P. Agrafiotou

Direct RP‐HPLC determination of underivatized amino acids with online dual UV absorbance, fluorescence, and multiple electrochemical detection

The combined use of a dual-UV detector, a fluorimetric one and of a multiple electrochemical (EC) detector equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 mum thick carbon microfibers, is proposed for the determination of 15 amino acids, two dipeptides and creatinine. This online coupling of the above detection modes could partially replace amino acid analysis by derivatization methods, since it solves problems concerning the direct detection of selected underivatized amino acids. Additionally, it was proved that the use of multiple-detection allows positive peak identification in a single chromatographic run, yields more information for free amino acids and solves in some cases the problem of chromatographic resolution. In order to optimize the detection conditions of the underivatized amino acids and related compounds by different detectors, their detection characteristics were determined by adequate preliminary experiments. The electro-oxidation characteristics of the underivatized compounds of interest were determined by hydrodynamic voltammetry using a flow cell with a macrodisc GCE and by ex-situ voltammetry using both a GCE of conventional size and a carbon fiber disk microelectrode. Important practical advantages of microfiber and microdisk electrodes with respect to macroelectrodes were demonstrated.

Multilinear gradient elution optimization in reversed-phase liquid chromatography based on logarithmic retention models: Application to separation of a set of purines, pyrimidines and nucleosides

The analytical solutions of the fundamental equation of the multilinear gradient elution are derived in two cases, when the dependence of the logarithm of the solute retention (lnk) upon the volume fraction of organic modifier (φ) is a three-parameter logarithmic expression, and when a simple linear relationship between lnk and lnφ is adopted. The derived theoretical expressions for retention times under multilinear gradient conditions are embodied to simple algorithms for fitting gradient data and especially for resolution optimization. Their performance was examined by using a mixture of 16 model compounds chosen among purines, pyrimidine and nucleosides in eluting systems modified by acetonitrile. It was found that the accuracy of the predicted gradient retention times is very satisfactory even if the simple logarithmic expression for the retention behavior of solutes, i.e. the linear dependence of lnk upon lnφ, is used.

Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids

The combined effect of the ion-pairing reagent concentration, Cipr, and organic modifier content, φ, on the retention under φ-gradient conditions at different constant Cipr was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D φ-gradient retention data obtained under the same φ-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant Cipr values in the eluent. The second approach is based on a retention model describing the combined effect of Cipr and φ on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different Cipr values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different Cipr values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by φ-variation.

Simple models for the effect of aliphatic alcohol additives on the retention in reversed-phase liquid chromatography

Four retention models for the effect of aliphatic alcohol additives on the retention of analytes in reversed-phase liquid chromatography have been developed following either a semi-thermodynamic treatment or an empirical approach. Their performance was tested using the experimental retention times of six non-polar analytes (alkylbenzenes) and ten o-phthalaldehyde derivatives of amino acids under different isocratic chromatographic runs when a small amount of ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol was added to methanol/water mixtures containing a constant amount of methanol. It was shown that for the structurally simple alkylbenzenes all the models can be adopted for retention prediction with good results. In contrast, just one out of four models, that with the fewest approximations, predicts satisfactorily the retention properties of amino acids derivatives. However, the most interesting feature is that this model can predict the effect of an alcohol-additive on the retention properties of solutes, even if this additive was not used in chromatographic runs done for the fitting procedure, provided that it belongs to the same homologous series of alkanols. This feature is also observed in all models described the retention of alkylbenzenes.

Retention prediction in reversed-phase liquid chromatography systems with methanol/water mobile phases containing different alkanols as additives

In an effort to gain enhancement of selectivity in reversed-phase liquid chromatography, retention was tuned in this study by introducing short and medium straight-chained-length alkanol additives (methanol (MeOH), ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol) at low concentrations in mobile phases containing MeOH as the main organic solvent. A six-parameter retention model considering simultaneously the contents of the main organic modifier and of the alcohol additive as well as of the number of alkyl chain of additive was developed by a direct combination of equations expressing separately a linear dependence of the retention upon each of these factors. The effectiveness of the above model was tested in the retention prediction of a mixture of six alkylbenzenes under isocratic conditions with mobile phases containing as an additive any member of the homologues series of alkanols (with 1-7 carbon atoms) at different low concentrations in a wide range of MeOH-water mixtures. The prediction was excellent in all cases even when the alkanol additives used in experiments for the fitting procedure are different than those used in chromatographic runs done for testing the prediction ability of the proposed model.

Modeling the effects of different mobile phase compositions and temperatures on the retention of various analytes in HPLC

A mathematical model is proposed for representing the combined effects of mobile phase solvent composition and temperature on the retention of various analytes in HPLC. The applicability of the model in describing the retention of four macrolides in aqueous mixtures of methanol and acetonitrile determined at 20-80 degrees C in various volume fractions of the organic modifiers was shown. The mean percentage deviation (MPD) was computed as an accuracy criterion in which the overall MPD of four analytes investigated in this work was 3.9+/-1.5% (N=72). The proposed model could be reduced to two simpler versions. The first version concerning the retention data of analytes in one organic modifier at various temperatures produced for the retention description of the above experimental system as well as for the retention of three benzodiazepines in aqueous mixtures of methanol at 25-40 degrees C an overall MPD of 3.6+/-1.8%. The more reduced version of the model for calculating the retention factor of one analyte in a given organic modifier at various temperatures produced an overall MPD of 1.7+/-1.1% for both the experimental systems studied. The accuracy of the proposed model is compared with recent models to predict the retention of an analyte with respect to solvent component of the mobile phase and the temperature of column in which the results were comparable. The main advantage of the proposed model is its capability to predict the retention of various analytes considering (i) temperature of the column, (ii) the mobile phase solvent composition, (iii) the chemical structure of the analytes and (iv) the nature of organic modifier.

Optimal Conditions for the Direct RP-HPLC Determination of Underivatized Amino Acids with Online Multiple Detection

The combined use of a dual-UV detector as well as a fluorimetric and a multielectrode electrochemical detector (equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 μm thick carbon microfibers) is proposed for the detection of the following 15 underivatized amino acids: L: -histidine (his), L: -cysteine (cys), creatine (crn), S-methyl-L: -cysteine (me-cys), DL: -homocysteine (hcy), L: -methionine (met), beta-(3,4-dihydroxyphenyl)-L: -alanine (dopa), L: -tyrosine (tyr), DL: -m-tyrosine (m-tyr), L: -a-methyl-dopa (me-dopa), L: -phenylanine (phe), DL: -alpha-methyltyrosine (me-tyr), 5-hydroxy-tryptophan (5htp), 3-nitro-L: -tyrosine (NO(2)Tyr) and L: -tryptophan (trp), as well as of two dipeptides: L: -cystathionine (cysta), L: -carnosine (car), and of creatinine (cre). A multilinear solvent (acetonitrile) gradient elution program, determined by a simple optimization algorithm, is required for the efficient reversed phase separation of the above mixture of 18 solutes within 27 min at a flow rate of 1.0 mL/min and at 25°C.

Separation optimization in reversed-phase liquid chromatography by using alkanol additives in the mobile phase: Application to amino acids

In an effort to enhance complex mixture separations by using small amounts of a homologous series of alkanols as additives in the mobile phases, it was proposed an optimization algorithm based on a sixth-parameter retention model. This model considers simultaneously the contents of the main organic modifier and of the alkanol additive in the mobile phase as well as of the number of alkyl chain of the additive. This model is in fact a modification of a previously one derived in a recently published paper for the retention description of a mixture of purely hydrophobic alkylbenzenes under isocratic conditions with mobile phases containing alkanol additives. The effectiveness of the new retention model as well as the optimization algorithm was successfully applied to the separation of ten o-phthalaldehyde (OPA) derivatives of amino acids. Indeed, the new retention model exhibited an excellent prediction performance since the obtained overall predictive error between calculated and experimental times was only 2.8% for all isocratic runs by using a variety of mobile phase compositions containing any alkanol homologue even different than those used in the starting/fitting experiments. Moreover, a perfect resolution of the above amino acid mixture was achieved within only 7.4 min in the chromatogram recorded using the optimal mobile phase determined by means of the simple optimization algorithm proposed in this study.