N-hexane Phase Research Articles

Gas chromatography–mass spectrometric analysis of bonded long chain fatty acids in a single zebrafish egg by ultrasound-assisted one-step transmethylation and extraction

Changes in the level of lipid composition in a single zebrafish egg have been involved in biological responses to chemical exposures. In this paper, an one-step transmethylation of lipids and simultaneous extraction of resultant fatty acid methyl esters followed by gas chromatography–mass spectrometric analysis was developed for the identification of bonded long chain fatty acids in a single zebrafish egg. The efficiency of transmethylation under different experimental conditions has been investigated. Dried egg homogenates were directly mixed with either 0.5 M NaOH or 1% H 2SO 4 or 4% HCl solution of methanol and then an n-hexane solution was added on the top. Ultrasonication of this immiscible liquid–liquid–solid system produces high velocity impacts between solid particles and liquid phases and thus promotes mass transfer among phases. It was demonstrated that ultrasound irradiation has strong effect on the alkaline-catalyzed transmethylation of lipids but cannot significantly change the acid-catalyzed transmethylation of lipids. With the aid of ultrasonication, transmethylation can be combined with simultaneous extraction of the resultant fatty acid methyl esters into n-hexane phase. This approach simplifies the sample preparation procedure, shortens the reaction time but improves the efficiency of the transmethylation of lipids and reduces sample losses, especially for small size samples. It has been applied to determine bonded fatty acids in a single zebrafish egg. In total, 28 fatty acids from a single zebrafish egg have been identified reproducibly.

Separation and purification of isofraxidin from Sarcandra glabra by microwave-assisted extraction coupled with high-speed counter-current chromatography

In this paper, a simple method based upon microwave-assisted extraction (MAE) coupled with high-speed counter-current chromatography (HSCCC) for the rapid isolation, separation and purification of isofraxidin from Sarcandra glabra (thunb.) was developed. The dry extract obtained from MAE was directly introduced into the HSCCC for separation and purification. The critical experimental parameters of MAE and HSCCC were optimized. 2 g of Sarcandra glabra sample was extracted with 20 mL 60% (v/v) ethanol aqueous at 65 °C for 20 min at 500 W of microwave irradiation, then the extracting solution was concentrated to 300 mg dry extract. The dry extract was dissolved with the lower phase of n-hexane–ethyl acetate–methanol–water (1:2:1:2, v/v/v/v), separated and purified by preparative HSCCC. 300 mg dry extract can be introduced into the HSCCC system in one-step and 1.20 mg of isofraxidin was obtained with purity over 99% in 2.5 h. The purity of isofraxidin was determined with HPLC and its chemical structure was identified with 1H NMR. The results indicate that the method is simple, fast and efficient. The method has good potential on the separation and purification of effective compounds from natural product.

Application of NP-TLC with Densitometric Detection for Separation and Quantitative Analysis of Unconjugated Bile Acids Presented in Human Bile

A simple and rapid method of separation and quantification of five bile acids such as: cholic acid (C), deoxycholic acid (DC), chenodeoxycholic acid (CDC), lithocholic (LC) and ursodeoxycholic (UDC) with the use of the NP-TLC method has been developed. The five bile acids were satisfactory separated on aluminum and glass plates precoated with silica gel 60 and silica gel 60F254 (E. Merck, Art. 1.05553, Art. 1.05554, and Art. 1.05715) using mobile phase n-hexane–ethyl acetate–acetic acid in different volume compositions. For the optimal conditions of separation the quantitative analysis of studied acids was elaborated on. The quantitative analysis was provided with the use of densitometric detection at λ = 360 nm. Densitometric analysis has been used to find the linear correlations between the peak area and amount of each bile acid spotted on the chromatographic plates. The statistical parameters of obtained linear regressions indicated that NP-TLC method with densitometric detection is statistically significant and can be used for separation and detection of C, DC, CDC, LC, and UDC in biological samples and also in pharmaceutical formulations.

Extraction of surfactin from fermentation broth with n-hexane in microporous PVDF hollow fibers: Significance of membrane adsorption

Abstract In order to avoid foaming behavior and the formation of stable emulsions in traditional extraction, non-dispersive extraction of surfactin from the fermentation broth of Bacillus subtilis ATCC 21332 culture with n-hexane was studied in microporous polyvinylidene fluoride (PVDF, pore size 0.2 μm) hollow fiber module. In this work, the broth was pretreated by acid precipitation and the precipitate was then dissolved in NaOH solution, and the treated broth was passed through the lumen side of the module and n-hexane was flowed across the shell side. Experiments were performed at a fixed pH of 8.0 and a flow rate of both phases of 2.5 mL min−1 but at different surfactin concentrations (300–3000 mg L−1). Under the conditions studied, it was shown that surfactin was adsorbed onto the surface of the fibers, instead of being extracted by n-hexane and transported through the pores of the fibers into bulk n-hexane phase. The adsorption capacity was determined and the adsorption dynamics was analyzed. The purity of surfactin desorbed from the fibers with ethanol was found to be higher than that obtained after solvent extraction with n-hexane.

An in situ high pressure FTIR study on molecular interactions of ketones, esters, and amides with dense phase carbon dioxide

The interactions between a variety of carbonyl compounds (ketones, esters, and amides) and dense phase CO 2 have been investigated by in situ high pressure FTIR measurements. The gas and liquid phase spectra of these compounds have also been measured to provide accurate reference data, and these have been compared with those obtained in n-hexane and dense phase CO 2 at a variety of pressures. The frequency of the carbonyl IR absorption is a measure of the strength of the carbonyl bond, and in the dense CO 2 medium, is in between that observed in the gaseous and liquid phases. The substituent effect on the ν(C O) absorption bands is the same for the gas, liquid, and dense CO 2 spectra and it is related to the electron donating nature of the substituents. When solutions of carbonyl compounds in n-hexane are diluted, the ν(C O) absorption bands are blue-shifted. In contrast, these bands in dense CO 2 are red-shifted with increasing pressure (dilution by CO 2). The red shift with increasing CO 2 pressure is larger in amides > esters > ketones.

Anti-fungal active fraction to Candida albicans from methanolic extract of camilen flowers (Matricaria chamomilla,L.)

The flowers of kamilen (Matricaria chamomilla, L.) has been used as antifungal in Indonesian traditional medicines. Previous study showed that the extract of flowers inhibited Candida albicans activity. In order to determine the active fraction, the using bioactive-guided fractionation method was used. The air-dried flowers was extracted from methanol. Methanolic extract was fractionated by using n-hexane, chloroform and ethyl acetate. n-Hexane fraction was separated by vacuum liquid chromatography (VLC) by busing silica gel H 60 with mobile phase n-heksan : ethyl acetate and chloroform : methanol by gradient polarity. VLC results fraction was separated by preparative thin layer chromatography with silica gel GF254 and mobile phase n-hexane : chloroform (1:1). The anti-fungal activities of Candida albicans was done by difuse method. Based on the TLC appearence , nine fractions were obtained (FK 1-9) from VLC, and FK-3 was the most active fraction in inhibiting Candida albicans. The result from preparative TLC of FK-3 was 5 subfractions (FP 1-5) Subfraction FP-3 and FP-5 were most active. that had inhibiting radical zone diameter 18 mm in 3% consentration. Key words : Kamilen ( Matricia chamomila, L), Candida albicans, anti-fungal.

Enantiomeric resolution of bupivacaine by high-performance liquid chromatography and chiroptical detection

This work reports a new analytical procedure for the separation and determination of the enantiomers of bupivacaine and the determination of the enantiomeric purity. The isomers were separated using a Chirex 3020 (250 mm × 4.6 mm) with a mobile phase of n-hexane:dichloroethane:ethanol (82:9:9, v/v/v) at a flow-rate of 1 ml min −1 and UV, polarimetric and circular dichroism (CD) detection. Obtained retention times were 5.93 and 7.53 min ( R and S) with a resolution of R s = 2.36. Precisions (RSD) were 1.83 and 2.02% (CD detection) and 3.07 and 1.26% (UV detection) for R- and S-enantiomers, respectively (at 10 μg level). Detection limits were 0.5 and 0.5 μg ( R and S) with CD detection, and 0.9 and 0.3 μg with UV detection. Polarimetric detection was inadequate to perform a quantitative method at similar concentration ranges as UV and CD because of poor sensitivity. A procedure for determination of enantiomeric purity using a conventional chromatographic column (RP18, Luna) coupled to a CD detector and anisotropy factor (CD/UV) as analytical signal was also developed. Obtained results show that RSDs of 6.7–1.6% were obtained in the range of 0–100% enantiomeric purity.

A Pharmacognostic Study on Sphaeranthus indicus

The study is aimed at development of Physico-chemical parameters and estimation of 7-hydroxy eudesmanolide, a major sesquiterpene lactone of Sphaeranthus indicus , (Family: Astraceae), commonly known as Gorakhmundi. S. indicus is a branched, hairy and strongly scented herb with spathulate, sessile leaves and pinkish purple flowers. Leaf shows both simple (uni-multicellular) and glandular (Club and clavate) type of trichomes. Stem shows a ring of deltoid vascular bundles and well-developed pith with few pitted cells. Root shows metaderm, and radially arranged fibers and secretory canal placed alternatively in cortical region. Powdered drug shows large number of various types of trichomes, pollen grains in pollen sacs and cruciferous stomata. HPTLC method was developed to quantify 7- hydroxy eudesmanolide in S. indicus using a mobile phase n-hexane: diethyl ether (3:7) and scanning the plate at 213 nm. The amount of 7-hydroxy eudesmanolide was found to be 0.0658% w/w. The present study is an effort to develop a pharmacognostic report for S. indicus .

Quasi-dynamic leaching characteristics of polychlorinated dibenzo- p-dioxins and dibenzofurans from raw and solidified waste incineration residues

Quasi-dynamic leaching characteristics of polychlorinated dibenzo- p-dioxins and dibenzofurans (PCDD/Fs) from raw and solidified air pollution control (APC) residues were examined via a nine-time multiple leaching test. The effect of injected activated carbon in the APC residues on the PCDD/F leachability was also evaluated. When humic acid solution was used as a leachant, the leaching concentrations of PCDD/Fs fluctuated between the first and the fifth leaching, followed by a gradual increase and then suddenly reached maximum values at the leaching sequences around seventh and eighth. This significant enhancement in PCDD/F leachability was mainly due to an increase in the release of highly chlorinated PCDD/Fs. Leaching of PCDD/Fs with n-hexane was, in contrast, primarily caused by the partitioning of hydrophobic PCDD/Fs between the APC residue surface and the liquid phase of n-hexane. Consequently, the largest leaching concentrations for n-hexane tests achieved at the first leaching, followed by a decrease and reached plateaus. Solidification/stabilization (S/S) decreased the PCDD/F leachability up to the fifth leaching by the use of humic acid solution. However, S/S increased the PCDD/F leaching concentrations and rates with n-hexane. The activated carbon in APC residues significantly inhibited the release of PCDD/F with n-hexane. The inhibiting effect provided by activated carbon was, however, less significant by the use of humic acid solution.

Diterpenoids and flavonoids from the fruits of Vitex agnus‐castus and antioxidant activity of the fruit extracts and their constituents

From the n-hexane fraction of the fruits of Vitex agnus-castus, two labdane-type diterpenes, vitetrifolin B and C, were isolated by means of multiple chromatographic separations, together with the previously identified rotundifuran, vitexilactone and the sesquiterpene spathulenol. From the EtOAc fraction, eupatorin was identified for the first time, besides the known casticin, penduletin, vitexin and orientin. The n-hexane, EtOAc and MeOH-H(2)O fractions of the MeOH extract of Agni-casti fructus were subjected to in vitro antioxidant assays. The EtOAc extract displayed a significant concentration-dependent effect when tested by 1,1-diphenyl-2-picrylhydrasyl (DPPH) free radical assay (IC(50) = 68 microg/mL) and against the autooxidation of a standard rat brain homogenate (IC(50) = 14 microg/mL). The MeOH-H(2)O fraction was less active with 3643 microg/mL (DPPH test) and IC(50) = 125 microg/mL (rat brain homogenate), while the n-hexane phase proved to be inactive. The main flavonoid constituents of the EtOAc extract, casticin, vitexin and orientin were assayed for antioxidant activity and found that only casticin possesses a marked lipid peroxidation inhibitory effect (IC(50) = 0.049 mm) compared with that of the positive control ascorbic acid (IC(50) = 0.703 mm).

Liquid–liquid equilibria and partitioning in organic–aqueous systems

Liquid–liquid equilibria at ambient temperature and pressure were measured for four ternary systems: n-hexane + THF + water, n-hexane + acetonitrile + water, n-hexane + 1,4-dioxane + water, and ethyl ether + 1,4-dioxane + water. Partitioning of 1-octene and 1-nonanal in these systems was also measured. The results were modeled with the UNIQUAC and NRTL g E models. The experiments showed an aqueous phase in the n-hexane–THF–water system that was 90% water on average, and an organic n-hexane phase in the n-hexane–acetonitrile–water system that was 96% n-hexane on average. 1,4-Dioxane distributes fairly equally to both phases in the n-hexane–1,4-dioxane–water and ethyl ether–1,4-dioxane–water systems.

Responses of enantioselective characteristics of imidazolinone herbicides and Chiralcel OJ column to temperature variations

Temperature affects not only the chromatographic characteristics of solute but may also alter the conformation of the stationary phase. However, temperature influences on enantioseparation of solute and conformation of chiral stationary phase (CSP) are seldom considered simultaneously. In this study, three temperature programs, a conventional heating procedure, a cyclic van’t Hoff program, and a step-temperature program, were employed to evaluate temperature effects on enantioseparation of five imidazolinone herbicides on Chiralcel OJ column and the conformational state of the stationary phase. The van’t Hoff plots of retention factor ( k′), distribution constant ( K) and separation factor ( α) for imazapyr ( 1), imazapic ( 2), imazethapyr ( 3), and imazamox ( 4) were linear within 15–50 °C. Nonlinear van’t Hoff plots of α were observed for imazaquin ( 5) with mobile phase of n-hexane (0.1% TFA)-2-propanol at 70/30 or 60/40 (v/v). The large molecular size of imazaquin ( 5) and van’t Hoff plots of α were therefore more sensitive at detecting conformational changes of the stationary phase. Small but irreversible conformational changes occurred at 5–10 °C with the solvent ratio of 60/40. During the cyclic van’t Hoff program, reversible conformational changes were observed at ≥15 °C. A switch was even visible at about 25 °C with the solvent ratio of 60/40 during the re-cooling cycle. The cyclic van’t Hoff temperature program showed that using OJ column may yield satisfactory results at 15–50 °C but not at ≤15 °C. The step-temperature program showed that fast temperature changes over the 5–50 °C range may be performed on the coated OJ phase, and the recovery of enantioselectivity was relatively fast. This study shows that simultaneous evaluation of temperature effects on the chromatographic behavior of a chiral analyte and the conformational state of the chiral stationary phase provides more insights for understanding mechanisms of enantioseparation and selecting optimal separation conditions.

Chiral separation of rac‐Ornidazole and detection of the impurity of (R)‐Ornidazole in (S)‐Ornidazole injection and raw material

(S)-Ornidazole is a subject of research as an antifertility agent in male animals at present. However, there seems to be no relative report on chiral separation for rac-Ornidazole, which has been used as an effective medicine for more than 30 years. In this article, the chiral separation of rac-Ornidazole on a Chiralcel OB-H column based on normal-phase high-performance liquid chromatography (NP-HPLC) is investigated and the methodology for detection of impurity of (R)-Ornidazole in (S)-Ornidazole injection and raw material is established. The novel mobile phase is utilized by mixing n-hexane, methanol and isopropyl alcohol (95:4:1, v/v/v) instead of the typical mobile phase of n-hexane and isopropyl alcohol, although the methanol, which offers a good resolution factor for the enantiomeric separation in this system, is not recommended on the Chiralcel OB-H column according to the instruction supplied by Daicel Chemical Ind., LTD (Japan).

Determination of benzo[a]pyrene in charcoal grilled meat samples by HPLC with fluorescence detection

In this study, an HPLC procedure for the quantitative determination of benzo[a]pyrene (BaP) in charcoal grilled meat samples has been applied to the analysis of the Turkish meat samples. The grilled meat samples were first treated in alkaline medium, then BaP was extracted into n-hexane phase, purified on XAD-2 column and eluted with n-hexane/dichloromethane mixture (9:1,v/v). Separation and quantitative determination of BaP has been carried out by a C18 reversed phase column mounted HPLC with a fluorescence detection of 254–355 nm (excitation–emission). The BaP levels determined in grilled and over-grilled lamb and beef meats were 43.80±1.80 µg/kg, 31.33±0.94 µg/kg and 62.60±3.72 µg/kg, 37.60±3.84 µg/kg, respectively.

Separation and Simultaneous Determination of Enantiomers of Tau-fluvalinate and Permethrin in Drinking Water

The chiral liquid chromatographic separation of two pyrethroid insecticides, tau-fluvalinate and permethrin, on different polysaccharide-based chiral stationary phases (CSPs) has been investigated. Complete separation of these compounds was achieved with Chiralcel OJ under normal phase mode and a mobile phase of n-hexane:ethanol (90:10, v/v). This method has been used for determining these compounds in drinking water at ppb levels after solid-phase extraction (SPE) with C-18 cartridges. Recoveries ranged between 103–113% and precision values (expressed as relative standard deviations) were lower than 10%.

Preparative isolation and purification of coumarins from Angelica dahurica ( Fisch. ex Hoffm) Benth, et Hook. f (Chinese traditional medicinal herb) by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarins from Angelica dahurica (Fisch. ex Hoffm) Benth, et Hook. f (Baizhi in Chinese) was successfully established by using n-hexane–methanol–water as the two-phase solvent system in gradient elution mode. The upper phase of n-hexane–methanol–water (5:5:5, v/v) was used as the stationary phase of HSCCC. The mobile phase used in HSCCC was the lower phase of n-hexane–methanol–water (5:5:5, v/v) and n-hexane–methanol–water (5:7:3, v/v) that was changed in gradient. Three major components including imperatorin, isoimperatorin and oxypeucedanine were isolated, each at over 98% purity as determined by high-performance liquid chromatography (HPLC). The peak fractions of HSCCC were identified by 1H-NMR and 13C-NMR.

Adsorption kinetics of some carotenoids at the oil/water interface

The kinetic analysis of the adsorption of two carotenoids (i.e., ethyl ester of β-apo-8′-carotenoic acid and β-carotene, all trans-isomers) from n-hexane solutions at the oil/water interface is presented for several carotenoid concentrations in the oil phase. A new kinetic approach is developed and it addresses the diffusion adsorption associated with a reversible interfacial reaction, which describes the reorientation of surfactant molecules between two conformations. This approach leads to a general analytical expression that contains four physical parameters and describes with high accuracy the experimental dynamic interfacial tensions for the two carotenoids, which independently adsorb from n-hexane phase to the n-hexane/water interface. The calculations give the characteristic times for the carotenoid adsorption at the oil/water interface in terms of diffusion relaxation and kinetic relaxation times. The results explain the long time effects on the adsorption of these carotenoids at the oil/water interface. The data are in substantial agreement with the molecular structure of these carotenoids and with the earlier data recorded for cholesterol adsorption at the n-heptane/water interface. Based on these findings, we propose a molecular mechanism for the interfacial transformation of carotenoid molecules at a hydrophobic/hydrophilic interface.

Improved simultaneous determination method of β-carotene and retinol with saponification in human serum and rat liver

Among the many simultaneous determination methods for carotenoid and retinoid, there are only a few reports including the saponification process. However, the yields of β-carotene and retinol were higher when using this process. In this study, the analytical conditions, including saponification, were investigated. The extraction solvent was n-hexane and the sample solvent was HPLC mobile phase in the β-carotene and retinol analysis. BHT as an antioxidant was added at concentrations of 0.125 and 0.025%, respectively, to ethanol and n-hexane phase in the extraction process for serum. The recovery rates were 99.7, 93.7 and 98.3% for β-carotene, retinol and retinyl palmitate in serum, respectively, and 107.1, 92.8 and 98.8% for β-carotene, retinol and retinyl palmitate in liver, respectively. The within-day coefficients of variation (C.V.) were 6.0% for serum and 4.7% for liver in the case of β-carotene, 7.1% for serum, and 5.1% for liver in the case of retinol. The between-day coefficients of variation were 2.7% for serum and 2.7% for liver in the case of β-carotene, and for retinol, 6.4% for serum and 2.7% for liver.

Direct separation and quantitative determination of clenbuterol enantiomers by high performance liquid chromatography using an amide type chiral stationary phase

Enantiomers of clenbuterol were directly separated by a new high performance chromatographic method on Chirex 3005 column. Several parameters such as mobile phase composition, column temperature and flow rate were studied. Baseline enantioseparation was achieved, using the optimized mobile phase of n-hexane–1,2-dicholoethane–methanol (54:38:8, v/v/v) at 17 °C and 1.0 ml/min, with the separation factor ( α) 1.43 and the resolution factor ( R S) 1.81. The mechanism of separation was also discussed. Standard linear calibration cures were established for the R- and S-enantiomers, over the range of 26.1–1045.8 and 5.7–229.6 nmol/ml, with the correlation coefficient of 0.9999 for both. The limits of detection were 0.47 and 1.04 nmol/ml for R- and S-enantiomers, respectively. Recovery and precision of the method were also evaluated, which had been successfully used to monitor and identify quantitatively the profile of the clenbuterol enantiomers in human serum.

Analysis of fatty acid ethyl esters in hair as possible markers of chronically elevated alcohol consumption by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS)

Fatty acid ethyl esters (FAEE) are products of the nonoxidative ethanol metabolism, which are known to be detectable in blood only about 24 h after the last alcohol intake. After deposition in hair they should be suitable long-term markers of chronically elevated alcohol consumption. Therefore, a method for the analysis of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate from hair was developed based on the extraction of the hair sample by a dimethylsulphoxide (DMSO)/ n-hexane mixture, separation and evaporation of the n-hexane phase and application of headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) to the extract. For use as internal standards, the corresponding D 5-ethyl esters were prepared. The HS-SPME/GC-MS measurements were automatically performed using a multi-purpose sampler. The detection limits of the FAEE were between 0.01 and 0.04 ng/mg and the reproducibility was between 3.5 and 16%. By application of the method to hair samples of 21 fatalities with known heavy alcohol abuse 0.045–2.4 ng/mg ethyl myristate, 0.35–13.5 ng/mg ethyl palmitate, 0.25–7.7 ng/mg ethyl oleate and 0.05–3.85 ng/mg ethyl stearate were measured. For social drinkers (30–60 g ethanol per week), the concentrations were about one order of magnitude smaller. For 10 teetotalers negative results or traces of ethyl palmitate were found. It was shown by supplementary investigations in single cases that FAEE are also present in sebum, that there is no strong difference in their concentrations between pubic, chest and scalp hair, and that they are detectable in hair segments after a 2 months period of abstinence. From the results follows that the measurement of FAEE concentrations in hair is a useful way for a retrospective detection of alcohol abuse.