Solvent Modifier Research Articles

Solubility of the diarrheic shellfish toxin okadaic acid in supercritical CO2

AbstractThe solubility of okadaic acid (OA) in supercritical CO2 was measured using a flow‐type apparatus with sequential sampling during dynamic nonrecirculating experiments at saturation conditions. Methanol and water were used as solvent modifiers of CO2. Collected OA was measured by high‐performance liquid chromatography with fluorimetric detection after derivatization with 1‐bromoacetylpyrene to obtain the labeled ester of the toxin. Solubility results were obtained with methanol concentrations ranging from 0 to 8.5% volume in the CO2 density range of 0.495 to 0.913 g/mL at 40, 60, and 73°C. Measured solubility of OA ranged from 0 to 15×10−6 mol/L, increasing with methanol concentration and fluid density and diminishing with temperature. Experiments with water‐modified CO2 up to 0.3% volume (near saturation) were done at 60°C; solubilities of OA up to 5×10−6 mol/L were measured. This is the first approach to handle the liposoluble diarrheic shellfish toxins with supercritical CO2. The study, with pure OA, provides useful information regarding the effects of pressure, temperature, and addition of modifiers on its solubility. Obtained results show that the toxin can be solubilized in this media and potential applications are suggested and being currently investigated.

Characterizing the selectivity of stationary phases and organic modifiers in reversed-phase high-performance liquid chromatographic systems by a general solvation equation using gradient elution.

Retention data for a set of 69 compounds using rapid gradient elution are obtained on a wide range of reversed-phase stationary phases and organic modifiers. The chromatographic stationary phases studied are Inertsil (IN)-ODS, pentafluorophenyl, fluoro-octyl, n-propylcyano, Polymer (PLRP-S 100), and hexylphenyl. The organic solvent modifiers are 2,2,2-trifluoroethanol (TFE); 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP); isopropanol; methanol (MeOH); acetonitrile (AcN); tetrahydrofuran; 1,4-dioxane; N,N-dimethylformamide; and mixed solvents of dimethylsulfoxide (DMSO) with AcN and DMSO with MeOH (1:1). A total of 25 chromatographic systems are analyzed using a solvation equation. In general, most of the systems give reasonable statistics. The selectivity of the reversed phase-high-performance liquid chromatographic (HPLC) systems with respect to the solute's dipolarity-polarity, hydrogen-bond acidity, and basicity are reflected in correspondingly large coefficients in the solvation equation. We wanted to find the most orthogonal HPLC systems, showing the highest possible selectivity difference in order to derive molecular descriptors using the gradient retention times of a compound. We selected eight chromatographic systems that have a large range of coefficients of interest (s, a, and b) similar to those found in water-solvent partitions used previously to derive molecular descriptors. The systems selected are IN-ODS phases with AcN, MeOH, TFE, and HFIP as mobile phase, PLRP-S 100 phase with AcN, propylcyano phase with AcN and MeOH, and fluorooctyl phase with TFE. Using the retention data obtained for a compound in the selected chromatographic systems, we can estimate the molecular descriptors with the faster and simpler gradient elution method.

Solubility of purified lutein diesters obtained from Tagetes erecta in supercritical CO(2) and the effect of solvent modifiers.

Purified lutein diesters deposited on commercial nonporous glass beads were solubilized in supercritical CO(2) in a computerized batch extractor, and their solubilities were compared to their solubilities in hexane. Densities of 0.7, 0.8, and 0.9 g/mL were evaluated without modifiers. Both pressure and temperature increased solubility, although temperatures >50 degrees C promoted carotenoid loss as determined by mass balance. Solubility was enhanced by the use of modifiers and was related to their log P. Chloroform (log P = 2) increased 2.8 times the amount of solubilized lutein diesters compared to pure CO(2) at the same extraction conditions (0.9 g/mL and 40 degrees C) to yield 65% of the amount extracted with hexane. Supercritical CO(2) extraction of lutein diesters could represent a cleaner technology as compared to the current industrial use of hexane with important ecological and health-related implications.

Improving the recovery of ionic solutes from aqueous media by modified thermospray liquid-liquid extraction

Our previously reported procedure for the extraction of charged compounds from aqueous samples by thermospray liquid-liquid extraction (TSLLE) was essentially a one-step extraction involving large sample volumes. In this report, recirculative extraction, analysis of small sample volumes, the halide ion effect, and the effect of solvency or solvent modification on the extraction efficiency of benzoic acid (BA) by TSLLE were investigated. Compared to the one-step procedure that resulted in an extraction efficiency of only 28% for BA in n-hexane, recirculative TSLLE resulted in a BA recovery of 65% after five extraction cycles. When applied to sample volumes of 5-10 mL, TSLLE extracted BAwith a precision of 2.8-6.1%. NaF, NaCl, and NaBr were also used to enhance analyte recovery. NaF gave the best recovery, 104%, for BA relative to the 88% obtained by batch processing. Some improvements in the extraction efficiency was observed when solvent modifiers such as methanol, ethanol, and 2-propanol were used.

Comparison of the enantioseparation of racemic uridine analogs on Whelk-O 1 and ChiralPak-AD columns

The commercially available, brush-type (S,S)-Whelk-O 1 chiral stationary phase (CSP) has been used to separate 10 racemates of structurally related uridine analogs, potentially anti-viral agents, under various mobile phase compositions, using various temperatures. The enantioseparation was evaluated by comparing the Whelk-O 1 column performance with that of ChiralPak-AD column, reported previously. The comparison involved the role of some distinctive structural features of the racemates, type and composition of the solvent modifiers, as well as effect of temperature on the chiral discrimination. Despite the fact that both columns separate almost all the uridine analogs, significant differences were observed in their chiral recognition, as revealed from their retention, selectivity, resolution and elution order. The chiral recognition processes, responsible for enantioseparation on the Whelk-O 1 column, were relatively more systematic and easier to manipulate than on ChiralPak-AD column. Enantioseparation on the latter are of more complex nature and frequently gave results that were contradictory to the expectations. On the other hand, the performance in the ChiralPak-AD column was superior to that of the Whelk-O 1column. Limitations in column handling and maintenance (pressure and temperatures) as well as limited solvent choice lead to the preference of the Whelk-O 1 column, in spite of its lower (but adequate) performance.

Reversed phase liquid chromatography with UV absorbance and flame ionization detection using a water mobile phase and a cyano propyl stationary phase: Analysis of alcohols and chlorinated hydrocarbons

The development of liquid chromatography with a commercially available cyano propyl stationary phase and a 100% water mobile phase is reported. Separations were performed at ambient temperature, simplifying instrumental requirements. Excellent separation efficiency using a water mobile phase was achieved, for example N=18 800, or 75 200 m −1, was obtained for resorcinol, at a retention factor of k′=4.88 (retention time of 9.55 min at 1 ml min −1 for a 25 cm×4.6 mm i.d. column, packed with 5 μm diameter particles with the cyano propyl stationary phase). A separation via reversed phase liquid chromatography (RP-LC) with a 100% water mobile phase of six phenols and related compounds was compared to a separation of the same compounds by traditional RP-LC, using octadecylsilane (ODS), i.e. C18, bound to silica and an aqueous mobile phase modified with acetonitrile. Nearly identical analysis time was achieved for the separation of six phenols and related compounds using the cyano propyl stationary phase with a 100% water mobile phase, as compared to traditional RP-LC requiring a relatively large fraction of organic solvent modifier in the mobile phase (25% acetonitrile:75% water). Additional understanding of the retention mechanism with the 100% water mobile phase was obtained by relating measured retention factors of aliphatic alcohols, phenols and related compounds, and chlorinated hydrocarbons to their octanol:water partition coefficients. The retention mechanism is found to be consistent with a RP-LC mechanism coupled with an additional retention effect due to residual hydroxyl groups on the cyano propyl stationary phase. Advantages due to a 100% water mobile phase for the chemical analysis of alcohol mixtures and chlorinated hydrocarbons are reported. By placing an absorbance detector in-series and preceding a novel drop interface to a flame ionization detector (FID), selective detection of a separated mixture of phenols and related compounds and aliphatic alcohols is achieved. The compound class of aliphatic alcohols is selectively and sensitively detected by the drop interface/FID, and the phenols and related compounds are selectively and sensitively detected by absorbance detection at 200 nm. The separation and detection of chlorinated hydrocarbons in a water sample matrix further illustrated the advantages of this methodology. The sensitivity and selectivity of the FID signal for the chlorinated hydrocarbons are significantly better than absorbance detection, even at 200 nm. This methodology is well suited to continuous and automated monitoring of water samples. The applicability of samples initially in an organic solvent matrix is explored, since an organic sample matrix may effect retention and efficiency. Separations in acetonitrile and isopropyl alcohol sample matrices compared well to separations with a water sample matrix.

Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments

The isocratic and gradient elution behaviour of β-endorphin and glucagon, two polypeptides known to exist in amphipathic α-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo–acetonitrile mobile phases. The effects of changes in the volume fraction, ψ, of the organic solvent modifier and temperature, T, on the magnitudes of the S and logko values of these two polypeptides, obtained from the plots of logarithmic capacity factor (logk ́) vs. ψ using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and logko values, obtained from the plots of logarithmic median capacity factor (logk) versus the median volume fraction of the organic solvent modifier ( ψ) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo–acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the logko and logko values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for β-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of logk vs. 1/T (corresponding to an apparent change in the median enthalpy of association, ΔHassoco, or an apparent change in the median entropy of association, ΔSassoco) substantially deviated from the values obtained for the thermodynamic parameters, ΔHassoco and ΔSassoco, derived from the logk′ vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.

Recent advances in HPLC pigment analysis of phytoplankton

Analysis of phytoplankton pigments is central to studies of marine ecology and climate research. This paper summarizes milestones in the development of methods of pigment analysis, and shows the use of HPLC technology in their verification. New advances in HPLC methods are discussed, with key developments being the use of polymeric C18 and monomeric C8 columns and pyridine as solvent modifier. These have allowed the resolution of divinyl chlorophylls a and b, and the discovery of new chlorophyll c pigments (both polar and non-polar) and new 4-keto fucoxanthin derivatives. The taxonomic value of pigments and pigment suites is examined. Methods of interpreting the percentage of algal types from field measurements of pigment ratios through the use of computer algorithms are discussed. Finally, prospects for future development are suggested.

Supercritical fluid extraction of organochlorine pesticides in eggs.

The efficacy of supercritical fluid extraction (SFE) for the recovery of 16 common organochlorine pesticides (OCPs) from liquid whole eggs was investigated by employing supercritical carbon dioxide (SC-CO(2)) without the use of a solvent modifier to minimize interfering coextractives. The OCPs tested included aldrin; alpha-, beta-, delta-, and gamma-BHCs; p,p'-DDD, -DDE, and -DDT; dieldrin; endosulfans I, II, and sulfate; endrin; endrin aldehyde; heptachlor; and heptachlor epoxide. The SFE conditions were as follows: 10000 psi (680 bar), 40 degrees C, SC-CO(2) flow rate of 3.0 L/min with an extraction time of 40 min for a total of 120 L of CO(2). The OCPs were trapped off-line in an SPE cartridge containing Florisil and then eluted by an acetone/hexane mixture and analyzed by gas chromatography-electron capture detection (GC-ECD). Recovery studies were carried out on homogenized eggs fortified at the 0.05, 0.10, and 0.20 ppm levels. At the lowest level, 0.05 ppm, recoveries ranged from 81.8 to 108.3%, with CVs < 9.8%. All recoveries were significantly higher than those obtained by an AOAC/FDA solvent extraction method. Eggs containing incurred endosulfan I were also effectively extracted by SFE. This study suggests that the application of SFE for the extraction of OCPs from eggs will result in significant savings in analysis time and lower solvent use and disposal costs compared to conventional solvent extraction procedures.

Supercritical fluid carbon dioxide extraction of α-and β-carotene from carrot (Daucus carota L.)

Carrots (Daucus carota L. var. Caro Pride) were extracted with supercritical fluid carbon dioxide (SFOC2) under various combinations of pressures and solvent modifiers at 40°C and analysed for their α- and β-carotene content by high pressure liquid chromatography. The SFCO2 extraction at 40°C and 60.6 MPa with 5% chloroform as modifier afforded 111.16 and 148.32 μg of α- and β-carotene per gram of dried carrot, respectively. Also, this method extracted 92.70% of the total carotenoids present in the dried carrots when compared to the solvent extraction using chloroform (100%). © 1997 John Wiley & Sons, Ltd.

Simultaneous Flame Ionization and Absorbance Detection of Volatile and Nonvolatile Compounds by Reversed-Phase Liquid Chromatography with a Water Mobile Phase

A flame ionization detector (FID) is used to detect volatile organic compounds that have been separated by water-only reversed-phase liquid chromatography (WRP-LC). The mobile phase is 100% water at room temperature, without use of organic solvent modifiers. An interface between the LC and detector is presented, whereby a helium stream samples the vapor of volatile components from individual drops of the LC eluent, and the vapor-enriched gas stream is sent to the FID. The design of the drop headspace cell is simple because the water-only nature of the LC separation obviates the need to do any organic solvent removal prior to gas phase detection. Despite the absence of organic modifier, hydrophobic compounds can be separated in a reasonable time due to the low phase volume ratio of the WRP-LC columns. The drop headspace interface easily handles LC flows of 1 mL/min, and, in fact, compound detection limits are improved at faster liquid flow rates. The transfer efficiency of the headspace interface was estimated at 10% for toluene in water at 1 mL/min but varies depending on the volatility of each analyte. The detection system is linear over more than 5 orders of 1-butanol concentration in water and is able to detect sub-ppb amounts of o-xylene and other aromatic compounds in water. In order to analyze volatile and nonvolatile analytes simultaneously, the FID is coupled in series to a WRP-LC system with UV absorbance detection. WRP-LC improves UV absorbance detection limits because the absence of organic modifier allows the detector to be operated in the short-wavelength UV region, where analytes generally have significantly larger molar absorptivities. The selectivity the headspace interface provides for flame ionization detection of volatiles is demonstrated with a separation of 1-butanol, 1,1,2-trichloroethane (TCE), and chlorobenzene in a mixture of benzoic acid in water. Despite coelution of butanol and TCE with the benzoate anion, the nonvolatile benzoate anion does not appear in the FID signal, allowing the analytes of interest to be readily detected. The complementary selectivity of UV-visible absorbance detection and this implementation of flame ionization detection allows for the analysis of volatile and nonvolatile components of complex samples using WRP-LC without the requirement that all the components of interest be fully resolved, thus simplifying the sample preparation and chromatographic requirements. This instrument should be applicable to routine automated water monitoring, in which repetitive injection of water samples onto a gas chromatograph is not recommended.

Effect of organic solvent modifier and nature of solute on the performance of bonded silica reversed-phase columns for the analysis of strongly basic compounds by high-performance liquid chromatography

The performance of eight silica-based RP-HPLC columns was examined with nine relatively high-p K a bases of different structure using isoeluotropic mixtures of either methanol, acetonitrile or tetrahydrofuran (THF) in combination with phosphate buffer pH 7.0. Meaningful evaluation of the columns in unbuffered methanol-water and acetonitrile-water mixtures could only be obtained for low-p K a bases, probably due to variable ionisation effects. Buffered acetonitrile generally produced worst peak shapes for the nine higher-p K a bases; further improvement may be gained in some cases using THF rather than methanol. Although in general high-p K a solutes with reduced steric hindrance gave the most asymmetric peaks, the nature of the solute chosen to probe activity could give considerably different results of relative column performance when using a given mobile phase.

Adaptation of high-performance liquid chromatographic methods to multiple spectral analyses

Adapting well-characterized existing liquid chromatographic separations for compatibility with mass spectrometric detection often requires changes in chemical and/or chromatographic conditions. Non-volatile solvent modifiers, such as phosphates or alkylsulfonates, have been widely used in chromatography because of their buffer or ion-pairing capacities, coupled with advantageous low-UV-wavelength absorption characteristics. Non-volatile components in the eluent stream are generally avoided in mass spectrometry as they quickly decrease the detector response. In addition to issues such as buffer capacity and effective ion-pairing, consideration should be given to the impact of stationary phase and column geometry. The work presented here illustrates advantages of a new high-purity, high-pore-volume stationary phase used in Waters Symmetry columns when attempting to accommodate a wide range of conditions imposed by different detection schemes. The effect of column geometry under a variety of typical LC-MS conversion situations is also included. The studies include resolution of critical hydrophilic pairs and stationary-phase loading performance relative to converting existing separations for LC-MS analysis while achieving maximum sensitivity and maintaining chromatographic fidelity.

Purification and Quantitative Analysis of Veratridine and Cevadine by HPLC

As part of a study to determine the variation in the effectiveness of commercial pesticide formulations of ester steroidal alkaloids from sabadilla (Schoenocaulon officinale Grey) seeds in the integrated pest management of citrus, a method to isolate and measure large quantities of alkaloids from natural starting materials was needed. The addition of the solvent modifier sodium dodecyl sulfate (SDS) to a previously published HPLC method greatly improved peak resolution by minimizing peak tailing. The use of the non-ester alkaloid papaverine as an internal standard permitted quantification of sabadilla alkaloids by HPLC, and this modified method was then used to monitor the breakdown of cevadine and veratridine, the two most abundant sabadilla alkaloids, in aqueous solutions as a function of time and pH.

Influence of organic solvent modifier and solvent strenght on peak shape of some basic compounds in high-performance liquid chromatography using a reversed-phase column

Comparative column effiency and peak assymmetry measurements were made for 16 pyridine derivatives using approximately isoeluotropic mixtures of methanol, acetonitrile and tetrahydrofuran (THF) in combination with phosphate buffer at pH 7.0 on a RP-HPLC column suitable for the analysis of basic compounds. On average, THF and methanol gave significantly better peak shape than acetonitrile, although considerable variations in behaviour for the different derivatives were noted. Methanolic mobile phases yielded straight line plots of log k′ vs. percentage of modifier; nevertheless the increase in k′ with decreasing methanol concentration is accompanied by a deterioration in peak shape which varies in extent depending on the nature of the probe compound.

Supercritical Fluid Extraction of Strychnine Using Two Different Sequential Organic Modifiers

Carbon dioxide supercritical fluid extraction (SFE) of strychnine from oat grain bait was accomplished using a methanol modifier during static extraction and a chloroform modifier during dynamic extraction. Extraction efficiency was better than with the use of either modifier alone. Analysis time, strychnine recoveries (82%), and reproducibility (SD = 7.8%) were not significantly different from the 85% recovery and 5.4% SD obtained with a routinely used organic solvent/solid extraction method. The supercritical fluid extraction method generated no hazardous waste as compared to the solvent/solid extraction method, which produced 50 mL of hazardous waste per sample. This two solvent modifier SFE method resulted in better precision and recovery of strychnine from oat bait than other reported SFE methods for the recovery of pesticides from treated grains.

Influence of solvent effects on the breakthrough volume in solid-phase extraction using porous polymer particle-loaded membranes

A general dependence of the breakthrough volume in solid-phase extraction, using a porous polymer particle-loaded membrane on the selection of the organic solvent added to water at the 1%(v/v) level to promote acceptable and even flow through the membrane is demonstrated. The selective sorption of the organic solvent by the polymer changes the stationary-phase volume and the system selectivity, resulting in changes in up to an order of magnitude in the breakthrough volume when either methanol, propan-1-ol, tetrahydrofuran, or acetonitrile are used as the organic-solvent modifier. A solvation parameter model is used to characterize the changes in selectivity of the sampling system incorporating the different solvent modifiers. Retention by the solvated porous polymer sorbent occurs because of the relative ease of cavity formation in the sorbent compared to the sample solvent while all polar interactions, especially hydrogen-bond interactions, tend to reduce retention by the sorbent, and result in smaller breakthrough volumes. In determining the optimum solvent to obtain an adequate breakthrough volume, the solute size is as important as the capacity of the solute for polar interactions. All relevant factors are taken into account through the solvation parameter model which allows the prediction of breakthrough volumes with different sample co-solvents with adequate accuracy for practical applications.

Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK

This study further examines the factors which affect the chromatographic response of carotenoids and contribute to analytical variation and inaccuracies in their quantitative determination. A method for the analysis of carotenoids in vegetables and fruits is described and data are presented for the carotenoid content of vegetables and fruits commonly consumed in the UK. The addition of a solvent modifier (triethylamine) to the mobile phase was shown to improve the recovery of carotenoids from the column from around 60% to over 90%. The linearity and reproducibility of the chromatographic response was investigated and the robustness and reproducibility of the method was measured using a reference vegetable material developed in the laboratory. Short and longer term reproducibility showed an average CV of around 8% for all carotenoids. Analysis showed that good sources (>1000 μg/100 g) of lutein were broccoli, butterhead lettuce, parsley, peas, peppers, spinach and watercress; of lycopene: tomatoes and tomato products; and of β-carotene: broccoli, carrots, greens, butterhead lettuce, mixed vegetables, parsley, spinach and watercress. There was little or no loss of carotenoids on cooking, green vegetables showed an average increase in lutein levels of 24% and in β-carotene levels of 38%. This study and previous studies in our laboratory have demonstrated that a number of factors affect the validity of the ‘peak response’ and are likely to contribute to within and between laboratory variation. It is suggested that the development and use of standard reference materials would significantly improve the quality of data.

Study of the competition between disproportionation and oxidation of cyclohexene in the liquid phase over solid catalysts

A study has been made of the liquid phase conversion of cyclohexene over metallic palladium and platinum catalysts under oxygen and nitrogen atmosphere. Special attention has been given to the effect of oxygen pressure, catalyst support, solvent and metal modifiers on the activity of metal catalysts and distribution of products. In every case the main reaction pathway is disproportionation of cyclohexene to benzene and cyclohexane. Formation of oxygenated products is low even at 2–6 atm of oxygen. Doping of Pd and Pt catalysts with metal modifiers strongly suppresses the conversion of cyclohexene and has no influence on the ratio of benzene/cyclohexane at experiments carried out in an inert atmosphere. An interpretation of these effects is presented.

Carbon dioxide supercritical fluid extraction of incinerator fly ash with a reactive solvent modifier

Carbon dioxide supercritical fluid extraction was used to extract polycyclic aromatics, halogenated phenols, halogenated aromatics and dioxins from a municipal incinerator fly ash matrix. The extraction solvent was modified with methanol or a reactive solvent modifier, N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA), which was added to the sample before extraction. Extracts, obtained at two temperatures and with three supercritical fluid formulations, were analyzed by the US Environmental Protection Agency contract laboratory program GC-MS procedure for semi-volatile organic compounds. Average recoveries of surrogate analytes with pure carbon dioxide and carbon dioxide modified with methanol were 50% (500 atm and 100°C). Average recoveries were 82% at 100°C and 54% at 30°C with BSTFA present. Carbon dioxide modified with methanol was found to be less efficient than carbon dioxide modified with BSTFA. Unlike earlier uses of reactive modifiers, the acidic and phenolic components were determined as the free acids and phenols. Hydrolysis of trimethylsilyl derivatives of phenols, produced by the modifier during the extraction, with methanol reproduced the free phenols. At 60°C the average hydrolysis yield of the four phenols was 96.7%. This hydrolysis step also allowed analysis of free acids by standard methods.