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.     

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.     

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.     

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.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

Preparation and evaluation of novel chiral stationary phases based on quinine derivatives comprising crown ether moieties

The C9‐position of quinine was modified by meta‐ or para‐substituted benzo‐18‐crown‐6, and immobilized on 3‐mercaptopropyl‐modified silica gel through the radical thiol‐ene addition reaction. These two chiral stationary phases were evaluated by chiral acids, amino acids, and chiral primary amines. The crown ether moiety on the quinine anion exchanger provided a ligand‐exchange site for primary amino groups, which played an important role in the retention and enantioselectivity for chiral compounds containing primary amine groups. These two stationary phases showed good selectivity for some amino acids. The complex interaction between crown ether and protonated primary amino group was investigated by the addition of inorganic salts such as LiCl, NH₄Cl, NaCl, and KCl to the mobile phase. The resolution results showed that the simultaneous interactions between two function moieties (quinine and crown ether) and amino acids were important for the chiral separation.     

Modeling the retention mechanism for high‐performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu²⁺(l ‐phenylalanine)₂ using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu²⁺(l ‐phenylalanine)₂ complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed‐phase column with an optimized mobile phase composed of 6 mmol/L of l ‐phenylalanine or N,N‐dimethyl‐l ‐phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.     

Retention behavior of a homologous series and positional isomers of aliphatic amino acids in hydrophilic interaction chromatography

The retention behavior of several series of free α‐ and ω‐amino acids and positional isomers of amino pentanoic acid in the hydrophilic interaction chromatography mode (HILIC) was studied. The study was carried out on three stationary phases followed by post‐column derivatization with fluorescence detection in order to describe the retention mechanism of the tested amino acids. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.5) and concentration of buffer in the mobile phase was investigated. The effect of the number of carbon atoms (n_C) in aliphatic chains of the individual homologue of α‐ and ω‐amino acids and the logarithm of the partition coefficient (logD) on retention was also a part of the presented study. A good correlation (r > 0.98) between the logk and logD values of amino acids or n_C, respectively, was observed. The described linear relationships were subsequently applied to predict the retention behavior of individual members of the homologous series of amino acids and to optimize the mobile phase composition in HILIC. The obtained results confirmed that the retention mechanism of α‐amino acids, ω‐amino acids and positional isomers of amino acids was based on the logD values and the number of carbon atoms in the aliphatic chains of amino acids. The elution order of ω‐amino acids and positional isomers of amino pentanoic acid was strongly dependent on the mobile phase pH in the investigated range whereas the retention factors of all α‐amino acids remained essentially unchanged on all tested stationary phases.     

Selectivity of dansyl amino acids in micellar liquid chromatography with an ion-exchange-induced stationary phase

Summary The retention behavior of dansyl amino acids in micellar liquid chromatography has been examined by using ionexchange-induced stationary phases. Several parameters affected the retention of the analytes, including the type and concentration of micellar agent and modifier ion and the concentration of acetonitrile in the mobile phase. The order of elution of dansyl amino acids obtained with the micellar mobile phase was very different from that observed in conventional reversed-phase liquid chromatography. Fluorescence intensities of some dansyl amino acids were enhanced by the micellar mobile phase.     

Investigating the Effect of Chromatographic Conditions on Retention of Organic Acids in Hydrophilic Interaction Chromatography Using a Design of Experiment

Small organic acids have shown significant retention on various stationary phases, such as amide, amino, aspartamide, silica and sulfobetaine phase commonly used in hydrophilic interaction chromatography (HILIC). This study investigated the effect of chromatographic conditions on the retention behavior of organic acids in HILIC using the tool of design of experiment (DOE). The results of the DOE study indicated that both the content of organic solvent (i.e., acetonitrile) and salt concentration in the mobile phase had significant effects on the retention of organic acids. Higher content of organic solvent in the mobile phase led to a significant increase in retention on all types of stationary phases. Increasing salt concentration also resulted in a moderate increase in retention; however, the effect of salt concentration varied with the type of stationary phase. The study also revealed that column temperature had less impact on retention than organic solvent content and salt concentration in HILIC.     

Prediction of the chromatographic behaviour for a series of diuretic compounds

Summary The proportion of organic modifier and the pH of the acetonitrile-water mixtures used as mobile phases were optimized in order to separate a group of diuretic compounds covering a wide range of physyco-chemical properties. The Linear Solvation Energy Relationship (LSER) formalism based either on the multiparameter π^*, β and α scales or the single solvent polarity parameterE _T ^N , have been used to predict their chromatographic behaviour as a function of the percentage of acetonitrile in the eluent. Moreover, correlation established between retention and pH of the aqueous-organic mobile phases have been used to predict the chromatographic behaviour of the diuretic compounds studied as a function of the eluent pH. Linear correlation between a function of the eluent pH. Linear correlation between the chromatographic retention and theE _T ^N polarity parameter of mobile phases containing different percentages of organic modifier has been obtained Based on the knowledge of the acid-base dissociation constant the relation between retention and mobile phase pH has also been linearized. These relationship allowed an important reduction of the experimental retention data needed for developing a given separation and a great improvement in chromatographic optimization schemes.     

Reversed-phase liquid chromatography analysis of alkyl-imidazolium ionic liquids II. Effects of different added salts and stationary phase influence

Two mixtures of four 1-alkyl-3-methylimidazolium ionic liquids (ILs) salts associated to the anions tetrafluoroborate or hexafluorophosphate were analyzed by reversed-phase liquid chromatography with three different stationary phases: Kromasil C(8), Zorbax Extend C(18) and Zorbax Sb-Aq. The effect on retention of various inorganic salts (NaCl, NaH(2)PO(4,) NaBF(4), NaClO(4) and NaPF(6)) added to acetonitrile/water mobile phases was studied. The three columns gave similar separation profiles. In all cases, the retention of ILs increased with the increasing affinity of the inorganic anions for the apolar stationary phases; a phenomenon called chaotropicity. The chaotropic anion order is Cl(-) approximately H(2)PO(4)(-) < BF(4)(-) approximately ClO(4)(-) < PF(6)(-). It is established that the presence of chaotropic anions in the mobile phase do not permit to differentiate between ILs associated to different anions. However, chloride or dihydrogenphosphate added salts do not fully screen the retention differences between ILs associated with different anions. Distorted and even split peaks may appear in the chromatogram depending on the nature and concentration of the injected ILs. In the RPLC analysis of imidazolium-based IL, it is recommended to add to the mobile phase significant amounts of a salt containing a chaotropic anion. This salt addition will improve the IL peak shapes and give reproducible retention factors. LODs in the low microgram range ( approximately 5 nmol) were obtained with the Kromasil C(8) column with a 50/50 acetonitrile-water mobile phase containing 0.01 M NaPF(6) added salt and 230 nm UV detection.     

The retention behaviour of amino acids in hydrophilic interaction liquid chromatography on zwitterionic stationary phases†

The retention behaviour of amino acids was studied in hydrophilic LC on zwitterionic stationary phases. Evaluation of the influences of acetonitrile/water content, ammonium acetate (NH₄Ac) concentration and mobile phase pH values was performed. Fourteen amino acids were tested and they were all retained to varying extents, with poorer retention in high water content eluents. The linear relationship between the logarithm of retention factor and log(water content) indicated that adsorption dominated or at least was partly involved in the separation mechanism. Electrostatic and hydrophilic interactions also contributed to the retention of these amino acids under different separation conditions with various mobile phase pH values and NH₄Ac concentrations. Thus, the overall retention mechanism could be explained as a combination of adsorption, electrostatic and hydrophilic interactions. The magnitude and contribution of each mechanism is dependent on the nature of the analyte and the separation conditions applied.     

Reversed-Phase High-Performance Liquid Chromatography of Unsubstituted Aminobenzoic Acids

Abstract

High-performance liquid chromatographic (HPLC) characteristics of three position isomers of aminobenzoic acids (potential metabolites of important anesthetic drugs), were delineated with respect to their interactions with various mobile phases and stationary phases. HPLC with five hydrocarbonaceous phase, β-cyclodextrin silica (CDS), macrophase MP-1 polymer (MP), macroporous polystyrene/divinylbenzene (MPD), octadecylsilica (ODS), and propylphenylsilica (PPS), yielded results explicable in terms of substituent effects derived from the bifunctional amino– and carboxy groups. For cases where mobile phases contained sulfonates or quaternary ammonium salts both having longer chain alkyls, retention of analytes on all but CDS appeared to proceed predominantly via an ion-pairing mechanism. The extent of the corresponding counter-ion effects decreased in the order: MPD > ODS > PPS > MP, while the analyte retention order paralleled thier pH₂ values. On the other hand, an inverse relationship between the magnitude of capacity factors (k′) and pK₁ values of the title compounds was observed in experiments that produced retention data incompatible with ion-pair interaction rationales. The unique HPLC results obtained with the CDS phase are compared with those obtained with other phases.     

Reversed phase liquid chromatography of alkyl-imidazolium ionic liquids

Eleven 1-alkyl-3-methyl imidazolium ionic liquid (IL) salts were analyzed in reversed phase mode with a Kromasil C18 column. The mobile phases were water-rich acetonitrile solutions (water content > or =70%, v/v) without any added salts. It is shown that it is possible to separate different ILs sharing the same cation and differing by the anion when salt-free mobile phases are used. When a buffer, acetate or phosphate salt, or any salt, such as sodium chloride or sodium tetrafluorobarate, is added to the mobile phase, the ILs differing only by their anions cannot be separated. ILs with different alkyl chains in the imidazolium cation are separated by mobile phases with or without added salts following a hydrophobic interaction behavior: log k is proportional to nC, the carbon number of the alkyl chain. Important differences in ion/stationary phase interactions are observed depending on the ionic content of the mobile phase. With salt-free mobile phases, the IL/C18 stationary phase interactions correspond to concave isotherms associated with fronting peaks for all ILs. With mobile phase containing 0.01 M of salt, tailing IL peaks correspond to convex adsorption isotherms. Also, the IL retention factor depends on the concentration and nature of the added salt. Hexafluorophosphate chaotropic anions can adsorb on the Kromasil C18 surface dramatically increasing the imidazolium cation retention factors.     

Direct liquid chromatographic enantioseparation of sotalol and other β-blockers using an α1-acid glycoprotein-based chiral stationary phase

The behaviour of an α₁-acid glycoprotein-based chiral stationary phase (Chiral AGP) towards changes in pH and organic modifier in the mobile phase was investigated in order to deduce suitable conditions for the liquid chromatographic enantioseparation of a series of β-adrenoreceptor blocking drugs. The effects of the pH of the mobile phase on retention, selectivity and resolution were studied. Methanol was the only non-ionic modifier tested in the mobile phase while different aliphatic carboxylic acids (C₄ to C₈) and alkanesulfonic acids (C₆ to C₈) were used as ionic modifiers. The influence of the nature and concentration of these modifiers on retention and enantioselectivity was investigated. Under these conditions, enantiomeric separations could be obtained for more than 70% of the β-blocking agents examined. The use of heptanoic acid as an ionic additive in the mobile phase has permitted the resolution of sotalol enantiomers. An enantioselective assay for sotalol was then developed and validated.     

Click regulation of cyclodextrin primary face for the preparation of novel chiral stationary phases

In this study, a series of novel CD chiral stationary phases were fabricated by immobilization of mono‐6^A‐deoxy‐N₃‐cyclodextrin onto silica surfaces followed by click regulation of CD primary face with 4‐pentynoic acid (acidic moiety), 2‐propynylamine (alkaline moiety) and L‐propargylglycine (chiral amino acid moiety), respectively. Enantioseparations of various kinds of racemates including dansyl‐amino acids, chiral lactides and diketones were conducted in reversed phase modes on these chiral stationary phases, where nearly forty diketones and chiral lactides were firstly separated on cyclodextrin stationary phases. 4‐Pentynoic acid moiety can make the retention ability decline while amine moiety significantly enhanced the retention ability of the stationary phases. For most of the studied analytes, the chiral amino acid moiety had the most positive effects on both the retention time and the resolution. The inclusion complexation between chiral analytes and cyclodextrins were also investigated by fluorescence method.     

Micelles for signal enhancement and novel selectivity of dansyl amino acids

Fluorescence intensity of various chemical species is enhanced in the microenvironment provided by micelles. Parameters which affect fluorescence intensities are examined by using dansyl (Dns) amino acids as the probe. The retention behavior of Dns-amino acids in micellar LC is examined by using ion-exchange-induced stationary phases. The type and concentration of micellar agent and modifier ion as well as concentration of acetonitrile in the mobile phase affect the retention and signal intensity of Dns-amino acids. The order of elution of Dns-amino acids obtained with the micellar mobile phase is very different from that observed in conventional reversed-phase LC. Fluorescence intensities of Dns-amino acids are enhanced by the micellar mobile phase.     

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.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.