High temperature liquid chromatography and liquid chromatography-mass spectroscopy analysis of octylphenol ethoxylates on different stationary phases

Temperature was investigated as active parameter in the liquid chromatography (LC) analysis of octylphenol ethoxylates. Significant differences in selectivity were observed when the oligomers were analyzed by reversed phase LC (RPLC) on silica-, zirconia- and polystyrene/divinylbenzene based stationary phases at low (ambient), medium and elevated temperature with acetonitrile/water as mobile phase. As ascertained by LC-mass spectroscopy (MS), in most cases the elution order of the oligomers was completely reversed comparing ambient and high temperature separations. On a graphitized carbon type column, the selectivity remained unchanged, regardless the analysis temperature. Also in normal phase LC, the elution order remained unaffected by temperature variations both for acetonitrile/water and methanol/water mixtures as mobile phase. Surprisingly, when reversed phase LC on a octadecylsilicagel column at different temperatures was repeated with methanol instead of acetonitrile as mobile phase ingredient, the reversal of elution order did not take place. Results are evaluated in terms of thermodynamic parameters.     

Characterization of different reversed phase systems in liquid adsorption chromatography of polymer homologous series

The interaction parameter of a given repeat unit in liquid adsorption chromatography (LAC) can be determined from the slope in a plot of the elution volumes versus the difference in elution volumes of subsequent monomers. In such a plot, the intercept represents the void volume, and from the slope the adsorption interaction parameter can be calculated. This parameter is independent of column dimensions and pore diameter and can thus be used as a measure of the interaction of a given repeat unit with the surface of a stationary phase in a given mobile phase composition. The interaction parameter can also be obtained from the slope in a plot of the logarithmic retention factors k versus the number of repeat units n or from the slope in a plot of the logarithmic difference in elution volumes of subsequent monomers versus n. The values obtained by the three different approaches are in good agreement. In a given mobile phase, the interaction parameter of a given repeat unit was found to be almost the same for various alkyl bonded columns from different producers. The retention of a given oligomer depends as well on the interaction parameter as on the pore dimensions (and thus the internal pore surface) of the stationary phase. The pore surface can be determined from the intercept in a plot of the logarithmic difference in elution volumes of (subsequent) nonfunctional monomers as a function of n. As the interaction parameter of a given repeat unit in a given mobile phase is the same for stationary phases with the same chemical nature, retention can be adjusted by selection of the pore surface. On a given stationary phase, there is a linear dependence between interaction parameter and mobile phase composition.     

Monofunctional polymers in liquid adsorption chromatography: determination of the interaction parameter in the range of weak interaction

The main physical characteristics of monofunctionals in adsorption chromatography - the adsorption interaction parameter of the repeat units c and the interaction parameter of specific end group q - are discussed. Both parameters are independent on column dimensions and pore diameter, and depend on mobile phase composition. In a plot of elution volumes V(n) vs. the difference DeltaV=V(n)-V(n-1) in elution volumes of consecutive non-functional or monofunctional oligomers, straight lines with the same slope are obtained for sufficiently high molar masses. The intercept of these lines yield the accessible volumes of functionalized and non-functionalized oligomers. In the range of weak interaction, the interaction parameter of the repeat unit can be determined using monofunctional chains with strongly adsorbing end group. Scope and limitations of this approach are studied using monoalkyl ethers of polypropylene glycol as model polymers.     

The rule parameters in liquid adsorption chromatography of polymer homologous series and their determination

The separation of polymer homologous series is governed by two rule parameters: the interaction parameter c of the repeat unit and the sorbent surface S (relation of pore volume to pore diameter). The interaction parameter is independent on column dimensions and pore diameter. In a plot of elution volumes Vn versus the difference DeltaV =Vn -Vn-1 in elution volumes of consecutive oligomers, straight lines are obtained, the intercept of which yields the accessible volume. Using the accessible volume as the hold-up volume in the calculation of the retention factor k, a strictly linear dependence of ln k versus the number of the repeat units n is observed (Martin's plot). The interaction parameter c can be determined experimentally by three different approaches: from the slope in a plot of V versus DeltaV, from the slope in Martin's plot (ln k versus n), and from the intercept in a plot of ln(DeltaV) versus n. The pore surface is obtained from the intercept in Martin's plot (ln k versus n) or the intercept in a plot of ln(DeltaV) versus n. The results obtained by the different approaches agree very well. The theoretical predictions are verified by comparison with the experimental data obtained on columns with different pore diameter and pore volume and thus, different pore surface at conditions corresponding to the same interaction parameter.     

Structural organization of ethyl esters based on hexafluoropropene oxide oligomers: Inverse gas chromatography study

Samples of ethyl esters of perfluorinated acids based on hexafluoropropene oxide oligomers with molecular masses of 2000, 3100, and 4500 are used as stationary phases, and their interactions with low-molecular-mass compounds (sorbates) of different natures are studied by inverse gas chromatography. The linear dependence of the specific retention volumes for alkanes, cyclohexane, aromatic hydrocarbons, tetrachloromethane, and trifluorotrichloroethane (CFC-113) on inverse molecular masses of the oligomer samples is ascribed to the quasi-biphasic structure of the liquid oligomers. The experimental dependences of specific retention volumes on inverse molecular masses of oligomer samples make it possible to estimate the main thermodynamic characteristics of sorbate interaction with individual quasi-phases and their solubility parameters directly related to the cohesion-energy density.     

Retention of functional polymers in liquid adsorption chromatography: Effect of the end groups in PEGs and their methyl ethers in different mobile phases

It is shown that the end group parameter, which describes the influence of the end group on retention, can be determined in RP chromatography from two chromatograms of a nonfunctional and a monofunctional sample, if a sufficient number of peaks with the same number of repeat units in each sample can be reasonably resolved and identified. The same procedure can also be applied for pairs of di‐ and monofunctional polymers. End group parameters have been determined in three different mobile phases: acetonitrile–water, acetone–water and methanol–water of different compositions. The temperature dependence of the interaction parameter of the repeat unit and the end group parameter has been found to be different, which allows a fine‐tuning of retention.     

Comparison of eluents in supercritical fluid chromatography as based on density and free volume

A comparative study of chromatographic properties of different supercritical eluents has been performed by considering the retention at equal density or at equal free volume for different mobile phases. At equal density, the temperature dependence of the capacity ratios of aromatic hydrocarbons is the same for different alkane mobile phases, whereas significant deviations are observed when carbon dioxide is employed as the mobile phase. At equal free volume, a comparison of the capacity ratios measured in carbon dioxide and in alkane eluents showed a pronounced similarity for the different mobile phases when the free volume was low, i.e., at high densities. With increasing free volume, the chemical differences of the alkanes on the one hand and carbon dioxide on the other become more apparent in their elution behavior. Furthermore, it is demonstrated that the free volume can also be used as a reference basis for the data of solvatochromic shift experiments.     

Adsorption effects in gel permeation chromatography: The application of binary mobile phases

Summary The influence of the adsorption properties of the column packing on the elution mechanism of macromolecules was investigated. Experimentally determined elution volumes of polystyrene standards in different mobile phases were compared with computed theoretical plots assuming (a) the pure steric exclusion mechanism; (b) mixed steric exclusion and adsorption mechanism.     

Determination of the accessible volume and the interaction parameter in the adsorption mode of liquid chromatography

The main physical parameters in liquid chromatography of oligomers-the accessible volume and the adsorption interaction parameter-are discussed. It is shown, that in liquid adsorption chromatography (LAC) there is a linear relation between elution volume and the distance of two subsequent peaks of a homologous series. From the intercept of the regression lines in such a plot the accessible volume can be easily determined at any mobile phase in LAC (corresponding to conditions of weak or strong adsorption) without any information about the molar mass of the peaks. From the slope of this dependence the adsorption interaction parameter of a given repeat unit can be obtained. The accurate determination of the accessible volume and the adsorption interaction parameter in the LAC regime is presented for PEG, PPG and fatty alcohols on various reversed phase columns with different pore size in methanol-water or acetone-water mobile phases. The difference between the void volume, the dead volume or hold-up volume (from the solvent peak position) and the accessible volume (obtained by this procedure) is discussed.     

Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides

The application of enhanced fluidity liquid (EFL) mobile phases to improving isocratic chromatographic separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC) mode is described. The EFL mobile phase was created by adding carbon dioxide to a methanol/buffer solution. Previous work has shown that EFL mobile phases typically increase the efficiency and the speed of the separation. Herein, an increase in resolution with the addition of carbon dioxide is also observed. This increase in resolution was achieved through increased selectivity and retention with minimal change in separation efficiency. The addition of CO₂ to the mobile phase effectively decreases its polarity, thereby promoting retention in HILIC. Conventional organic solvents of similar nonpolar nature cannot be used to achieve similar results because they are not miscible with methanol and water. The separation of nucleosides with methanol/aqueous buffer/CO₂ mobile phases was also compared to that using acetonitrile/buffer mobile phases. A marked decrease in the necessary separation time was noted for methanol/aqueous buffer/CO₂ mobile phases compared to acetonitrile/buffer mobile phases. There was also an unusual reversal in the elution order of uridine and adenosine when CO₂ was included in the mobile phase.     

Application of size exclusion chromatography with surfactant eluent to the study of polymer–surfactant interactions: oligomeric and micellar chromatographic effects

Complexation of sodium dodecyl sulphate (SDS) with a wide range of molecular weights of poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO) has been studied by size exclusion chromatography using aqueous SDS eluent. A multi-angle laser light scattering detector and a differential refractometer were applied to give direct measurement of the molecular weight of complexes without reference to elution volume, since the latter is not a reliable indicator of the complex size. Background light scattering from micellar eluents hampered quantitative size measurements, but was minimal in sub-micellar eluent, where saturated binding was observed for polymers larger than 1000 g mol⁻¹. Multiple peaks and voids were observed in the elution profiles of low molecular weight polymers (up to a mass of 600 g mol⁻¹) in eluent at micellar concentrations. Several sources contribute to this behavior, including micellar chromatographic separation of the PEG oligomers due to their different distribution coefficients between the micellar and water phases. Preliminary results are reported for distribution coefficients of individual oligomers in a 600 g mol⁻¹ PEG sample. Three distinct binding behaviors are observed with increasing degree of polymerization of PEG: no interaction for small glycols, equilibrium partitioning of intermediate oligomers in and out of micelles, and binding of micelles to the larger polymers.     

Gel permeation chromatography with mixed eluents: Polymer solution as mobile phase

The GPC elution behaviour of a polymer was studied when a solution of another polymer in a liquid was used as an eluent. In ternary systems containing two polymers, GPC results are influenced by the thermodynamics of polymer incompatibility. The incompatibility manifests itself both in the slope of the dependence of elution volume upon the concentration of injected polymer and in the shift of elution volumes extrapolated to zero concentration. Experimental data for systems containing poly(styrene) and poly(methyl methacrylate) have been accounted for qualitatively by theoretical views on coil shrinkage resulting from polymer incompatibility. The concentration effects of the injected polymer and the polymer in the mobile phase on elution volume are compared; further parameters affecting the separation in the systems with polymer solution as eluent are discussed.     

Measurement of the elution strength and peak shape enhancement at increasing modifier concentration and temperature in RPLC

Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in the experimental factor. The approaches were applied to the separation of a set of flavonoids with aqueous micellar mobile phases of the surfactant Brij-35 (polyoxyethylene(23)dodecanol), in comparison with acetonitrile–water mixtures, using an Eclipse XDB-C18 column. The particular interaction of each flavonoid with the oxyethylene chains of Brij-35 molecules (adsorbed on the stationary phase or forming micelles) changed the elution window, distribution of chromatographic peaks and partitioning kinetics, depending on the hydroxyl substitution in the aromatic rings of flavonoids. At 25?°C, peak shape with Brij-35 mobile phases was significantly poorer with regard to acetonitrile–water mixtures. At increasing temperature, the efficiency of Brij-35 increased, approaching at 80?°C the values obtained at equilibrium conditions, already reached with acetonitrile at 25?°C.     

Retention and separation studies of cholesterol and bile acids using thermostated thin-layer chromatography.

The influence of temperature on retention and separation of cholesterol and bile acids, using reversed-phase thin-layer chromatography, was studied. As mobile phases methanol-water mixtures of various compositions were used. Chromatographic experiments were performed using vapor-saturated chambers at temperatures ranging from 5 to 60 degrees C. A linear relationship between R(M) values and temperature (1/T) as well as mobile phase composition was observed. The elution order of steroids under the conditions investigated was discussed. Each chromatogram was evaluated using simple optimization parameters and the best chromatographic conditions for the separation of multicomponent samples were chosen.     

Separation of free sterols by high temperature liquid chromatography

Increasing the column temperature accelerates markedly elution in HPLC. The separation of five free sterols was studied on three packing materials that can withstand high temperatures. These stationary phases included graphitic carbon, a polymeric C18 silica, and a zirconia-based adsorbent. Measurements of retention data were made at up to 150 degrees C with mobile phases of different compositions. Since the columns tested afford different retention mechanisms, a variety of elution patterns were observed, with some being more advantageous than others for certain sterol separations. Effects observed include some selectivity improvements and some elution order reversals. The separation of free sterols in selected fruit juices is also presented. Albeit at the expense of a longer analysis time, the graphitic carbon column produced the best separation of the sterols in this study.     

Computer-assisted transfer of programmed elutions in reversed-phase high-performance liquid chromatography

Computer-assisted procedures were used to simulate modifications in chromatograms caused by the transfer of elution programmes between instruments with significantly different dwell volumes. Moreover, for the first time the same approach was used to modify the elution programmes to match the chromatograms produced in the different instruments. The process may consist of making minor modifications to gradient programmes or transforming the original gradient programme into a stepwise gradient profile and/or the simultaneous programming of flow and solvent composition. The combination of these approaches has been shown to have an enormous potential for producing matched chromatograms in instrumental systems with dwell volumes that differ by several millilitres. The efficiency and robustness of the proposed procedure is demonstrated with a variety of compounds (two different mixtures of 10 and 11 analytes), mobile phases (methanol and acetonitrile gradients), flow rates (0.5-1.5 mL/min range), temperatures (35-45 degrees C interval) and gradient profiles (linear, multilinear, curved and stepwise).     

Adsorption equilibria of proline in hydrophilic interaction chromatography

The adsorption behavior of proline under hydrophilic interaction chromatography conditions was investigated from six aqueous solutions of acetonitrile. Proline adsorption isotherms were recorded at each mobile phase composition by frontal analysis and inverse method. The BET model was found to be the best choice to describe the nonlinear behavior of proline adsorption under hydrophilic interaction chromatography conditions. The adsorption isotherm parameters were derived from two independent parameter estimation methods. The parameters derived from regression analysis of the frontal analysis data and from overloaded elution bands were found to be in good agreement with the excess isotherm of water. The mobile phase composition at which the maximum excess adsorption of water was observed corresponded to the maximum saturation capacity measured for proline.     

Temperature-responsive chromatography using poly(N-isopropylacrylamide) hydrogel-modified silica.

Poly(N-isopropylacrylamide) (PNIPAAm) has the sharpest phase transition of the class of thermo-sensitive N-alkyl acrylamide polymers. We developed a new method of HPLC using packing materials modified with cross-linked poly(N-isopropylacrylamide) (PNIPAAm) hydrogel. A temperature-responsive surface was prepared by polymerization of NIPAAm in the presence of a cross-linker on the silica support. The surface properties and functions of the stationary phases change in response to the external temperature. Therefore it easily changes the interaction of a solute with the surface with a constant aqueous mobile phase. A temperature-responsive elution behavior was observed on the separation of steroids and PTH-amino acids. The method is expected to be applicable to separation in the pharmaceutical and biomedical fields.     

Determination of alcohols by high-performance liquid chromatography with fluorimetric detection after pre-column derivatisation with carbazole-9-N-acetic acid and chromatographic behaviour of alcoholic derivatives

Alcohols were derivatised to their carbazole-9-N-acetic acid (CRA) esters with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) as the dehydrating agent. Studies on derivatisation conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives. The retention behaviour of alcohol derivatives was investigated by varying mobile phase compositions (ACN-water and MeOH-water). The parameters from the equation log k'=A-BX were evaluated by retention data of derivatives using an isocratic elution with different mobile phases. The results indicated that the parameters derived allowed computation of retention factors in good agreement with experiments. At the same time, a general equation was derived that makes possible predictions of partition coefficient in binary mobile phases with different proportions of organic solvent to water based on some simple regression analysis. The LC separation for the derivatised alcohols containing higher carbon alcohols showed good reproducibility on a reversed-phase C18 column with gradient elution. The detection limits (excitation at 335 nm, emission at 360 nm) for derivatised alcohols (signal-to-noise ratio=3:1) were in the range of 0.1-0.4 pg per injection.     

Micellar and aqueous‐organic liquid chromatography using sub‐2 μm packings for fast separation of natural phenolic compounds

The objective of the present work was to investigate the chromatographic behavior of natural phenolic compounds in micellar and aqueous‐organic LC using a short column packed with 1.8 μm particles. Firstly, the effect of ACN and SDS on elution strength and selectivity was examined by isocratic submicellar (0–30% ACN/5% 1‐butanol/1–6 mM SDS) and micellar (0–30% ACN/5% 1‐butanol/40–60 mM SDS) systems. The varied concentrations of two modifiers in the mobile phases revealed different eluting power. Then, the application of organic modifier gradient was discussed in both submicellar and micellar LC using mobile phases of 4 mM SDS/5% 1‐butanol or 50 mM SDS/5% 1‐butanol containing ACN gradient from 0 to 30%, respectively. For micellar system, the separation was found to be better in gradient than isocratic elution. Additionally, the sensitivity of aqueous‐organic LC was examined. The mobile phase was a mixture of ACN and water employing gradient elution at a flow rate of 0.5 mL/min, with analysis time below 9 min. It was found that separation efficiency was significantly better compared with micellar LC. Besides, the aqueous‐organic LC has been applied to separation of various phenolic compounds in Yangwei granule or Radix Astragali samples.