Reversed-phase high-performance liquid chromatography of human haemoglobin chains

A reversed-phase high-performance liquid chromatographic method for the separation of human haemoglobin chains has been devised. Using a LiChrospher 100 CH-8/2 column and a ternary eluent (acetonitrile-methanol-0.155 M NaCl, pH 2.7) improved resolution was achieved between (delta beta) Lepore, beta A, beta S, alpha, G gamma and A gamma chains within a 60-min linear gradient. The A gamma T chain can also be separated by increasing the gradient time and decreasing the flow-rate. Silanophilic interactions play an important role in the retention mechanism, and NaCl addition was necessary in order to suppress adsorption on free silanols. Increasing the methanol concentration to 10% caused a slight increase in chain retention, probably owing to solvation of the stationary phase. The recovery was 82% and the reproducibility of retention times was as good as +/- 1.5%. Quantitation of chains is likely to be possible by peak area measurement. Owing to its sensitivity, the proposed method may be useful in the diagnosis of haemoglobinopathies and in the study of haemoglobin variants.     

Reversed-phase high-performance liquid chromatography of protamines

The protamines from the gonads of the sturgeonAcipenser stellatus have been separated by high-performance liquid chromatography. The proteins were eluted with mixtures of water and ethanol having a gradient of ethanol concentrations in the presence of trifluoroacetic acid (TFA). The influence of the concentration of TFA and the temperature of the column on separation was studied. The quantitative (95–98%) isolation of the protamines from the column was achieved at a temperature of 30°C and a 0.15% concentration of TFA.     

Reversed-phase high-performance liquid chromatography of protamines

The protamines from the gonads of the sturgeonAcipenser stellatus have been separated by high-performance liquid chromatography. The proteins were eluted with mixtures of water and ethanol having a gradient of ethanol concentrations in the presence of trifluoroacetic acid (TFA). The influence of the concentration of TFA and the temperature of the column on separation was studied. The quantitative (95–98%) isolation of the protamines from the column was achieved at a temperature of 30°C and a 0.15% concentration of TFA.Moscow Technological Institute of the Meat and Dairy Industry. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 744–748, November–December, 1986.     

Reversed-phase high-performance liquid chromatography of dinucleoside polyphosphates

The reversed-phase high-performance liquid chromatographic separation of purine dinucleoside polyphosphates on octadecyl- and phenyl-bonded silica packings using phosphate-based eluents was studied. The effects of pH, ionic strength and the content of the organic modifiers methanol and acetonitrile in the mobile phase on the retention and other chromatographic parameters are reported. The data obtained were used to establish an isocratic assay for diguanosine and diadenosine polyphosphates.     

Reversed-phase high-performance liquid chromatography peptide mapping of bovine somatotropin

Reversed-phase high-performance liquid chromatography peptide mapping techniques have been used to examine the primary structure of bovine somatotropin (bSt) isolated from Escherichia coli modified by recombinant DNA techniques (rbSt) and from bovine pituitary (pbSt). Peptide fragments arising from tryptic digestion of bSt were separated on Baker wide pore C4 or C8 columns with linear gradients (acetonitrile-water with 0.1% trifluoroacetic acid). Major peaks eluted in a consistent order, but significant day-to-day variation in retention times was observed. Isolated peptide fragments were analyzed via acid hydrolysis followed by formation and separation of the phenylthiocarbamyl amino acids. These correspond to expected tryptic fragments.     

Reversed-phase high-performance liquid chromatography of thiol—bimane derivatives

Fluorescent labelling of thiols with monobromobimane followed by RP-18 reversed-phase HPLC with aqueous acetic acid/acetone mixtures and fluorescence detection (λ(ex) = 380 nm;λ(em) = 470 nm) allows their selective and sensitive identification in various preparations.     

Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides

Biologically active peptides synthesized by the solid phase methodology of Merrifield were purified by reversed-phase high-performance liquid chromatography using newly developed preparative radially compressed cartridges fitting Waters Assoc . Prep LC 500 liquid chromatograph. Cartridges were handpacked with Vydac C18, C4 or diphenyl derivatized silicas (pore size 300 A) of different particle sizes (10-20 micron). Large scale purification of gram amounts of gonadotropin releasing hormone analogs (agonist and antagonist) as well as amidated human pancreatic tumor growth hormone releasing factor (a 40-peptide) illustrate the resolutive power of this technique applied to the isolation of more than 300 synthetic peptides in our laboratory over the last two years. Difficult separations were achieved by changing supports (C18, C4, diphenyl) as well as mobile phase composition: (triethylammonium phosphate pH 2.25 or 6.5, 0.1% trifluoroacetic acid, ammonium acetate pH 6.5 and acetonitrile). Protected amino acids and peptides amenable to normal-phase chromatography on Vydac spherical underivatized silica were purified economically by the reversed-phase mode. It is understood that this general, convenient and versatile strategy may be applicable to the preparative scale isolation of any other class of compounds usually separated on reversed-phase high-performance liquid chromatography.     

Cystine-rich proteins of human hair: characterization and structure as revealed by reversed-phase and size-exclusion high-performance liquid chromatography

The human hair cystine-rich proteins have been separated through the combined use of reversed-phase and size-exclusion chromatography into more than fifty components. These have been grouped, based on molecular weight, into six families of closely related members. The families range in molecular weight from less than 6500 for the low-molecular-weight components to more than 67 000 for the high-molecular-weight components, with average intermediate values for the other families of 8000, 11 500, 15 500 and 19 000. The results also suggest an organized structure of the hair matrix proteins. The combined use of reversed-phase and size-exclusion high-performance liquid chromatography in these studies presents an example where the quaternary structure of a multi-component protein can be largely deduced from its chromatographic behaviour.     

Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids

A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.     

Reversed-phase systems for the separation of pentapeptides by high-performance liquid chromatography

Summary Reversed-phase systems using octyl modified silica as such and as a support for dynamically coated ion-exchangers, were investigated for their ability to separate pentapeptides. Normal reversed-phase adsorption with C-8 bonded silica in combination with citrate bufferpropanol-1 mixtures were found useful for the separation of a number of pentapeptides. The separation of pentapeptides differing widely in retention can be speeded up by applying an organic modifier and/or sodium citrate gradient. A solvent generated cation-exchange system with sodium dodecylsulfate as surfactant showed a high selectivity for the pentapeptides under investigation and is better for analytical purposes than the normal reversed-phase adsorption systems investigated. With respect to the detection of pentapeptides with fluorescamine, the use of dry pyridine as a basic buffer and as diluent for the fluorescamine was also investigated. Compared to the commonly used diluent acetone, pyridine is better when using acidic eluents of moderate buffer strength. At pH>6 no significant differences in sensitivity between acetone and pyridine could be noticed.     

Reversed-phase high-performance liquid chromatography fingerprint profiles of thirty-nine mosses with chemometric

Thirty-nine land and aquatic mosses were extracted by double maceration and ultrasonic extraction techniques using the mixture of 80% ethanol and water. Obtained extracts were analyzed using the reversed-phase-high-performance liquid chromatography–diode array detector with the Kinetex C18 chromatographic column and mobile phase consisting of acetonitrile–water–0.1% formic acid mixture (gradient 5–100%, 60 min) with detection wavelength of 280 nm. Next, obtained chromatograms were preliminary processed with the smoothing, noise reduction, background subtraction, and alignment using the SpecAlign program (version 2.4.1). The chemometric analysis was performed to obtain the fingerprint chromatograms of selected mosses. The principal component analysis and the cluster analysis (with paired group algorithm and correlation coefficient—r, as similarity measure) confirm the chemical similarity or differences between studied Bryophyta sp.     

Reversed-phase high-performance liquid chromatography on porous copolymers of different chemical structure

The influence of chemical structure of porous polymers on the chromatographic properties of high-performance liquid chromatography columns was studied. Columns were packed with four different porous copolymers: di(methacryloyloxymethyl)naphthalene-divinylbenzene containing ester functional groups, 4,4'-bis(maleimido)diphenylmethane-divinylbenzene with imide groups, di(4,4'-dimethacrylphenyl)sulfone-divinylbenzene which contains sulphonyl groups, and styrenedivinylbenzene with any functional groups. Using the alkyl aryl ketone scale, the retention indices of five homologous series (alkylbenzenes, alkyl aryl ketones, N-alkylanilines, alkyl aryl ethers, alkylbenzoates) and column test compounds (toluene, nitrobenzene, p-cresol, 2-phenylethanol, N-methylaniline) were calculated. Their values were used for comparison of the selectivities of the studied polymeric packings.