Preparative Reversed-phase High-performance Liquid Chromatography Research Articles

Isolation of recombinant hirudin by preparative high-performance liquid chromatography

The purification of recombinant hirudin varaint 2-Lys 47 (rHV2-Lys 47), produced by a genetically engineered yeast strain, is described. rHV2-Lys 47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth aftr cell harvesting by centrifugation. Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys 47 in the presence of acetone, removed most of the contaminants from the culture medium. The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases. Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Liquid secondary ion mass spectrometry was used to confirm its primary structure. The isolated product was tested in an inhibition assay of human α-thrombin and proved to be fully active.

Isolation and analysis of peptidic fragments of α-gliadin using reversed-phase high-performance liquid chromatography

Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

Preparative high-performance liquid chromatography of peptides on a new reversed-phase packing material, kromasil™ C 18

Preparative reversed-phase high-performance liquid chromatography has found wide use in the production ofpeptides for pharmaceutical formulations. Purity of the substance and overall economy of the chromatographic system are the most important criterias. In this sense optimized, silica particles and production process with capability to separately control parameters important to chromatography, are essential to high-performance chromatography. Kromasil™ C 18 packing material was tested and evaluated in respect of its selectivity, flow and pressure properties, resolution, load capacity, recovery, adsorption effects, mechanical strength and chemical degradation.

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.

High-speed preparative reversed-phase high-performance liquid chromatography of synthetic oligonucleotides

A rapid method is described for the purification and analysis of synthetic oligonucleotides, based on reversed-phase high-performance liquid chromatography. Volatile buffers and a short column (40 mm × 4.6 mm) packed with Nucleosil 300-5 C 4 were employed. Monitoring the column effluent with an UV detector provides an excellent means of controlling product quality. The method is suitable for the purification of crude synthesis products, as well as for desalting and removing gel contaminants from oligonucleotides eluted from polyacrylamide gels. The total time required per sample is less than 25 min.

Hemoglobin-based blood substitutes: characterization of five pyridoxal 5'-phosphate derivatives of hemoglobin

Si fractions (I-VI) of pyridoxal 5'-phosphate (PLP) hemoglobin (Hb), prepared by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205] have been isolated and purified by anion-exchange high-performance liquid chromatography (HPLC). Total phosphate analyses indicate that I is HbA, II and III are double-lebelled, IV and V are tetra-labelled and VI contains 6 mol of phosphorus per mol of hemoglobin. The purified components have been resolved into their α and β chains by preparative reversed-phase HPLC using a macroporous C 4 support. Phosphate analyses indicate that the β chains of II, III and IV each contain one phosphate per chain while the β chains of V and VI wach contain two phosphates. The α chains of IV and VI were found to be monophosphate-labelled. Reversed phase HPLC analysis of the tryptic peptides of the β chains indicates that the label is bound exclusively to the 1-valine residue in II, III and IV while both the 1-valine and the 82-lysine are labelled in V and VI. Similarly, modification of the 1-valine residue of the α chains of IV and VI was detected. Components II and III have the same molecular formula. Evidence is presented which shows that they are interconvertable and that they correspond to the PLP 2-Hb species and component V is Benesch's PLP 4-Hb [J. Biol Chem., 257 (1983) 1320 and references cited therein]. Component IV is III with one additional PLP per α chain and similarly VI is V with monolabelled α chains. Both IV and VI are hitherto unknown pyridoxal 5'-phosphate derivatives of hemoglobin. The oxygen affinities of the components were determined and were found to decrease with increases in the extent of pyridoxal '-phosphate labelling. This effect, however, reaches a maximum in V since VI which contains 6 mol of phosphate per mol of hemoglobin has a higher affinity than V.

Application of preparative reversed-phase high-performance liquid chromatography to the isolation of insulin-like growth factor II from human serum

Substantially purified insulin-like growth factor II (IGF-II) was prepared from human serum. Initial enrichment using ion-exchange chromatography on DEAE Sephadex A50, followed by gel permeation chromatography on Sephadex G-75 in 1% formic acid produced material suitable for application to a preparative reversed-phase high-performance liquid chromatographic (HPLC) column containing LiChroprep RP-18. The latter step gave about 90-fold purification with a recovery of about 70% IGF-II bio-activity. Finally, a small reversed-phase HPLC column achieved a 17-fold purification with similar yield of activity. Overall, the four steps gave IGF-II of about 90% purity in yield of 12%.

Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase.

The role of the Tyr10-Ser11-Lys12-Tyr13 region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe13]glucagonamide, [Phe10]glucagonamide, [Phe10]glucagon, [Phe10,13]glucagon, [Pro11]glucagon, [Pro11,Gly12]glucagonamide, [Ala11]glucagon, and [Oac11-13]glucagonamide. These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected N alpha-tert-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse-phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by alpha-chymotryptic peptide mapping with HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [125I]glucagon from glucagon receptors. The results of these studies indicate that while the biological "message" region of glucagon is located elsewhere, the 10-13 region has multiple roles in the glucagon-glucagon receptor interaction: this region provides functional groups for direct binding interaction with the receptor, and this region interacts with the receptor in such a way as to allow the "transduction message" portion of glucagon to interact and activate the receptor.

Preparative reversed-phase high-performance liquid chromatography of iodinated insulin retaining full biological activity

Insulin monoiodinated in Tyr A14, A19, B16 and B26 can be separated from insulin and diiodoinsulins using reversed-phase high-performance liquid chromatography on LiChrosorb RP-18 columns. Monoiodoinsulins with high and low specific activities were isolated from a number of buffer systems without any reduction in binding affinity and biological activity in isolated rat fat cells. The reason for the previously observed reduction in the binding affinity was probably column bleeding, i.e., chemical degradation of the column support.

Preparative purification of Escherichia coli heat-stable enterotoxin

Heat-stable enterotoxin (ST a) isolated from bovine Escherichia coli strains was purified to homogeneity by growing the bacterial strains in a chemically defined medium, desalting, and concentrating the culture filtrate by batch adsorption chromatography on Amberlite XAD-2 resin, batch adsorption chromatography on reversed-phase silica, and preparative reversed-phase high-performance liquid chromatography. This rapid preparative purification scheme gave high recovery yields of pure ST a which exhibited biochemical homology to ST a purified by more complicated procedures.

ChemInform Abstract: IMPROVED ANTAGONISTS OF LUTEINIZING HORMONE‐RELEASING HORMONE MODIFIED IN POSITION 7

The structure-activity relationship of position 7 in the antagonist [N-Ac-D-Nal1,D-pClPhe2,D-Trp3,D-Arg6,D-Ala10]-LH-RH has been investigated by the incorporation of a series of amino acids at this position. The analogues were prepared by solid-phase peptide synthesis. All purifications were performed in two stages: gel permeation followed by preparative reversed-phase high-performance liquid chromatography. The analogues were assayed in the standard rat antiovulatory assay using a 40% propylene glycol-saline vehicle. The results demonstrated that position 7 requires a hydrophobic aromatic amino acid for greatest antiovulatory activity. The compound [N-Ac-D-Nal1,D-pClPhe2,D-Trp3,D-Arg6,Phe7,D-Ala10]-LH- RH caused 65% blockade of ovulation at the 500-ng dose and is approximately twice as active as the parent analogue in this assay system. The enhanced activity may indicate the stabilization of the active conformation via intramolecular hydrophobic or tau-tau interactions.

Isolation, separation and detection via high-performance liquid chromatography of acidic and neutral acetylated rearrangement products of opium alkaloids

The isolation, separation and detection of acidic and neutral acetylated rearrangement products of opium alkaloids, which are major sources of heroin processing impurities, are described. Preparative reversed-phase high-performance liquid chromatography with the aid of a micro-computer has been utilized for the isolation of product obtained from the action of acetic anhydride on thebaine and noscapine alkaloids. Results obtained show that a high-speed C 18 analytical column (3 μm particle size, 3.0 cm × 4.6 mm I.D.) containing a monomeric bonded phase can be used to develop separations for a polymeric bonded-phase preparative column (10 μm particle size, 25 cm × 22 mm I.D.). An optimized reversed-phase separation was developed for the acetylated thebaine and noscapine compounds and for acetylated rearrangement products from three other opium alkaloids: morphine, codeine and norlaudanosine. Ultraviolet, fluorescence and electrochemical detection were compared for these compounds. A detection system optimized in terms of sensitivity and selectivity was developed utilizing two variable-wavelength UV detectors, programmable fluorescence detector, and an electrochemical detector in series.

Macroscale high-performance liquid chromatographic separation and instrumental identification of components of diethylaminoethyl murine epidermal growth factor

Murine epidermal growth factor (m-EGF), a polypeptide produced as a chromatographically homogeneous peak on diethylaminoethyl (DEAE) cellulose by the method of Savage and co-workers, and characterised as a single compound, has been shown by ourselves and several other groups to be a mixture. The present contribution extends our previously reported work and discuss the separation of this material, termed DEAE—m-EGF, into its components by preparative ion-pair reversed-phase high-performance liquid chromatography (RP-HPLC) on C 18 μBondapak in quantities up to 50 mg per run. Isocratic elution was used and the mobile phase was acetonitrile—water (26:74, v/v, 0.04 M in triethylamine acetate); pH was 5.6, temperature 40°C, and detection was by ultraviolet absorption at 254 nm, and (for some runs) by differential refractometry. Seven significant peaks, four major, three minor, were detected. Of the major peaks, two designated α- and β-EGF, constituted 70% of the total mass and were the most important to our work. Each of the eluted peaks was recovered by lyophilisation, and this product checked for homogeneity by ion-pair RP-HPLC on a C 18 μBondapak analytical column, with utraviolet detection as before. All recovered peaks were found to be homogeneous by this criterion. These chromatographically homogeneous compounds were investigated by modern physicochemical instrumentation to determine their structure. The molecular weight of each of the species was determined by fast atom bombardment mass spectrometry. High-field proton magnetic resonance at 270 MHz provided structural and conformational information. Polarimetry and ultraviolet absorption were also used to characterise the compound. α-EGF, for example, had a molecular weight of 6040 corresponding to the 53 amino acid residue peptide previously designated EGF; β-EGF had a molecular weight of 5930. This molecular weight differential of 110 suggested the hypothesis that β-EGF was a 52 residue peptide corresponding to α-EGF minus the terminal asparagine at position 1. Proton magnetic resonance difference spectroscopy (β spectrum subtracted from α) provided powerful confirmatory evidence for this hypothesis. All materials recovered from RP-HPLC were tested in the sheep and found to retain their biological activity.

Reversed-phase high-performance liquid chromatography of durum wheat gliadins: Relationships to durum wheat quality

Durum wheat gliadins have been analyzed by RP-HPLC (reversed-phase high-performance liquid chromatography) for the first time. Gliadins extracted from each particular durum variety were resolved to give a complex chromatographic pattern that was unique to that variety. However, four chromatographic fractions, which were eluted under hydrophobic conditions (45·2, 45·6, 47·4 and 49·1% acetonitrile, respectively), could be used to classify durum varieties into two groups. Proteins in these fractions were purified by preparative RP-HPLC and analyzed by aluminium lactate-polyacrylamide gel electrophoresis (AL—PAGE), which indicated that two of these fractions contained gliadin bands 45 or 42 that have been shown previously to be correlated closely with gluten strength and weakness, respectively. Therefore, RP—HPLC can be used also to screen for gluten quality characteristics in durum varieties in a way analogous to that in which electrophoresis may be used. A rapid, highly-automated RP-HPLC analytical procedure was developed to screen for durum varieties with unacceptably weak gluten characteristics. Each determination takes less than 10 min, permitting approximately 200 analyses per 24 h. Because of its convenience and rapidity, RP-HPLC of durum wheats has considerable potential as an alternative to electrophoresis as an analytical tool for screening for gluten traits when large numbers of samples must be screened, as in early generation of wheat breeding.

Analysis of fatty acid exthoxylates by preparative high-performance liquid chromatography

A method for the determination of poly(ethylene glycols) and poly(oxyethylene) mono- and dialkyl esters in fatty acid ethoxylates by preparative reversed-phase high-performance liquid chromatography is described. The complete separation of these non-ionic compounds has been obtained on a 500 x 20 mm I.D. column packed with C 18 bonded phase with acetone-water as the mobile phase. The separated compounds were subsequently determined by gravimetry.

Purification of D&C Red. No. 33 by preparative high-performance liquid chromatography

Preparative reversed-phase high-performance liquid chromatography (HPLC) was used to purify a composite sample of D&C Red No. 33. A number of contaminating impurities were isolated fromthe dye and the purified material was essentially free of residual aromatic amines. A comparison was made between purification by preparative HPLC and recrystallization.

Identification of betanin degradation products.

Betanin in solution, upon heating, was found to hydrolyze to betalamic acid and cyclodopa-5-O-glycoside. This reaction was monitored by an analytical high performance liquid chromatography (HPLC) method. The products were isolated by preparative reversed-phase HPLC or column chromatography using anion-exchange resins. Derivatives of betalamic acid (anilide, semicarbazone, condensation with L-proline) and cyclodopa-5-O-glycoside (hexacetate) were prepared as evidence to support the identification of these decomposition compounds. Formation of decarboxylated betanin was proposed based on the identification of CO2, chromatographic properties and the light absorption characteristics of the decarboxylated product.

Studies on iodinated compounds. II. High performance liquid chromatographic studies on the synthesis and purification of monoiodocarnosine.

Conditions for the synthesis and purification of monoiodocarnosine (MIC) were examined by high performance liquid chromatography (HPLC). When carnosine (β-alanyl-L-histidine) was iodinated to obtain MIC, the maximum yield was attained by the equimolar reaction of carnosine with I2 in 0.5 N NaOH solution. The synthesized MIC was separated by preparative reversedphase HPLC with H2O-MeOH (90 : 10) as the eluent and then purified by recrystallization. Chemically pure MIC was obtained in 72% yield in a short time.

Preparative reversed-phase high-performance liquid chromatography of monensin A and B sodium salts

Preparative reversed-phase high-performance liquid chromatography of monensin A and B sodium salts

Crystal structure of the [Pt(9-methylguanine) 4] 2+ cation

The [Pt(9-methyl-guanine) 4] 2+ cation is the first example of a Pt 2+ compound to have four purine ▪ bases as ligands. This compound is especially interesting as it was suggested earlier that the anti-tumor drug cis-dichlorodiammineplatinum(II) could lose all four ligands in order to interact with the purine bases of the DNA double helix [1]. The title compound was prepared by heating K 2PtCl 4 with excess 9-methylguanine (ratio 1:6) under reflux for several hours. The reaction mixture was separated by preparative reverse-phase high-performance liquid chromatography to remove excess ligand and some minor impurities possibly due to the formation of other Pt-guanine complexes. [Pt(9-Methylguanine) 4] 2+Cl −(CF 3COOH) − crystallizes as colorless bricks in the triclinic space group P 1 , with a = 16.234(8) Å, b = 13.475(7) Å, c = 10.856(5) Å, α = 103.80(4)°, β = 91.40(4)°, γ = 115.50(3)°, Z = 2. The structure consists of a Pt(9-MeG) 2+ 4 cation in which each purine ligand is coordinated through its N(7) nitrogen atom (Fig. 1) to result in a PtN 4-square plane. The purine ligands are in the usual alternating head to tail arrangement with the adjacent C(6)O(6) pointing in opposite directions. The average PtN distance is 2.036 Å. The current R-value is 7.8% with 4475 independent reflections.