C.A. Bishop

High-pressure liquid chromatography of peptides and proteins : II. The use of phosphoric acid in the analysis of under-ivatised peptides by reversed-phase high-pressure liquid chromatography

Abstract The chromatographic properties of a range of peptides varying in size from di- to decapeptide have been investigated by reversed-phase high-pressure liquid chromatography. A new set of conditions, namely, the addition of phosphoric acid to the mobile phase, has been found to have very real advantages in the analysis of underivatised peptides. These conditions allowed marked alterations in retention times, improvement in reproducibility and excellent resolution of peptides differing by as little as a single amino acid. A major advantage of phosphoric acid is that it can be used successfully in the range 195–220 nm which makes it compatible with the use of variable wavelength UV monitors as sensitive detectors in high-pressure liquid chromatography. In addition, the use of phosphoric acid permits the significant lowering of concentrations of organic solvents in the mobile phase, thus reducing the possibility of denaturation or precipitation.

The use of high pressure liquid chromatography (hplc) for peptide mapping of proteins. IV.

Abstract This paper describes the use of high pressure liquid chromatography (hplc) for the rapid analysis at high sensitivity of peptide mixtures from proteolytic digestlon of proteins. A reversed phase system has been developed which consists of a μ-Bondapak-fatty acid analysis column and an eluant which contains a hydrophilic ion-pairing reagent such as phosphoric acid. Examples described in this paper include the elution profile of a thermolysin digestion of 10 pmol of acyl carrier protein, which was resolved into 13 components in 20 min. In addition, the chromatographic system can be used to follow the progress of a digestion and to monitor the purity of the proteolytic enzyme.

High-pressured liquid chromatography of amino acids, peptides and proteins : V. Separation of thyroidal iodo-amino acids by hydrophilic ion-paired reversed-phase high-performance liquid chromatography

Abstract The separation of thyroidal iodoamino acids has been carried out by high-performance liquid chromatography in phase systems consisting of chemically bonded C 18 -hydrophobic supports as the stationary phase and water—organic solvent mixtures containing phosphoric acid or other ion-pairing reagents as the mobile phase. Under conditions of hydrophilic ion-pair formation, excellent resolution of the iodoamino acids is observed. This method permits the rapid separation and, hence, analysis of mixtures containing thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine and related compounds in ca . 30 min with sensitivity, using a UV monitor at 210 nm, at the 1–10-pmole level.

High-pressure liquid chromatography of peptides and proteins XI. The use of cationic reagents for the analysis of peptides by high-pressure liquid chromatography

This report describes the effect of different cationic reagents (tetraalkylammonium, alkylammonium and inorganic salts) on the retention times of di- to pentapeptides chromatographed on a reversed-phase support (i.e. a μBondapakalkylphenyl column). Several trends are apparent with these reagents which can be explained on the basis of either ion-pairing or ion-exchange interaction of the reagent with the peptide sample. Reagents which generate in solution small highly solvated cations, e.g. Li, Na or Mg salts, give retention times similar to those obtained for ammonium salts. Tetraethylammonium salts give a modest increase in retention times relative to ammonium salts. By contrast, hydrophobic cations with long or bulky carbon chains. e.g. tetrabutylammonium or dodecylammonium ions, cause substantial decreases in retention times, resulting in very rapid elution of all peptides examined from the reversed-phase column. These observations are consistent with the composite interplay of ion-pair partition and dynamic ion-exchange effects for the cationic reagents. The use of a mixture of dodecylammonium acetate and sodium dodecylsulphate for the analysis of peptides and proteins is described. It is anticipated that such a chromatographic system will he useful for the analysis of proteins which readily aggregate.

High-pressure liquid chromatography of peptides and proteins : VI. Rapid analysis of peptides by high-pressure liquid chromatography with hydrophobic ion-pairing of amino groups

Abstract This report describes the use of hydrophobic ion-pairing reagents in the rapid analysis of peptides by reversed-phase high-pressure liquid chromatography. It was found that combination of a hydrophobic anion such as hexanesulphonate with the cationic groups (R P + of a peptide resulted in a decreased polarity of the sample. This change in polarity resulted in an increased retention time on a μBondapak-alkylphenyl column. In addition, the use of the different ion-pairing reagents allowed dramatic changes in the selectivity of the reversed-phase system. This is demonstrated with peptides which range from tri- to heptapeptides using the following ion-pairing reagents: phosphoric acid, sodium hexanesulphonate and sodium dodecycl sulphate.

High pressure liquid chromatography in the analysis of underivatised peptides using a sensitive and rapid procedure

High pressure liquid chromatography (HPLC) has been used in the analysis of PTH amino acids [ 1,2] PTC-peptides [3] some growth promoting peptides [4] and peptide antibiotics [5-71. A major improvement would be achieved if no derivatisation of the peptides was necessary and a rapid, sensitive, low cost method could be employed. To achieve this HPLC was tested on an analytical scale with the possibility that methods developed could be scaled up to a preparative system. Because of the relative complexity of even small peptide molecules in terms of partition coefficient, polarity, hydrophobicity, and bulkiness [8] a series of column supports were investigated with a view to finding a system which both retained samples and allowed their ready elution. The reverse phase mode (liquid-liquid chromatography) was found to best satisfy these criteria. The most common and economical polar solvents used for reversed phase liquid-liquid chromatography, water, methanol and acetonitrile have the additional advantage of allowing spectrophotometry in the range 205-225 mm. In this range the peptide bond has sufficient absorption to allow quantitative detection in the nmol range.

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

Abstract In recent publications we have described the analysis of peptides and proteins by reverse-phase high-pressure liquid chromatography (1–3). These studies demonstrated that the addition of 0.1% phosphoric acid to the mobile phase allowed the rapid and reproducible analysis of peptidic compounds. In addition phosphoric acid allows uv detection to be used at wavelengths down to 190 nm, thus permitting high sensitivity detection of underivatized samples. It is the purpose of the report to show that this chromatographic system allows the facile analysis of a variety of amino acids. The high sensitivity of the method is demonstrated by the analysis of 0.1 ng of tyrosine.

Separation of the apoprotein components of human very low density lipoproteins by ion-paired, reversed-phase high performance liquid chromatography

A number of crude apolipoprotein samples isolated from human very low density lipoproteins (VLDL) were analyzed by reversed phase high performance liquid chromatography. The mobile phase consisted of a 1% solution of the polar ion-pairing reagent triethylammonium phosphate. A slow, nonlinear gradient of acetonitrile (37–42%) was used to elute the apolipoproteins. The order of elution was as follows: apolipoprotein Cx, apolipoprotein C-I, apolipoprotein C-III2, apolipoprotein C-III1, apolipoprotein C-III0 and apolipoprotein C-II. This order is consistent with the known polarity of the proteins, i.e., the most nonpolar, apolipoprotein C-II, was the last to be eluted, whereas apolipoprotein C-I, with the lowest nonpolar surface area eluted first. The recovery of the individual apolipoproteins was 80–95% and the individual peaks were characterized by amino acid analysis, UV absorption spectra and chromatography of pure protein standards.

High Performance Chromatography of Amino Acids, Peptides and Proteins XXIII · Peptide Mapping by Hydrophilic Ion-paired, Reversed-Phase High Performance Liquid Chromatography for the Characterisation of the Tryptic Digest of Haemoglobin Variants

Abstract The application of high performance liquid chromatography on reversed-phase columns for the tryptic mapping of haemoglobin variants is reported. The effect of flow rate and gradient shape on resolution and column efficiencies has been examined using acetonitrile-water-orthophosphoric acid elution systems. With these conditions it is possible to recognise sequence changes for variant haemoglobins including HbC, HbS, HbE, HbJ Cambridge and HbD Punjab.

High-Speed Gel Permeation Chromatography of Human Thyroglobulin and Sheep Liver Aldehyde Dehydrogenase

Abstract This paper reports the application of high speed gel permeation techniques for the fractionation of human thyroglobulin preparations on chemically bonded, polar phase microparticulate silicas. Both resolution and recovery were found to be dependent on the ionic strength and pH of the eluent. At high salt concentrations significant hydrophobic interactions may occur between the support matrix and the protein solutes. The sheep liver enzyme, aldehyde dehydrogenase, was found to exhibit similar salt-and pH-dependent effects under these chromatographic conditions.