Retention In Reversed-phase Liquid Chromatography Research Articles

Temperature and solvation effects on homologous series selectivity in reversed phase liquid chromatography

Although the theoretical basis for temperature effects in reversed phase liquid chromatography (RPLC) has been well established, temperature is an under-utilized parameter for enhancing resolution in RPLC systems. However, the use of subambient operating temperatures can bring about significant increases in selectivity which are useful in separating solutes with subtle differences in their morphology. We have been examining the effects of temperature, particularly in the subambient range, and mobile phase solvation on RPLC solute selectivity for a homologous series of non-polar compounds on monomeric C 18 stationary phases. By using solute sets consisting of structural homologs, we have examined whether linear free energy relationships are operant, as well as measured enthalpic and entropic contributions to retention and selectivity over a wide temperature range. The solute homologous series sets include flexible chain solutes, such as the methylene series consisting of the C 1–C 6 n-alkylbenzenes rod-like solutes, such as the phenylene series consisting of benzene, biphenyl, p-terphenyl and p-quaterphenyl and plate-like solutes, such as the fused-ring series consisting of benzene, naphthalene, anthracene and 2,3-benzanthracene. This work is relevant in better understanding RPLC retention mechanisms as well as solute partitioning in other organized assemblies, and also is resulting in improved separation methods for many structurally similar compounds of environmental and pharmaceutical importance.

Structure-Activity Relationships of 2-Chloro-2′-arabino-fluoro-2′-deoxyadenosine and Related Analogues: Protein Binding, Lipophilicity, and Retention in Reversed-Phase LC

Abstract Plasma protein binding of 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine (CAFDA), 2-chloro-2′-deoxyadenosine (CdA), 2-fluoro-1-β-D-arabinofuranosyladenine (F-araA) and structurally related analogues 2-chloro-adenosine (2-Cl-Ado), 5′-chloro-5′-deoxyadenosine (5′-Cl-5′-dAdo), as well as parent nucleosides 2′-deoxyadenosine (dAdo), 1-β-D-arabinofuranosyladenine (araA) and adenosine (Ado) was determined and correlated with lipophilicity expressed as the logarithm of partition coefficient in n-octanol/water system (log Po/w). Drug binding to human serum albumin (HSA) was utilized since it is considered to exemplify nonspecific binding of small molecules to other macromolecules. Percentage of drugs bound to HSA increased from 3.5 % to 27 % following the order of increase in lipophilicity (log Po/w increased from -0.970 to 0.498). A similar correlation was observed when protein binding was correlated with retention in reversed-phase liquid chromatography (RP-LC) (capacity ratios, k', increased from 0.36 to 1.15), but the elution order of some compounds did not follow the parallel increase in either protein binding or lipophilicity. The introduction of a fluorine at the 2′-arabino position of CdA not only increased the acid stability of CAFDA, but also resulted in a higher binding to HSA (27.0% for CAFDA versus 24.3% for CdA) and much higher lipophilicity (log P of 0.498 for CAFDA compared to 0.025 for CdA).

Retention Behavior of Poly(L-Tryptophan)S and Poly(D,L-Tryptophan)S in Reversed-Phase Liquid Chromatography

Abstract The reversed-phase liquid chromatographic retention behavior of poly(l-tryptophan)s and poly(d,l-tryptophan)s whose molecular weight ranged from 5.4 kD to 37.25 kD is examined using a C-8 chemically bonded stationary phase and binary mobile phases of tetrahydrofuran (THF)-water and ternary mobile phases of THF-water-methanol. The retention of poly(l-tryptophan)s is compared with that of poly(d,l-tryptophan)s. Linear-Solvent-Strength (LSS) model is examined in describing the retention behavior. The effect of adding a third solvent to the binary mobile phase is also discussed.

Hydrophobicity parameters of 2-chloro-2′-deoxyadenosine and some related analogues and retention in reversed-phase liquid chromatography

Hydrophobicity parameters expressed as the logarithm of the partition coefficient (log P) and ionization constants (p K a) for 2-chloro-2′-deoxyadenosine and some related nucleoside analogues (2-fluoroarabinosyladenine, 2-chloroadenosine and 5′-chloro-5′-deoxyadenosine) are reported. The effects of methanol concentration and the pH of the mobile phase on the retention of the studied compounds in a reversed-phase system were examined. The relationship between hydrophobicity parameters and the retention in the HPLC system was investigated. A fairly good linear correlation was obtained when log k′ values ( r = 0.960) and/or log k′ w values (extrapolated from the relationship between log k′ and the percentage of methanol to 100% water) ( r = 0.959 and 0.967, respectively) were correlated with log P values.

Calculation of retention in reversed-phase liquid chromatography: IV. ChromDream software for the selection of initial conditions and for simulating chromatographic behaviour

A computer-aided method to assist the chromatographer in the determination of initial conditions in reversed-phase HPLC has been developed. ChromDream software predicts the optimum methanol, acetonitrile or tetrahydrofuran concentration in a mobile phase on the basis of the structural formulae of the compounds. The effects of the concentration of an organic solvent in water on retention and chromatograms of compound mixtures are simulated. Reversed-phase column parameters responsible for selectivity and retention are calculated. Volume and energy increments for more than 100 volume fragments and more than 30 bond dipoles have been derived. An approach for the selection of reference compounds for column calibration is proposed.

UNIFAC model as a heuristic guide for estimating retention in reversed-phase liquid chromatography

The utility of the UNIFAC activity coefficient determination method for understanding the magnitude of solute-solvent interactions in reversed-phase liquid chromatography (RPLC) is discussed. UNIFAC-computed partition coefficients for the transfer of various homologous series of solutes from aqueous mixtures of methanol, acetonitrile and tetrahydrofuran to hexadecane, octane, butane and benzene were used to investigate the effects of the carbon number, the type of functional group of the solutes, the composition of the mobile phases, the chain length of alkyl-bonded stationary phases and temperature on retention in reversed-phase liquid-liquid partition chromatography. Although UNIFAC is not accurate enough to be useful for the quantitative determination of retention in RPLC, it is useful in explaining a wide variety of issues of general importance in RPLC.

Revisionist look at solvophobic driving forces in reversed-phase liquid chromatography

Based on the use of alkylbenzenes as test solutes, most of the free energy of retention in reversed-phase liquid chromatography (RPLC) is shown to arise from net attractive (exoergic) processes in the stationary phase, and not from net repulsive (endoergic) processes in the mobile phase. The classical view of the “passive” role of bonded phase ligands is challenged. However, it is also shown that variations in retention upon changing the mobile phase are dominated by alterations in the net processes in the mobile phase. Furthermore, it is shown that the free energy of transfer of a methylene group from the mobile phase to a bonded reversed phase over a wide range in mobile phase composition, is similar but not equal, to the free energy of transfer of a methylene group from the same mobile phase to pure bulk hexadecane. This observation is in accord with the partition model view of the mechanism of RPLC. Finally, by comparison of measured and computed activity coefficients, the regular solution theory is shown to be a grossly inadequate model of interactions in water and hydro—organic mixtures. It should not be used to model retention in aqueous mobile phases.

Modification of reversed-phase liquid chromatographic retention characteristics for metal—tetraphenylporphyrin complexes using octane as a mobile phase additive

The effect of the addition of octane to a methanolic mobile phase on the reversed-phase liquid chromatographic separation of a series of porphyrin compounds is described. The capacity factors of tetraphenylporphyrin (TPP) and five metal—TPP complexes decreased with increase in the octane content of the mobile phase. Selective sorption of octane on the surface of ODS occurred. The addition of octane to the mobile phase caused an alteration in the sequence of elution, improved the resolution of some TPP complexes and reduced the time required for analysis.

Relationships between the reversed-phase liquid chromatographic retention characteristics and physico-chemical parameters of some β-casomorphin peptides

Log k′ w and slope values for ten β-casomorphin peptides were calculated in ten different reversed-phase liquid chromatographic systems using methanol as organic modifier. The differences between the retention behaviours of C 8, C 18 and C 30 columns were fairly small the eluent composition had a greater impact than the length of the hydrophotic ligand on the retention of peptides. A quaternary amino column showed considerably different retention characteristics. The results suggest that each substructure in the peptides makes a similar contribution to the overall retention of the peptide. The retention parameters of peptides mainly correlated with their electronic and steric characteristics. This finding emphasizes the importance of the electrostatic interactions between the free silanol groups on the surface of the reversed-phase support and the polar substructures of peptides.

Hydrophobic substituent constants derived by intracolumn bilogarithmic analysis of reversed-phase liquid chromatographic retention of 4,4'-disubstituted benzanilides and benzamides

The reversed-phase liquid chromatographic (RPLC) retention of substituted benzanllides and benzamides was studied to determine hydrophobic substituent contributions to retention. Multiple linear regression analysis was used to evaluate functional group retention increments, τ, of aromatic substituents (CI, CH 3 , OCH 3 , and NO 2 ) on octadecyl-bonded sillica stationary phases as the molecular environment of the substituent and organic modifier conentration of the mobile phase were varied. The data illustrate the sensitivity of RPLC toward descrimination among subtile variations in molecular environment.

Temperature dependence of retention in reversed-phase liquid chromatography. 1. Stationary-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been investigated by examining the temperature dependence of retention, with emphasis on the role of the stationary phase in the retention process. Both chromatographic temperature studies and differential scanning calorimetry were used to examine the role of alkyl chain bonding density on the retention mechanism in RPLC. Phase transitions of reversed-phase stationary phases were observed at bonding densities greater than 2.84 mumol/m2. Thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase (delta H degrees and delta S degrees) were calculated for low bonding density columns, and comparison of these values to previously reported values for the partitioning of a nonpolar solute from the bulk organic liquid to water indicated that the chromatographic retention process is not well-modeled by bulk-phase oil-water partitioning processes. In addition, this data showed that the entropic contribution to retention becomes more significant with respect to the enthalpic contribution as the stationary-phase bonding density is increased, providing additional support that partitioning, rather than adsorption, is the relevant model of retention.

Temperature dependence of retention in reversed-phase liquid chromatography. 2. Mobile-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been examined over a wide temperature range with emphasis on the role of the mobile phase. van't Hoff plot shapes were used to assess the retention mechanism, and the data showed evidence of the hydrophobic effect when water-rich and/or hydrogen-bonded mobile phases such as methanol/water were used. However, different van't Hoff plot shape was observed with acetonitrile/water mobile phases, indicating a change in the retention mechanism. These data showed that the hydrophobic effect, which had previously been proposed as the driving force for retention, is not a satisfactory explanation for the retention process in all RPLC systems.

Control of the retention selectivity of rare earth octaethylporphyrins in reversed-phase high-performance liquid chromatography using amines as mobile phase additives

The reversed-phase liquid chromatographic retention behaviour of the complexes of octaethylporphyrin (OEP) with eleven trivalent rare earth (REs), viz., Y, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu, on an octadecyl-bonded silica gel column is described. All these complexes can be eluted without demetallation with a methanol-water mixture that contains a small amount (about 1%) of acetylacetone and an amine. The retention order of the RE-OEP complexes depends on the amine used. With a mono- n-alkylamine and a di- n-alkylamine, the retention of the RE-OEP complex decreases with increasing atomic number of the REs within the lanthanide series, whereas the reverse tendency occurs with a trialkylamine and a dialkylamine possessing branched alkyl side-chains or alcoholic side-chains. In all instances, the retention of the Y complex is close to that of the Dy complex. Successful separation of the OEP complexes of Lu, Yb, Tm, Er and Sm in 20 min is demonstrated with dihexylamine.

Microscopic order as a function of surface coverage in alkyl-modified silicas: spin probe studies

The chemically modified surface plays an important role in retention in reversed-phase liquid chromatography. One surface factor that might affect retention is solute expulsion due to partial ordering of the grafted chains at sufficiently high grafting densities The study has shown by the use of the eletron paramagnetic resonance technique of spin probing that spectroscopic evidence existe for an increaee in molecular order of the bonded octadecyl phase as a function of grafting density

Solvatochromic hydrogen bond donor acidity of cyclodextrins and reversed-phase liquid chromatographic retention of small molecules on a β-cyclodextrin-bonded silica stationary phase

Values of the Kamlet-Taft hydrogen bonding (HB) donor acidity (α) and dipolarity/polarizability (π *) parameters for α-, β- and λ-cyclodextrins (CDs) are reported. They possess HB donor acidities in the range 0.21-0.12 and dipolarity/polarizability parameters of about 0.43. Both values are smaller than those for the aliphatic alcohol analogues. The reversed-phase liquid chromatographic retention behaviour of some small molecules on the β-CD bonded silica was compared with that on an octadecylsilylsilica based on the linear solvation energy relationship. It was found that he factors affecting retention on the two stationary phases are very different in that on the ODS column cavity formation and type A HB interactions determine retention whereas on the β-CD column dipolar and type A HB interaction determine retention. Differences in the retention properties of β-CD and ODS phases are rationalized in terms of types of solute-stationary phase interactions involved in the retention process.

Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition

Models describing the concomitant effects of pH and organic modifier concentration on retention in reversed-phase liquid chromatography are established. Two different octadecyl-modified silica columns were used. The retention behaviour of several acidic, basic and neutral solutes were studied, using methanol as the organic modifier. The suggested models accurately describe retention as a function of pH and composition. A unified formalism for retention modelling that is applicable to all ionogenic solutes is also established. This formalism is tested with the modelling of retention for several weak bases. The resulting (general) model describes retention accurately and is applicable to all solutes studied.

Selectivity studies on branched and linear alkyl bonded stationary phases in reversed-phase liquid chromatography

Reversed-phase liquid chromatographic retention characteristics for the sixteen acyclic C1−C5 N-alkylbenzamide congeners were measured on various branched and linear, alkyl bonded hydrocarbon stationary phases. Retention factors, k′, were determined in acetonitrile-water mobile phase compositions on ethyl, n-octyl, n-dodecyl, n-octadecyl, 1-ethyladamantyl, 4-butyloctyl, and 2,4,4-trimethylpentyl stationary phases. Statistical analysis of the two main effects investigated — type of stationary phase and percentage of organic modifier (acetonitrile) in the mobile phase — described greater than ninety percent of the variability in the data for most of the comparisons. Selectivity effects due to variation in the mobile phase dominated the results.

Method for characterization of selectivity in reversed-phase liquid chromatography : V. Calibration of the retention scale for chromatographic systems with low concentrations of organic solvents in the mobile phase

The method for the characterization and prediction of selectivity and absolute retention in reversedphase liquid chromatography based on two indices (a lipophilic and a polar one) has been previously applied to chromatographic systems with mobile phases containing 50% and more of one or two organic solvent(s). Because of a high retention, the homologues series of n-alkylbenzenes is not suitable for the direct calibration of the retention scale in mobile phases with lower concentrations of organic solvents, and the extrapolation of the calibration parameters based on the data for this series in mobile phases with low water content yields predicted retention data that are subject to significant errors. To circumvent this inconvenience and to expand the application possibilities of the above method to mobile phases with higher concentrations of water, three homologous series were tested as potential candidates for calibration standards: 3- n-alkyl-6-methyluracils, 3-alkoxycarbonyl-2-pyrazolines and alkan-2-ones. All three homologous series tested were found to be suitable substitutes for n-alkylbenzenes as calibration standards in mobile phases containing 25–50% methanol in water. The selection of suitable calibration standards for acetonitrile—water mobile phases is more difficult because of a curvature of the dependence of log k′ on the concentration of acetonitrile at low concentrations of this solvent in aqueous mobile phases. Here, the alkoxycarbonyl pyrazolines and alkan-2-ones proved useful as calibration standards in mobile phases containing 30–50% acetonitrile. The agreement between the predicted and experimental k′ was within 10% or better of the experimental k′.

Correlation analysis in liquid chromatography of metal chelates : II. One-dimensional retention-mobile phase composition (physicochemical property) models in reversed-phase liquid chromatography

The applicability of linear correlation models for describing the effect of mobile phase composition and properties on the retention of metal chelates in reversed-phase liquid chromatography is demonstrated. The macroscopic (dielectric constant, surface tension, viscosity, concentration of organic modifier, distribution constant in an n-octanol-water system, etc.) and microscopic (characteristics of donor and acceptor ability) parameters of binary water-organic mixtures are used as variables. Although each of the studied parameters alone is somewhat limited in application, together they allow retention values to be estimated and the chromatographic behaviour of metal chelates to be predicted. The dominant influence of mobile phase composition and proton-donating ability on chelate retention in reversed-phase liquid chromatography is confirmed.

Reversed-phase liquid chromatographic retention of geometrical isomers of tris(β-diketonato)—chromium(III) and tris(β-diketonato)—cobalt(III) : Comparison with liquid—liquid partition in a dodecane—(methanol—water) system

The retention of the fac and mer isomers of chromium(III) and cobalt(III) complexes with eight asymmetrical β-diketones in the octadecyl-bonded silica (ODS)—(methanol—water) system was studied. The retention order for two geometrical isomers depends on the structure of the complexing β-diketone; the retention order, fac< mer, is found for complexes of the β-diketones which possess a fluorinated functional group in each molecule, whereas the reverse order is found for complexes of β-diketones without a fluorinated moiety. The capacity factors ( k′) of the chromium(III) complex isomers were compared with their partition coefficients ( P) between dodecane and methanol—water of an identical composition to the mobile phase used in the chromatographic separation. It was found that the transfer of a complex from a methanol—water phase occurs more easily to ODS than to dodecane. The retention sequence for the fac and mer isomers of each metal complex is the same as the increasing order of P values. The difference in k′ between the mer and fac isomers is smaller than the difference in the P value.