchange resin (pore size: 130 nm) based on a styrene‐divinylbenzene copolymer.

Ｗａｎｄｏａｎ炭液化油中の重質極性成分の化学構造に関する考察 重質極性成分の水素化分解により生成する無極性成分の化学構造

The stepwise hydrocracking of heptane-soluble polar fraction (Po) and asphaltene (As) isolated from wandoan coal-derived residue was carried out over a presulfided Ni-Mo/Al2O3 catalyst at 375 °C to investigate their chemical structures. Five nonpolar samples (Po/NP, Po/Po/NP, As/NP, As/As/NP, As/Po/NP) were obtained from the hydrocracking products as shown in Fig. 1.The nonpolar products (Po/NP, As/NP) from the first hydrocracking of Po and As had lower molecular weights than those (Po/Po/NP, As/As/NP, As/Po/NP) from the second hydrocracking of the recovered polar materials. Each nonpolar product was separated into seven compound-types by HPLC equipped with a semipreparative Bondapak-NH2 column. Fr.3 and Fr.6 were analyzed by field ionization mass spectrometry (FIMS). The same compound-type fractions were shown to con-sist of similar polycyclic hydroaromatic structures, although those from the second hydrocracking have more naphthenic rings which resulted in higher molecular weights.

Isolation of a specific membrane protein by immunoaffinity chromatography with biotinylated antibodies immobilized on avidin-coated glass beads

Avidin-coated, solid glass beads have been used as an immobilization support for attaching biotinylated antibodies. These beads have been packed into analytical, semi-preparative and preparative columns and used to isolate the B27 histocompatibility anigen (HLA) from human lymphocytes. The beads provided a suitable column material for all three chromatographic procedures and, depending on the size of the immunoaffinity column, B27 antigen could be isolated in nanogram to microgram quantities. Polyacrylamide gel electrophoresis demonstrated the presence of only a single band in the immunoaffinity peaks isolated by all three procedures. Enzyme-linked immunosorbent assay analysis of these immunoaffinity-isolated materials revealed that they were biologically active and could be used to determine the levels of anti-B27 antibodies in clinical studies.

Direct electrophilic iodination of 2-thiouracil using iodo-gen™

5-Iodo-2-thiouracil (ITU) is of interest due to its ability to bind specifically to the pigment melanin during melanogenesis and is of potential value in the diagnosis and treatment of malignant melanoma. Radioiodinated ITU was prepared directly from 2-thiouracil in a two-phase reaction using Iodo-Gen™ in 0.05 M phosphate buffer pH 7.0. The identity radiochemical purity and stability of the product were checked by reversed-phase high pressure liquid chromatography (HPLC). ITU labeled with 123I, 125I or 131I has been produced in millicurie amounts and isolated on a semi-preparative reversed-phase HPLC column. Production time was 2–3 h, overall radiochemical yields averaged 80%; the radiochemical purity was greater than 98%. Specific activities on the order of 20 Ci/mmol have been obtained.

Improved high-speed counter-current chromatograph with three multilayer coils connected in series : I. Design of the apparatus and performance of semipreparative columns in 2,4-dinitrophenyl amino acid separation

A compact desktop model of a high-speed counter-current chromatograph holds three identical multilayer coils in the symmetrical positions around the rotary frame to maintain perfect balance of the centrifuge system without the use of a counterweight. These multilayer coils are connected in series to make up a total capacity of 400 ml while the unique gear arrangement on the rotary frame establishes a twist-free mechanism of the flow tubes so that continuous elution can be performed without the use of rotary seal. The high performance of the present system was successfully demonstrated in separations of 10–250 mg of 2,4-dinitrophenyl amino acid mixtures in a two-phase solvent system composed of chloroform—acetic acid—0.1 M hydrochloric acid (2:2:1, v/v/v).

Attempts on the semi-preparative resolution of racemic esters of aminoacids on a chiral liquid chromatographic column derived from N-formylisoleucine

A semi-preparative (250mm×10mm i.d.) chiral liquid chromatographic column incorporating a chiral material derived from N-formylisoleucine has been found to be suitable for the resolution of milligram or larger quantities of two aminoacid esters. The method should be capable of being used for other racemic materals on an even larger scale.

Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods

Samples of 7 foods commonly eaten in the Northeast of China (i.e. fried and broiled fishes and broiled meat) were tested for mutagenicity on Salmonella typhimurium TA98 with S9 mix. The basic fractions of the samples were mutagenic, inducing 33–2930 revertants/g of cooked food. Fried walleye pollack (a kind of cod fish heated on a stainless steel pan) showed the highest mutagenicity, so attempts were made to isolate mutagens from the basic fraction of this food. The mutagens were purified by treatment with blue cotton and HPLC on a semi-preparative ODS column and analytical cation exchange and ODS columns. 5 mutagens were isolated and identified as 2-amino-3-methylimidazo[4,5- f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5- f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQ), 2-amino-3,4,8-trimethylimidazo[4,5,- f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). 1 g of fried fish was estimated to contain 0.16 ng of IQ, 0.03 ng of MeIQ, 6.44 ng of MeIQx, 0.10 ng of 4,8-DiMeIQx and 69.2 ng of PhIP. MeIQx and PhIP accounted for 24% and 4.7%, respectively, of the total mutagenicity. The other 3 heterocyclic amines were each responsible for only 0.3–1.2% of the total mutagenicity.

Scale-up from analytical to preparative reversed-phase high-performance liquid chromatography of soybean glycinin

Analytical (50 μg) reversed-phase high-performance liquid chromatography (RP-HPLC) of fractions from soy protein purifications has been scaled up to injections in the preparative (1–200 mg) range to obtain sufficient quantities of glycinin subunits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis characterization. We describe here the glycinin separations obtained by using analytical, semi-preparative, and preparative columns with an analytical gradient HPLC system. Parameters such as injection volume, flow-rate, and detector sensitivity are noted. Our results suggest that analytical RP-HPLC separationis of proteins can be conveniently scaled up in most HPLC systems.

Isolation and identification of the glucuronide conjugate of 2-hydroxydesipramine by preparative liquid chromatography

A semi-preparative column liquid Chromatographie procedure for the isolation and purification of milligram quantities of the glucuronide conjugate of 2-hydroxydesipramine, a major metabolite of desipramine, is presented. Urine from patients receiving desipramine was collected and passed through a column of XAD-2 resin. The methanolic extract was chromatographed on a reversed-phase octadecyl semi-preparative column followed by further purification on a silica gel column of the same dimension, yielding a product 95% pure. Fast atom bombardment and themospray mass spectroscopy, as well as ultraviolet photodiode-array spectroscopy and hydrolysis with β-glucuronidase confirmed the identification and purity of 2-hydroxydesipramine glucuronide. This important glucuronide metabolite will be a useful tool as an authentic standard for pharmacokinetic and metabolism studies and for determining its pharmacological characteristics in laboratory animals.

Isolation and characterization of thirteen new salt-soluble proteins from barley by reversed-phase high-performance liquid chromatography

Thirteen new proteins from barley (Hordeum vulgare L.) have been isolated and characterized. These proteins and seven other previously known components were isolated from a 0.5-M NaCl extract of endosperm by single-step, reversed-phase, high-performance liquid chromatography, using a Vydac C4 semi-preparative column. The purity of the isolated proteins was analyzed by polyacrylamide gel electrophoresis at pH 3.2 or in sodium dodecyl sulfate, by two-dimensional gel electrophoresis and was further confirmed by partial NH2-terminal sequencing. The NH2-terminal amino-acid sequences of fourteen of the components were determined, from 5 to 27 cycles, by automated liquid-phase sequencing. According to the sequence data and predictions of secondary structure, different groups of homologous proteins were established. Based on the presented results, two thionins, one trypsin inhibitor and one α-amylase/subtilisin inhibitor are included among the purified proteins.