Ion Interaction Chromatographic Separation of Amino Acids Using a Basic-Tetraalkylammonium Salt Mobile Phase

Abstract

A basic mobile phase containing a tetraalkylammonium (R₄N⁺) salt was used to enhance the retention of free amino acids (AA) in their anion form on a polystyrene divinylbenzene copolymeric (Hamilton PRP-1) nonpolar stationary phase adsorbent. Major variables, which can be readily manipulated to alter this retention and resolve complex AA mixtures, are: structure and concentration of R₄N⁺ salt, type and amount of organic modifier in the mobile phase solvent, concentration and selectivity of the counteranion present, and mobile phase pH and ionic strength. Mobile phase gradients based on a pH change, or an ionic strength change and their combination, while all other variables are constant, were evaluated for the separation of complex AA mixtures. Detection was accomplished by absorbance or fluorescence after a post-column ortho-phthalaldehyde reaction.     

Ion-Interaction Chromatographic Separation of Free Amino Acids

Abstract

An extensive study of the HPLC separation of 20 free amino acids by the addition of alkanesulfonate salts to the mobile phase was previously reported (1). This paper describes modifications in the procedure that improves the separation and resolution of the 20 free amino acids. Mobile phase variables (type and concentration of alkanesulfonate salt, organic modifier concentration, mobile phase pH, and mobile phase ionic strength), and stationary phase variables (particle size, type of packing) which can affect amino acid separation, resolution and selectivity were studied. Two stationary phases were compared, the 5 μm Hamilton PRP-1 and Phase Separations 3 μm, ODS-2. Longer chain alkanesulfonate salts (octane and decanesulfonate salts) were evaluated as mobile phase additives. A mobile phase gradient of increasing per cent organic modifier was necessary for separating complex mixtures of polar and nonpolar-basic amino acids. It is now possible to separate 19 of 20 free amino acids with this ion-interaction chromatographic procedure.     

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.     

Adsorption of a Hydrophobic Chelating Agent and its Chelate-Metal Complex on RP-Columns

Abstract

The reversed-phase adsorption of the hydrophobic chelating agent 4-dodecyldiethylenetriamine, in the presence of zinc ions, was measured at different compositions of the mobile phase. The effect of the following parameters was analyzed: proportion of metallic ion to chelating agent, concentration of chelating agent, volumic fraction of organic modifier, pH and salt concentration. Results show that, depending on the composition of the mobile phase, the chelating agent can exist in two different forms in both phases: “free” and/or forming a chelate-metal complex. The adsorption of the latter is stronger than that of the free triamine. This phenomenon, which has not been reported before, plays undoubtedly an important role on retention in Reversed-Phase Ligand Exchange Chromatography and should be considered in the study of the retention mechanism.     

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-     

The Quantitation of Heroin and Selected Basic Impurities Via Reversed Phase HPLC. I. The Analysis of Unadulterated Heroin Samples

Abstract

Methodology was developed employing reversed phase liquid chromatography for the simultaneous determination of heroin, O³-monoacetylmorphine, o⁶-monoacetylmorphine, acetylcodeine, noscapine and papaverine in unadulterated illicit powders. An HS-5 C18 column was used with a gradient system using methanol and a hexylamine phosphate buffer at pH 2.2. This method, suitable for automated analysis, used a multi-mode detection scheme via the use of a photodiode array detector. In order to arrive at the optimum chromatographic conditions in terms of selectivity and stability, a study was performed on the effect of various mobile phase parameters on log k′ for heroin, various impurities, and common adulterants. The mobile phase parameters included amine concentration, organic modifier type, and eluent pH.     

OJ手性柱分离芳基烷酸类非甾体解热镇痛药的色谱保留特性和拆分机理

使用纤维素类手性柱OJ柱, 分离布洛芬、依托度酸、非诺洛芬钙、酮洛芬、洛索洛芬等5个芳基烷酸类非甾体解热镇痛药对映体, 并通过溶质计量置换保留模型和热力学研究对OJ手性柱手性分离机理进行探讨. 结果表明, OJ手性柱可在正相条件下分离系列芳基烷酸类非甾体解热镇痛药, 对映体的色谱保留和分离度可以通过改变流动相中醇类置换剂和浓度、有机酸性改性剂和浓度、柱温等因素调节.     

The Separation of Organic Analyte Cations on a Low-Capacity Cation Exchange Column Using Indirect UV Detection

Abstract

The retention of organic analyte cations on a low-capacity cation exchange column using indirect UV detection was studied. It was found that a combination of cation exchange/reversed-phase interactions affected the retention of organic analyte cations provided the analytes have both a cationic charge site and a hydrophobic center. The factors that influenced the organic analyte cation retention were: concentration of organic modifier, concentration of UV absorbing analyte, pH, and mobile phase ionic strength. Elution orders for several of the organic analytes studied on the low-capacity cation exchange column were different than those observed on silica-based strong cation exchange columns.     

Demixing effects in thin-layer chromatography with NH2-modified silica gel precoated plates

Summary Demixing effects in thin-layer chromatography have been investigated with NH₂-modified silica gel precoated plates and for frequently used hydroorganic binary solvents of various compositions with salt addition.The position of the solvent demixing front depends on: (1) solvent composition, (2) nature of the organic modifier and (3) salt concentration. Whatever the organic modifier, for a given water percentage in the developing solvent, solvent demixing is found to occur at the same concentration of NaCl.     

A Simple Two-Membered Model for Retention in RP-IPC with Hydrophobic Counterions

Abstract

The retention behavior of some organic anions in the presence of tetralkyl ammonium ions in the mobile phase is analized under conditions where these hydrophobic counterions do adsorb on the surface of a reversed-phase packing. The study comprises the effects of the counterion concentration in the mobile and in the stationary phase, the salt concentration in the eluent and the salt type. Experimental results are discussed in view of previously proposed mechanisms and 2 simple two-member expression relating corrected capacity factor to adsorbed counterion concentration is deduced. The retention model indicates that solute is present in the stationary phase at two different levels; first into a diffuse ionic cloud associated electrically to the ionic surface and, second, at the surface of the packing where it forms ion pairs with the adsorbed counterions.     

Roles of Organic Modifiers in Micellar Electrokinetic Capillary Chromatography

In the present work, four organic modifiers, viz. urea, dioxane, methanol, and tetrahydrofuran, were comparatively and systematically studied in terms of their effects on electrokinetic migration behavior and the retention mechanism of homologous solutes in MECC. The results showed that the electroosmotic mobility, μ_eo, and the electrophoretic mobility of a micelle, μ_ep,mc, decrease linearly with increasing organic modifier concentration. The ability of organic modifiers to lower μ_eo is greater than their ability to lower μ_ep,mc. The negative values of the slopes of these linear relationships, D_eo and D_ep,mc, increase along the series the order urea < methanol < dioxane < tetrahydrofuran. The logarithm of the capacity factor (ln k′) of uncharged homologous solute, which is mainly determined by the hydrophobic interaction, decreases linearly with increasing organic modifier concentration, due not only to the decrease in the partition coefficient but also to the decrease in the phase ratio. A linear relationship was observed between the slope of the plot of ln k′ vs. organic modifier concentration and the carbon number of homologous compound. The slope of such a relationship can characterize the hydrophobicity of the organic modifier. The hydrophobicity of such organic modifiers is also found to increase along the series urea < methanol < dioxane < tetrahydrofuran.     

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

Separation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.     

Ruthenium (II) 1,10-Phenanthroline Salts as Mobile Phase Additives for the Separation and Indirect Detection of Free Amino Acids

Abstract

Ruthenium (II) 1,10-phenanthroline, Ru(phen)²⁺ ₃, salts are used as ion interaction reagents in a basic mobile phase for the retention, resolution, and indirect photometric detection (IPD) of free amino acids on a polystyrene divinylbenzene (Hamilton PRP-1) column. Mobile phase Ru(phen)²⁺ ₃ concentration and pH and type and concentration of organic modifier and counteranion affect retention and IPD. Underivatized amino acid elution order is influenced by side chain structure typical of ion exchange processes. Detection limits for the separation and detection of free amino acids using an isocratic elution condition are about 0.1 nmole for lower retained amino acids and 0.25 nmole for higher retained amino acids for a 3:1 signal:noise ratio. Gradient elution is possible but at higher detection limits.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

Influence Of Organic Modifiers On The Retention Characteristics Of Graphitized Carbon Column

Abstract

The retention time of α-(1,1,3,3-tetramethylbutyl)phenyl ethylene oxide oligomer surfactants was determined on a porous graphitized carbon column (PGC) in eluents containing acetonitrile, methanol, ethanol, 2-propanol, tetrahydrofuran, dioxane and 2-ethoxyethanol as organic modifier. Linear relationships were calculated between the logarithm of the capacity factor and the concentration of organic modifier in the eluent. Ethylene oxide oligomers were well separated on the PGC column in acetonitrile-water eluent mixtures. Oligomers were not separated in eluents containing 2-propanol, tetrahydrofuran. dioxane and 2-ethoxyethanol as organic modifier. This result was explained by the supposition that the bulky organic modifiers occupy the active adsorption centers on the PGC surface decreasing in this manner the separation efficiency of the support.     

Fast Analysis of Capillary Electrochromatography with Non-Porous ODS as the Stationary Phase

ABSTRACT

High-speed capillary electrochromatography was developed on both short and long packed columns with 2 μm non-porous ODS as the stationary phase. Factors that affect the analysis time of samples, such as voltage, electrolyte concentration, pH and organic modifier concentration in the mobile phase, were studied systematically. Fast analysis of aromatic compounds within 13 seconds was realized with column efficiency of 573,000 plates/m and a R.S.D.% of the retention times of all components in 8 consecutive injections below 1.0%, which demonstrated the high efficiency and high reproducibility of such a technique. In addition, DNPH derived aldehydes and ketones in both standards and environmental samples were separated with high speed.     

LC-EC of Endorphins

Abstract

Reversed phase liquid chromatography with electrochemical detection (LC-EC) was used to separate a series of endorphin standards. Chromatographic conditions were manipulated so that methionine- and leucine-enkephalin were clearly resolved from other endorphins of similar hydrophobicity using an isocratic mobile phase. The most significant factors affecting endorphin retention were the concentration and type of organic modifier in the isocratic mobile phase. Hydrodynamic voltammograms were performed for methionine- and leucine-enkephalin to assess their electroactivity. Both enkephalins were oxidized with a glassy carbon electrode only at high potentials (>+.90V vs Ag/AgCl). The effect of these high potentials on the sensitivity of electrochemical detection of endorphins was evaluated.     

Void volume determination and experimental probes in reversed phase chromatography

Summary In reversed-phase liquid chromatography with n-alkyl bonded silica, the dead volume (V₀) of the column is theoretically indeterminate owing to adsorption of organic modifier on n-alkyl chains and of water on silanol groups. With binary mobile phases, retention volumes of the mobile phase components and of their deuterated species are relaeed to the adsorption isotherms and V₀ by equations which can be solved with some assumptions on the adsorbed layer composition. Methanol-water and acetonitrile-water systems are studied. As the experimental excess isotherm shows a linear part in the concentration range 50–80% in organic modifier, the hypothesis of an adsorbed layer of constant composition in this range is possible. When increasing the water content of the mobile phase, adsorption of water occurs up to saturation of silanol groups. Then the assumption of a constant water content for a mobile phase having more than 50% of water is applied. With the hypothesis of a constant adsorbed content of organic modifier when the eluent has more than 80% of organic modifier, V₀ and the absolute isotherms are calculated over the entire range of mobile phase composition. Experimental retention behavior of the mobile phase components are totally explained by these V₀ determinations. The retention times of commonly used V₀ markers are compared with V₀ values. It is shown that, when buffering the eluent, no visible effect on the distribution equilibrium is observed, so that injection of concentrated potassium nitrate is a convenient method to measure V₀. With a few solutes with are UV detectable it is possible to measure V₀ whatever the mobile phase composition in methanol-water and acetonitrile-water systems.     

Separation of cytochromes c by reversed-phase high-performance liquid chromatography

Six kinds of cytochrome c of different origin, i.e., bovine, chicken, dog, horse, rabbit and tuna, were subjected to separation by reversed-phase high-performance liquid chromatography on three commercial packing materials; octadecyl-, octyl- and cyanoalkyl-silicas. The effects of reversed-phase material, mobile phase and temperature on the separation of cytochromes c were examined. The parameters of the mobile phase were the organic modifier, the pH, the salt concentration and additives. Under optimal conditions, five of the six cytochromes c were resolved in 10 min. The relative retention values cannot be explained in terms of the relative lipophilicities of the side-chains of the amino acid residues.     

Simultaneous Optimization of Surfactant Concentration and Organic Modifier in Micellar Liquid Chromatography. Application to the Separation of Phenolic Compounds

ABSTRACT

The separation selectivity of eighteen phenolic derivatives in micellar high-performance liquid chromatography was studied as a function of parameters on which it depends: surfactant and organic modifier concentration. The surfactant used in this study was cetyltrimethylammonium bromide, and as organic modifier methanol. An iterative regression optimization strategy for these two parameters has been used. The equation that best explains the experimental

results is 1/k' = A +Bμ + Cφ + DμΦ. We propose the use of this model in conjunction with the appropriate factorial design to predict the solute retention behaviour in micellar liquid chromatography with hibrid eluents.