Enthalpic Partition-Assisted Size Exclusion Chromatography: 1. Principle of Method

A novel high performance liquid chromatographic (HPLC) method viz. “enthalpic partition assisted size exclusion chromatography” deliberately combines entropic and enthalpic partition mechanisms. It enables separation of homopolymers according to their molar mass with increased selectivity, as well as discrimination of polymer species differing in their nature/composition. Enthalpic partition of macromolecules takes place between the mobile phase and the stationary “liquid” of a different chemical nature, which is immobilized within pores of an appropriate carrier (a bonded phase). The extent of enthalpic partition depends on the accessibility of bonded phase for macromolecules and on the difference of polymer solubility in the mobile phase and in the solvated bonded phase. The enthalpic partition in favor of column packing arises from better solubility of polymer solutes in the solvated stationary phase compared to the mobile phase. Macromolecules are “pushed” into the solvated stationary phase and their retention volumes (V_R) increase. In the area of high molar masses, the extent of enthalpic partition as rule raises with the increasing size of macromolecules. However, under properly chosen experimental conditions the enthalpic partition may rapidly diminish with the sample molar mass (M), likely due to the solubility changes and/or due to partial exclusion of macromolecules from the pores. As result, the corresponding retention volumes sharply drop within a narrow range of M with the increasing size of macromolecules. This results in the log M vs. V_R dependences, which resemble in their form that for size exclusion chromatography but are much more flat indicating highly selective separations of homopolymers according to their molar masses. In this way, enthalpic partition “assists” entropic partition (size exclusion). Polymer species, which do not undergo enthalpic partition, elute from the HPLC column in the conventional size exclusion mode and can be discriminated from the partitioning species. Enthalpic partition assisted size exclusion chromatography can be utilized in separation and characterization of various homopolymers, and polymer blends.     

Retention mechanism of a cholesterol-coated C18 stationary phase: van't Hoff and Linear Solvation Energy Relationships (LSER) approaches

This study examines the effect of temperature on the dynamic cholesterol coating of a C18 stationary phase and the effect of this coating on the retention mechanism. In general, an increase in temperature results in a decrease in the mass of cholesterol coated on the stationary phase. Typically, an increase in temperature from 25°C to 55°C results in a nearly 60% reduction in the mass of cholesterol loaded. The inclusion of temperature, along with loading solvent composition and cholesterol concentration in the loading solvent, allows for loading a targeted amount of cholesterol on the stationary phase over an order-of-magnitude range. In addition to loading studies, the retention mechanism of small non-ionizable solutes was examined on cholesterol-coated stationary phases. A van't Hoff analysis was performed to assess retention thermodynamics, while a LSER approach was used to examine retention mechanism. With 50/50 water/organic mobile phases, the addition of cholesterol results in an increase in the entropic contribution to retention, with a decrease in the enthalpic contribution. The opposite trend is seen with 40/60 water/organic mobile phases. LSER system constants are also affected by a cholesterol coating on the stationary phase, with some changing to favor elution and others changing to favor retention.     

Temperature changes of sorption parameters of di-n-alkylketones and methylcyclohexanones in capillary gas chromatography

The gas chromatographic behavior of di-n-alkylketones and isomeric methylcyclohexanones at variable temperatures in the isothermal runs has been studied using three capillary columns coated with SE-30, OV-225, and PEG-40M/KF stationary phases. The retention indices and their temperature increments were determined, and the partial molar free energies of the compounds under study were calculated. A linear dependence of the values of the partial molar free energy of sorption on the homologue number in di-n-alkylketones is not observed for the first member on the nonpolar SE-30 phase and for the third member on the polar OV-225 and PEG-40M/KF phases. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 328–332, February, 1997.     

Evaluation of ternary mobile phases for reversed-phase liquid chromatography: Effect of composition on retention mechanism

The effect of varying mobile phase composition across a ternary space between two binary compositions is examined, on four different reversed-phase stationary phases. Examined stationary phases included endcapped C8 and C18, as well as a phenyl phase and a C18 phase with an embedded polar group (EPG). Mobile phases consisting of 50% water and various fractions of methanol and acetonitrile were evaluated. Retention thermodynamics are assessed via use of the van’t Hoff relationship, and retention mechanism is characterized via LSER analysis, as mobile phase composition was varied from 50/50/0 water/methanol/acetonitrile to 50/0/50 water/methanol acetonitrile. As expected, as the fraction of acetonitrile increases in the mobile phase, retention decreases. In most cases, the driving force for this decrease in retention is a reduction of the enthalpic contribution to retention. The entropic contribution to retention actually increases with acetonitrile content, but not enough to overcome the reduction in the enthalpic contribution. In a similar fashion, as methanol is replaced with acetonitrile, the v, e, and a LSER system constants change to favor elution, while the s and c constants change to favor retention. The b system constant did not show a monotonic change with mobile phase composition. Overall changes in retention across the mobile phase composition range varied, based on the identity of the stationary phase and the composition of the mobile phase.     

Linear Solvation Energy Relationships in the Determination of Specificity and Selectivity of Stationary Phases

The retention of fifty structurally different compounds has been studied using linear solvation energy relationships. Investigations were performed with the use of six various stationary phases with two mobile phases (50/50 % v/v methanol/water and 50/50 % v/v acetonitrile/water). Packing materials were home-made and functionalized with octadecyl, alkylamide, cholesterol, alkyl-phosphate and phenyl molecules. This is the first attempt to compare all of these stationary phases synthesized on the same silica gel batch. Therefore, all of them may be compared in more complex and believable way, than it was performed earlier in former investigations. The phase properties (based on Abraham model) were used to the classification of stationary phases according to their interaction properties. The hydrophilic system properties s, a, b indicate stronger interactions between solute and mobile phase for most of the columns. Both e and v cause greater retention as a consequence of preferable interactions with stationary phase by electron pairs and cavity formation as well as hydrophobic bonds. However, alkyl-phosphate phase has different retention properties, as it was expressed by positive sign of s coefficient. It may be concluded that most important parameters influencing the retention of compounds are volume and hydrogen bond acceptor basicity. The LSER coefficients showed also the dependency on the type of organic modifier used as a mobile phase component.

     

Linear Solvation Energy Relationships in the Determination of Specificity and Selectivity of Stationary Phases

The retention of fifty structurally different compounds has been studied using linear solvation energy relationships. Investigations were performed with the use of six various stationary phases with two mobile phases (50/50?% v/v methanol/water and 50/50?% v/v acetonitrile/water). Packing materials were home-made and functionalized with octadecyl, alkylamide, cholesterol, alkyl-phosphate and phenyl molecules. This is the first attempt to compare all of these stationary phases synthesized on the same silica gel batch. Therefore, all of them may be compared in more complex and believable way, than it was performed earlier in former investigations. The phase properties (based on Abraham model) were used to the classification of stationary phases according to their interaction properties. The hydrophilic system properties s, a, b indicate stronger interactions between solute and mobile phase for most of the columns. Both e and v cause greater retention as a consequence of preferable interactions with stationary phase by electron pairs and cavity formation as well as hydrophobic bonds. However, alkyl-phosphate phase has different retention properties, as it was expressed by positive sign of s coefficient. It may be concluded that most important parameters influencing the retention of compounds are volume and hydrogen bond acceptor basicity. The LSER coefficients showed also the dependency on the type of organic modifier used as a mobile phase component.     

Factors influencing retention and resolution of substituted alkylbenzenes in reversed-phase liquid chromatography

Summary The retention data of isomeric xylenes, ethyltoluenes and diethylbenzenes, and of mesitylene, benzene, toluene and ethylbenzene were obtained on a reversedphase column using methanol-water and ethanol-water mobile phases at four different temperatures. This database was used to relate the dependence of solute retention and resolution on the polarity of the mobile phase, solute dipole moment, and column temperature. The additivity of the free energy of the transfer of solute molecules or solute segments from the stationary phase to the mobile phase, was examined for the isomeric compounds. For this, the logarithm of the net retention volume was substituted for the free energy. Deviations from the additivity of free energies indicate that the separation of isomeric substituted alkylbenzenes is governed by their differential interactions with both the polar mobile phase and nonpolar stationary phase. Among the disubstituted alkylbenzenes,ortho-isomers favor the mobile phase more andpara-isomers tend to prefer the stationary phase more. Themeta-isomers are found to follow the additivity rule closely. These trends are amplified as the polarity of the mobile phase is increased indicating that these isomers are resolved better in water-rich mobile phases. These findings are substantiated by analogous results from gas-liquid chromatographic retention data, estimation of dipole moment effects, and examination of the entropic and enthalpic contributions to the net retention volume.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

The effects of temperature and mobile phase on the retention of aliphatic carboxylic acids in hydrophilic interaction chromatography on zwitterionic stationary phases

The influence of the mobile phase and temperature, on the retention behavior of seven aliphatic acids (pyruvic, gluconic, 2‐oxoglutaric, tartaric, malic, oxalic, and citric acid) in hydrophilic interaction liquid chromatography on zwitterionic stationary phases with sulfobetaine and phosphorylcholine ligands is investigated. In agreement with the van't Hoff model, most acids show linear ln k versus 1/T plots. However, the retention of structurally symmetrical oxalic and tartaric dicarboxylic acids is almost independent of temperature, or slightly increases at rising temperature. The experimental parameters of the van't Hoff plots suggest positive entropic contributions to the retention of these symmetrical acids, possibly connected with changes in molecular symmetry on their adsorption. The type of the zwitterionic stationary phase and the mobile phase composition (the molar concentration of acetate buffer and the volume fraction of acetonitrile) affect the retention and the selectivity of the separation of the acids.     

A simple TLC and HPTLC method for separation of selected steroid drugs

Chromatographic properties of five steroid drugs: cortisone, hydrocortisone, methylprednisolone, prednisolone and norgestrel have been studied by normal-, reversed-phase and hydrophilic neutral cyano-bonded silica stationary phase with five binary mobile phases (acetonitrile-water, acetonitrile-DMSO, acetonitrile-methanol, acetone-petroleum ether, acetone-water) in which the concentration of organic modifier was varied from 0 to 100% (v/v). This study reports the optimization of steroid hormones separation. Chromatographic retention data and possible retention mechanisms are discussed. Separation abilities of mobile and stationary phases were studied using the principal component analysis method. The best separation of methylprednisolone and prednisolone is with a chromatographic system included silica gel as stationary phase and mixture of acetonitrile and DMSO (10:90 v/v). These two anti-inflammatory drugs can be fast separated from norgestrel when CN is used as stationary phase and acetone and water (40:60 v/v) as mobile phase. The highest values of the parameter Δ(ΔG°) and alfa for cortisone and hydrocortisone was observed in case of using CN as stationary phase and water-acetonitryle (40:60 v/v) as mobile phase.      

Temperature dependence of the free energy of sorption ofn-alkanes and energy contribution of their methylene groups on columns with fullerene C60

The partial molar free energy, enthalpy, and entropy of sorption of C₁₁−C₂₃ n-alkanes were calculated on the basis of the GC data obtained on the glass capillary column coated with fullerene C₆₀ (Ful-60) as stationary phase. The thermodynamic parameters ofn-alkane sorption on a column with Ful-60 and a fused silica capillary column with polydimethylsiloxane OV-1 were determined and compared. The enthalpy-entropy compensation effect for the sorption ofn-alkanes on Ful-60 and OV-1 was found. A linear dependence of the partial molar free energy ofn-alkane sorption on the temperature of analysis and carbon chain length was found. The free energy contributions of the methylene groups were calculated, and their temperature dependences were studied. The differences in the temperature dependences of the energy contributions of methylene groups ofn-alkanes on Ful-60 and OV-1 were revealed. The entropy contribution is 68–82% of the enthalpy contribution which indicates a substantial role of the number of contacts with Ful-60 in retention ofn-alkanes. The ability of Ful-60 for dispersive interactions is similar to those of nonpolar liquid phases and substantially differs from that for carbon adsorbents. Fullerene columns were shown to be convenient for analysis of highly boiling organic substances in aqueous and organic solutions. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1490–1495, August, 1999.     

Comparative separation of biologically active components inRhizoma chuanxiong by affinity chromatography with α1-acid glycoprotein and human serum albumin as stationary phasesglycoprotein and human serum albumin as stationary phases

Summary Affinity chromatography with α₁-acid glycoprotein (AGP) and human serum albumin (HSA) stationary phases was applied to screen and analyze the biologically active components ofRhizoma chuanxiong. Five major peaks and a number of small peaks were resolved based on their affinity for AGP and HSA, respectively, and three of them, identified as ferulic acid, chuanxiongzine and ligustilide via standard compounds, are regarded as effective components. The effects of acetonitrile concentration, pH, inorganic salt concentration and temperature on the retention behaviors of five major components on the two stationary phases were also investigated. It was observed that hydrophobicity is the major contributor to retention on both stationary phases, and ferulic acid has a weak electrostatic interaction with HSA. It demonstrated that the chromatograms ofRhizoma chuanxiong on the two stationary phases have concise and different fingerprinting characteristics. The amount of ferulic acid, chuanxiongzine and ligustilide inRhizoma chuanxiong determined using the AGP column are as much as 0.064%, 0.021% and 2.00% by weight.     

Energetics of some sulphur heterocycles

The present work is part of a broader research program on the energetics of formation of heterocycles, aiming the study of the enthalpic effects of the introduction of different substituents into heterocycles. In this work we present the results of the thermochemical research on sulphur heterocycles of the type substituted thiophenes with different kind of substituents, mainly alkyl, ester, acetyl, carboxamide, acetamide, carbonitrile and carboxaldehyde. The standard (p ^o=0.1 MPa) molar enthalpies of formation, in the condensed phase, at T=298.15 K, of a large number of substituted thiophenes, were derived from their standard massic energies of combustion, measured by rotating-bomb combustion calorimetry, while the standard molar enthalpies of vaporization or sublimation of those compounds were obtained either by high temperature Calvet Microcalorimetry, or by the temperature dependence of their vapour pressures determined by the Knudsen effusion technique. The standard molar enthalpies of formation, of the studied sulphur heterocycles in the gaseous phase, were then derived. The results are interpreted in terms of structural contributions to the energetics of the substituted thiophenes, the internal consistency of the results is discussed and, whenever appropriate and possible, empirical correlations are suggested for the estimation of standard molar enthalpies of formation, at T=298.15 K, of substituted thiophenes. A Table of enthalpic increments for different group substituents in positions 2 or 3 of the thiophene ring has been established.     

Analysis of normal and modified nucleosides in urine samples by high‐performance liquid chromatography with different stationary phases

The main aim of the present work was to study the retention behavior and quantification of nine nucleosides with the use of octadecyl, alkylamide, cholesterol and alkyl‐phosphate stationary phases. The influence of organic solvent and buffer concentration on the separation of these compounds was under investigation. The retention factor had the highest values for the octadecyl and cholesterol packing materials. Complete separation of all the studied nucleosides was achieved in case of cholesterol stationary phase. The optimized separation method was applied for the quantification of nucleosides in the urine samples. Calibration plots showed good linearity (R² > 0.999) and the limits of detection were in a range of 0.3–0.5 µg/mL, while the limits of quantitation were >0.9 µg/mL. Accuracy was in the range of 5–11%. Copyright © 2014 John Wiley & Sons, Ltd.     

Combined effects of mobile phase composition and temperature on the retention of homologous and polar test compounds on polydentate C8 column

Combined effects of temperature and mobile phase on the reversed phase chromatographic behavior of alkylbenzenes and simple substituted benzenes were investigated on a Blaze C₈ polydentate silica-based column, showing improved resistance against hydrolytic breakdown at temperatures higher than 60 °C, in comparison to silica-based stationary phases with single attachment sites. For better insight into the retention mechanism on polydentate columns, we determined the enthalpy and entropy of the transfer of the test compounds from the mobile to the stationary phase. The enthalpic contribution dominated the retention at 80% or lower concentrations of methanol in the mobile phase. Entropic effects are more significant in 90% methanol and in acetonitrile–water mobile phases. Anomalies in the effects of mobile phase on the enthalpy of retention of benzene, methylbenzene and polar benzene derivatives were observed, in comparison to regular change in enthalpy and entropy of adsorption with changing concentration of organic solvent and the alkyl length for higher alkylbenzenes. The temperature and the mobile phase effects on the retention are practically independent of each other and – to first approximation – can be described by a simple model equation, which can be used for optimization of separation conditions.     

Gradient elution chromatography of polymers on reversed-phase columns with tetrahydrofuran as an eluent component

Summary The chromatographic separation of poly(styrene-co-acrylonitrile) samples (SAN) with an elution gradient iso-octane/tetrahydrofuran is solubility governed. This has been proven by the correspondence between the volume fraction of precipitant as estimated from elution data and as measured by turbidimetric titration, by the molar mass dependence of retention, and by the temperature effect on retention. In addition to these arguments data are presented which have been obtained on columns with different packing materials ranging from bare silica through CN bonded phase material to hydrocarbonaceous stationary phases. The eluent composition at peak position is nearly independent of the stationary phase used. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

A comparative study of hydrophobicity of octadecyl and alkylamide bonded phases based on methylene selectivity

The conformational dynamics of home‐made monomeric and polymeric (octadecyl – C18 and alkylamide – AP) stationary phases have been investigated by analyzing the dependence of retention on the reciprocal temperature for different organic compounds. Theoretical considerations were based on the results of methylene selectivity determination. Two selected PAHs – naphthacene and benzo[a]anthracene, whose structures differ in the position of the ring – were chosen as solutes to study the thermodynamics of retention behavior. Standard enthalpies (ΔH ⁰) were obtained from the slope of the plots of the capacity factor logarithm (ln k) against the reciprocal temperature (1/T), i. e. van't Hoff plots, in a temperature range from 285 to 315 K. The conformation of the stationary phase (monomeric and polymeric) structure and the length of bonded ligands with their specific functional (N‐acylamide) groups had a significant effect on retention.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Selectivity of stationary phases for the resolution of polycyclic aromatic hydrocarbons

Summary Relative retentions, heats and entropies of solution for 16 PAH in seven different stationary phases were measured under standard isobaric conditions using GC. The experimental conditions to obtain reproducible retention data at non-linear adsorption isotherms are discussed and standard conditions are recommended using the linear relationship between retention time and 1/log h (where h is the peak height). A linear relationship was established between the relative molar heats of solution and the number of carbon atoms in the PAH-molecule. This relationship is valid only for non-polar stationary phases. Certain effects of the isomeric structures of PAH are studied using the relative molar heats and entropies of solution. The latter function is very sensitive to the spherical form of a molecule. Different aspects of isomeric selectivity are discussed and it is concluded that the better way for resolving complex PAH-mixtures is rather an improvement in column efficiency than searching for new stationary phases.     

Separation of polycyclic aromatic hydrocarbons with a C60 bonded silica phase in microcolumn liquid chromatography

A chemically bonded C₆₀ silica phase was synthesized as a stationary phase for liquid chromatography (LC) and its retention behavior evaluated for various polycyclic aromatic hydrocarbons (PAHs) using microcolumn LC. The results indicate that the C₆₀ bonded phase offers selectivity different from that of octadecylsilica (ODS) bonded phases in the separation of isomeric PAHs. With the C₆₀ phase, PAH molecules having a partial structure similar to that of the C₆₀ molecule, e.g. triphenylene and perylene, were retained longer than with ordinary ODS stationary phases. The results also show that good correlation exists between the retention data with this C₆₀ bonded phase and with C₆₀ itself as the stationary phase.