Superior gas-liquid chromatography of methyl cholanoate acetates on cyanopropylphenylsiloxane liquid phases

The resolution and recovery of mixtures of the methyl ester acetates of synthetic and natural bile acids are excellent on gas-liquid chromatographic columns prepared with 1–3% OV-225 on 100–200 mesh Gas Chrom Q. The columns are operated isothermally at 250–260°C with helium as carrier gas and conventional gas chromatographic equipment. Under these conditions, complete separation of the major bile acids of rat and human bile is obtained in 30–60 min. This method of analysis is superior to that based on the trifluoroacetyl or trimethylsilyl derivatives, using OV-225 or other liquid phases.     

Two-column gas chromatography of trimethylsilyl derivatives of biochemically significant compounds

Gas chromatographic (GC) retention data as methylene unit (MU) values are presented for the trimethylsilyl (TMS) derivatives of 250 biochemically significant compounds on two different GC stationary phases (OV-1 and OV-17). A ΔMU value is calculated for the difference in MU values on the moderately polar OV-17 and the nonpolar OV-1 columns. Some relationships between ΔMU values and structure are discussed; in general highly polar compounds have large ΔMU values and those compounds forming derivatives containing several TMS groups have low ΔMU values. Compounds in the following categories were studied: amino acids; organic acids; aromatic acids, amines, and glycine conjugates; and purines, pyrimidines, and other nitrogen heterocyclic compounds.     

A multiple-column approach to the methylation analysis of plant cell-walls

A procedure has been developed for the improved analysis of the neutral-sugar glycosidic linkages in plant cell-walls, utilising capillary g.l.c. and columns of three different phases for the separation of the products of methylation analysis. Retention coefficients are reported for a wide range of partially methylated alditol acetates on columns of CP-Sil88 and a bonded phase (BP-1) equivalent to OV-1. Using these phases and SP-1000, all cases of co-chromatography can be resolved. Computers were used to process the large amounts of data produced, to identify peaks and to assist in merging the results obtained using the three phases.     

Gas chromatographic analysis of free fatty acids. Part 1. Principles and methodic variants

Proceeding from a literature search the problems faced by the today's gas chromatographer concerned with analysis of free fatty acids are summarized. In terms of column technology self-made OV-1/FFAP mixed phase capillary columns are well suited for adequate tailing — free elution of these polar molecules. Whereas fatty acids having two and more carbon atoms can be analyzed in an underivatized state on acidic capillary columns, the involving of formic acid and dicarboxylic acids cells for deactivation procedures.     

The acetylation of apiitol in the determination of apiose

The complete acetylation of apiitol required 9 h when acetic anhydride at 120 degrees was used and sodium acetate was the catalyst. Both apiitol pentaacetate and apiitol tetraacetate were detected before acetylation was complete. When the reaction was done in dimethyl sulfoxide, with 1-methylimidazole as the catalyst, a third compound was observed, and identified as 1,2,4-tri-O-acetyl-3-C-(acetoxymethyl)-3-O-(methylthiomethyl)-D-glycero- tetrito l [3-O-(methylthiomethyl)apiitol tetraacetate] by gas-liquid chromatography and mass spectrometry. In N,N-dimethylformamide, with 1-methylimidazole as catalyst, the acetylation of apiitol was essentially complete in 4 h at 85 degrees, and the formation of methylthiomethyl ether was avoided. A method for preparing alditol acetates using 1-methylimidazole as the catalyst, and suitable for samples containing apiose as well as ordinary sugars, is described. The separation of apiitol pentaacetate from xylitol pentaacetate by gas-liquid chromatography proved difficult. However, a virtually complete separation of the peracetates of apiitol and xylitol as well as complete separation of those of rhamnitol, fucitol, arabinitol, mannitol, galactitol, glucitol, and myo-inositol, plus apiitol tetraacetate and 3-O-(methylthiomethyl)apiitol tetraacetate, was accomplished with a 30 m x 0.53 mm (i.d.) SP-2380 column in 49 min, and on a 30 m x 0.75 mm (i.d.) SP-2330 column in 82 min. A complete separation of apiitol and xylitol pentaacetates as well as four other alditol peracetates was obtained with a 60 m DB-1 column in 15.2 min, however this column did not resolve the acetates of fucitol and arabinitol. A variety of other columns and column conditions were ineffective.     

Steroids in porcine follicular fluid: analysis by HPLC, capillary CG and capillary CG/MS after purification on SEP-PAK C18 and ion exchange chromatography

Steroids in porcine follicular fluid have been concentrated by reverse phase chromatography in SEP-PAK C18 and purified further on the cation exchanger SP-Sephadex C-25. Fractionation into unconjugated neutral and phenolic steroids, glucuronides and sulfates was carried out on triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). The unconjugated neutral fraction was analysed by high pressure liquid chromatography (HPLC) on a C18 radial cartridge 5 mm I.D.; 10 mu, or on a C18 5 mu RESOLVE column, and by capillary gas chromatography (GC) on a 12 M OV-1 cross linked fused silica column. Testosterone, progesterone and androstenedione were the major steroids detected by HPLC monitored at 254 nm, although 17- hydroxy-, 20 alpha-dihydro- and 20 beta-dihydroprogesterone were also present. Pregnenolone, pregnanediol, dehydroepiandrosterone, 17-hydroxypregnenolone and androsterone were detected by capillary CG as their 0-methyloxime trimethylsilyether derivatives. Further confirmation of structure was provided by complete mass spectral data or by selective ion monitoring (SIM).     

Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopropane fatty acids and alk-1'-enyl ethers by capillary column chromatography

The positional isomers of the cyclopropane fatty acids of Clostridium butyricum phospholipids have been analyzed by capillary column gas-liquid chromatography. Greater than 95% of the methylenehexadecanoic acids was the 9,10 isomer. On the other hand, 60-70% of the hexadecenoic acid precursors was the Delta(7) isomer, and the remainder was the Delta(9) isomer. Of the methyleneoctadecanoic acids 75-80% was the 11,12 isomer, with the remainder being the 9,10 isomer. There were approximately equal amounts of the Delta(9)- and Delta(11)-octadecenoic acids in the phospholipids. This study reveals a surprisingly strong specificity of the cyclopropane synthetase for the (n-7) series of monoenoic fatty acids. An analysis by capillary column chromatography of the monoenoic and cyclopropane aldehyde dimethylacetals derived from the plasmalogens (1-alk-1'-enyl-2-acyl-glycero-phosphatides) of C. butyricum revealed the presence of the same positional isomeric mixtures of the 16- and 18-carbon monoenoic residues in approximately the same ratios as were found in the fatty acids. In the formation of the cyclopropane alk-1'-enyl ethers there was also specificity for the (n-7) series, but it was not as strong as that seen in the fatty acids. The ratio of the 7,8 isomer to the 9,10 isomer was higher in the methyl-enehexadecanals than in the corresponding fatty acids. This paper extends the use of Golay capillary columns to the analysis of the positional isomers of plasmalogen aldehydes as their dimethylacetal derivatives.     

Gas chromatographic-mass spectral characteristics of aporphine and tetrahydroprotoberberine alkaloids

The gas chromatographic (GC) and mass spectrometric (MS) properties of the silyl derivatives of aporphine and tetrahydroprotoberberine alkaloids are described. Selected representatives of these chemical classes of pharmacologically active bases were chromatographed on polar (OV-17) and nonpolar (OV-1) columns as their trimethylsilyl derivatives. The aporphines were eluted before the tetrahydroprotoberberines on both the polar and nonpolar columns. Simultaneous resolution of mixtures of aporphines and tetrahydroprotoberberines was readily achieved on the OV-1 column. An SE-30 column, used for combined GC-MS analysis, gave a similar resolution of these alkaloids. The mass spectra observed for the silylated 1,2,9,10-substituted aporphines were similar to those of underivatized aporphines, while the mass spectra of the silylated 1,2,10,11-substituted aporphines differed markedly from the spectra of the underivatized alkaloids. Although the mass spectra of the silylated derivatives of the 2,3,9,10- and 2,3,10,11-substituted tetrahydroprotoberberines were identical, these isomeric derivatives were separated by gas chromatography.     

Improved method for simultaneous determination of alcohols, volatile fatty acids, lactic acid or 2,3-butanediol in biological samples

A rapid and sample procedure was developed to determine, by gas-liquid chromatography, the concentrations of C₂---C₄ alcohols, C₂---C₆ volatile fatty acids (VFA) and lactic acid or 2,3-butanediol in fermentation liquids. both lactic acid and 2,3-butanediol are oxidized to acetaldehyde by periodic acid and acetaldehyde was eluted before ethanol. A complete separation of the alcohols and acids was performed in <15 min on a column packed with 80/100 Chromosorb WAW, having GP 10% SP-1200/1% H₃PO₄ as the liquid phase. The method was suitable for the analysis of rumen fluid and fermentation products from microbial cultures. The detection limits for all compounds were <0.13 nmol · injection⁻¹.     

Estimation of plasma free fatty acids as the trimethylsilyl (TMS) esters

Quantitative estimates of free fatty acids in total lipid extracts of plasma were obtained by glc on nonpolar columns following trimethylsilylation. The presence of other lipid esters in the reaction mixture had no effect upon the yield of the trimethylsilyl (TMS) esters or upon their resolution on the glc column. Routine quantitations by gas-liquid chromatography (glc) were obtained on 2 ft × 1/8 in. o.d. stainless steel columns packed with 3% OV-1 on 100–120 mesh Gas Chrom Q by means of temperature programming in the range 175–350°C with tridecanoin as internal standard. High resolution glc of the TMS esters of fatty acids was done on a 6 ft × 1/8 in. o.d. glass column packed with 1% SE-30 on Gas Chrom Q. In both instances the fatty acids were resolved on the basis of carbon number and by the presence or absence of double bonds. On gas chromatography/mass spectrometry (GC/MS), TMS esters of fatty acids were shown to yield proportionally greater amounts of high mass fragments, including the parent ions, than their methyl or ethyl esters, which has special advantages for the detection and characterization of polyunsaturated fatty acids.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Steroids and hypertension. 1--Gas-liquid chromatography and gas chromatography mass spectrometry of 3,15- and 3, 16-dihydroxy-17-oxosteroids

In order to analyse and quantitate the urinary 16-oxysteroids known or thought to be associated with hypertension, we have established for six 16-oxy-C19 reference steroids the following parameters: elution volume on lipophilic gel columns, gas chromatographic retention data expressed as methylene unit values of trimethylsilyl ether and O-methoxime trimethylsilyl ether derivatives on OV-1 and OV-17 packed columns and on SE-30 capillary column, and mass spectra of these compounds. These reference steroids were: 3 alpha, 16 alpha-dihydroxy-5 alpha-androstan-17-one, 3 alpha, 16 alpha-dihydroxy-5 beta-androstan-17-one, 3 beta, 16 alpha-dihydroxy-5 alpha-androstan-17-one, 3 beta, 16 alpha-dihydroxy-5-androsten-17-one, 3 beta, 16 beta-dihydroxy-5-androsten-17-one, 3 beta, 17 beta-dihydroxy-5-androsten-16-one and 3 alpha, 15 alpha-dihydroxy-5 beta-androstan-17-one. The proposed method was shown to be applicable to the specific analysis of 16-oxy-C19-steroids in biological samples since it achieved the selective isolation of these compounds from other steroids and their quantitative elution in a single fraction. The analysis of the urinary steroids of two patients with arterial hypertension demonstrated an elevated rate of 3 beta, 16 alpha-dihydroxy-5-androsten-17-one.     

Gas chromatographic determination of the optical purities of amino acids using N-trifluoroacetyl menthyl esters

Nineteen α-amino acids and one β-amino acid were resolved as their N-TFA menthyl esters by gas-liquid chromatography. Neutral and basic amino acids were well resolved on some conventional packed columns. For the complete resolution of acidic amino acids, a capillary column was used. The results of the applications to some biochemical substances showed that this method is of value for practical usage. The mechanism of the resolution was also investigated and showed that the difference between the derivatives of the enantiomeric pairs in the ability to form intermolecular hydrogen bond contributes essentially to the resolution.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside

Described herein is the synthesis of eight positional isomers of methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside. The compounds were generated simultaneously from methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography (HPLC). The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography (GLC) retention indices on three different capillary columns.     

Analysis of bile acids in serum and bile by capillary gas-liquid chromatography

Various liquid phases for glass capillary columns have been evaluated for gas-liquid chromatographic analysis of methyl ester trimethylsilylether derivatives of bile acids from serum and bile. Bile acid analysis is rapid and exhibits high separation efficiency with a 20 X 0.3 mm glass capillary column whose internal surface is covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20000 as liquid phase according to Grob et al.     

The Minor Chemical Components of the Absolute Oil from the Flower of Jasminum sambac L. Wu Cheng-(L.) Aiton

The concrete extracted from the flowers of Jasminum sambac is a precious perfume. The absolute has been analysed by CC-MS with two capillary columns of non-polar (OV-101) and polar (PEG-20 M) stationary phase respectively. The Kovats indices of the peaks on OV-101 were determined also. The results were highlighted on minor components and are shown in Tables 1 and 2. Several important components are: 2,6-dimethyl heptanal; methyl n-methyl anthranilate; bergmotene; torreyol; cis-3-hexenyl butyrate; cislinalool oxide; 2H-pyran-3-ol, 6-ethenyl tetrahydro-2,2,6-trimethyl and a few sesquiterpenes. Together with those previously determined, 50 components of the absolute have been identified, their contents have been determined also. These results offer quite detailed information to the compounding and technology in perfumery.     

Glass capillary columns applied to prostaglandins measurement: a useful tool for gas-chromatography mass spectrometry analysis

The difficulties to analyse prostaglandins (PG) by gas-liquid chromatography are mainly due to the lack of sensitivity of the gas-chromatograph itself (higher than 200 ng) and to the poor resolution of the packed columns. Therefore the use of glass capillary columns which has been applied with success for other biological compounds was tempting. We describe a comparison of the preparation of the columns and their use for PG analysis of standards and of human semen. A complete resolution of PG-1 from PG-2 series was achieved. The sensitivity was multiplied 100 fold with a flame ionisation detector when compared to packed columns and was equal to the one obtained with electron capture detectors without the inconveniences of this technique. The successful coupling of glass capillary columns to a mass spectrometer leads to promising results and allows profile studies of primary PG and their metabolites as seen with human semen.     

Glass capillary or fused-silica gas chromatography—mass spectrometry of several monosaccharides and related sugars: Improved resolution

The gas chromatographic separation of several monosaccharides and related sugars derivatized by methoxylation and trimethylsilylation reactions was optimized with glass capillary (SP-2250) and fused silica (SP-2100) columns. Individual sugars included aldoses, ketoses, polyols, acidic forms and N-acetylated amino sugars. Peaks were detected by selected ion monitoring (SIM). The fused silica column gave complete resolution of all peaks (two per hexose and one per hexitol) arising from glucose, galactose, mannose, fructose, sorbitol, mannitol and dulcitol. The resolution of these sugars with the glass capillary column was not as good, but full differentiation was possible on the basis of SIM. Because the fused silica column gave a better resolution of 33 sugars tested and was more easily installed than the glass capillary column, it was utilized for quantitative analysis. A deuterated algal sugar mixture used for quantitation by isotope dilution was found to contain glucose, galactose, mannose, xylose, arabinose, ribose and rhamnose. Full recoveries were obtained of various amounts of glucose, galactose, mannose, fructose and xylose added to human serum.     

Steric analysis of 3-, ω4-, ω3- and ω2-hydroxy acids and various alkanols by gas-liquid chromatography

D-Phenylpropionate (PP) derivatives of racemic hydroxy acid methyl esters and alkanols were prepared and the gas-liquid chromatographic separation of diastereoisomers was investigated using QF-1 as stationary phase. Good separations were obtained for the diasteroisomeric PP-derivatives of methyl 3-, 15-, 16-, and 17-hydroxyoctadecanoates. Less separation was observed for methyl 2- and 14-hydroxyoctadecanoates as PP-derivatives and there was no visible separation for PP-derivatives of 4-, 7-, or 13-hydroxyoctadecanoic acid methyl esters. The use of optically active 15-, 16- and 17-hydroxyoctadecanoic acids showed, that in these cases, the diastereoisomers containing L-hydroxy acids had shorter retention times than the ones containing D-hydroxy acids. On the other hand, the D-phenylpropionate derivative of methyl 3D-hydroxydecanoate had shorter retention time than the derivative of its L-enantiomer. PP-derivatives of 3-hexanol, 3-heptanol, 3-octanol, 2-octanol and 2-eicosanol could be resolved by gas-liquid chromatography.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydroribitol

The eight positional isomers of methylated and benzoylated 1,5-anhydroribitol were generated simultaneously from 1,5-anhydroribitol by sequential partial methylation and benzoylation, and the four meso isomers and two enantiomeric pairs of isomers so-formed were isolated in chemically pure form by high-performance liquid chromatography. The corresponding acetates were obtained by debenzoylation of the pure isomers and acetylation. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.