Chromatographic behaviour in reversed-phase high-performance liquid chromatography with micellar and submicellar mobile phases: effects of the organic modifier

Continuing our earlier study of the retention behaviour in reversed-phase systems with aqueous mobile phases containing surfactants in concentrations lower (submicellar systems) and higher (micellar systems) than the critical micellar concentration (CMC), we investigated the chromatographic behaviour of various non-ionic solutes in mixed aqueous-organic micellar and submicellar mobile phases and their dependence on the methanol concentration. CMC values were measured for two cationic surfactant and one anionic surfactant in mixed aqueous-methanolic solvents, and were found to increase slightly with increasing methanol concentration. Depending on the character of the surfactant, a limiting concentration of methanol was found, above which micelles do not occur anymore. Sorption isotherms of the surfactants on an octylsilica gel column were measured as a function of the concentration of methanol in aqueous-methanolic solvents. A modified Langmuir equation was used to describe the distribution of the surfactants between the stationary and the mobile phases in the concentration range below CMC. The retention of several polar solutes was measured on an octylsilica gel column both in micellar and submicellar mobile phases containing methanol. The dependencies of the capacity factors of the solutes studied on the concentration of methanol in the mobile phase can be suitably described by the same form of equation as that conventionally used for aqueous-organic mobile phases that do not contain surfactants, but the slopes of the dependencies for a given solute are different in the two ranges of surfactant concentrations. The ratio of the two slopes is controlled by the interaction with micelles and is approximately equal to, below or above 1, depending on whether the solutes do or do not associate with the micelles, or are repulsed from them. Simultaneous control of the concentrations of the organic solvent and of the surfactant in the mobile phase can be used for fine tuning the selectivity of separation as a complement to commonly used adjusting concentrations of two organic solvents in ternary aqueous-organic mobile phases. These effects are illustrated by practical examples of submicellar HPLC with mobile phases containing methanol.     

Retention model for the resolved enantiomers of felodipine on chiral-AGP using micellar mobile phases

A retention model for the chiral separation of an uncharged solute, felodipine, on CHIRAL-AGP, using a micellar mobile phase is proposed. The model assumes the presence of two stereoselective sites and each enantiomer was found to interact with different sites. Addition of a chiral aliphatic alcohol, (+)-(S)-2-octanol, preferentially interacted with the binding site for (?)-(S)-felodipine. The monomeric form of the micellar agent (Tween® 20) competed with the enantiomers for the adsorption sites, and the formation of a 1:1 complex between the enantiomers and the micelles was assumed. The retention of the solutes was effectively controlled by adding small quantities (<1.63 × 10^?3 M) of the nonionic detergent Tween 20 to the mobile phase. Baseline separation was achieved by addition of 1.0 mM n-octylamine to the mobile phase; 8.14 × 10^?4 M Tween 20 in phosphate buffer pH 7.0. The separation factor (α = 1.74) was unaffected by the detergent concentration in the presence of 1.0 mM n-octylamine. © 1995 Wiley-Liss, Inc.     

Effect of temperature on the reversal in the retention order of the enantiomers of mosapride on Chiral-AGP

The retention order of the enantiomers of mosapride could be controlled by column temperature and mobile phase pH. In the presented paper, temperature studies have been used to study the thermodynamics of the reversal in retention order. A linear relationship was obtained plotting the logarithm of the capacity factor versus the inverted column temperature. However, at higher mobile phase pHs, the logarithm of the separation factor versus the inverted column temperature showed a non-linear behaviour and at the highest mobile phase pH used (pH=7.4), an optimum in the separation factor was observed. The plots showed that the thermodynamics for the two enantiomers of mosapride differ in the studied mobile phase pH interval. Thermodynamic values, enthalpy and entropy were calculated and showed that at a low mobile phase pH, the enantiomeric resolution was caused by differences in enthalpy between the two enantiomers. However, at a higher mobile phase pH, the chiral discrimination was a result of entropy effects. High correlation was obtained between experimental and predicted separation factors at different mobile phase pHs.     

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

It is well established that salt enhances the interaction between solutes (e.g., proteins, displacers) and the weak hydrophobic ligands in hydrophobic interaction chromatography (HIC) and that various salts (e.g., kosmotropes, chaotropes, and neutral) have different effects on protein retention. In this article, the solute affinity in kosmotropic, chaotropic, and neutral mobile phases are compared and the selectivity of solutes in the presence of these salts is examined. Since solute binding in HIC systems is driven by the release of water molecules, the total number of released water molecules in the presence of various types of salts was calculated using the preferential interaction theory. Chromatographic retention times and selectivity reversals of both proteins and displacers were found to be consistent with the total number of released water molecules. Finally, the solute surface hydrophobicity was also found to have a significant effect on its retention in HIC systems.     

Optical resolution of glycerin sulfides and glycerin selenides on the chiral stationary phase of cellulose‐tris(3,5‐dimethylphenylcarbamate)

Cellulose‐tris(3,5‐dimethylphenylcarbamate) was prepared after a reported method and was coated onto an aminopropylated mesopore spherical silica gel. The final product was used as a chiral stationary phase of high performance liquid chromatography for the enantioseparation of a series of glycerin sulfides and glycerin selenides. Mixtures of hexane and 2‐propanol were used as mobile phases. The effects of 2‐propanol concentration in the mobile phase on the retention and resolution were investigated. Some enantiomers of the glycerin monosulfides and monoselenides could be separated satisfactorily, but none of the disulfides could be separated. The structural features of the solutes that influence chiral separation were discussed. Chirality 11:598–601, 1999. © 1999 Wiley‐Liss, Inc.     

Separation of icosenoic acids,monohydroxyicosenoic acids,and prostaglandins by high-pressure liquid chromatography on a silver ion-loaded cation-exchange column

Prostaglandins and monohydroxy fatty acids derived from 8,11,14-icosatrienoic acid and arachidonic acid have been separated by high-pressure liquid chromatography using a cation-exchange column loaded with silver ions. The retention times in a variety of solvent systems have been determined for prostaglandin E₁(PGE₁), PGF_1α, PGD₂, PGE₂, PGF_2α, 6-oxoPGF_1α, 15-hydroxy-8,11,13-icosatrienoic acid, 5-hydroxy-6,8,11,14-icosatetraenoic acid, 8-hydroxy-5,9,11,14,-icosatetraenoic acid, 9-hydroxy-5,7,11,14-icosatetraenoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid, 12-hydroxy-5,8,10,14-icosatetraenoic acid, 15-hydroxy-5,8,11,13-icosatetraenoic acid, 8,11,14,-icosatrienoic acid, and arachidonic acid. The mechanisms involved in the interaction of solutes with the stationary phase have been investigated. Retention times on silver ion columns appear to be determined by a combination of interactions between (a) the silver ions of the stationary phase and double bonds of the solute and (b) polar groups of the stationary phase and polar groups of the solute. The relative contributions of these two types of interactions to the retention of solutes can be varied over a wide range by altering the composition of the solvent. In this way the selectivity of the stationary phase can be controlled in order to optimize the separation of any given group of solutes. The maximum separation of solutes on the basis of the number of double bonds they possess is obtained by using polar solvents containing low concentrations of acetonitrile. As the polarity of the mobile phase is reduced or the concentration of acetonitrile increased, the selectivity of the stationary phase tends to resemble that of normal-phase chromatography on silicic acid.     

Binding of substituted phenol and aniline derivatives to the corn protein zein studied by high-performance liquid chromatography

The interaction of 12 substituted phenol, three aminophenol and four substituted aniline derivatives with the corn protein zein was studied on zein-coated silica and alumina stationary phases by high-performance liquid chromatography using bidistilled water as mobile phase. Solutes were eluted from the zein-coated supports with different retention times indicating that they bind to the protein with different forces. They were more strongly retained on silica-based than on alumina-based support proving that the original adsorptive character of the support remains even after impregnation. The retention of solutes on both zein-coated stationary phases significantly depended on the steric and electronic parameters of solutes and was independent of the calculated and measured lipophilicity parameters, indicating that hydrophobic forces are not included in the interaction of zein with these class of solutes. It has been concluded that the interaction is governed by steric and electrostatic forces.     

Mobile phase effects on enantiomer resolution using molecularly imprinted polymers

The aim of this study was to rationalise retention behaviour of a chiral solute on molecularly imprinted polymer (MIP) HPLC stationary phases in terms of variation of the mobile phase. It is generally held that the most important interaction governing the separation of enantiomers on such materials is H-bonding, and that retention times increase with decreasing H-bonding potential of the mobile phase. Previous studies have largely concerned mobile phases containing chloroform with acetic acid as a polar modifier. Boc-L-Phenylalanine (Boc-L-Phe-OH) MIPs were prepared, processed, and packed into HPLC columns, which were then used to investigate the retention characteristics of Boc-L-Phe-OH and Boc-D-Phe-OH with a range of mobile phases. The main observations were as follows: (1) in chloroform-based mobile phases there was generally a linear relationship between the H-bond donator factor of the polar modifier and capacity (K′). Results also indicated a hydrogen bond donor parameter value for a polar modifier at which retention became concentration independent; (2) For given values of K′_L, K′_D varied depending on the polar modifier, indicating that enantiomer resolution was solvent dependent; (3) Using mobile phases based on solvents of lower polarity/H-bonding potential than chloroform, substantial increases in K′ were observed, although enantioselectivity was greatly reduced. Chirality 9:238–242, 1997. © 1997 Wiley-Liss, Inc.     

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewiński competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.     

Monitoring the reactions of cisplatin with nucleotides and methionine by reversed-phase high-performance liquid chromatography using cationic and anionic pairing ions

Methodology, based on reversed-phase high-performance liquid chromatography, is described for monitoring the reactions of cisplatin with DNA, nucleotides, and methionine. Cisplatin was determined in DNA ultrafiltrates on solvent-generated anion exchangers which were prepared by coating the surface of a reversed-phase column with hexadecyltrimethylammonium bromide. These systems were also applicable to studies on the reactions of cisplatin with nucleotides. The retention of the nucleotides studied (5'-AMP, 5'-GMP, 5'-CMP, and 5'-TMP) was described by means of an ion-exchange model and was manipulated by controlling the phosphate concentration in the mobile phase and its pH. The results indicate that cisplatin interacts predominantly with adenosine and guanosine groups on the DNA molecule and that binding is limited by the rate of conversion to an aquated intermediate. Whereas reversed-phase HPLC systems employing cationic pairing ions were applicable to the analysis of mixtures containing cisplatin and anionic solutes, systems employing alkyl sulfonates were required to monitor the reaction of cisplatin with methionine which produces cationic products. Retention, in this latter system, was optimized by the addition of acetonitrile to the mobile phase and by controlling the concentration and chain length of alkylsulfonate in the mobile phase. Although an octadecylsilylsilica, reversed-phase column was preferred for the analytical separation of the methionine-platinum complexes, a polystyrene-divinylbenzene colume was preferred for preparative work.     

Effect of the solute molecular structure on its enantioresolution on cellulose tris(3,5-dimethylphenylcarbamate)

The effects of the molecular structures for 13 structurally similar chiral solutes on their HPLC retention and enantioresolutions on a commercially important polysaccharide-based chiral stationary phase, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) are studied. Among these 13 solutes, only methyl ephedrine (MEph) shows significant enantioresolution. The retention factors of these chiral solutes vary significantly from 0.7 to 3.2 in n-hexane/2-propanol (90/10, v/v) at 298 K. The retention factors of some simpler non-chiral solutes having similar but fewer functional groups than their chiral counterparts are also studied under the same conditions and are compared to those of the chiral solutes. The H-bonding interactions between the functional groups of the solute and the C=O and NH functional groups of the polymer are probed with attenuated total reflection-infrared spectroscopy (ATR-IR) for the polymer, for binary sorbent-solute systems. The CDMPC IR amide band wavenumbers change significantly, indicating H-bonding interactions of the polymer C=O and NH groups with the solutes. The elution orders predicted for the enantiomers of these chiral solutes using molecular dynamics (MD) simulations of the polymer-solute binary systems are consistent with the HPLC results. The CDMPC cavity nano-structure and the potential interactions with chiral solutes are proposed based on HPLC data, IR data, and the simulations. The results are consistent with the three-point attachment model and support the hypothesis that significant enantioresolution requires at least three different synergistic interactions which can be a combination of steric hindrance, H-bonding, or pi-pi interactions.     

Effect of Chromatographic Conditions on Enantioseparation of Bovine Serum Albumin Chiral Stationary Phase in HPLC and Thermodynamic Studies

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA‐CSP) by high‐performance liquid chromatography (HPLC) in reversed‐phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N‐(2, 4‐dinitrophenyl)‐serine (DNP‐ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R_s) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N‐(2, 4‐dinitrophenyl)‐proline (DNP‐pro) decrease along with increasing 1‐propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods. Chirality 25:487–492, 2013. © 2013 Wiley Periodicals, Inc.     

Determination of a prostaglandin D2 antagonist and its acyl glucuronide metabolite in human plasma by high performance liquid chromatography with tandem mass spectrometric detection--a lack of MS/MS selectivity between a glucuronide conjugate and a phase I metabolite

A method for the determination of a prostaglandin D(2) receptor antagonist (I, a compound being evaluated for the prevention of niacin induced flushing) and its acyl glucuronide metabolite (II) in human plasma is presented. The method utilized high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization mode. The product ion was a radical cation generated via a homolytic bond cleavage. A chemical analog of the drug was used as internal standard (III). The acyl glucuronide metabolite (II) was detected using the same precursor-to-product ion transition used for the parent compound after chromatographic separation of I and II. Drug and metabolite were extracted using semi-automated, 96-well format solid phase extraction (SPE), and chromatography was performed using a reverse phase analytical column with an isocratic mobile phase. The chromatographic retention factor (k') of II was found to be highly sensitive to mobile phase formic acid concentration. An adjustment in mobile phase formic acid concentration improved the chromatographic separation between II and a mono-hydroxylated metabolite after an unexpected lack of MS/MS selectivity between the two molecules was observed. The dependence of retention factor on formic acid concentration (k' increased as formic acid concentration decreased) was thought to indicate polar interactions between II and the stationary phase. The stability of II in spiked human plasma was determined. The rate of hydrolysis back to parent compound was relatively low (approximately 0.1 and 0.5% per hour at room temperature and 4 degrees C, respectively) indicating that significant changes in analyte concentrations did not occur during sample processing. The concentration range of the assay was 10-2500 ng/mL for both drug and glucuronide metabolite.     

Comparison of partial least-square method and canonical correlation analysis in a quantitative structure–retention relationship study

The retention of 7 monotetrazolium and 9 ditetrazolium salts was determined on alumina and reversed-phase (RP) alumina layers using n-hexane-1-propanol and water-1-propanol mixtures as eluents. The retention capacity and the specific surface area of solutes in contact with the stationary phases were calculated. The relationship between retention characteristics and physicochemical parameters of solutes was elucidated by canonical correlation analysis and partial least-square regression analysis. Both methods found significant relationships between the chromatographic and physicochemical parameters, however, the results were different according to the method applied. Calculations suggested that the retention on both alumina and RP alumina layers is of mixed character, hydrophobic, electronic and steric parameters are equally involved in the retention.     

Mathematical Modeling of Size Exclusion Chromatography

A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk‐fluid phase, interfacial film mass‐transfer between the stationary and mobile phases, and diffusion of solutes within the macro pores of the packing particles. Differential equations of the process model were solved by the finite difference method. Characteristics of the column and the packing particles (bed void volume fraction, particle porosity, accessible particle porosity) were obtained experimentally, as well as retention times of different molecules with known molecular weights. Experiments were performed with two different columns containing two different packing materials, Superdex 75 HR 10/30 and BioSep SEC S2000, respectively. The model has been validated by comparing theoretical and experimental retention times for the different columns.     

Enantiomeric Separation of Original Heterocyclic Organophosphorus Compounds in Supercritical Fluid Chromatography

New and original heterocyclic α-enamido phosphine chiral solutes were prepared: four structurally similar racemates with the chirality center placed on the phosphorus atom, and four other related pairs of enantiomers with chirality borne by the carbon atoms of the phospholane ring. The structural variations were placed on an aliphatic heterocycle (six- or seven-member rings) and on the carbamate function (methyl or t-butyl). Their separation was achieved on a commercial cellulose tris-(3,5-dimethylphenylcarbamate) stationary phase (Lux Cellulose-1, Phenomenex) in supercritical fluid chromatography (SFC). The effects of molecular structure on SFC retention and enantioresolution were studied. Among these eight pairs of enantiomers, some reversal of elution order between similar compounds was observed. The effect of changing the organic solvent (methanol and ethanol) and its proportion (between 5 and 40%) in the mobile phase was investigated. Retention data were collected over the temperature range 0–50 °C, and the results interpreted from thermodynamic aspects. Chirality, 2013. © 2013 Wiley Periodicals, Inc.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

In vitro survival and proliferation of porcine primordial germ cells

Primordial germ cells (PGC) collected from the genital ridge of Day 25 porcine embryos were cultured on STO feeder cells in medium with or without supplemented growth factors. The effects on porcine PGC proliferation of leukemia inhibitory factor (LIF), LIF + stem cell factor (SCF) or LIF + SCF + basic fibroblast growth factor (bFGF), growth factors shown to be essential for in vitro survival and proliferation of murine PGC, were tested. After histochemical staining, both freshly collected and cultured PGC expressed alkaline phosphatase activity. With or without supplemented growth factors, porcine PGC survived and proliferated in culture for at least 5 d. None of the growth factors tested markedly enhanced in vitro growth of porcine PGC. These results suggest that growth factors provided by either the STO feeder layer or the cultured PGC themselves are sufficient to support in vitro survival and proliferation of porcine PGC. With the support of STO cells, addition of growth factors shown to be essential for the in vitro growth of murine PGC is not required for survival and proliferation of cultured porcine PGC.