An Estimation Technique of Molecular Weights of Bio-Oligomers by Reversed-Phase Liquid Chromatography

Abstract

A new simple and accurate method for molecular weight estimation of oligomers by reversed-phase liquid chromatography was developed and has been proposed to use in the investigation of bio-oligomers. Capacity factors (k′) of 24 peptides and proteins ranging in molecular weight from 200 to 70000 were measured independently under isocratic eluting conditions with slightly different acetonitrile contents in the mobile phase. Profiles of plots of the log k' values of the peptides and proteins against the acetonitrile contents were on straight lines with different slopes. These slopes were characteristic for each oligomer and found to be proportional to the two/thirds power of the molecular weight. This elution behavior of oligomers in reversed-phase liquid chromatography can be explained in terms of the solvophobic theory. The estimated molecular weights of several bio-oligomers were more accurate than those obtained by size-exclusion chromatography. when the range of molecular weights was limited from 10³ to 2×10⁴.     

HPLC of Peptides and Peptide Diastereoisomers on ODS-and Cyanopropyl-Silica Gel Column Packing Materials

Abstract

The use of hplc for the separation of various peptide diastereoisomers without the need for sample pre-treatment is described. The effect of the concentration of acetonitrile in the mobile phase on the retention of peptides on ODS- and Cyanopropyl-bonded phases is discussed and compared with the effect of methanol. The use of temperature and pH to control the relative retention of peptides on cyano phase is also discussed.     

Elution Behavior of Oligomers on a Polyvinyl Alcohol Gel Column with Chloroform,Methanol, and Their Mixtures

Abstract

Elution phenomena of size exclusion chromatography (SEC) plus superimposed adsorption effects for oligostyrenes, epoxy resins, methylated melamine-formaldehyde resin prepolymers, p-cresolformaldehyde resin prepolymers, and phenol-formaldehyde resin prepolymers were investigated. SEC and superimposed adsorption effects could be elucidated from a concept of solubility parameter. Minimum retention volumes of these oligomers were obtained with the mobile phases of chloroform/methanol, 80/20 or 60/40 (v/v), and separation was expected to be mostly performed by SEC. The solubility parameter of polyvinyl alcohol gels was estimated to be between 21 and 23 from the above results. Elution for normal phase chromatography was in the order of increasing molecular weight and that for reversed-phase chromatography was in the order of decreasing molecular weight. These are reversed phenomena to those for low-molecular weight compounds. Solubility of sample solutes to mobile phase must be considered. Methanol mobile phase-polyvinyl alcohol gel system might be exception.     

Exclusive Use of High Pressure Liquid Chromatography for the Determination of the Complete Amino Acid Sequence of the 12K Fragment of Avian Sarcoma Virus Structural Protein p27

Abstract

Using exclusively high pressure liquid chromatography for the protein and peptide separation complete primary structure of the 12,000 molecular weight (12K) amino terminal (1–87 residues) fragment obtained by mild acid hydrolysis of p27 (Avian Sarcoma Virus structural protein) has been determined. The sequence was established by direct degradation of the native molecule and its (12K) peptides isolated by molecular exclusion and reverse phase HPLC.     

Multimode Retention Behavior of Azaarenes in Size Exclusion Chromatography with Poly(Divinylbenzene)

Abstract

The size exclusion retention behavior of azaarenes on poly(divinylbenzene) is described. With dichloromethane as the mobile phase a strong nonsize effect was evident and retention was found to be governed by the pK_a of the eluate. By changing the mobile phase to N-methylpyrrolidinone, retention became independent of pK_a and components eluted in order of decreasing molecular weight. Some degree of nonsize behavior was still evident and separation proceeded through a multimode mechanism. LogMW for both the azaarenes and PAH. However, in the case of the azaarenes, a less than ideal correlation coefficient (-.705) suggests that retention is not purely size-dependent.     

The Preparative Separation of Synthetic Peptides on Reversed-Phase Silica Packed in Radially Compressed Flexible-Walled Columns.+

Abstract

The rapid and efficient separation of multigram amounts of unprotected synthetic peptides on octadecylsilica, packed in flexible walled polyethylene columns, is described. The mobile phase used was 0.05% trifluoroacetic acid with methanol as the organic modifier. An advantage of this eluant system was that the salt free peptide could be isolated simply by lyophilisation of the sample after chromatography. The following peptides were purified with this system: glycylgly-     

Size Exclusion Chromatography of Poly(vinylpyrrolidone)

Abstract

Poly(vinylpyrrolidone) and poly(ethylene oxide) separate by hydrodynamic volume on Toyo Soda TSK-PW columns in a mixed solvent mobile phase of 50:50 (v/v) MeOH/H₂O containing 0.1M LiNO₃. From this separation a single universal calibration curve based on hydrodynamic volume [η]M can be obtained. Accurate weight average molecular weights of PVP were obtained by both SEC/LALLS and universal calibration showing good agreement between the two methods. SEC/LALLS overestimates the number average molecular weight for broad distribution polymers due largely to the lack of sensitivity of the LALLS detector to the low molecular weight portion of the polymers, while the universal calibration method slightly underestimates the number average molecular weight as compared to osmometric values.     

Phase-distribution chromatography (PDC) of polystyrene

A chromatographic technique is described where the stationary phase is a layer of a very high molecular polystyrene fraction (M = 10⁷) on glass beads (ballotines). The mobile phase is cyclohexane passing the column at a constant temperature below the theta-temperature. A polystyrene sample of sufficiently low molecular weight (M ≤ 10⁶) injected as a small plug at the top of the column is fractionated because the distribution between the mobile and the stationary phase depends on the molecular weight. Since the large molecules preferentially remain in the stationary phase, the smaller molecules leave the column first. The fractionation effect is inverse to that found in GPC experiments. The separation efficiency is rather good and can be described by a simple thermodynamic theory.     

Ion Exchanger Liquid Chromatography of N-Acetylaspartic Acid and Some N-Acetylaspartyl Peptides

Abstract

A high performance liquid chromatographic (HPLC) procedure for the rapid separation of N-acetylaspartic acid, N-acetyl-aspartyl-glutamic acid and N-acetylaspartyl-glutamyl-aspartic acid is described. The procedure utilizes a pellicular ion-exchange column support, and ionic strength gradient with mobile phase solutions buffered to pH 5.0 and a UV detector operated at 210 nm. Reproducibility and quantitative capabilities are also discussed. The method has been used for a tentative estimation of N-acetylaspartic acid and N-acetylaspartyl peptides in a rat brain synaptosomal extract.     

Metabolic Profiling Using Reversed Phase High Performance Liquid Chromatography: Analysis of Urine from Patients with Rheumatoid Arthritis

Abstract

Optimal conditions are described for the reversed phase HPLC separation of UV absorbing constituents including peptides and proteins in urine. Metabolic profiles have been obtained for urinary constituents in the molecular weight range > 1000, 1000–10,000, and > 10,000 for healthy controls, patients with rheumatoid arthritis and osteoarthritis. A computer program was employed to compare individual and groups of profiles. Considerable variation was encountered in the patterns of constituents and comparison of the metabolic profiles revealed no diagnostically significant differences between the various groups of subjects.     

Purification of Solid-Phase Synthesized Peptides on the Coil Planet Centrifuge

Abstract

The analytical flow-through coil planet centrifuge, an instrument for countercurrent chromatography, performs the preparative purification of synthetic peptides. Various two-phase solvent systems have been tried with either phase mobile to purify many synthesized peptides. A series of N-terminal fragment peptides of cholecystokinin octapeptide (CCK 26–33) were synthesized by solid-phase techniques and purified on the coil planet centrifuge. The peptides were sulfated and chromatographed again. For hydrophobic peptides, purification is effected in solvent systems with a mobile aqueous phase. The n-butanol, acetic acid and water system (4:1:5 by volume) with the lower phase mobile was utilized. For sulfated peptides, the neutral system, 0.2 M ammonium acetate and n-butanol was generally applied.     

Liquid Chromatographic Analysis of Some N-Alkyl-3,4-methylenedioxyamphetamines

Abstract

The liquid chromatographic separation of a series of low molecular weight N-alkyl derivatives of 3,4-methylenedioxyamphetamine (MDA) is reported. The N-methyl-, N-ethyl- and N,N-dimethyl-derivatives of MDA have appeared as street drugs in recent years. These compounds are becoming significant forensic problems due to their unique pharmacological properties and relative ease of chemical synthesis. These amines were synthesized via reductive amination of the corresponding ketone with alkylamines. The UV absorption spectra for these compounds produced almost equally intense absorbance at 234 and 285 nm. The compounds were separated by reversed-phase (C₁₈) HPLC procedures using a mobile phase of aqueous methanol at low pH.     

Factors Affecting the Separation of Arginine Vasopressin Peptide Diastereoisomers by HPLC

Abstract

Experimental conditions and parameters involved in HPLC separations of the peptide hormone arginine vasopressin and some of its diastereoisomers on several reverse phase columns were investigated. The effects of percent carbon loading on an octadecyl reverse phase column, carbon chain length of the bonded phase, concentration of the buffer, and organic solvent were examined. Using the appropriate solvent systems, arginine vasopressin was separated from each of its diastereoisomers with most solvent systems studied, but the order of elution of the diastereoisomers was dependent on the column employed. Separation of the peptides was only part of the goal. A continuing study to understand the interactions of the peptides with the stationary phase as a function of structure was also undertaken. This lead to the conclusion that the choice of column as well as solvent affect the separation of peptide diastereoisomers and that both the eluting strength of the mobile phase and the stationary phase chemical composition must be considered.     

Separation of Free Amiimo Acids on Reverse Stationary Phases Using an Alkyl Sulfonate Salt as a Mobile Phase Additive

Abstract

Alkylsulfonate (RSO₃ ^?) salts were evaluated as mobile phase additives for the separation of free amino acids on reverse stationary phases using an acidic mobile phase where the amino acids are cations. The enhanced amino acid retention is the result of two major interactions, one being retention of the RSO₃ ^? salt on the stationary phase and the other an ion exchange selectivity between the amino acid analyte cation and the RSO₃ ^? countercation, or other countercations in the mobile phase. Major mobile phase variables are: type and concentration of RSO₃ ^? salt (the studies focused on C₈SO₃ ^? salts), presence of organic modifier, type of countercation present, and mobile phase pH and ionic strength. Alkyl modified silica and polystyrenedivinyl-benzene copolymeric reverse stationary phases were compared. A mobile phase gradient, increasing per cent organic modifier was shown to be best, is necessary for separating complex mixtures of polar and nonpolar or basic amino acids. The procedure is applicable to the identification and/or determination of amino acids in mixtures or in peptides after hydrolysis.     

Preparative Liquid Chromatographic Separation of Amino Acids and Peptides on Amberlite XAD-4

Abstract

Amberlite XAD-4, a polystyrene-divinylbenzene copolymic reversed phase adsorbent which has a 750 m²/g surface area and 50Å porosity, was used as the stationary phase for the preparative liquid chromatographic separation of amino acids and peptides. Mixtures of > 40 mg and > 100 mg sample load were separated on 8.0 and 20.5 mm i.d. columns, respectively. Mixed solvent and acidic and basic solutions which cannot be used with silica and alkyl-modified silica, were evaluated as mobile phases. Mixtures of amino acids, diastereomeric di-and tri-peptides, diastereomeric dipeptides obtained from the reaction of tert-butyloxycarbonyl-L-amino acid-N-hydroxysuccinimide esters with D, L-amino acids, and enkephalin peptides were separated. Major and minor sample components were isolated.     

Pseudophase Liquid Chromatography: Applications to TLC

Abstract

The technique of pseudophase liquid chromatography is described. In this type of separation, a substance does not partition to the bulk mobile phase but rather to discreet aggregates or other substances (i.e., micelles or cyclodextrins) which are dissolved in the mobile phase. Advantages of this technique include high selectivity, low cost, and low volatility and toxicity of the mobile phase. Appropriate previous TLC separations are reviewed and new data on the separation of quinones are presented.     

Separation of peptide diastereomers using CEC and a hydrophobic monolithic column

A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di‐ and tripeptides composed only of L ‐configured amino acids migrated faster than peptides containing D ‐amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and ¹⁶[D ‐Lys]‐tetracosactide could also be separated by CEC on the hydrophobic monolith.     

On-Stream HPLC Determination of Residual Ethylene Oxide and Propylene Oxide in Glycerol Based Polyol Synthesis

Abstract

A simple automated HPLC scheme was developed to monitor synthetic polymerization reaction on-stream. Chromatographic conditions that affect the separation of the reaction species from the reaction product and base catalyst using high-performance aqueous gel permeation chromatography were also studied. Reactor samples are automatically drawn from the reaction vessel through a micro-loop sampling valve/actuator assembly and transferred to the chromatographic system via stainless steel tubing without interruption of the mobile phase flow. The resulting chromatograms, monitored by a refractive index detector, of reactants and products are continuously measured at intervals during the reaction automatically and unattended. Application of this system for the reaction of propylene oxide and ethylene oxide to high molecular weight polyols is presented.     

The Utility of Cyclodextrin in Mobile Phase for High-Performance Liquid Chromatographic Separation of Isomeric Estrogens

Abstract

Cyclodextrin was used as a component of mobile phase for the separation of isomeric estrogens by reversed-phase high-performance liquid chromatography. The positional isomers of catechol and guaiacol estrogens were distinctly resolved by the addition of β-cyclodextrin in the mobile phase. The separation of estriol 16- and 17-glucuronides requires usually a prolonged time. The use of β-cyclodextrin in the mobile phase, however, reduced the retention time considerably.

The effect of β-cyclodextrin concentration in the mobile phase on the detector response was also investigated. The response of a fluorescence detector was raised with an increasing concentration of β-cyclodextrin, while that of an electrochemical detector was significantly depressed.     

Reversed Phase Thin Layer Chromatography of Amino Acids

Abstract

The use of reversed phase layers for the thin layer chromatography of amino acids is described. Only when a modifier was added to the mobile phase was clear separation of the amino acids achieved. Ion paring with trifluoroacetic acid overcame problems with streaking and poor separation on C₂ or C₁₈ reversed phase layers. All amino acids could not be separated with a single mobile phase. Thus, three different combination of acetonitrile-0.4% trifluoroacetic acid were used to separate eighteen amino acids with derivatization. No derivative was required.