Milton T. W. Hearn

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-performance liquid chromatography of amino acids, peptide and proteins : XX. Investigation of the effect of pH and ion-pair formation on the retention of peptides on chemically-bonded hydrocarbonaceous stationary phases

Abstract The effects of different anionic and cationic reagents on the retention of a series of dipeptides chromatographed chromatographed on μBondapak C 18 reversed-phase supports are described. At low pH, hydrophobic anionic reagents result in increased retention of the peptide sample whereas hydrophobic cationic reagents cause decreased retention. These trends can be explained on the basis of either ion-pairing or ion-exchange interactions of the reagent with the protonated peptide. Equations based on the ligand adsorption model for ion-pair reversed-phase chromatography accurately describe the dependency of the capacity factor on the counter-ion concentration as well as on the pH. Hyperbolic and parabolic dependenciens of the capacity factor on the counter-ion concentration were observed depending on te hydrophobic nature oft he counter-ionic reagent. for short alkyl chain reagents, ion-pairing formation appears to govern retention whereas for long chain reagents ion-exchange mechanisms dominate. A local minimum in capacity factor occurs when the pH of the mobile phase corresponds to the pI of the peptide. Although it is possible to predict elution conditons based on pH from a minimum set of capacity factor and pH measurements for closely related peptides, such as those present in a crude synthetic preparation or in a fraction isolated from natural sources, there is little practical advantage in using pH variation as the sole method to influence selectivity. However, large selectivity differences can be easily achieved by the addition at suitable pH of low concentrations, e.g. 5 m M , of suitable reagents that can either undergo ion-pair formation with the peptides or modify the stationary phase to a dynamic ion-exchanger. These methods can be

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-performance liquid chromatography of amino acids, peptides and proteins : XXXVI. Organic solvent modifier effects in the separation of unprotected peptides by reversed-phase liquid chromatography

Abstract The influence of the organic solvent modifier on peptide and polypeptide retention to octadecylsilica supports has been examined. Over a wide range of volume fractions of the organic solvent modifier, Ψ s , unprotected peptides do not show linear dependencies of their logarithmic capacity factors on the composition of binary hydro-organic solvent eluents. Instead, bimodal plots are observed with minima characteristic of the peptide and the organic solvent. The influence of acidic amine buffers on peptide retention behaviour to silica-bonded C 18 hydrocarbonaceous stationary phases has also been further investigated. With mobile phases of low water content, it is possible to obtain on these alkylsilica supports, elution orders characteristic of a normal or polar phase elution mode. This irregular retention behaviour has been discussed in terms of the participation of multiple retention processes in the interaction of ionised peptides with chemically-bonded alkyl-silicas. The influence of flow-rate on column efficiencies for peptides separated under reversed-phase conditions has been studied. The results confirm that column efficiencies for small peptides decrease with increasing flow-rate and eluent viscosity. The significance of the dependence of ln k ′ on Ψ s for the isolation of peptides and polypeptides from biological sources is discussed.

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.

Application of 1,1′-Carbonyldiimidazone-activated agarose for the purification of proteins : II. The use of an activated matrix devoid of additional charged groups for the purification of thyroid proteins

Abstract The use of 1,1′-carbonyldimizale-activated agarose for biospecific affinity chromatography is described. Activation of agrose with this carbonylating reagent gives a matrix devoid of additional charged groups. Conditions for the coupling of a rannge of ligands and leashes have been evaluated. The efficient purification of bovine trypsin, human thyroglobulin and sheep thyroid membrane glycoproteins demonstrates the suitabliliy of the new activated matrix for affinity chromatography.

Preparative flatbed electrofocusing in granulated gels with natural pH gradients generated from simple buffers

Abstract Techniques are reported for preparative electrofocusing in natural pH gradients formed from simple buffers using flatbeds of granulated gels. The stability and reproducibility of the gradients permit excellent resolution of complex mixtures of proteins. Examples reported in this paper include the preparative separation and recovery of biologically active serum and thyroid proteins.

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.