Separation of phosphohydroxyamino acids by high-performance liquid chromatography

A high-performance liquid chromatography system has been developed which resolves O-phosphoserine, O-phosphothreonine, and O⁴-phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O-phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases.     

Separation of neutral oligosaccharides by high-performance liquid chromatography

We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H₂O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of ³H by reduction with NaB³H₄ permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.     

Separation of dansyl hydrazine-derivatized oligosaccharides by liquid chromatography

A sensitive method has been developed for the rapid separation of neutral oligosaccharides derivatized with dansyl hydrazine by liquid chromatography on silica gel modified with 1,4-diaminobutane. Malto-oligosaccharides from corn syrup in amounts ranging from 7 to 280 nmol and ovalbumin-derived oligosaccharides in amounts ranging from 0.25 to 250 nmol were easily detectable and gave linear responses over the respective ranges.     

Separation of anionic oligosaccharides by high-performance liquid chromatography

We have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the amnionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since the latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study we demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (alpha 2.3 vs alpha 2.6) and/or location of alpha 2,3- and alpha 2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.     

Separation of proteins by high-speed pressure liquid chromatography

Two chromatographic systems for separation of proteins by high-speed pressure liquid chromatography are described. Molecular size exclusion chromatography was achieved by the use of porous silica deactivated by Carbowax-20M to prevent protein adsorption. Protein separations were successful provided the salt concentration in the eluting buffer was relatively high.The second system described is adsorption chromatography of proteins on deactivated Porasil. This technique involves elution of the proteins from the gel by means of a salt and pH gradient. In both systems the total time required for the chromatography is less than 1 hr.     

Separation of partially methylated mannitols by liquid chromatography

To facilitate the methylation linkage analysis of complex carbohydrates containing radioactively labeled mannose residues, an HPLC micromethylation technique has been developed which effectively resolves all of the partially methylated mannitol standards prepared using an under-methylation protocol. The method was applied to the linkage analysis of the nine mannose residues of the dolichol-derived oligosaccharide Glc3Man9GlcNAc2 isolated from BHK-21 fibroblasts. This technique should prove widely applicable to the methylation linkage analysis of radiolabeled complex carbohydrates.     

Separation of galactolipid molecular species by high-performance liquid chromatography

A technique for the separation, detection, and quantification of molecular species of monogalactosyldiglyceride and digalactosyldiglyceride is described. Use of the technique to analyze the molecular species composition of the galactolipids isolated from Dunaliella salina chloroplasts is presented. The results indicate that the respective compositions of the two lipids are quite different. This suggests that the enzymes involved in galactolipid metabolism are very specific with respect to acyl chain composition and pairing, or that extensive retailoring of constituent acyl chains occurs following formation of digalactosyldiglyceride from monogalactosyldiglyceride.     

Separation of the natural retinoids by high-pressure liquid chromatography

A reverse phase high-pressure liquid chromatography system for rapid separation of various retinoids (vitamin A and its analogs) with little or no degradation is described. This method permits detection of as little as 22 pmol of retinoic acid. The procedure has been applied to the study of retinoic acid metabolism in vitamin A-deficient hamsters.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Separation of porphyrin isomers by high-performance liquid chromatography.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.     

Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an unusual phycocyanin from a marine cyanobacterium, red algal B- and R-phycoerythrins, and a cryptomonad phycoerythrin. The subunit sizes in these proteins range from about 7.5 to 30 kDa. Sample recovery was in excess of 85% in all cases. On-line spectroscopic analysis with a multiple diode array detector allowed determination of the type and number of bilins carried by each subunit.     

Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

A rapid and sensitive reversed-phase HPLC procedure is reported which allows the simultaneous separation and quantitation of LTC₄, 11t-LTC₄, LTD₄, LTB₄, 12epi,6t,8c-LTB₄, 6t-LTB₄ + 12epi,6t-LTB₄, two trihydroxy-eicosatetraenoic acids tentatively identified as 20-OH-LTB₄ and 20-OH,12epi,6t,8c-LTB₄ and several not yet identified 15-series leukotrienes produced by the cytosol of porcine polymorphonuclear leukocytes.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

Separation and quantitation of dolichyl esters by high-performance liquid chromatography

An HPLC procedure for the isolation and quantitation of total and individual dolichyl esters in tissues has been developed. The purified lipid extracts are subjected to sequential reversed-phase, straight-phase, and reversed-phase HPLC, which yield complete resolution and high recovery of the individual dolichyl esters. The isoprenoid distribution in the esterified fraction was similar to that of the free alcohol fraction in liver, kidney, and spleen. All fatty acids present in the total fraction were also recovered in all the individual polyisoprenoids. Dolichyl esters thus appear to differ from other lipid esters in tissues in containing a broader range of fatty acids.     

Separation of amino acid phenylthiohydantoin derivatives by high-pressure liquid chromatography

Separation of the phenylthiohydantoin (PTH) derivatives of all 20 common amino acids is accomplished in approximately 11 min with excellent resolution by using high-pressure liquid chromatography. The chromatography is achieved at 50 degrees C on an Altex reversed-phase PTH-C18 column in an ammonium acetate-buffered acetonitrile, pH 4.5, mobile phase. Simple isocratic and linear gradient steps are used. Retention times for the various PTH-amino acids are very reproducible. Because the baseline is flat and free of background noise, PTH-amino acids can be detected in the low picomole range. The simplicity of this chromatographic system allows it to be easily automated.