Reversed Phase High Performance Liquid Chromatography Using Carboxylic Acids in the Mobile Phase

Abstract

This report describes the use of different carboxylic acids as mobile phase modifiers. The effect on retention of acid chain length, pH, and eluent composition for a series of phenylalkanols, phenol, and the amines aniline, N-methylaniline, and benzylamine is discussed. The retention of both neutral and positively charged compounds is influenced by the dissociation equilibrium of the carboxylic acid in the mobile phase. By using l-pentanol to coat excess exposed silanol groups on the reversed phase column used, the inflection in the retention of both neutral and charged solutes as pH is changed occurs at the pK_a of the acid in the mobile phase. In addition, by using an acid and amine with the same or similar pK_a values, selective ion-pairing of this pair over others with dissimilar pK_a values can be promoted. Application of this technique to the selective retention of amino acids and peptides was unsuccessful.     

Quantitative Determination of Methyl Red Adsorption on Stationary Phases Used in Liquid Chromatography

Abstract

The method described is a modification of the dye adsorption method, first published by Shapiro and Kolthoff. It is reproducible and allows the estimation of the relative amount of the reachable free silanol groups on silica gels and reversed phase materials which are frequently used as stationary phases in liquid chromatography. Results obtained with commercially available thin layer and high performance liquid chromatographic materials, as well as with self-made reversed phase materials, are reported.     

Separation Mechanism of Copolymers from Styrene and Methyl Methacrylate According to Composition by Liquid Adsorption Chromatography

Abstract

Silica gel and a mixture of chloroform and ethanol were used as the stationary and the mobile phases, respectively. At the isocratic elution mode, the copolymers tended to adsorb on the column with increasing MMA content in the copolymers, with increasing column temperature, and with decreasing ethanol content in the mobile phase. The copolymers appeared always at the position proportional to the interstitial volume in the column, otherwise they adsorbed on the column. The equilibrium distribution of the copolymers between the mobile phase and the stationary phase cannot be considered in this system. Hydrogen bonding of carbonyl groups in MMA units of the copolymers to silanol groups on the surface of silica gel was considered to be the main interaction between the copolymers and the adsorbent. The hydrogen bonding increases with increasing the number of carbonyl groups per unit surface of copolymer coil in solution and with increasing free silanol groups on the silica surface. Ethanol in the stationary phase controls the content of the free silanol groups on the surface and is controlled by the content of ethanol in the mobile phase in addition to column temperature. This phenomena can be explained by introducing the term of the adsorption energy of a copolymer segment with the silica surface. The adsorption energy is proportional to the strength of the hydrogen bonding. Above the critical adsorption energy which is defined to be equal to the dissolution energy of the copolymer to the mobile pahse, the copolymers adsorb on the surface of silica gel and the desorption of the copolymers advances when the adsorption energy approaches to the critical adsorption energy. No adsorption of the copolymers is possible when the adsorption energy is equal or below the critical adsorption energy.     

The Significance of Metallophilic and Silanophilic Interactions is Reversed Phase Hplc

Abstract

The poor peak shape encountered for amines in reversed phase high performance liquid chromatography (RPLC) is widely recognized. In order to improve chromatographic efficiency, the mechanism of the broadening must be understood. In addition to the silanol groups on the support surface, raetal sites have been implicated as possible adsorption sites for amines.

We have investigated the relative role of metallophilic and silanophilic interactions in the non-ideal behavior of amines and proteins. Stainless steel frits appear to have a deleterious effect on column efficiency via both mechanical and chemical interactions. The use of stainless steel meshes or screens is recommended. The residual metal sites on the support material have been found to play an insignificant role in solute retention or peak broadening. Minimization of silanophilic interactions is therefore the key to increasing efficiency in the separation of basic amines.     

Liquid Chromatographic Characteristics of Ethylenethiourea with HPLC Carbon,Chiral, Polymer and Reverse-Phase Bonded Silica Columns

Abstract

Of the columns investigated, the graphitised carbon column provided the best chromatographic characteristics for the highly water-soluble compound ethylenethiourea (ETU). The stability of the carbon column in strongly acidic media permitted the incorporation of the phosphoric acid electrolyte into the 5% acetonitrile-in-water mobile phase. ETU eluted from the column in 200 s as a sharp symmetrical peak at a mobile phase flow rate of 1 mL/min and a column temperature of 35°C. The k' value was 1.72. ETU peak retention times and responses showed excellent repeatability with coefficients of variation of 0.28 and 1.40%, respectively, for 6 replicates with the high performance liquid chromatographic-electrochemical system using the graphitised carbon column. Although ETU eluted as a sharp symmetrical peak with the cyclodextrin chiral columns, their instability at low pH required post-column addition of the phosphoric acid electrolyte solution. ETU chromatographed poorly or degraded on the polymer columns. The chromatographic separation of ETU on the C-8 reverse-phase bonded silica column appeared to be due mainly to residual silanol groups. With the NH₂ bonded silica column ETU eluted immediately after the injection solvent.     

The Use of Decanol for Improving Chromatographic Stability in Isocratic Non-Aqueous Reversedphase Analysis of Carotenoids by High-Pressure Liquid Chromatography

Abstract

A non-aqueous isocratic solvent system for reversed-phase analysis of a full range of carotenoids from polar to non-polar is described. The major advance in the method is the use of n-decanol added at 0.1% by volume as modifier to a mobile phase of ethyl acetate-acetonitrile. The presence of the modifier rapidly conditions new columns and avoids problems with trace solvent impurities, both of which would otherwise give unsatisfactory chromatographic behaviour. In the absence of n-decanol, activation of residual silanol groups in the reversed-phase packing can cause a drastic deterioration in column behaviour. The method is rapid, versatile and simple. Information on separation conditions, solvent effects, column conditioning and carotenoid response curves is given.     

HPLC of Steroids in Non-aqueous Mobile Phase at Subambient Temperature

Abstract

Increased resolution of steroid mixtures was found in high performance liquid chromatography of steroids at subambient temperatures. With aqueous mobile phase using a reversed phase column it was not possible to decrease temperatures below ?10°C due to increased viscosity. This report describes further increase in resolution at ?50°C using a non-aqueous mobile phase for the separation of steroids. Retention times were shorter several fold while resolution was improved.     

Resolution of D,L-Dansyl Amino Acids by HELC with a Cu(II)-L-proline Eluant

Abstract

Highly fluorescent dansyl derivatives of D and L amino acids were resolved on a reversed phase column by using an aqueous eluant containing copper(II)-L-proline.     

Polyvinylacetate Gel Permeation Chromatography of H-Coal Liquids and Model Compounds. Comparison with Rigid Gel,Reversed Phase and Normal Phase Chromatography

Abstract

This paper reports the application of polyvinylacetate gel (Fractogel) for size fractionation of the H-Coal liquids and model aromatic hydrocarbons. The four H-Coal liquids were each divided into three classes according to solubility in hexane, benzene, and pyridine. A model mixture of eight compounds when applied to the Fractogel column yielded four fractions; a similar performance was given by a prepacked, Toyo Soda microparticulate column containing styrene-divinylbenzene copolymer. Regarding the twelve H-Coal solvent fractions, the chromatograms obtained from the Fractogel column were analogous to those from the Toyo Soda column. By making further use of model compounds, the Fractogel results are compared with those obtained from reversed phase chromatography on a Partisil ODS column and those from normal phase chromatography on a LiChrosorb silica column. These comparisons reveal the usefulness in separating certain aromatic hydrocarbons by the Fractogel column. Furthermore, judging from the chromatograms of a hexane-soluble H-Coal fraction obtained by the reversed phase and normal phase methods, the ODS column will complement the Fractogel column in fractionating the H-Coal liquids.     

Buffer Choice for Tuning the Selectivity in Reverse Phase Chromatography

Abstract

Retention behaviour of ionogenic species in high-performance liquid chromatography on reversed phase materials was studied, specifically dependence of buffer quality applied to mobile phases. The buffers' effect on retention of organic acids, amino acids and dipeptides is quantified by modelling capacity factors as a function of pH-values. At constant ionic strength, increasing capacity factors were observed going from phosphate to less polar citrate buffer, modification of accessible silanol groups of the stationary phase being responsible for this effect. Application of citrate buffer for separation of a seven-component mixture is demonstrated on the basis of a computerized search for optimum chromatographic performance. The evaluated factor levels (pH, methanol content and ionic strength) differ from those found using phosphate buffer-containing mobile phases.     

Preparation of a polyglycol-C8 bonded phase and its characteristics

Summary A new bonded phase containing both non-polar C₈ and polar polyglycol chains was synthesized in two steps. Silica was first silanized with octyltrimethoxysilane and then the intermediate product was reacted with tetraethylene glycol to introduce the polar group. The bonded phase exhibits a selectivity different from that of C₈ or C₁₈ reversed phases with a mixed-mode retention mechanism. The silanol effect on the polyglycol-C₈ bonded phase was examined with the test mixture proposed by Engelhardt and co-workers. The experimental results indicate that the new bonded phase displays less silanol effect. The observed tailing of some ionizable species resulted mainly from their partial dissociation at the pH values of eluents close to thepK _a values of the solutes. Some potential applications of the bonded phase are also given.     

Differential Measurement of Cortisol and Cortisone in Human Saliva by HPLC with Uv Detection

Abstract

Both cortisol and its dehydro metabolite cortisone are present in normal human saliva. A method for differential Measurement of both compounds in 1 ml samples of saliva by HPLC/UV is described. the method uses an extraction column having a cyclodextrin bonded phase to retain the compounds of interest while allowing elution of interfering compounds. A steroid-bearing fraction is eluted from the cyclodextrin column, dried, reconstituted in a weak mobile phase, and injected on a reversed phase HPLC/UV system provided with an injector-mounted reversed phase extraction column. Samples containing corticosteroid concentrations as low as 0.5 ng/ml can be effectively analyzed by this method.     

Determination of Melphalan and Chlorambucil in Tablet Dosage form Using High-Performance Liquid Chromatography and Amperometric Detection

Abstract

We developed a liquid chromatographic method for the measurement of melphalan and chlorambucil in tablets, using a reversed phase C18 column with isocratic elution and amperometric detection. For melphalan the potential was set at +0.95V and chlorambucil at + 0.90V. The method is simple, sensitive and may also be applicable to pharmacokinetic studies.     

High Performance Liquid Chromatographic Determination of N-Methyl-2-pyrrolidone in Water

Abstract

N-Methyl-2-pyrrolidone was determined in water using High Performance Liquid Chromatography. A determination required less than 10 minutes using an octadecyl reversed phase column and isocratic elution with methanol: water (10/90, V/V). Detection was at 205 nm. Sub-part-per-billion concentrations were determined without precon-centration by injecting a 500 μL sample into the chromatograph.     

Test compounds for detecting the silanol effect on the elution of ionized amines in reversed‐phase LC

The effectiveness of several basic compounds for testing silica‐based stationary phases was reviewed by applying them to recent columns for reversed‐phase HPLC. Most octadecylsilylated (C18) stationary phases, prepared as a base‐deactivated material from high‐purity silica gel with endcapping, provided excellent peak shape and column efficiency for the bases including benzylamine and amitriptyline that once caused problems and were subsequently employed for testing silanol activities. However, a cyclic tertiary amine, dextrometorphan, was eluted as an acceptable peak from only a few columns at neutral pH. Such a more sensitive probe is expected to contribute to further improvement of the stationary phase for reversed‐phase HPLC.     

Rapid Determination of Aldehydes in Air Analyses

Abstract

A simple analytical method for aldehyde determinations in ambient air is described. The method involved trapping the aldehydes as their DNPH derivatives in acetonitrile solution and then direct injection and analysis of the resulting solution on a reversed phase HPLC column with simultaneous detection at 254 and 360 nm. Several comparison studies are described.     

A Simultaneous Analysis by High Performance Liquid Chromatography of Bamipine Combined with Tricyclic Antidepressants and/or Antipsychotics in Dosage Forms

Abstract

A reversed phase high-performance liquid chromatographic method is described for the simultaneous determination of antihistamines, tricyclic antidepressants and anti-psychotics in pharmaceutical formulations and in spiked placebos. The separation was performed on an octadecyl-silica column using acetonitrile: tetrahydrofuran: 0.015 M aqueous ammonium acetate (53:42: 5) as mobile phase. The presence of ammonium acetate both shortens the elution time and improves the symmetry of the chromatographic peaks. Measurements were made at 251 nm.     

Rapid Analysis for Codeine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid codeine in pharmaceutical preparations and in body fluids. The minimum detectable concentrations for body fluids were 5ppb and 7ppb respectively for urine and whole blood with an analysis time of under 5 min. A RP-8 Spheri-5 guard column and a RP-8 Lichrosorb-10 column were used and codeine was detected at its absorption maximum wavelength of 212 nm using an eluting system of methanol: 0.5% w/v aqueous ammonium acetate (70:30) at a pH of about 7.0.     

High Performance Liquid Chromatographic Method for Quantitation of a New Antidiabetic Agent in Plasma

Abstract

An HPLC procedure for the detection and quantitation of a new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), in dog plasma was developed. The drug and internal standard were extracted from plasma using a reversed phase C₁₈ extraction column (Sep-pak). Separation was accomplished on a ERC-ODS-1161 reversed-phase column with a mobile phase of acetonitrile/0.1M phosphate buffer, pH 6.6 (30/70). Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. A linear relationship between concentration and peak height ratio (A4166/internal standard) was obtained. The method has been successfully used for analysis of plasma samples from beagle dogs following oral administration of A4166.     

HPLC Determination of Cyclosporine in Whole Blood

Abstract

A rapid sensitive method for measuring cyclosporine concentration in whole blood by HPLC has been developed. The pre-chromatography isolation steps are convenient and rapid and are based on salting out acetonitrile with simultaneous extraction of cyclosporine from the blood. A reversed phase column is used with detection by absorbance at 200 nm.