Preparative Reversed-phase High-performance Liquid Chromatography Research Articles

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection ( λ ex=330 nm; λ em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.

Isolation and quantification of tuliposides and tulipalins in tulips (Tulipa) by high-performance liquid chromatography.

The content of tuliposides and tulipalins were determined in Tulipa species and cultivars by reversed-phase high-performance liquid chromatography (RP-HPLC), using a water:methanol gradient as mobile phase. The compounds were detected by a diode array detector employed at 208 nm. The investigation revealed, in addition to 1- and 6-tuliposide A, tuliposide D and the lactonized aglycones tulipalin A and (-)-tulipalin B, the new tuliposide F and 6-tuliposide B, the latter being a new acyl derivative of the known 1-tuliposide B. All compounds were isolated by preparative RP-HPLC and identified by NMR and mass spectroscopy. The predominant compounds were 6-tuliposide A and B present in amounts up to 1.5% and 1.3% of fresh weight, respectively. 6-Tuliposide A and tulipalin A seem to be the major allergens in tulips, although tuliposide D and F may also contribute to the allergenic properties. Tulipalin A and (-)-tulipalin B occur in intact tulips and are not only produced in response to fungal attack or after excision of the plants. A few species were found to have very low allergen content and a relatively high level of tuliposide B, indicating it should be possible to breed non-allergenic and disease-resistant tulips.

Regiospecific analysis of fractions of bovine milk fat triacylglycerols with the same partition number.

Bovine milk fat was fractionated using preparative reversed-phase high-performance liquid chromatography. The conditions consisted of two successive linear gradients of acetonitrile and tert-butylmethylether, followed by a final isocratic mixture of the two eluants, leading to triacylglycerols grouped by their partition number (PN). Fractions corresponding to partition numbers 32 to 50 were isolated and analyzed for fatty acid distribution between sn-1,3 and sn-2 positions by Grignard degradation. Results showed that the fatty acid distribution in milk fat triacylglycerols is nonrandom. The distribution of short-chain fatty acids, stearic (predominantly at sn-1,3 position) and palmitic (predominantly sn-2 position), did not change with triacylglycerol size. Medium-chain fatty acids were predominantly located at sn-2 position, but their proportion at this position decreased with triacylglycerol size. Oleic acid distribution was also size-dependent in that it was located in high proportions at sn-2 position in smaller triacylglycerols and vice versa. Results also showed that the sn-2 position was more unsaturated than sn-1,3 position in the PN range from 32 to 40, but it was more saturated in triacylglycerols with higher PN.

Isolation and identification of a metabolite of cidofovir from rat kidney

Cidofovir is an acyclic nucleotide analog with potent and broad-spectrum antiviral activity against adenoviruses and herpesviruses including cytomegalovirus (CMV). Cidofovir undergoes intracellular phosphorylation by host enzymes to cidofovir phosphate and cidofovir diphosphate (the active form). An unidentified metabolite has been observed previously in rat tissues and in urine of rabbits, rats and monkeys dosed with cidofovir. In the present study, this metabolite was isolated from rat kidney following an intravenous dose of 100 mg kg −1 cidofovir. The metabolite (metabolite I) was separated from cidofovir and impurities using extraction on anion-exchange resin followed by preparative normal and reversed-phase high-performance liquid chromatography (HPLC). The isolated metabolite I was subjected to proton, 13C and phosphorus nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization mass spectroscopy, and confirmed to be cidofovir–phosphocholine. The uptake of cidofovir by rat kidney was saturated at an intravenous dose of 100 mg kg −1, probably as a result of saturation of the renal tubular secretion pathway. However, the relative abundance of cidofovir phosphocholine was not affected by dose.

Isolation and Structure Elucidation of the Major Degradation Products of Cefaclor in the Solid State

Cefaclor is a β-lactam antibiotic that degrades slowly under normal storage conditions to several minor products. To obtain samples large enough to permit structure elucidation, cefaclor was allowed to degrade at 40°C (75% relative humidity) and at 85°C. The profile of degradation products formed under these conditions is qualitatively similar to the profile of degradation products observed in samples of cefaclor aged for 14years at room temperature, although some products found in the sample degraded at 85°C are not formed at the lower temperatures. Using preparative reversed-phase high-performance liquid chromatography (rp-HPLC) and a combination of spectroscopic methods, we have isolated and characterized 17 of these degradation products. Some of these products were also isolated from studies of aqueous degradations. The major products appear to have arisen from five distinct pathways: (1) isomerization of the double bond in the dihydrothiazine ring; (2) decarboxylation; (3) ring contraction of the cephem nucleus to thiazole structures; (4) oxidative attack at carbon 4 of the dihydrothiazine ring; and (5) intramolecular attack of the primary amine of the side chain on either the β-lactam carbonyl to form 3-phenyl-2,5-diketopiperazines or the "masked aldehyde” at carbon 6 to form 2-hydroxy-3-phenylpyrazine derivatives. The pathway involving oxidation at carbon 4 is particularly important at ambient temperatures and is unique to the solid-state degradation.

Isolation and identification of hevein as a major IgE-binding polypeptide in Hevea latex

Background: Polypeptides in Hevea latex are known as the major cause of latex type I sensitivities. So far, only a few of them have been characterized. Methods: Proteins with a molecular weight lower than 10 kd in fresh Hevea latex were separated by ultrafiltration and further characterized by liquid chromatography on-line–coupled electrospray mass spectrometry. Hevein in this fraction was then purified by preparative reverse-phase high-performance liquid chromatography and characterized by matrix-assisted laser desorption ionization mass spectrometry and protein sequencing. Skin prick tests, enzyme-linked allergosorbent tests, and inhibition immunoblotting were performed to show the allergenicity of the purified hevein. Results: Hevein, a 4.7 kd polypeptide, is the predominant component in the fraction with latex proteins of smaller than 10 kd. Specific IgE antibodies to hevein were detected by enzyme-linked allergosorbent test in 48 of 64 (75%) sera from health care workers allergic to latex and in three of 11 (27%) sera from patients with spina bifida and hypersensitivity reactions to latex. Inhibition immunoblotting demonstrated that the preincubation of 14 sera and a serum pool from patients allergic to latex with purified hevein completely inhibited IgE binding to the 20 kd protein, which has been recently reported to be a major allergen in latex (prohevein). Skin prick testing showed a positive reaction to hevein in 17 of 21 (81%) patients with latex allergy. Conclusions: The results clearly demonstrate that hevein is an important latex allergen, and the IgE-binding capacity of prohevein in latex is mostly attributed to hevein, the N-terminal domain of prohevein. ( J Allergy Clin Immunol 1997;99:402–9.)

Purification and Characterisation of HighMrGlutenin Subunit 20 and its Linked y-type Subunit from Durum Wheat

HighMrglutenin subunit 20 and its linked y-type subunit, present in the durum wheat cultivar Lira, were purified by preparative reversed-phase high-performance liquid chromatography (RP–HPLC). Amino acid and N-terminal sequence analysis of subunit 20y confirmed that it corresponded to a y-type subunit. Moreover, the number and position of the cysteine residues in subunit 20 were determined by alkylation with the fluorogenic reagent 7-fluoro-4-sulfamoyl-2,1,3,-benzoxadiazole (ABD-F) and subsequent enzymic digestion with trypsin. N-terminal amino acid sequence analysis of the fluorescent peptides showed that subunit 20 had only two cysteine residues, one in the N-terminal region and the other in the C-terminal domain.

Disulphide bonds in wheat gluten: cystine peptides derived from gluten proteins following peptic and thermolytic digestion.

Gluten from the wheat variety Rektor was extracted with 70% aqueous ethanol. The insoluble portion (whole glutenin) was partially hydrolysed with trypsin at pH 6.5 and separated on a Sephadex G25 column. The high molecular weight fraction 1 was further hydrolysed with pepsin at pH 2.0. To remove low molecular weight proteins, a portion of whole glutenin was extracted with dilute acetic acid. The residue (enriched glutenin), which contained mostly LMW and HMW subunits of glutenin, was hydrolysed with thermolysin at pH 6.5. The peptic and tryptic hydrolysates were separated on a Sephadex G25 column and the peptide fractions with the highest cystine content were separated further by reversed-phase high-performance liquid chromatography (RP-HPLC). Cystine peptides were detected by differential chromatography (RP-HPLC prior to and after reduction of disulphide bonds) and then isolated by preparative RP-HPLC. After reduction, cysteine peptides were alkylated and analysed for their amino acid sequence. Altogether, 19 cystine peptides were characterized and assigned to known sequences of gluten proteins; 16 peptides confirmed the positions of disulphide bonds present in LMW subunits and gamma-gliadins, as described previously. For the first time, a cystine peptide has been isolated, representing an intermolecular disulphide bond between the y-type of HMW and LMW subunits. Furthermore, a cystine peptide was assigned to gamma-gliadins; thus, all cysteine residues of gamma-gliadins are documented by at least one cystine peptide. One peptide analysed came from the alpha-amylase inhibitor CM 16. Altogether the results indicate that the intramolecular linkages of gluten proteins are not formed at random, but are strongly directed.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a new genetic variant of bovine beta-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis.

Various components of the beta-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants beta A1, beta A2, beta A3, and an unknown component previously denoted beta X [S. Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these components had relative molecular masses of 16 higher than the corresponding ones in the beta A1 map. The main differential peaks represented the 114-169 fragments of beta A1 and beta X, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145-156 sequence showed the 16 relative molecular mass difference. In beta X this sequence contained a Leu residue at position 152 instead of the Pro-152 in beta A1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence of the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named beta-casein F-5P.

Purification of a Recombinant Human Respiratory Syncytial Virus Chimeric Glycoprotein Using Reversed-Phase Chromatography and Protein Refolding in Guanidine Hydrochloride

FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu2+-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

Preparative reversed-phase high-performance liquid chromatography of soybean proteins and characterization of fractions by gel electrophoresis

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPreparative reversed-phase high-performance liquid chromatography of soybean proteins and characterization of fractions by gel electrophoresisWalter J. Wolf, Robert E. Peterson, and Mardell L. SchaerCite this: J. Agric. Food Chem. 1992, 40, 10, 1809–1816Publication Date (Print):October 1, 1992Publication History Published online1 May 2002Published inissue 1 October 1992https://doi.org/10.1021/jf00022a016RIGHTS & PERMISSIONSArticle Views134Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-Alerts Get e-Alerts

A new type of mitogenic factor produced by Streptococcus pyogenes

A new type or mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein an SDS-PAGE, The molecular weight of the purified mitogenic factor was determined to be 25.370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gin-Thr-Gin-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu-Asn-Glu-Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.

Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins

A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofluoresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4′,5′-dibromo-4,5,6,7-tetrachlorofluorescein and 2′,4′,5′-tribromo-4,5,6,7-tetrachlorofluorescein, were isolated and characterized by ~H NMR and chemical ionization mass spectrometry.

Liquid chromatographic isolation and structural elucidation of methoxymethylene dimethyl dodecatrienes catalytically synthesized from methanol and three moles of isoprene

The palladium-catalysed reaction of isoprene with methanol yields methoxy dimethyl octadienes (“methoxy dimers”) and dimers of isoprene. To obtain additional information about the mechanism of catalysis this paper is aimed at identifying the higher boiling side-products of this reaction. After distillation in high vacuum the residue of the reaction was separated by analytical and preparative reversed-phase and normal-phase high-performance liquid chromatography, yielding at least six compounds with the formula C 16H 28O (“methoxy trimers”) plus trimers and tetramers of isoprene, all proven by mass spectra. The structure of two methoxy trimers was elucidated by 1H and 13C NMR spectra. Up until now they have not been described in the literature and they show an unexpected position of the methoxy group.

Characterization of ethanol-extractable reduced subunits of glutenin separated by reversed-phase high-performance liquid chromatography

Wheat flour (cv. Rektor) was defatted and extracted stepwise with buffered 0-4M NaCl and 70% (v/v) aqueous ethanol. The glutenin containing residue was further extracted with buffered 70% (v/v) ethanol at 4 °C under reducing conditions. The last extract (E1), containing the ethanol-extracted reduced subunits of glutenin, was fractionated by preparative reversed-phase high-performance liquid chromatography on C18 silica gel into 25 components. Based on amino-acid compositions and hydrophobicities, most proteins were classified into five groups. The first two groups (peaks 2-4 and 5-7), which had similar compositions and were characterized by high contents of Glx, Pro and Phe, corresponded to the ω5- and ω1,2-gliadins. The third group (peaks 8-17) was the major group, and contained low-molecular-weight (LMW) subunits of glutenin characterized by compositions that were similar to α- and γ-gliadins, but with a higher content of Ser and lower contents of Asx and Ala. The last two minor groups (peak 20-21 and 22-25) were either identical with or closely related to the γ-gliadins. N-terminal sequence analysis of the four major components from the LMW subunit group revealed unique sequences, not present in other types of gluten proteins.

Halocyamines: novel antimicrobial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian Halocynthia roretzi

Two novel antimicrobial tetrapeptide-like substances, halocyamine A and B, were isolated from the solitary ascidian Halocynthia roretzi by a procedure including extraction steps, chromatographies on coarse and fine HP-20 columns, and preparative reversed-phase high-performance liquid chromatography. The structures of halocyamine A and B were determined to be L-histidyl-L-6,7-dihydroxyphenylalanylglycyl-6-bromo-8,9-didehy drotryptamine and L-threonyl-L-6,7-dihydroxyphenylalanyl-L-histidyl-6-bromo-8,9- didehydrotryptamine, respectively, by spectral analyses and degradation studies. Besides antimicrobial activities against several kinds of bacteria and yeasts, both of them showed cytotoxic activities against neuronal cells cultured from rat fetal brain, mouse neuroblastoma N-18 cells, and human hepatoma Hep-G2 cells. They were only detected in the "morula"-like cells, which are of the most abundant cell type among the hemocytes of H. roretzi.

Structural features of aquatic fulvic acids. Analytical and preparative reversed-phase high-performance liquid chromatography separation with photodiode array detection

La chromatographie HPLC preparative et analytique a permis de separer les acides fulviques d'une eau en fractions hydrophiles et hydrophobes. Les constituants des fractions sotn identifies par spectrometrie UV visible

Preparative reversed-phase high-performance liquid chromatography in the synthesis of viscosin, a cyclic depsipeptide

The importance of peptides in biochemical research and the facility with which they can be prepared by solid-phase techniques highlights the need for continuing research in the chromatographic purification of these molecules on a preparative scale. In this regard, synthesis of the cyclic depsipeptide antibiotic, visconsin, has provided the opportunity to demonstrate the use of radial compression cartridge technology in the reversed-phase purification of three key peptide intermediates on a scale of several hundred milligrams. Superior resolution of linear and cyclic peptide mixtures on a Bondapak C 18 radial compression cartridge by using an aqueous acetonitrile solvent system containing 0.1% trifluoroacetic acid contributed significantly to the first total synthesis of viscosin, and demonstrates the applicability of this system to the purification of peptide mixtures.

Isolation and quantitative analysis of hydroxylysine glycosides

Methods are described for the isolation of galactosyl hydroxylysine and glucosylgalactosyl hydroxylysine from collagen and for the quantitative analysis of these hydroxylysine glycosides. The isolation procedure, based upon gel filtration and preparative ionpaired reversed-phase high-performance liquid chromatography, is simple and rapid in comparison with existing methods, allowing the production of tens of milligrams of each glycoside in a single day. Quantitative analysis of the hydroxylysine glycosides is effected by reversed-phase high-performance liquid chromatography with precolumn derivatization (usingo-phthal-dialdehyde) and fluorescence detection.

Amino acid compositions of avenins separated by reversed-phase high-performance liquid chromatography

The prolamins from wheat (gliadin), rye (secalin), barley (hordein) and oats (avenin) were extracted with 70% aqueous ethanol. Reversed-phase high-performance liquid chromatography (RP-HPLC) on C 18 -silica gel revealed clear differences in elution patterns. Crude avenin from the oat variety Flamingsregent was sepoarated by preparative scale RP-HPLC into thirty components. The amino acid compositions indicate that the first group of minor components (peaks 1–14) comprised non-avenin proteins. The second group (peaks 15–30) was the predominant group and corresponded to avenin proteins. These were classified into three subgroups differing mainly in the content of hydrophobic amino acids (Leu, Ile, Val, Phe). Within these groups, the avenins had similar compositions and differed only in the content of a few amino acids. Avenins appeared not to be related to the prolamins of other cereals on the basis of the amino acid composition data.