Influence of mobile phase composition on surface diffusion in reversed-phase liquid chromatography

Influence of column temperature and mobile phase composition (phi) on surface diffusion was studied in reversed-phase liquid chromatography using a C(18)-silica gel column and methanol-water mixtures. The temperature dependence of surface diffusion coefficient (D(s)) was explained by considering that of the molecular diffusivity, suggesting the presence of a kind of correlation between surface and molecular diffusions. The influence of phi on D(s) was accounted for by considering the restriction energy (E(r)) for surface diffusion. Physical meanings of the linear correlation between E(r) and the enthalpy change due to sample retention are discussed in connection with the mechanism of surface diffusion.     

Effect of the mobile phase composition on the adsorption behavior of tryptophan in reversed-phase liquid chromatography

Single-component adsorption isotherm data of l-tryptophan on a C18-bonded silica column were acquired by frontal analysis (FA), with aqueous mobile phases containing 2.5, 5, and 7.5% of acetonitrile (ACN) or 7, 10, 15, and 20% of methanol (MeOH). Most of these isotherms have two inflection points and three different parts. The low and the high concentration parts exhibit langmuirian behavior. The intermediate part exhibits anti-langmuirian behavior. The inflection points shift toward higher concentrations with increasing mobile phase concentration in ACN or MeOH, which causes the differences in the isotherm profiles. The nature of the organic modifier and its concentration affect only the isotherm profile and the numerical values of its parameters, not the nature of the best model, which is the bi-Moreau model in all cases. The isotherm profiles depend on the experimental conditions because they affect the intensity of the adsorbate-adsorbate interactions. Overloaded band profiles of tryptophan were recorded with the seven mobile phase compositions. They were used to determine the best values of the isotherm coefficients by the inverse method (IM) of chromatography. There is an excellent agreement between the values of these parameters obtained by FA and by IM. Increasing the concentration of either ACN or MeOH in the mobile phase causes a slight decrease in the saturation capacities of the low and the high energy sites, and in the adsorption constant of the low energy sites. The adsorption constant of the high energy sites increases with increasing concentration of either solvent or is little affected. The adsorbate-adsorbate interaction constants of both low and high energy sites increase for both solvents. Saturation capacities of the high energy sites are higher for ACN than for MeOH.     

Mobile phase effects in reversed-phase chromatography VI. Thermodynamic models for retention and its dependence on mobile phase composition and temperature

Summary The physico-chemical framework is examined by comparing the predictions of three models for the combined effects of the composition of the hydroorganic mobile phase and the column temperature on the retention ofn-alkylbenzenes on hydrocarbonaceous bonded stationary phases. The well-mixed model leads to expressions for the dependence of retention on three factors which are equivalent to those derived previously from linear extrathermodynamic relationships. The diachoric model stems from the assumption that the mobile phase is microscopically heterogeneous and the displacement model is identical to the retention model most widely used in chromatography with polar sorbents and less polar solvents. Over limited ranges of mobile phase composition and temperature, each model does describe retention behavior. However, only the wellmixed model describes retention well over the entire range of mobile phase composition and temperature studied here. The success of the well-mixed model, and its limits, give insight into the role of the organic solvent in determining the magnitude of chromatographic retention on non-polar stationary phases with hydro-organic eluents.Dedicated to Professor S. R. Lipsky on the occasion of his 60th birthday.     

Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropic salts

Sodium hexafluorophosphate, perchlorate and trifluoroacetate were applied as ion-ion interaction reagents in reversed-phase liquid chromatography. The separation of chosen alkaloids was performed by changing the kind of the organic modifier (methanol, acetonitrile, tetrahydrofuran), concentration of the ion-ion-interaction reagents and the concentration of phosphate buffer at constant pH (2.7) in the mobile phase. Obtained results were analyzed in connection to a dynamic ion-exchange model of retention and ion-ion interaction effects. The perturbation method was applied to test proposed retention theories. The formation of ion-complexes controlling the retention in chaotropic systems was confirmed. On the basis of the relationships of capacity factors (k) versus salt concentrations derived experimentally, absolute increases in capacity factors, the desolvation parameters and the limiting retention factors were calculated and compared for all the investigated compounds in eluent systems studied. The selectivity of the proposed mobile phases was compared on the basis of the separation of alkaloid mixture.     

Influence of stationary phase solvation on shape selectivity and retention in reversed-phase liquid chromatography

In the past few decades, shape selectivity has drawn a great deal of attention from chromatographers. The chemistry and characteristics of bonded stationary phases such as phase type, length of bonded phase, surface coverage, and silica surface material have an effect on the shape selectivity of the columns. Although the effects of bonded phase shape selectivity are relatively well understood, one remaining question is the effect of intercalated solvent on shape selectivity. The intercalation of organic modifier and water molecules into the stationary phase is believed to introduce more rigidity into bonded alkyl chains in RPLC. The use of gas chromatography (GC) opens a new dimension to approach this question. C18 columns 4 cm in length were prepared in our laboratory and used in both LC and GC experiments. Shape selectivity and thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase have been determined as a function of monomeric octadecyl stationary phase bonding densities over the range of 1.44-3.43 micromol/m2 and a polymeric phase (nominal surface coverage 4.77 micromol/m2). Comparing LC and GC experiments, we observed: (a) similar relationships between shape and phenyl selectivities with monomerically bonded C18 phase densities; (b) different correlation of thermodynamic quantities (DeltaH degrees , DeltaS degrees , and DeltaG degrees ) versus bonded phase densities. The effects of high temperature and residual silanol groups are sources of difficulty in elucidation of the intercalated mobile phase role in selectivity and retention for GC measurements.     

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

Predicting retention in reverse‐phase liquid chromatography at different mobile phase compositions and temperatures by using the solvation parameter model

The prediction capability of the solvation parameter model in reverse‐phase liquid chromatography at different methanol‐water mobile phase compositions and temperatures was investigated. By using a carefully selected set of solutes, the training set, linear relationships were established through regression equations between the logarithm of the solute retention factor, logk, and different solute parameters. The coefficients obtained in the regressions were used to create a general retention model able to predict retention in an octadecylsilica stationary phase at any temperature and methanol‐water composition. The validity of the model was evaluated by using a different set (the test set) of 30 solutes of very diverse chemical nature. Predictions of logk values were obtained at two different combinations of temperature and mobile phase composition by using two different procedures: (i) by calculating the coefficients through a mathematical linear relationship in which the mobile phase composition and temperature are involved; (ii) by using a general equation, obtained by considering the previous results, in which only the experimental values of temperature and mobile phase composition are required. Predicted logk values were critically compared with the experimental values. Excellent results were obtained considering the diversity of the test set.     

Effect of the mobile phase composition on the isotherm parameters and the high concentration band profiles in reversed-phase liquid chromatography

Single-component adsorption isotherm data were acquired by frontal analysis for phenol on a C18-Kromasil packed column, under reversed-phase liquid chromatography conditions, using various methanol-water solutions (30-60%, v/v, methanol). The isotherm model accounting best for these data was the biLangmuir model. With increasing methanol content, the two saturation capacities decrease, particularly that of the high-energy sites, the adsorption constant of the low-energy sites decreases significantly and that of the high-energy sites decreases strongly. These results allow a quantitative investigation of the properties of the high-energy sites (which are not necessarily the so-called active sites), a feature rarely discussed yet. The band profiles calculated with the numerical values of the isotherm model parameters derived by fitting the frontal analysis data to the model and using the equilibrium-dispersive model agree very well with the experimental band profiles in the whole concentration range.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Convenient estimation of the mobile phase volume for water-rich eluents in reversed-phase liquid chromatography

Summary Oxalodihydroxamic acid is proposed as a UV-detectable substitute for D₂O for the convenient estimation of the mobile phase volume (V_m) of water-rich hydro-organic eluents in reversed-phase liquid chromatography. The retention volume of oxalodihydroxamic acid deviates less than 6% from that of D₂O in 0 to 50% methanol-and acetonitrile-water eluents on C₁, C₈, and C₁₈ reversed-phase supports; it is independent of the amount injected from 10ng to 20μg when monitored at 254nm, although the sensitivity is about four-fold greater at 219 nm. The pH should be maintained between about 2 and 4. The deviation from the D₂O retention volume on supports of typical porosity is attributed, at least in part, to a size-exclusion effect. Simple hydroxamic acids can be used as a homologous series for the estimation of V_m over most of the methanol- and acetonitrile-water concentration ranges by the linearization of retentions of homologous series method. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Dimethylformamide as a mobile phase component in reversed-phase liquid column chromatography on octadecylsilica

Summary N,N-Dimethylformamide selectively accelerates the elution of weakly basic compounds and reduces their tailing in reversed-phase liquid chromatography with aqueous organic mobile phases. This effect is demonstrated with two types of octadecylsilica packings for representative solutes, covering aromatic amines and N-heterocyclic compounds such as pyridine, pyrimidine and purine derivatives.     

Retention prediction in ternary solvent reversed-phase liquid chromatography systems based on the variation of retention with binary mobile phase composition

An extension of the treatment adopted in a recent paper [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33] was used to derive expressions describing the variation of solute retention k with composition in ternary reversed phase liquid chromatography, RP-LC, solvent systems. The equation of the partition model obtained in this way for a ternary mobile phase was identical to that previously derived using the solubility parameter concept. This equation as well as two new expressions of In k versus organic modifiers content were tested in a variety of ternary solvent systems in order to examine the possibility of predicting retention behavior of solutes under ternary solvent mixture elution conditions from known retention characteristics in binary mobile phases. It was demonstrated the superiority of both new equations derived in this paper to that previously proposed and applied to date in ternary solvent mixtures.     

Modeling the effect of solvation on solute retention in reversed-phase liquid chromatography

The retention of a homologous series of alkylbenzenes was determined on octyl and octadecyl reversed-phase columns in several polar organic liquids. Free energies of transfer were calculated by the SM5.0R classical solvation model for each organic liquid tested and for several alkanes. The relationships between the measured retention factors and the calculated free energies of transfer were then investigated. Although the natural logarithms of the retention factor and the calculated free energies of transfer were linearly correlated, the obtained free energies of transfer of the solutes did not completely explain the retention behavior of the solutes. Nonetheless, even in these pure organic liquids, the energetics of RPLC retention behaved very similarly to those of partitioning.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for anionic analytes and mobile phase components on polystyrene polymers using an alkaline mobile phase

Summary Retention models for anionic analytes and mobile phase compounds in reversed-phase ion-pair chromatographic systems have been studied. The solid phase was a polystyrene-divinylbenzene copolymer. The analytes were mono- and divalent anions monitored by indirect detection technique. The mobile phase was highly alkaline and contained a detectable anion, sulfanilic acid.Expressions for the ion-pair retention of solutes and mobile phase components have been developed assuming Langmuir distribution of ion-pairs to a solid phase with one kind of binding site. The validity of the expressions and the origin of system peaks have been confirmed by determination of distribution constants using linear regression.     

Methylammonium formate as a mobile phase modifier for totally aqueous reversed-phase liquid chromatography

Although alkylammonium ionic liquids (ILs) such as ethylammonium nitrate and ethylammonium formate have been used as mobile phase "solvents" for liquid chromatography (LC), we have shown that the IL methylammonium formate (MAF), in part because of its lower viscosity as compared to other ILs, can be an effective replacement for methanol (MeOH) in reversed-phase LC. Plots of log retention factor versus the fraction of MeOH and MAF in the mobile phase indicate quite comparable solvent strength slope values of 2.50 and 2.05, respectively. Using a polar endcapped C18 column, furazolidone and nitrofurantoin using 20% MAF-80% water could be separated in 22 min but no baseline separation is possible using MeOH as the modifier, even down to 10%. Suppression of silanol peak broadening effects by MAF is important, permitting a baseline separation of pyridoxine, thiamine, and nicotinamide using 5% MAF-95% water at 0.7 mL/min. Using 5% MeOH-95% water, severe peak broadening for thiamine is evident. The compatibility of MAF as a mobile phase modifer at the 5% level for LC with mass spectrometry detection of water-soluble vitamins is also shown.     

Comparative study of some commercial chemically bonded phases in classical reversed-phase chromatography and in ion-pair reversed-phase liquid chromatography

Summary The behaviour of various octadecyl commercial bonded phases are compared in classical reversed-phase chromatography and in ion-pair reversed-phase chromatography. Great differences are exhibited by the packings studied according to the polarity of the solutes. Whereas hydrocarbonaceous bonded phases show very similar selectivity versus apolar or weakly polar solutes, great differences are observed when analyzing more polar solutes even when ion-pair reversed-phase chromatography is performed.Presented at the 14th International Symposium on Chromatography London, September, 1982     

A new method for the determination of mobile phase volume in normal and reversed-phase liquid chromatography

Summary A new method for the determination of the mobile phase volume (V _m) in liquid chromatography is presented based on the model regarding the retention of ionic solutes in the presence of eluent electrolytes. TheV _m value can be determined by measuring the retention volumes of two ions that have the same charge in two eluent electrolyte systems. Compared with the methods using isotopically labelled eluent components or inorganic salts asV _m markers, the method presented is proved to give more reasonableV _m values for both normal and reversed-phase liquid chromatography. As well as in binary mixed solvent systems, theV _m values in single solvent systems can be determined by this method.     

Comparison of different retention models in normal- and reversed-phase liquid chromatography with binary mobile phases

The dependence on mobile phase composition of the retention of selected test analytes in different normal- and reversed-phase chromatographic systems is studied. The aim of this study is to compare the performance of six valuable retention models reported in the literature with a new empirical equation, which is first introduced in this study. All of these models are compared for different thin-layer chromatographic and high-performance liquid chromatographic systems by use of three criteria: the sum of the squared differences between the experimental and theoretical data, approximation of the standard deviation, and the Fisher test.