Characterisation of sorbate geometrical isomers

trans,trans Isomers of sorbic acid, its potassium salt and ethyl sorbate isomerise under UV irradiation. All four geometrical isomers of the acid, salt and ester were separated using high-performance liquid chromatography on a nonpolar reversed-phase column (C18) and the ester also by gas chromatography on a VOCOL capillary column. The limit of detection and the interval of linearity were determined for all chromatographic methods. Individual isomers were identified with NMR analysis. Resolved chemical shifts of protons adjacent to the double bonds enabled qualitative and quantitative determination of isomers in the mixture. Antimicrobial activity of potassium sorbate isomers was tested on yeast Saccharomyces cerevisiae. Results show that the pure trans,trans isomer has a higher antimicrobial activity than the mixture of isomers.     

多维高效液相色谱分离模式组合

简述了多维高效液相色谱法的特点及发展简况,重点对分子排阻色谱／反相色谱、离子交换色谱／反相色谱、正相色谱／反相色谱、分子排阻色谱／离子交换色谱、液固色谱／反相色谱、亲合色谱／反相色谱、非手性柱／手性柱等的联用模式及实际应用进行了概括和总结。     

Utilisation of reversed-phase high-performance liquid chromatography as an alternative to silver-ion chromatography for the separation of cis- and trans-C18:1 fatty acid isomers

Silver ion-high-performance liquid chromatography (HPLC) has been commonly used for the separation and the analysis of trans-18:1 isomers in partially hydrogenated oils and milk fat. This paper describes an easy HPLC method using two reversed-phase columns. The cis- and trans-18:1 fatty acids isomers as methyl esters were eluted as two separate fractions. The collected fractions were analysed by gas chromatography (GC). The purity of the two fractions were tested by GC-MS and GC-Fourier transform IR.     

Gas chromatographic method for analysis of conjugated linoleic acids isomers (c9t11, t10c12, and t9t11) in broth media as application in probiotic studies

A gas chromatography (GC) procedure is assayed for analysis of conjugated linoleic acid (CLA) isomers cis-9, trans-11-octadecadienoic (c9t11); trans-10, 12 cis-octadecadienoic (t10c12); and trans-9, trans-11-octadecadienoic (t9t11) in culture broth by GC using NaOH-BF3 in methanol for methylating and a long capillary (100 m) high-polarity column. Repeatability of the method is assessed; the coefficient of variation for CLA isomers ranges from 4.62 for c9t11 to 8.19 for t9t11. Recovery ranges between 88.01 and 89.76, with a mean value of 89.06 for all CLA isomers studied. This method may be considered advantageous for analysis of CLA isomers in probiotics cultures samples.     

Separation of the Geometric Isomers of 4-Octyl-1,1a,2,3,4,5,6,6a-Octahydro-Cyclopropa[f]Indene-1-Carboxylic Acid Ethyl Ester and their Positional Isomers by Reversed-Phase HPLC

A high-performance liquid chromatography (HPLC) method for the separation of geometric and positional isomers is described. A gas chromatography-mass spectrometry (GC-MS) method for the detection and evaluation of various isomers is described. The developed method was able to separate the isomers in the purity range of 97.96% to 99.3% (GC analysis). Revised: 16 November 2005 and 9 March 2006     

Determination of Fe-55 and Ni-63 using semi-preparative ion chromatography – a feasibility study

 The feasibility of semi-preparative high-performance ion chromatography (HPIC) for the analysis of the activation products Fe-55 and Ni-63 in samples of different sources was studied. A mixed anion/cation exchanger column was applied to the separation of Fe(III), Ni(II) and Co(II) using 1 mmol/L PDCA-solution as an eluent. After fractionation of the eluate, Fe(III)- and Ni(II)-fractions were analysed by low-level liquid scintillation counting (LSC). The method was applied to the analysis of corrosion products originating from the primary circuit of a shut-down nuclear power plant. The limits of detection are dependent on the loading of metal ions on the column and are therefore given in terms of specific activities. For a typical sample composition they were found to be 0.011 Bq per mg Fe for Fe-55 and 0.054 Bq per mg Ni for Ni-63. An assessment of semi-preparative HPIC connected to off-line and on-line LSC as compared with standard radiochemical procedures is given. Received: 10 May 1996 / Revised: 2 July 1996 / Accepted: 6 July 1996     

Separation of technical 4-nonylphenols and their biodegradation products by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography (GC x GC) coupled to time-of-flight mass spectrometry (TOF-MS) was applied to improve the separation of 4-nonylphenol isomers and their biodegradation products. The structurally similar nonylphenol isomers were separated by combining a 30 m long semi-polar column and a short polar capillary. Both were coupled via a custom-made liquid nitrogen cryogenic modulator. The advanced GC resolution of coeluting isomers, additionally supported by fast scanning TOF-MS, provided clearer, non-interfered mass spectra of individual isomers. Thus, identification of components is facilitated as shown for isomeric 4-nonylphenols and metabolites of their biodegradation by Clavariopsis aquatica, an aquatic fungus. GC x GC-TOF-MS analysis enabled the separation of about 40 alkylphenol isomers included in technical 4-nonylphenol. During biodegradation the variety of emerging compounds increased with longer reaction time. The comprehensive analysis indicated a broad spectrum of hydroxylated, carboxylated nonylphenolisomers and additionally, chlorinated aromatic compounds produced and released from the fungal culture.     

Multilayer effects in liquid adsorption chromatography with mixed mobile phases

This paper describes the theory of liquid adsorption chromatography (LSC) with mixed mobile phase, involving formation of multilayer surface phases. An equation describing the dependence of the capacity ratio upon the mobile phase composition is derived and theoretical curves illustrating the same dependence are calculated accordingly by assuming different thicknesses of the surface phase.     

Methods for analysis of conjugated linoleic acids and trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-layer chromatography/gas chromatography, and silver-ion liquid chromatography

Conjugated linoleic acids (CLA) are octadecadienoic acids (18:2) that have a conjugated double-bond system. Interest in these compounds has expanded since CLA were found to be associated with a number of physiological and pathological responses such as cancer, metastases, atherosclerosis, diabetes, immunity, and body fat/protein composition. The main sources of these conjugated fatty acids are dairy fats. Rumen bacteria convert polyunsaturated fatty acids, especially linoleic and linolenic acids, to CLA and numerous trans- containing mono- and diunsaturated fatty acids. It has been established that an additional route of CLA synthesis in ruminants and monogastric animals, including humans, occurs via delta9 desaturation of the trans-18:1 isomers. To date, a total of 6 positional CLA isomers have been found in dairy fats, each occurring in 4 geometric forms (cis,trans; trans,cis; cis,cis; and trans,trans) for a total of 24. All of these CLA isomers can be resolved only by a combination of gas chromatography (GC), using 100 m highly polar capillary columns, and silver-ion liquid chromatography, using 3 of these 25 cm columns in series. Complete analysis of all the trans-18:1 isomers requires prior isolation of trans monoenes by silver-ion thin-layer chromatography (TLC), followed by GC analysis using the same 100 m capillary columns operated at low temperatures starting from 120 degrees C. These analytical techniques are required to assess the purity of commercial CLA preparations, because their purity will affect the interpretation of any physiological and/or biochemical response obtained. Prior assessment of CLA preparations by TLC is also recommended to determine the presence of any other impurities. The availability of pure CLA isomers will permit the evaluation and analysis of individual CLA isomers for their nutritional and biological activity in model systems, animals, and humans. These techniques are also essential to evaluate dairy fats for their content of specific CLA isomers and to help design experimental diets to increase the level of the desired CLA isomers in dairy fats. These improved techniques are further required to evaluate the CLA profile in monogastric animals fed commercial CLA preparations for CLA enrichment of animal products. This is particularly important because absorption and metabolism will alter the ingested-CLA profile in the animal fed.     

Resolution of positional isomers by capillary electrochromatography

Electroseparations have been very successful in increasing efficiencies and reducing analysis times. The analytical technique originally applied to open tube capillaries (capillary electrophoresis) has been used as a basis to develop renewed interest in electrochromatography. This paper describes the use of capillary electrochromatography to separate two positional isomers and describes a comparison between gas chromatography (GC), capillary electrochromatography (CEC) and nano-HPLC. The resolution of these isomers is quite crucial, since one of the isomers is the impurity in a pharmaceutically active drug.     

The isolation and identification of polychloro-2-(chloro-methylsulphonamide) diphenyl ether isomers and their metabolites from eulan wa new and fish tissue by gas chromatography—mass spectrometry

The active ingredients of Eulan WA New have been isolated and identified by nuclear magnetic resonance and mass spectrometry. The gas chromatography thermal decomposition products of the polychloro-2-(chloromethylsulphonamide) diphenyl ether isomers have been monitored and the chromatogram of Eulan WA New has been characterized. A method for the extraction, clean-up, and analysis of the isomers and their metabolites in fish tissue by g.c.—m.s. has been developed.     

Chromatographic analysis of binary mixtures of isomeric complexes

Summary Mixed ligand complexes of Cr(III), Co(II), Cu(II) and Zn(II) with the p-diethylaminoanil of phenylglyoxal and either thiourea or ammonia, isolated as binary mixtures of isomers, were separated on thin layers of alumina or silica gel and on paper strips. Separation of mixtures in bulk was by column chromatography whereas quantitative analysis including determination of isomeric compositions of isolates was by TLC. Resolved isomers were identified using correlations of their R_F with spectral properties.     

A microplate solid scintillation counter as a radioactivity detector for high performance liquid chromatography in drug metabolism: validation and applications

Sensitive radioactivity detection following high performance liquid chromatography (HPLC) separation remains a challenge in many drug metabolism studies with radiolabeled compounds. In this work, solid scintillation counting (SSC) after fraction collection into 96-well plates was evaluated as an off-line radioactivity detection method, in comparison with conventional liquid scintillation counting (LSC). The impact of counting time and biological matrix on the quantification of radiolabeled metabolites and parent drug in samples from animal and human absorption, distribution, metabolism and excretion (ADME) studies was investigated. Three different approaches were used to test whether reliable quantification by off-line SSC detection, which requires an approximately constant counting yield during the entire chromatographic run, can be realized: (i) the measurement of radioactivity-spiked biological blank samples without HPLC separation as an extreme case of biological background, (ii) the measurement of radioactivity-spiked HPLC fractions of biological blank samples and (iii) the comparison of radiochromatograms obtained by off-line SSC and LSC of real samples from ADME studies with radiolabeled compounds. Situations in which variations in SSC yield during an HPLC run are likely to lead to significant errors in quantitation were identified and are discussed. However, examples from a number of animal or human ADME studies showed that in the majority of cases off-line SSC provides very similar quantitative data, compared with the reference method of off-line LSC radioactivity detection. Approaches for validation of the off-line SSC approach in critical cases are discussed. The main advantages of off-line SSC, compared with off-line LSC, are lower detection limits and a substantially higher throughput. Several applications of off-line SSC detection in ADME studies are shown.     

Separation and characterization of three positional isomers of dimaltosyl-cyclomaltoheptaose (dimaltosyl-beta-cyclodextrin).

A mixture of maltosylcyclomaltoheptaoses (maltosyl-beta-cyclodextrins, G2-beta CDs) was prepared from maltose and beta-cyclodextrin (beta CD) through the reverse action of Klebsiella pneumoniae pullulanase. Three positional isomers of dimaltosyl-beta CD in the mixture were separated by high-performance liquid chromatography on a reversed phase column and a graphitized carbon column. Their molecular weights were measured by fast-atom bombardment mass spectrometry, and the structures were established by methylation analysis, hydrolysis with glucoamylase to the known compounds, three positional isomers of diglucosyl-beta CD, and 13C-nuclear magnetic resonance spectroscopy.     

(4-环戊二烯基苯甲酸-铁-甲苯)六氟磷酸盐键合硅胶固定相的制备及色谱性能

基于二茂铁独特的分子结构及其作为新型液相色谱分离基质的潜质,制备了新型(4-环戊二烯基苯甲酸-铁-甲苯)六氟磷酸盐键合硅胶固定相,利用红外光谱、元素分析、热重分析等手段对其进行了表征。分别选取多环芳烃、萘胺位置异构体、硝基苯胺位置异构体、硝基咪唑类化合物和有机磷化合物溶质作为探针,对其色谱性能进行了评价。结果表明,该色谱固定相能提供 π-电子转移、 π-π 作用、偶极-偶极作用及与底物的静电相互作用等多种作用力,并探讨了可能的分离机理。     

Superposition of permeation and adsorption effects in liquid chromatography

Summary A generalized treatment of the superposition of separations based on gel-permeation chromatography (GPC) and liquid-solid chromatography (LSC) is presented. Special emphasis is given to the fact that restricted-pore entry behaviour in GPC automatically eliminates part of the surface accessible for adsorption in LSC. Therefore the simple additivity rule for K_GPC and K_ads, applied so far exclusively to adsorptive GPC interpretation, cannot be used in general and special care has to be taken if information about adsorptivities is to be deduced from the chromatographic experiment.     

Luminescence determination of benzoquinoline isomers in complex samples

The room-temperature phosphorescence (r.t.p.) and fluorescence spectra of benzoquinoline isomers are investigated. The isomers can be resolved into the linear or angular subgroups on the basis of their fluorescence and r.t.p. spectra by using conventional fixed excitation. Second-derivative and synchronous scanning techniques can be combined to improve the selectivity of the r.t.p. and fluorescence methods. These simple luminescence techniques were used to estimate three benzoquinoline isomers in a coal tar fraction. Direct analysis of this complex sample allowed acridine to be estimated and upper limits to be provided for benzo(h)quinoline and phenanthridine; the presence of three other isomers was not detected. Comparative studies with data obtained by high-performance liquid chromatography are reported.     

Free solution capillary electrophoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with L- and D-Marfey's reagents

Separation of amino acid enantiomers and peptide isomers has been made possible through the use of Marfey's reagent and high-performance capillary electrophoresis (HPCE). Samples of amino acids and peptides were first derivatized with Marfey's reagent and subsequently analyzed by HPCE. Different modes of separation were investigated including free solution and micellar electrokinetic chromatography. The use of micellar electrokinetic chromatography in combination with L- and D-Marfey's reagent offered unequivocal means to confirm the presence of D-amino acid in an unknown sample. This method is also particularly useful for the analysis of peptide isomers.     

High-performance liquid chromatographic resolution of reverse isomers of 1,2-diacyl-rac-glycerols as 3,5-dinitrophenylurethanes

The resolution of reverse isomers remains a major unsolved problem in glycerolipid chromatography. We have investigated the separation of the reverse isomers of 1,2-diacyl-rac-glycerols under a variety of high-performance liquid chromatography (HPLC) conditions. The reverse isomers of diacylglycerols having various pairs of acyl groups including short and highly unsaturated chains, which were prepared by partial Grignard degradation of the corresponding triacylglycerols, were chromatographed as 3,5-dinitrophenylurethanes. Excellent resolution was achieved for the reverse isomers of very different pairs of acyl groups, such as acetate-palmitate and docosahexaenoate-palmitate, by chiral-phase HPLC on columns containing (R)- and (S)-1-(1-naphthyl)ethylamine polymeric phases, reversed-phase HPLC on a highly efficient C18 column (4 microm particle size) and silver ion HPLC on a silver loaded cation-exchange column. The chiral-phase HPLC also permitted complete enantiomer resolution for all the reverse isomers examined. No satisfactory resolution by any of the HPLC methods, however, was obtained for the reverse isomers possessing minor differences in chain lengths and degree of unsaturation, such as laurate-palmitate and oleate-linoleate. The limitations of resolution and characteristics of elution are described.     

Comparison of available analytical methods to measure trans-octadecenoic acid isomeric profile and content by gas-liquid chromatography in milk fat

Accurate quantification of trans-fatty acids (TFAs) could be achieved by infrared spectroscopy or by gas-liquid chromatography (GLC). Accurate quantification by GLC should be achieved using specific highly polar capillary columns such as 100 m CP-Sil 88 or equivalent. A pre-fractionation of cis and trans-fatty acids could be performed by silver-ion thin-layer chromatography (Ag-TLC), silver-ion solid-phase extraction (Ag-SPE), or by high-performance liquid-chromatography (HPLC). A pre-fractionation step allows accurate determination of the isomeric profile but it is not essential to achieve quantification of total trans-18:1 isomers nor to determine the level of vaccenic (trans-11 18:1) acid in dairy fat. TFA content could also be calculated in milk fat based on the TAG profile determined by GLC. In this paper, different GLC methods suitable to measure the total of trans-18:1 isomers, vaccenic acid and trans-18:1 acid isomeric distribution in milk fat were compared. Pre-separation of cis- and trans-18:1 isomers by Ag-TLC followed by GLC analysis under optimal conditions was selected as the reference method. Results obtained using alternative methods including pre-separation by HPLC followed by GLC analysis, direct quantification by GLC or calculation from the triacylglycerol (TAG) profile were compared to data acquired using the reference method. Results showed that accurate quantification of total trans-18:1 isomers and vaccenic acid could be achieved by direct quantification by GLC under optimal chromatographic conditions. This method represents a very good alternative to Ag-TLC followed by GLC analysis. On the other hand, we showed that pre-fractionation of fatty acid methyl esters (FAMEs) by HPLC represents a good alternative to Ag-TLC, even if some minor isomers are not selectively purified using this procedure.