Studies on the effect of alcohols on the chiral discrimination mechanisms of amylose stationary phase on the enantioseparation of nebivolol by HPLC

The chiral recognition mechanism of amylose CSPs has been described by achieving the enantiomeric resolution of (+/-)-nebivolol on Chiralpak AD and Chiralpak AD-RH columns with methanol, ethanol, 1-propanol, 2-propanol, 1-butanol as mobile phases at different flow rates. The energies of interactions of methanol, ethanol, 1-propanol, 2-propanol and 1-butanol with both phases were calculated. The (+)-RRRS enantiomer eluted first when using methanol, ethanol and 1-propanol, while the elution order was reversed when using 2-propanol and 1-butanol as the mobile phases. It has been concluded that the reversal elution order observed was due in part to the chiral cavities on the amylose CSP which were responsible for the bondings of different magnitude between chiral stationary phase and enantiomers, which are influenced with the type of alcohol used as mobile phase on the conformation of the 3,5-dimethyl phenyl carbamate moiety on the pyranose ring system of the amylose.     

Effect of alcohols and their interaction with ethylene on the ripening of epidermal pericarp discs of tomato fruit

Ethanol concentrations that were induced in pericarp discs of mature-green tomato fruit (Lycopersicon esculentum Mill, cv Castlemart) either by anaerobic metabolism or by exposure to ethanol vapor inhibited ripening without increasing the rate of ion leakage. Inhibition of ripening (i.e. lycopene synthesis) of excised tomato pericarp tissue by ethanol vapor was reversed by increasing concentrations of the plant hormone ethylene. A Lineweaver-Burk plot indicated noncompetitive interaction between ethanol and ethylene. Methanol and n-propanol also inhibited lycopene synthesis without significantly increasing ion leakage. The similar inhibitory effects of methanol, ethanol, and n-propanol at concentrations which did not stimulate ion leakage, and the relationship between activity and lipophilia of the alcohols suggest that their mode of action was through disruption of membranes associated with ethylene action.     

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.     

Separation of albendazole sulfoxide enantiomers by chiral supercritical-fluid chromatography

The enantioseparation of albendazole sulfoxide (ABZSO) by chiral supercritical-fluid chromatography (SFC) on two columns, based on the polysaccharide derivatives Chiralpak AD and Chiralcel OD, was studied. The effect of different modifiers, methanol, ethanol, 2-propanol, and acetonitrile, was examined. The results showed that ABZSO can be separated on both columns, using an alcohol-type modifier. Using the Chiralpak AD column, the best results were obtained with 2-propanol and, in the case of the Chiralcel OD, with methanol.     

Application of chiralcel OJ in supercritical fluid chromatography for the resolution of different groups of frequently used drug racemates

Supercritical fluid chromatography was applied to evaluate the direct chiral discriminative properties of Chiralcel OJ, a tris(4-methylbenzoate) cellulose column towards frequently administered drugs. Two groups of acidic drugs, profen and barbiturate derivatives, and a few basic drugs of the benzodiazepine type were analysed. The effect on enantioselectivity of carbon dioxide when using methanol are acetonitrile as primary modifier was studied. Acetonitrile proved to be a good alternative for methanol, especially for the profen compounds that were not well resolved using methanol. The results were compared to normal phase chromatographic applications on the same column. SFC was not necessarily superior to high-performance liquid chromatography. Chirality 9:126–132, 1997. © 1997 Wiley-Liss, Inc.     

Enantioseparation of hydrochloride salts using carbon dioxide-based mobile phases with on-line polarimetric detection

Most HPLC enantioseparations of amine analytes are performed using normal-phase systems containing mobile phases of heptane with ethanol (or 2-propanol) and an amine additive. Since salt-forms of amine analytes are usually insoluble in normal-phase eluents, free-base forms are synthesized for preparative chromatography. It would be highly desirable to directly chromatograph salt forms of amine analytes using mobile phases of carbon dioxide (CO(2)) and methanol (MeOH). Such separations would be readily suitable for preparative chromatography, since most amine salts are highly soluble in MeOH. In this article, advantages are shown for the use of supercritical fluid chromatography (SFC) instrumentation with tandem UV and polarimetric detection for confirming enantioseparation as well as for determining optimum preparative column injections. Examples are shown for racemic mixtures of propranolol HCl (I), thioridazine HCl (II), tramadol HCl (III), and flurbiprofen (IV), all of which resolved on Chiralpak AD-H chiral stationary phase using mobile-phase systems of CO(2) and MeOH without the use of basic or acidic additives.     

Measurement of thymine dimers in DNA by thin-layer chromatography: II. The use of one-dimensional systems

A number of chromatographic solvents and thin-layer chromatographic matrices were tested for their effectiveness in separating thymine dimers from thymine by one-dimensional chromatography. One combination, viz., watersaturated ethylacetate:n-propanol on silica gel thin layers was found to be particularly useful.     

Separation of Alkyl β-d-Glucosides and n-Alcohols by Using a Porous Trimethylolpropane Trimethacrylate Homopolymer Gel

Alkyl β-D-glucosides and n-alcohols were separated by high-performance liquid chromatography on a porou gel of a trimethylolpropane trimethacrylate homopolymer, using a mixture of water with methanol or acetonitrile as the eluent. The effect of the methanol and acetonitrile concentrations on the separability was examined, and the optimum concentration of each to separate alkyl glucosides and alcohols with alkyl chain lengths of six to twelve was determined. A model for analyzing the elution characteristics of the solutes for various levels of methanol or acetonitrile is proposed, based upon the chemical potential of the solutes and eluent at the interface between the gel and eluent phases. Conventional column chromatographic separation of an alkyl glucoside and an alcohol was also performed by using gel with a larger particle diameter.     

Investigation of the separation of heterocyclic aromatic amines by reversed phase ion-pair liquid chromatography coupled with tandem mass spectrometry: the role of ion pair reagents on LC-MS/MS sensitivity

Reversed phase ion-pair chromatography (RP-IPC) of seven heterocyclic aromatic amines encompassing quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP) and carboline derivatives (AalphaC, Harman, Norharman) was carried out with formate as counter ion in an aqueous eluent with acetonitrile as organic modifier. TSKgel ODS-80TS was used as the stationary phase. With the aim of acquiring a better insight into the mutual influence of ion-pair reagent and the organic modifier upon solute retention, the study was performed by using an experimental design approach able to evidencing the effect of the simultaneous variation of the two factors. A model for the chromatographic behavior of the amines is proposed that includes classical ion-pair mechanism involving formate in the case of MeIQx, PhIP, Harman and Norharman. A competitive ion-exchange mechanism was hypothesized to govern retention of quinoline compounds, whereas electrostatic interactions and hydrogen bond formation with the silanols of the stationary phase were judged to be responsible for the retention of AalphaC. Further, the chromatographic behavior of the analytes using the formic acid-ammonium formate buffer in the mobile phase was compared with that observed using acetic acid-ammonium acetate buffer. The method based on the use of RP IPC with tandem mass spectrometry when the eluent contained formate buffer at pH 2.8 exhibited higher detectability with respect to that achieved using the acetate buffer.     

Structural features affecting chiral resolution of cannabimimetic enantiomers by amylose 3,5-dimethylphenylcarbamate chiral stationary phase

The effect of structural features of six pairs of enantiomers of cannabimimetic compounds on their chromatographic resolution on an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase was studied using various compositions of n-hexane with 2-propanol and ethanol. Structural analysis by molecular mechanics was also performed to verify that the 3D conformation within this family of compounds was preserved with substitution. The homologous enantiomeric pairs showed better resolution when there was an additional OH group near the chiral centers (position 7 on the cannabinoid structure). Better resolution was observed also for the enantiomeric pair that had the smaller alkyl side chain. These differences indicated that the additional OH group contributed to a better discrimination of the enantiomers by the chiral sites of the stationary phase and that the bulkier alkyl side chain reduced it. The chromatographic resolution of two enantiomeric pairs of nonclassical cannabinoids HU-249 and HU-250, HU-255 and HU-256, was compared both in ethanol and 2-propanol. Both enantiomeric pairs showed relatively high resolution and selectivity, but the rigid benzofuran analogs (HU-249 and HU-250) exhibited better resolution using 2-propanol, in spite of the flexibility of the open chain analog (HU-255 and HU-256) and its additional OH group. The elution order of all the cannabinoids was (+)/(?) using both solvents. Unusual solvent effects were displayed by one enantiomeric pair, Δ⁶-THC, which was resolved easily using 2-propanol, but whose elution order reversed with 1% ethanol in the mobile phase. Partial separation was obtained at 5% ethanol [elution order (+)/(?)] and full separation was obtained at 0.5% ethanol [elution order (?)/(+)]. © 1995 Wiley-Liss, Inc.     

Separation of basic,hydrophilic peptides by reversed-phase ion-pair chromatography: II. Analytical applications with particular reference to phosphoserine peptides

Phosphoserine peptides, obtained by phosphorylation of synthetic precursors with cyclic AMP-dependent protein kinase, can be efficiently separated from the corresponding non-phosphorylated peptides and from each other by ion-pair high-performance liquid chromatography. All experiments were performed under isocratic conditions on a C₁₈ column, using phosphate buffers with pH 3.2–4.5, n-hexane sulfonic acid as counter ion, and ethanol as organic modifier.     

Reversed-phase high-performance liquid chromatography method for simultaneous analysis of two liposome-formulated short interfering RNA duplexes

Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol (mobile phase A) and 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50 × 2.1 mm i.d., 2.5 μm particle size), and separation was performed at 60 °C. Quantitation was achieved with ultraviolet (UV) detection at 260 nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110 μg/ml. Accuracy of the method was determined by replicate analysis (n = 5) at four concentrations (R² > 0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling.     

Comparative assessment of Cladophora, Spirogyra and Oedogonium biomass for the production of fatty acid methyl esters

The use of alternative fuels for the mitigation of ecological impacts by use of diesel has been focus of intensive research. In the present work, algal oils extracted from cultivated biomass of Cladophora sp., Spirogyra sp. and Oedogonium sp. were evaluated for the lipase-mediated synthesis of fatty acid monoalkyl esters (FAME, biodiesel). To optimize the transesterification of these oils, different parameters such as the alkyl group donor, reaction temperature, stirring time and oil to alcohol ratio were investigated. Four different alcohols i.e. methanol, ethanol, n-propanol and n-butanol were tested as alkyl group donor for the biosynthesis FAME and methanol was found to be the best. Similarly, temperature 50 C and stirring time of 6 h were optimized for the transesterification of oils with methanol. The maximum biodiesel conversions from Cladophora (75.0%), Spirogyra (87.5%) and Oedogonium (92.0%) were obtained when oil to alcohol ratio was 1: 8.     

Indirect- and direct-acting mutagenicity of diesel,coal and wood burning-derived particulates and contribution of polycyclic aromatic hydrocarbons and nitropolycyclic aromatic hydrocarbons

Particulates exhausted from two types of diesel engines (DEPs), burning-derived particulates from three types of coal (CBPs) and burning-derived particulates from three types of wood (WBPs) were separated into four fractions by silica-gel column chromatography using n-hexane, n-hexane–dichloromethane (3:1, v/v), dichloromethane and methanol, as the corresponding eluents. The indirect-acting mutagenicity of each fraction was assayed by the Ames test using the Salmonella typhimurium TA100 strain with S9 mix and the direct-acting mutagenicity was assayed using the S. typhimurium TA98 strain without S9 mix. The polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs) of each fraction were determined by high-performance liquid chromatography (HPLC). Both direct- and indirect-acting of mutagenicities were the highest in samples of DEPs. The contributions of PAHs in samples of WBPs and NPAHs in DEPs were the largest, respectively.     

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

Isolation of Indole-3-Acetyl Amino Acids using Polyvinylpolypyrrolidone Chromatography

Amino acid conjugates of IAA can be chromatographed on polyvinylpolypyrrolidone (PVPP) using either acidic methanol or aqueous buffers as eluents. In the aqueous system, elution of the compounds is pH-dependent, and the pattern obtained suggests that hydrophobic interactions contribute substantially to the chromatographic behavior of IAA peptides on PVPP. Purification of soybean seed extracts by PVPP chromatography produced fractions containing indole-3-acetylaspartate and indole-3-acetylglutamate, based on chromatographic and mass spectral analysis, as well as three other indolic compounds, tentatively identified as N-acyl tryptophan derivatives. PVPP chromatography provides an effective preliminary purification of IAA peptides from plant extracts prior to their separation by other techniques such as high performance liquid chromatography.     

2-Propanol in the mobile phase reduces the time of analysis of CLA isomers by silver ion-HPLC

Individual isomers of octadecadienoic acid (C18:2) with conjugated double bonds (conjugated linoleic acids; CLA) exert different biological activities. Their distribution in food and tissues differs. Therefore, the separation of the various positional and geometric isomers is important. The time of analysis using silver ion-high performance liquid chromatography can extend up to 90 min. The aim of this study was to reduce this time. The time of analysis reduced from ca. 90 min onto 45 to 35 min, respectively, by the addition of 0.05% or 0.1% (v/v) 2-propanol to the mobile phase [acetonitrile (0.1%; v/v) and diethyl ether (0.5%; v/v) in n-hexane]. There was no effect on resolution of the 17 individual CLA isomers of the CLA mixture. Regarding the lowest coefficient of variation and an adequate baseline separation the use of 0.05% 2-propanol in the mobile phase is recommended, without any disadvantages and adverse effects on the service life of columns. In conclusion, adding 0.05% or 0.1% 2-propanol to the mobile phase shortens the time of analysis of CLA isomers, saves solvents and reduces costs.     

Analysis of Volatile Components in Rice Bran

Volatiles stripped from rice bran are analyzed by gas chromatography and thin-layer chromatography either directly or after converted into 2, 4-DNPH’s or 3, 5-DNB’s. Most of prominent peaks revealed by a direct gas chromatography of the volatiles condensates behave as alcohols against classification reagents and are identified as methanol, ethanol, n-propanol, sec-butanol, isobutanol, n-butanol, isopentanol, n-pentanol and n-hexanol, respectively. Ethanol and methanol, being major components in the condensates are also detected by thin-layer chromatography of their 3, 5-DNB’s. Carbonyls identified by a flash exchange gas chromatography of 2, 4-DNPH’s of the volatiles are ethanal, propanal, isobutanal, acetone, n-butanal, isopentanal, n-pentanal and n-hexanal. Thin-layer chromatography of 2, 4-DNPH’s reveals presence of ethanal, acetone, isopentanal or/and isobutanal and n-hexanal in the volatiles. The experimental data have been integrated to furnish a basis for extensive comparison with other rice products and by-products.     

The use of 2,3,4,6-tetra-O-benzyl-α-d-glycopyranosyl iodides in α-glycoside synthesis

2,3,4,6-Tetra-O-benzyl-α-d-gluco- and -α-d-galactopyranosyl iodides react rapidly in acetonitrile and other solvents with limited excesses of methanol, ethanol, 2-propanol, and cyclohexanol in the presence of 2,6-lutidine. The products obtained from the lower alcohols, contain a high proportion of α glycoside. The highest ratios of α- to β-anomer are obtained at low concentrations of alcohol. The high rates, stereoselectivity, and lack of side-reactions observed suggest that the alcoholysis of glycosyl iodides should be tested as a route to oligosaccharide synthesis.     

Headspace gas chromatography with capillary column for urine alcohol determination

A headspace gas chromatographic method using a fused-silica capillary column Poraplot Q has been developed and validated for the detection and quantification of ethanol in urine. Under optimized conditions, ethanol was properly separated from acetaldehyde, acetone, isopropanol, methanol and n-propanol. Limits of detection (LODs) and quantification (LOQs) were 0.008 and 0.010 g/l, respectively. The precision studies within-run and between-run, using spiked urine samples (0.08, 0.8 and 2.0 g/l) showed maximum coefficients of variation 5.9 and 6.5%, respectively. Results of ethanol recovery varied from 91.6±0.8 to 103.3±1.8% over the concentration range from 0.01 to 3.20 g/l. The method was appropriate for the detection of ethanol in urine samples. This matrix can be used for monitoring alcohol abuse in the workplace and used in alcohol rehabilitation programs.