Separation of guinea-pig pancreatic juice proteins by reversed-phase high-performance liquid chromatography

Using a Protesil 300 octyl reversed-phase column with a multistage water-acetonitrile solvent gradient system, it was possible to obtain a well-resolved separation of the nine major proteins present in guinea-pig pancreatic juice. The protein present in each peak of the pancreatic juice chromatogram could only be identified by molecular weight analysis as the acetonitrile denaturated the enzymes and altered their isoelectric points. However, using sodium dodecyl sulphate gel electrophoresis the protein fractions obtained by high-performance liquid chromatography were characterised. Preliminary work has indicated that this system may be capable of separating other complex biological protein mixtures, i.e., saliva.     

Separation of porphyrin isomers by high-performance liquid chromatography

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.     

Analysis of cellulase proteins by high-performance liquid chromatography

A new procedure using high-performance liquid chromatography for the rapid separation of cellulase proteins is described. The cellulase components of Trichoderma reesei are fractionated on a DEAE anion-exchange column using a phosphate buffer at pH 6.2. Activities of the individual components obtained from T. reesei QM6a, a wild strain, and several mutant strains have been determined. Each system examined contained beta-glucosidase, at least two exo-beta-1,4 glucanases and five endo-beta-1,4 glucanases with the endo-beta-1,4 glucanases accounting for 20--36% and the exo-beta-1,4 glucanases for 64--80% of the soluble protein.     

Separation of friend erythroleukaemic cell histones and high-mobility-group proteins by reversed-phase high-performance liquid chromatography

A procedure for the rapid separation of histones and high-mobility-group (HMG) proteins from Friend erythroleukaemic cells (line F4N) by reversed-phase high-performance liquid chromatography is reported. By using a Nucleosil 300-5 C₄, column and a multistep water—acetonitrile gradient containing 0.1% trifluoroacetic acid, the HMG-1 and HMG-2 proteins, several his subtractions including H1⁰, H4, H2B, two H2A variants and two H3 subtractions were separated. Under changed conditions, by applying a varied acetonitrile gradient system, even two H2B variants were fractionated. The methods described seem to be a real alternative to the time-consuming polyacrylamide gel electrophoresis.     

Separation of cytochromes c by reversed-phase high-performance liquid chromatography

Six kinds of cytochrome c of different origin, i.e., bovine, chicken, dog, horse, rabbit and tuna, were subjected to separation by reversed-phase high-performance liquid chromatography on three commercial packing materials; octadecyl-, octyl- and cyanoalkyl-silicas. The effects of reversed-phase material, mobile phase and temperature on the separation of cytochromes c were examined. The parameters of the mobile phase were the organic modifier, the pH, the salt concentration and additives. Under optimal conditions, five of the six cytochromes c were resolved in 10 min. The relative retention values cannot be explained in terms of the relative lipophilicities of the side-chains of the amino acid residues.     

Separation of pyrimidine derivatives by reversed-phase high-performance liquid chromatography

Chromatographic behavior and separation conditions of pyrimidine derivatives were studied by high-performance liquid chromatography using a reversed-phase column and a multiwave UV detector.     

Separation of polypeptide antibiotics by reversed-phase high-performance liquid chromatography

Summary The influence of different reversed-phase packings and the addition of acidic modifiers to the mobile phase was observed on the separation of basic and neutral polypeptide antibiotics by gradient elution. A dependence of pore size, coverage, reaction type and endcapping of the packings was not observed. Nevertheless, not all reversed-phase packings were suitable for the separation of polypeptides, especially of basic molecules. The addition of phosphoric or perchloric acid to the mobile phase prevented adsorption of the basic polypeptide antibiotics on the stationary phase.     

Separation of pyrethroid enantiomers by chiral high-performance liquid chromatography

The separation of enantiomers of pyrethroid insecticides has been systematically studied using a commercially available Pirkle type 1-A chiral-phase high-performance liquid chromatography column. Useful resolution was obtained for compounds with a variety of acid and alcohol moieties, and containing one to four chiral centres. The chromatographic behaviour of the diastereomers of some of these insecticides on a cyano-bonded column was also examined.     

Separation of potential flavoring compounds by high-performance liquid chromatography

High-performance liquid chromatography separated successively and quantitatively the food flavoring agents pyrimidines, purines and nucleosides, followed by nucleotides, then by polyphenols and finally by pyrazines with a reversed-phase octadecylsilica (μBondapak C₁₈) column and various proportions of methanol, water, acetic acid and tetrabutylammonium phosphate (PIC A). The polar solvent (solvent A) was water—acetic acid—PIC A (97.5:1.5:1.0) and the relatively non-polar solvent (solvent B) was methanol—acetic acid—PIC A (97.5:1.5:1.0). Purines, pyrimidines, and nucleosides were eluted with solvent A. Nucleotides were eluted with a mixture of solvents A and B (9:1). Polyphenols were separated with a gradient starting at 10% solvent B and finishing at 25% solvent B, and finally the pyrazines were removed successively from the column with a gradient starting at 25% solvent B and finishing at 45% solvent B. The resolution and reproducibility were excellent for more than 50 compounds. By this method beverages could be analyzed directly, without solvent extraction, for flavoring compounds.     

Separation of tubulin subunits by reversed-phase high-performance liquid chromatography

When properly solubilized with trifluoroacetic acid (TFA), alpha- and beta-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters muBondapak C18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the beta-tubulin subunit eluting before the alpha. Column temperature above ambient improve both the resolution and the yield; less polar solvent systems do not. Tubulins not freely soluble in aqueous TFA may be solubilized in 6 M guanidine-hydrochloric acid with no change in retention time. Other columns with shorter carbon chain lengths and larger pore size produce a single, unresolved tubulin peak. Reversed-phase HPLC analysis provides an independent comparative evaluation of organelle-specific tubulins, with characteristic retention time differences observed between homologous ciliary and flagellar outer doublet tubulin subunits and also between them and their cytoplasmic counterparts.     

Separation of bromo- and iodosubstituted thyronines by high-performance liquid chromatography

Summary A reversed-phase high performance liquid chromatographic method on C-18 bonded silica is described for the separation of trisubstituted iodo/bromothyronines, which exert thyroid hormone activity. The composition of the mobile phase has been systematically optimized resulting in a ternary mixture of methanol/acetonitrile/water acidified with trifluoroacetic acid. The applicability of ultraviolet absorption detection, amperometric detection and off-line radioimmunoassay was investigated. The latter method allows detection of the different iodo/bromothyronines down to 40–120 ng/l mobile phase; this sensitivity is high enough for application to thyroid hydrolysates in order to clarify the question as to whether bromine can substitute iodine in the biosynthesis of thyroid hormones.     

Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Separation of retinyl esters by non-aqueous reversed-phase high-performance liquid chromatography

Rapid separation and sensitive quantitation of vitamin A esters can be achieved by use of an acetonitrile-dichloromethane (80:20) mobile phase with a 5-microns C18 column (15 cm X 4.6 mm) and absorbance detection at 325 nm. Either a Waters Resolve or a Rainin Microsorb column was used satisfactorily. Retinyl palmitate is eluted at about 7 min (capacity factor, k' = 5.5) at a flow-rate of 1.5 ml/min; retinyl palmitate and retinyl oleate, which are usually difficult to separate, are well resolved (resolution 1.2). Sensitivity (at a signal-to-noise ratio of 10:1) is 8 pmol retinyl palmitate (equivalent to 2.5 ng retinol). Quantitation of total retinyl esters is identical to that determined by a gradient high-performance liquid chromatographic technique over the range 30-1000 ng retinyl esters. Retinyl ester peaks in rat liver extracts were identified by their characteristic light absorption spectra, susceptibility to saponification, and by co-chromatography with authentic standards. Nine vitamin A ester peaks were identified and quantitated in rat liver extracts. A 10-microns Whatman Partisil 10/25 ODS-2 column was used with the same mobile phase to obtain partial resolution of retinyl esters (resolution 1.05 between retinyl oleate and retinyl palmitate; k' = 11.0 for retinyl palmitate) and improved retention for retinol (k' = 2.5, compared with k' = 0.6 for retinol on the 5-microns column).