LiChrosorb NH2 Column Research Articles

Chiral separation of ketoprofen on an achiral NH2 column by HPLC using vancomycin as chiral mobile phase additive

A high-performance liquid chromatographic method for chiral separation of ketoprofen racemate was developed. (R)- and (S)-ketoprofen enantiomers were separated on a LiChrosorb NH2 column (250 mm × 4.6 mm, i.d 5 µm) at 20 °C, using 2-propanol/potassium dihydrogen phosphate buffer (pH 6.0, 0.05 M) (50:50 v/v). Containing vancomycin as the mobile phase, at a flow rate of 0.8 ml min−1 and detection wavelength of UV, the detector was set at 310 nm. Under these conditions, ketoprofen enantiomers could be separated with a selectivity factor (α) of 2.172 and a resolution (Rs) of 4.78 using extremely low concentrations of the vancomycin chiral additive.

Analysis of ginseng saponins by HPLC with photoreduction fluorescence detection

A new high performance liquid chromatographic procedure is described for the analysis of ginseng saponins. Ginseng saponins were separated on Lichrosorb NH2 column and anthraquinone-2,6-disulfonate (AQDS) solution was added to the column effuent. The effluent was passed through 1.5 m-PTFE capillary coiled around 10 W-UV lamp to reduce AQDS to highly fluorescent 9,10-dihydroxyanthracene-2,6-disulfonate which was detected by fluorescence detector. The detection limit for the ginsenoside Rg1 by this method was found to be about 350 ng, the dynamic linear range was 102 and the correlation coefficient of the calibration curve was 0.9999.

High-performance liquid chromatographic determination of amygdalin in ume extract.

A simple and reliable method for the determination of amygdalin (AM) in ume (Japanese apricot) extract by high-performance liquid chromatography (HPLC) was investigated. The sample was extracted with hot water (80°C) and cleaned up by use of C18 and NH2 Sep Pak cartridge. The sample solution was chromatographed on a LiChrosorb NH2 column with a mobile solvent of acetonitrile-water (43 : 7). AM was detected with a UV monitor set at 210 nm. The peak corresponding to AM was able to be confirmed by the treatment with emulsin. By this method, the recoveries of AM were over 92.5% and the coefficients of variation (C.V.) were less than 5% at levels of 100μg/g and 1000μg/g. The detection limit corresponded to 20μg/g of AM in ume extract. Analyses of 6 commercial ume extracts revealed 53-1207μg/g of AM in all samples.

Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis. 3-O-methyl-D-galactose and N-acetyl-D-galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein.

Hemocyanin from the freshwater snail Lymnaea stagnalis is a high-molecular-mass copper-containing glyco-protein which functions as oxygen carrier in the hemolymph. To release the carbohydrate chains, the protein was digested by pronase followed by hydrazinolysis and reduction. The oligosaccharide-alditols were purified by gel permeation chromatography on Bio-Gel P-4, followed by HPLC on a Lichrosorb-NH2 column. Using 500-MHz 1H-NMR spectroscopy, in conjunction with sugar, methylation and deamination analysis, the following seven novel primary oligosaccharide structures could be unravelled. (Formula: see text).

Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA.

The proper glycosylation of erythropoietin is essential for its function in vivo. Human erythropoietins were isolated from Chinese hamster ovary cells transfected with a human erythropoietin cDNA and from human urine. Carbohydrate chains attached to these proteins were isolated and fractionated by anion-exchange high performance liquid chromatography (HPLC) and HPLC employing a Lichrosorb-NH2 column. The structures of fractionated saccharides were analyzed by fast atom bombardment-mass spectrometry and methylation analysis before and after treatment with specific exoglycosidases. Both erythropoietins were found to contain one O-linked oligosaccharide/mol of the proteins, and its major component was elucidated to be NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH (where NeuNAc represents N-acetylneuraminic acid) in both proteins. The N-linked saccharides of recombinant erythropoietin were found to consist of biantennary (1.4% of the total saccharides), triantennary (10%), triantennary with one N-acetyllactosaminyl repeat (3.5%), tetraantennary (31.8%), and tetraantennary with one (32.1%), two (16.5%), or three (4.7%) N-acetyllactosaminyl repeats. All of these saccharides are sialylated by 2----3-linkages. Tetraantennary with or without polylactosaminyl units are mainly present as disialosyl or trisialosyl forms, and these structures exhibit the following unique features. alpha 2----3-Linked sialic acid and N-acetyllactosaminyl repeats are selectively present in the side chains attached to C-6 and C-2 of 2,6-substituted alpha-mannose and C-4 of 2,4-substituted alpha-mannose. We have also shown that the carbohydrate moiety of urinary erythropoietin is indistinguishable from recombinant erythropoietin except for a slight difference in sialylation, providing the evidence that recombinant erythropoietin is valuable for biological as well as clinical use.

High‐performance liquid chromatographic determination of oligosaccharides in leguminous seeds

AbstractA simple and reliable high‐performance liquid chromatographic (h.p.l.c.) method has been developed for the determination of sucrose, raffinose and stachyose in leguminous seeds. A LiChrosorb NH2 column (Merck) and an acetonitrile+water solvent (65:35, by vol) at a flow rate of 1 ml min−1 were used for the separation. An interference type refractive index detector (Tecator) was used for the detection. With this detector the smallest amount of oligosaccharide detectable was 10 ng. With a traditional deflection type refractive index detector the smallest amount of oligosaccharide detectable was 240 μg. The oligosaccharides were extracted by placing the whole, dry seeds in boiling water for 30 min, blending the seeds and water, placing the whole suspension in a shaking bath at 60°C for 60 min, and removing the solid material by centrifuging. The extract was deproteinated by adding 65 parts of acetonitrile to 35 parts of extract (by vol), placing the mixture at 5°C for 60 min, and filtering off the resulting proteinaceous precipitate before injection into the chromatograph. The entire procedure has been successfully applied to soya beans, chick peas, garden peas and red kidney beans with recoveries of added raffinose in the range 97–102%.

Determination of serum diclofenac by high-performance liquid chromatography by electromechanical detection.

Diclofenac was isolated from plasma by double extraction with organic solvent. Chromatography was performed on Lichrosorb NH2 column with acetonitrile and perchloric acid (0.0025 mol/l; 35:65, v/v) as the mobile phase (flow rate 0.9 ml/min). The electrochemical detector was operated at +900 mV and sensitivity of 5-10 nA. The sensitivity of the method was 10 micrograms/l and the coefficients of variation below 10% in the entire range of the concentration in a dose interval.

Determination of Vicine and Convicine in Fababeans by High Pressure Liquid Chromatography

High pressure liquid chromatography has been applied for the quantitative determination of vicine and convicine, two glucosides present in fababeans. The following method has been developed: extraction of vicine and convicine by 0.1N NaOH, precipitation of proteins by adjusting the pH to 4.2 with 1N HC1, adequate dilution of the extract before its injection into a Waters high pressure liquid chromatograph. A LiChrosorb NH2 column and a mixture of H2O/acetonitrile in the ratio of 30/70 (v/v) at a flow rate of 2mL/min were used for the separation. Vicine, convicine and α-galactosides were detected by an ultraviolet detector (λ=273nm) and a differential refractometer, respectively. Identification and quantitative estimation were based on known reference samples. Treatment of fababean extracts with β-glucosidase led to a disappearance of the glucoside peaks that correspond in retention time to those of reference glucosides. The proposed method is reliable, rapid, and may be used as a technique for analysis of glucosides and α-galactosides for plant breeding and processed fababean products.

Separative determination of ascorbic acid and erythorbic acid in animal tissues by high-performance liquid chromatography.

A method for the separation and determination of ascorbic acid (AsA) and erythorbic acid (ErA) in animal tissues is described. It employs high-performance liquid chromatography on a LiChrosorb-NH2 column in conjunction with a mixture of acetonitrile, acetic acid and water as an eluant. Application of the method, which is sensitive, rapid and simple, to the analyses of AsA and ErA in animal tissues such as liver, adrenals, spleen, kidneys and heart gave satisfactory results. Dehydro-AsA and dehydro-ErA in rat liver could be determined after reduction with H2S. The method was shown to be useful for the routine analyses.

Separative determination of ascorbic acid and erythorbic acid by high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) was applied to the determination of ascorbic acid (AsA) and erythorbic acid (ErA). The apparatus was a Shimadzu model LC-2P Liquid Chromatograph equipped with a UV detector set at 254 nm. The separation was achieved on a LiChrosorb-NH2 column which was pre-treated with 0.1 M ammonium monophosphate solution using a mixture of acetonitrile, acetic acid and water (87:2:11, v/v) as an eluant. The HPLC method has the following advantages: AsA and ErA are quantitated after being distinctly separated, analysis time per one sample is short, and AsA or ErA levels as low as 1.0 X 10(-2) microgram are detectable. Recovery experiments with dehydro-AsA and dehydro-ErA, involving reduction with H2S, give satisfactory results.

Hydrocarbon group-type analysis of coal liquids by high-performance liquid chromatography

A rapid method is described for the determination of hydrocarbon groups in coal liquids. Analysis is carried out by high-performance liquid chromatography on a LiChrosorb NH2 column with n-hexane as the mobile phase. The separation is controlled only by the number of double bonds in a given hydrocarbon molecule. It is essentially independent of the molecular mass for a large variety of compounds investigated. Example of the separation of hydrogenated derivatives of anthracene and of a coal liquid fraction are presented.

High Pressure Liquid Chromatographic Determination of Vitamin D3 in Instant Nonfat Dried Milk

Vitamin D3 was determined in commercially fortified instant nonfat dried milk by using normal phase high pressure liquid chromatography (HPLC). The sample was extracted with dichloromethane with sodium phosphate tribasic solution added. The sample was cleaned up by using a Sep-Pak silica cartridge and then a microparticulate column containing 10 micrometer Partisil-10 PAC packing material. The final analysis was performed by using a normal phase HPLC system with 10 micrometer LiChrosorb NH2 column. Recovery of vitamin D3 at levels as low as 10000 IU/kg was 97.7% with a standard deviation of 3.9%.

High Pressure Liquid Chromatographic Determination of Vitamin D3 in Mineral Feed Premixes

Abstract A simple and rapid quantitative method is described for determining the presence and amount of vitamin D3 (cholecalciferol) in mineral feed premixes by high pressure liquid chromatography. The samples were extracted with dichloromethane–pentane–methanol (46+46+8) after treatment with ammonium hydroxide, cleaned up by silica Sep-pak, and analyzed by normal phase high pressure liquid chromatography: 10 μm LiChrosorb NH2 column, hexane-isopropanol (98+2) mobile phase, ultraviolet detection at 254 nm. Recoveries were better than 95% in the range 25 000–100 000 IU/kg.