Eluotropic Strength Research Articles

Ultra-trace-level determination of polar pesticides and their transformation products in surface and estuarine water samples using column liquid chromatography–electrospray tandem mass spectrometry

A method is developed for the determination of polar pesticides and their transformation products [atrazine, deethylatrazine, deisopropylatrazine, hydroxyatrazine, diuron, 3,4-dichlorophenylmethylurea, 3,4-dichlorophenylurea (DPU), monuron, bentazone, anthranil-isopropylamide, chloridazon, metolachlor] in surface, estuarine and sea water samples at the low ng/l level. Solid-phase extraction is combined off-line with column liquid chromatography–electrospray ionization tandem mass spectrometric detection (LC–ESI-MS–MS). The applicability of two solid-phase materials, i.e., LiChrolut EN cartridges and graphitized carbon black extraction disks, is evaluated. The influence of the organic solvent used in gradient LC, as well as the amount of co-extracted humic material on the ESI process is studied. The eluotropic strength of the organic solvent was found to have a distinct effect on the sensitivity of ESI-MS if coupled with LC gradient separations. Methanol gave much better results than acetonitrile and phenylurea compounds are more susceptible to solvent changes than triazines. Co-extracted humic material causes signal suppression in ESI-MS–MS detection. The degree of suppression depends upon the sample pH and the nature of the samples, i.e., surface or estuarine water. Detection limits in LC–ESI-MS–MS ranged from 0.2 to 2 ng/l, with the exception of DPU (8 ng/l). The applicability of the procedure was demonstrated by analyzing surface and estuarine water.

Methylene Selectivity and Eluotropic Strength Variations in Subcritical Fluid Chromatography with Packed Columns and CO2-Modifier Mobile Phases

This work has consisted of better understanding the chro-matographic behavior in subcritical fluid chromatography with CO(2)-acetonitrile and CO(2)-methanol mobile phases. To that end, the changes of solute solubility in the mobile phase have been studied by determination of methylene selectivity of a homologous series. Moreover, eluotropic strengths have been calculated for mobile phases containing from 5 to 45% modifier. To carry out the studies in all ranges of the subcritical area, experimental designs have been used. The influences of analytical temperature (from 10 to 30 °C) and outlet pressure (from 10 to 20 MPa) have also been investigated both on solubility and on eluotropic strength. A more general comparison of elu-otropic strengths of subFC and HPLC has been achieved.

Influence of mobile phase composition on methylene group selectivity and homologous compound retention in near-critical mobile phases

A number of near-critical mobile phases were evaluated with respect to methylene group selectivity and overall eluotropic strength on a polymeric column. Binary mobile phases, consisting of mixtures of carbon dioxide and 1,1,1,2-tetrafluoroethane (HFC-134a), showed strong temperature and compositional dependence on both retention and selectivity. Binary mobile phases, consisting of mixtures of carbon dioxide and conventional modifiers such as alcohols, acetonitrile and tetrahydrofuran, also showed a strong temperature dependence on methylene group selectivity and relative retention. In contrast to the behavior observed using a polymeric column, a bonded silica-based stationary phase displayed significant deviations from the Martin rule, even with high order homologs. Of all the systems evaluated in this study, a binary mixture of carbon dioxide and 2-methoxyethanol proved to be optimal for obtaining a high degree of methylene group selectivity and strong eluotropic strength.

Comparison of 1,1,1,2-tetrafluoroethane and carbon dioxide-based mobile phases for packed column supercritical fluid chromatography

Highly polar, homogeneous mobile phases have a number of advantages over carbon dioxide-based mixed mobile phases for supercritical fluid chromatography. While these fluids have been demonstrated to have very high eluotropic strengths in capillary systems, their utility for packed columns lies in their ability to overcome the deleterious effects of residual silanol groups present on the bonded-phase surface. A particularly promising fluid, 1,1,1,2-tetrafluoroethane (HFC-134a), is characterized with respect to eluotropic strength, selectivity, efficiency and temperature effects. These results are compared to those obtained with carbon dioxide and methanol-modified carbon dioxide mobile phases on a silica column. HFC-134a displays different selectivities, efficiencies, eluotropic strength and temperature dependencies on retention than either of the two reference mobile phases.

Temperature effects for chiral separations using various bulk fluids in near‐critical mobile phases

As supercritical fluid chromatography becomes more accepted as a facile means for the separation of chiral compounds, the need for mobile phases that can readily solubilize these polar compounds grows. Prior studies suggest that HFC-134a may prove suitable due to its very high eluotropic strength compared to carbon dioxide-based mobile phases. A comparison is made between ethanol-modified carbon dioxide, HFC-134a, and decafluoropentane as to their relative eluotropic strength, selectivity, and efficiency for three chiral compounds using a Whelk O-1 chiral bonded phase. The bulk component of the mobile phase was found to have relatively little effect on chiral selectivity over the range of 5° to 95°C. Chirality 9:693–698, 1997. © 1997 Wiley-Liss, Inc.

Temperature effects for chiral separations using various bulk fluids in near-critical mobile phases

As supercritical fluid chromatography becomes more accepted as a facile means for the separation of chiral compounds, the need for mobile phases that can readily solubilize these polar compounds grows. Prior studies suggest that HFC-134a may prove suitable due to its very high eluotropic strength compared to carbon dioxide-based mobile phases. A comparison is made between ethanol-modified carbon dioxide, HFC-134a, and decafluoropentane as to their relative eluotropic strength, selectivity, and efficiency for three chiral compounds using a Whelk O-1 chiral bonded phase. The bulk component of the mobile phase was found to have relatively little effect on chiral selectivity over the range of 5° to 95°C. Chirality 9:693–698, 1997. © 1997 Wiley-Liss, Inc.

Affinity purification of phospholipase A2 on immobilized artificial membranes containing and lacking the glycerol backbone.

Immobilized artificial membranes (IAMs) are chromatography surfaces containing monolayers of phospholipid ligands. etherIAM.PCC10/C3 contains the glycerol backbone whereas delta GIAM.PCC10/C3 lacks the glycerol backbone. Affinity purification of PLA2 on these IAM surfaces demonstrated that the surface structural differences were not important for phospholipase A2 (PLA2) binding. This suggests that the chromatographically important binding event involves the PLA2 surface and the monolayer of polar choline headgroups on the IAM surface. After sample loading, short-chain alkylsulfonates were used as low eluotropic strength detergents to remove contaminating proteins, and PLA2 were eluted with CH3CN (30%). Octyllysophosphatidylcholine (0.5%) can replace CH3CN to elute PLA2 from IAM surfaces. The PLA2 purity after IAM chromatography depends on the protein loading; analytical-scale loadings (0.8 mg protein/g IAM) resulted in a PLA2 purity of ca. 70% based on densitometric scans of proteins in polyacrylamide gels after electrophoresis. Preparative loadings of 3.21 mg protein/g IAM resulted in 48% PLA2 purity. Purification of PLA2 to electrophoretic homogeneity was achieved using an IAM column followed by a strong anion-exchange column. These results suggests that IAMs may be used to develop purification methods for PLA2 enzymes obtained from diverse biological specimens.

Effect of Various Modifiers on Selectivity in Packed-Column Subcritical and Supercritical Fluid Chromatography

A number of common mobile phase modifiers were characterized by chromatographing monofunctional probe solutes on a cyano-bonded stationary phase under subcritical and supercritical conditions. Solvatochromic analysis techniques were used to characterize the relative selectivity, efficiency, and eluotropic strengths of the various modified mobile phases. Analysis of the combined results suggests that modifier choice may be made in a rational manner to either enhance or mask a particular type of molecular interaction. This control is necessary for challenging applications such as chiral separations. The relative effects of each modifier property change depending on whether the chromatographic system is subcritical or supercritical, thus making knowledge of the phase equilibria associated with the mobile phase essential.

The optimization of step-gradient elution conditions in liquid chromatography: Application to pesticide residue analysis using coupled-column reversed-phase LC

A computer spreadsheet application has been developed for the optimization of step-gradient elution conditions as applied in coupled-column RPLC for online clean-up and separation in the analysis of pesticide residues. The procedure is based on the experimentally determined retention behaviour of the analytes as a function of mobile phase composition. Retention and peak volumes of the analytes eluting under isocratic and one- and/or two-step gradient conditions can be calculated. For a given set of analytes, the spreadsheet systematically calculates the resolution of the least efficiently separated pair of peaks in the space defined by the eluotropic strengths of the mobile phases and the time at which the step-gradient takes place. The performance of the proposed procedure was tested and evaluated for the analysis of nine polar pesticides for which in a previous study adequate conditions had been found by computer-aided chromatogram simulation. the optimum conditions predicted by the procedure were in good agreement with those established experimentally and by simulation. The simultaneous production of data on retention, peak volume and resolution for a group of analytes under selected one- and/or two step-gradient elution conditions makes the calculation procedure highly productive and flexible in searching for and/or adjusting appropriate separation conditions.

Mobile phase effects on membrane protein elution during immobilized artificial membrane chromatography

The eluotropic strength of different mobile phases for eluting membrane proteins from immobilized artificial membrane (IAM) chromatrography surfaces was studied. Two protein mixtures containing bovine pancreatic PLA 2 were used in this study. Protein mixture I was PLA 2 obtained from Sigma which contained ∼5–10 major protein bands in electrophoretic gels. Protein mixture II was obtained from fresh bovine pancreatic tissue and contained >100 proteins including the target protein, PLA 2. After adsorbing Sigma PLA 2 to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA 2. Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during ion-exchange chromatography, (iii) salt conditions used during hydrophobic interaction chromatography, (iv) acetonitrile gradients used during reversed-phase chromatography, and (v) a two-step gradient consisting of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels, PLA 2 from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA 2 from protein mixture II was purified in one step (660-fold increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70–100%.

Improved chiral stationary phase for ß-blocker enantioseparations

The previously described α-Burke 1 chiral stationary phase (CSP) was designed for the chromatographic separation of the enantiomers of ß-blockers. Difficulties with the reproducibility of the free radical addition reaction, used in the attachment of the chiral selector to the chromatographic support, have required the development of an alternative silane immobilization process (α-Burke 2 CSP). While the enantioselectivity afforded by this new CSP is generally equivalent to that of the original CSP, the α-Burke 2 CSP demonstrates longer analyte retention, necessitating the use of mobile phases of greater eluotropic strength. The increased retention of the new CSP presumably results from a greater surface density of functional selectors, an interpretation which is supported by the observation that the preparative capacity of the α-Burke 2 CSP is greater than that of the original. Some of the factors influencing the retention and separation of a group of 23 ß-blockers on the α-Burke 2 CSP are discussed.

Hydrofluorocarbon and perfluorocarbon mobile phases for capillary supercritical fluid chromatography

AbstractThe eluotropic strength of several hydrofluorocarbon and perfluorocarbon supercritical fluids is examined using capillary supercritical fluid chromatography. The results obtained are compared with carbon dioxide, fluoroform, and carbon dioxide modified with 5% methanol. Various naphthalene derivatives are used to determine differences in functional group selectivity between these fluids. Specific hydrogen bonding interactions are observed between hydrogen bond donating hydrofluorocarbon fluids and naphthalene derivatives with hydrogen bond accepting functional groups. Unexpectedly, perfluorocarbon fluids are much stronger in eluotropic strength than carbon dioxide, fluoroform, or methanol‐modified carbon dioxide.

Functional group selectivity using fluoroform as a mobile phase for supercritical fluid chromatography

AbstractThe eluotropic strenght and chromatographic selectivity of fluoroform is compared to that of carbon dioxide and methanol‐modified carbon dioxide. Naphthalene derivatives were used as probe solutes in capillary supercritical fluid chromatography to determine the effects of fluid type, pressure, and temperature on selectivity and retention. Fluoroform is much stronger in eluotropic strengh than carbon dioxide or methanol‐modified carbon dioxide at the same temperature and pressure. Under the same reduced conditions, fluoroform is lower in eluotropic strength than carbon dioxide. Specific interactions between hydrogen bond accepting functional groups and fluoroform are observed. Functional groups capable of accepting hydrogen bonds contribute less of an increase in retention when using fluoroform while those capable of dispersive interactions contribute less of an increase in retention when using carbon dioxide.

Comparison of the elution profiles of a weakly basic drug on cyanopropyl-bonded silica cartridges from different manufacturers and different batches

A comparison is made of the recoveries obtained for the solid-phase extraction (SPE) of an aqueous 1 μg ml -1 solution of papaverine on nine different batches of cyanopropyl-bonded silica (CN) phases from three different suppliers. The elution profile with up to 3 ml of methanol or methanol containing 0.1% propylamine was set up, whereby intra- and inter-day variations were taken into account. For each volume level and both eluents, significant differences could be demonstrated between the mean recoveries obtained on different batches of CN sorbents. If the volume of methanol used for the elution is increased, or if the eluotropic strength is increased by addition of propylamine, then the differences in mean recoveries are smaller. The nine sorbents could be divided into three groups according to the relative importance of the apolar dispersion forces and the ionic interactions for the adsorption of papaverine. In addition to between-batch and between-manufacturer variations, the SPE method with 1 ml of 0.1% propylamine in methanol as eluent yielded differences in recovery of ±10% from day to day.

Application of the split-peak effect to study the adsorption kinetics of human serum albumin on a reversed-phase support

The adsorption step of human serum albumin on a reversed-phase support was analyzed by studying the “split-peak” effect in mass-overload conditions. This behavior is characterized by the occurrence of a first non-retained fraction and is described by an analytical expression in the case of a Langmuirian adsorption isotherm. The method was applied to determine the column loading capacity, the number of mass-transfer units and the apparent adsorption rate constant measured at a given flow-rate. The nature of the organic modifier influences the split-peak effect: it increases with the eluotropic strength of the organic solvent added to the buffer. Compared to the results with pure buffer, it is the association of two effects, the decrease of the column loading capacity and that of the apparent adsorption rate constant, which increases the split-peak effects observed when methanol and 2-propanol are added to the eluent. These results allow us to gain a better understanding of the role of the organic solvent in the elution behavior of proteins in reversed-phase high-performance liquid chromatography.

Practical use of an optimization strategy in reversed-phase high-performance liquid chromatography for the separation of a limited subset of components in a reaction mixture

A step-by-step development of a high-performance liquid chromatographic method for the optimized separation of a largely unknown sample is presented. A limited subset of two main components is separated from a ten-component reaction mixture of pyrroloquinoline quinone and cyclopropanol, using tetrahydrofuranwater binary eluents buffered to pH 2.5 with triethylamine phosphate on a reversed-phase column packed with 5-μm ODS-Hypersil. After the definition of the separation problem, the mobile phase parameters are selected rationally on the basis of a systematic gradient scouting procedure. The optimization search area is defined in accordance to the complexity of the sample mixture (two out of ten components are of interest) by utilizing a statistical approach, while the selectivity optimization is carried out using binary eluents of variable eluotropic strength.

Interpretation of normal-phase solvent strength scales based on linear solvation energy relationships using the solvatochromic parameters π, α and

An analysis of normal-phase solvent eluotropic strength (ε0) and solute adsorption energy (S0) was carried out by use of linear salvation

Comparison of retention mechanisms of homologous series and triglycerides in non-aqueous reversed-phase liquid chromatography

The retention behaviour of saturated homogeneous triglycerides was compared to that of single chain homologous compounds in reversed-phase liquid chromatography on silica-based octadecyl bonded phases with non-aqueous binary eluents containing chloroform. For both series, in all eluents and at all investigated temperatures, a discontinuity (1–6% slope change) in log k′ vs. the carbon atom number, n c, of each solute hydrocarbonaceous chain was observed for a given critical number of carbon atoms, n c, crit ≈ 12–13. Similar discontinuities were observed in Δ H° and Δ S° vs. n c for the same n c, crit value. These and other phenomena (existence of two convergence points for log k′ vs. n c plotted at different eluent compositions; existence of two convergence temperatures in log k′ vs. 1/T plotted for several members of a given solute series) reflect the mechanism of penetration of the solute aliphatic chains into the bonded layer. The eluotropic strength of acetonitrile—chloroform mobile phases was determined and seen to have a larger rate of variation with the solvent composition than that of some other non-aqueous binary eluents. The ratio of the slope of plots of log k′ vs n c curves for triglycerides and single chain homologous series is not a whole number but has a value lying between 2 and 3. This explains why triglyceride retentions cannot be predicted from retention data for the fatty acid methyl esters. More importantly, this indicates that the triglycerides may interact with the stationary phase using various conformations, one, two or three chains penetrating into the bonded layer. These conformations, in dynamic equilibrium with each other, contribute differently to the retention. This offers, in principle, the possibility to separate unsaturated triglycerides having the same number of carbon atoms and of double bonds but differing in the distribution of the unsaturations along the chains, if the double bonds located in a non-penetrating chain can selectively interact with a mobile phase component.

Prediction of high-performance liquid chromatography capacity factors following column calibration : Influence of different solvents and columns on the separation of a series of aromatic amides

The rate of change of log k′ (where k′ is the capacity factor) with the percentage organic modifier in the mobile phase was determined for acetanilide and 25 substituted acetanilides. A strongly positive correlation ( r > 0.989) was found between the slope and y-intercept values for each compound when methanol or tetrahydrofuran were used as organic modifiers on both Spherisorb ODS-2 and LiChrosorb RP-18 columns. Using the slope and intercept data for acetanilide and the 4-substituted methyl, ethyl and propyl amides, and a k′ value measured for each of the 26 compounds at a single mobile phase composition, we were able to predict the slopes and intercepts of all the compounds studied, and thus their elution behaviour. The predicted data were compared with the slopes and log k 0 values calculated from the experimental results ( k 0 is the capacity factor that would be predicted in the absence of an organic modifier) and indicated that calibration of a column in this manner would allow accurate prediction of the chromatographic behaviour of a solute. Prediction of the percentage of organic modifier required in the mobile phase to achieve a particular k′ value was possible to within 3.5% or less of that actually found to be necessary. When acetonitrile was used as the organic modifier, curvilinear plots of log k′ were obtained for all compounds. The elution behaviour of the 4-bromo, 4-chloro, 4-nitro and 4-chloroaceto compounds revealed selectivity differences between methanol and tetrahydrofuran, suggesting that direct replacement of one organic modifier by another of similar eluotropic strength is not always viable.

The Surface Tension of Mixtures of Methanol, Acetonitrile, Tetrahydrofuran, Isopropanol, Tertiary Butanol and Dimethyl-Sulfoxide with Water at 25°C

Abstract Surface tension data of binary mixtures of methanol, acetonitrile, tetrahydrofuran, isopropanol, tertiary butanol and dimethylsulfoxide with water have been measured. Best fit polynomal functions are reported for in [sgrave] vs. volume fraction of an organic modifier. The reported data is precise and accurate to about 2% and is consistent with the available literature data. The sequence of eluotropic strength based on the solvophobic theory in RP-HPLC should be dimethylsulfoxide < methanol < acetonitrile <THF (tetrahydrofuran) <IPA (isopropanol) < tertiary butanol based on the results of this study.