Gel permeation chromatography of oxygenated components of cigarette smoke condensate

Oxygenated neutral constituents of a tumor-inhibiting fraction of cigarette smoke condensate were separated from interfering phenolic compounds by a combination of silicic acid and gel chromatography. Silicic acid adsorption and Bio-Beads gel chromatography separated aliphatic from aromatic ketones, and gel chromatography on Sephadex LH-20 resolved aromatic ketones from phenols. Identifications were achieved by studying individual fractions by g.c. and g.c.—m.s.     

Fractionation of cigarette smoke condensate for chemical and biological testing

Large-scale fractionation of cigarette smoke condensate (CSC) was carried out by gel filtration and silicic acid column chromatography, and selected fractions and their subfractions were tested for tumorigenicity by mouse-skin bioassay. The weak-acid fraction was separated into four subfractions, the polynuclear aromatic hydrocarbon (PAH) fraction into two subfractions, and the polar neutral lipid fraction into three subfractions. Also, combinations of subfractions were examined for synergic effects and portions of all active material were subjected to chemical analyses by gel filtration, column, thinlayer and gas chromatography, and ultraviolet and mass spectrometry. A weak-acid subfraction (F-63) in which catechol was concentrated, and which comprized 3.27% CSC was shown to be tumorigenic, as were combinations of this sub fraction with the active PAH (F-67) sub fraction and a polar, neutral lipid (F-70) sub fraction, representing 0.01 and 0.05% CSC, respectively. The results indicated that catechol may be a potent cocarcinogen and that the PAH in CSC interact with other components to exert a tumorigenic effect.     

Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia

An approach for the separation and identification of components in a traditional Chinese medicine Psoralea corylifolia was developed. Ion-exchange chromatography (IEC) was applied for the fractionation of P. corylifolia extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further separated on an ODS column with detection of UV absorbance and atmospheric pressure chemical ionization mass spectrometer (APCI/MS), respectively, and also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes. Totally more than 188 components in P. corylifolia extract were detected with this integrated approach, and 12 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS. The obtained analytical results not only demonstrated the powerful resolution of integration IEC fractionation with reversed-phase liquid chromatography (RPLC)-APCI/MS and MALDI-TOF/MS for analysis of compounds in a complex sample, but also exhibited the superiority of APCI/MS and MALDI-TOF/MS for identification of low-mass compounds, such as for study of traditional Chinese medicines (TCMs) and metabolome.     

Resolution of Polynuclear Aromatic Hydrocarbons by Packed Column GLC

Abstract

Specific GLC stationary phases have been identified which allow the resolution of certain polynuclear aromatic hydrocarbon (PAH) isomers with packed six foot columns. Phenanthrene and benz(a)anthracene can be measured in the PAH fraction of tobacco smoke condensate without significant interferences from anthracene and chrysene, respectively.     

Determination of benzo[a]pyrene in cigarette smoke condensate by liquid chromatography on amberlite xad-2

A method is described for the determination of benzo[a]pyrene in cigarette smoke condensate which utilizes chromatographic fractionation on Amberlite XAD-2. PAH are initially separated by step-wise gradient elution, progressing from reverse to normal-phase modes of operation. Other separation steps involve automated column chromatography on silica gel and thin-layer chromatography on 20% acetylated cellulose. Benzo[a]pyrene is finally determined by u.v. spectrophotometry and liquid scintillation counting of ¹⁴C—benzo[a]pyrene tracer. Results obtained compare favorably with those of the more traditional liquid—liquid extraction methods.     

Quantitative Bestimmung von Benzo(a)pyren in Zigarettenrauchkondensaten durch Hochdruckflüssigkeits-Chromatographie (HPLC)

Following a clean-up procedure by liquid-liquid distribution and column chromatography on a layer-column silica gel/aluminium oxide, a benzo(a)pyrene concentrate from cigarette smoke condensate was separated by high pressure liquid chromatography. As selective separating system served a column with cross-linked cellulose acetate. Benzo(a)pyrene was quantitatively determined with a fluorescence detector at a detection limit of 10–30 ng/5 μl injection volume. A determination was carried out from the same benzo(a)pyrene concentrate on a reverse-phase system.     

Determination of an experimental plant growth regulator on wheat and cotton plants by reversed-phase ion-pair partition high-performance liquid chromatography

After extraction from spiked wheat and cotton samples, and preliminary clean-up by anion-exchange chromatography, the plant growth regulator, 2-benzoxazolinylidenma-lononitrile, was determined by reversed-phase ion-pair partition h.p.l.c. wih tetrabutyl-ammonium as the pairing ion. In extracts, the solute could be separated from interferences by varying the tetrabutylammonium concentration; the average recovery of the compound from wheat plants (0.13–55.6 ppm) was 96.5% and from cotton plants (0.15–134.6 ppm) 104.4% by the h.p.l.c. method. These recoveries were confirmed by the scintillation counting method with the radiolabelled compound. In a dissipation study, a biological half-life of 1.5–2.0 days was found for the compound on cotton plants. Reversed-phase ion-pair partition h.p.l.c. is a promising technique for the determination of trace levels of ionic (or ionizable) compounds in environmental samples.     

Evaluation of octadecyl-bonded alumina for separations of proteins and peptides by reversed-phase high-performance liquid chromatography

Summary A recently-developed octadecyl-bonded alumina (ODA) stationary phase was evaluated for the separation of peptides and proteins by reversed phase high performance liquid chromatography. Using standard water-acetonitrile mobile phase gradients containing 0.1 % trifluoroacetic acid, the average peak capacity obtained for the separation of a mixture of ribonuclease a, cytochrome c, lysozyme and carbonic anhydrase on an ODA column are similar to that obtained on a widely used octadecylsilane (ODS) column. However, overall chromatographic resolution of the components of this mixture on ODA is inferior to that obtained on ODS. Cytochrome c peak areas were found to be 50% smaller on the ODA column than on ODS. On the other hand, both peak capacities and resolutions of octapeptide mixtures were found to be generally superior on the ODA column, and peak areas for a representative octapeptide were found to be virtually identical for both ODA and ODS columns. The differences in the results obtained on the ODA and ODS columns for these separations are attributed to the smaller pore size and unique fused-microplatelet shape of the ODA particles. Comparisons of the separations of the tryptic digest of cytochrome c on the ODS and ODA columns demonstrate that the ODA phase is potentially as useful as ODS for peptide mapping applications.     

A Chemical Study of Burley Tobacco Flavour (Nicotiana tabacum L.). I. Volatile to medium-volatile constituents (b.p. ≦ 84°/0.001 Torr

The fraction b.p. ≦ 84° (0.001 Torr) from Burley tobacco condensate was carefully investigated using fractional distillation and preparative column and gas liquid chromatography aided by GLC/MS coupling. Among the 193 compounds thus separated and characterized by their spectral data, 81 were newly identified tobacco constituents. Most of the compounds isolated in the course of this work display flavouring properties which make them highly suitable for improving the flavour and aroma of tobacco and tobacco smoke.     

The Identification of Polynuclear Aromatic Hydrocarbon (PAH) Derivatives in Mutagenic Fractions of Diesel Particulate Extracts

Abstract

The soluble organic fraction (SOF) of particulate matter from diesel exhaust (from point sources, ambient air, etc.) contains hundreds of organic constituents. Normal-phase high pressure liquid chromatography (HPLC) has been used to separate the SOF into sub-fractions suitable for subsequent chemical analysis and bioassays. These fractions consist of non-polar(PAH), moderately polar (transition) and highly polar constituents. The non-polar fractions have been well characterized and consist of PAH and aliphatic hydrocarbons. The specific compounds present in the transition and polar fractions are for the most part unknown. This analytical information has been difficult to obtain since these compounds are highly labile, polar, of low volatility and in very low concentrations when compared to the bulk of material found in the SOF. Mutagenicity tests using the Ames Salmonella typhimurium assay indicate that the transition fraction accounts for most of the mutagenicity when compared to the non-polar (PAH) and polar fractions.

A variety of chromatographic and mass spectrometric techniques are described that have been used to determine the composition of the HPLC fractions. More than one hundred species have been identified in the transition fraction of diesel particulate matter using high resolution gas chromatography (HRGC)/high resolution mass spectrometry (HRMS), HPLC and direct-probe high resolution mass spectrometry. It has been found that the transition fraction contains mostly PAH derivatives consisting of hydroxy, ketone, quinone, carboxaldehyde, acid anhydride and dihydroxy derivatives of PAH. Three nitro-PAH species have been tentatively identified and 1-nitropyrene positively identified in the transition fraction. The 1-nitropyrene was found to account for approximately 45% and 30% of the direct-acting mutagenicity observed for the transition fraction and total extract, respectively. The HPLC separation procedure was shown to give better than 95% recovery of the mass and mutagenic activity. The problem of PAH oxidation during the analytical procedures and possible effect on bioassay results are discussed.     

Preparation of a high performance liquid chromatography column based on N-(3,5-dinitrobenzoyl)-aminoundecyl silica for the resolution of liquid crystal mixtures

A bonded silica stationary phase (SP 1) was prepared by connecting N-(3,5-dinitrobenzoyl) aminoundecylsilane to silica gel. The stationary phase was applied in resolving a liquid crystal mixture with a reversed phase high performance liquid chromatography (HPLC) mode and the chromatographic resolution results were compared with those on an octadecylsilane (ODS) column. From the comparison of the resolution results on SP 1 and the ODS column, we found the new stationary phase was better than the ODS column in resolving a liquid crystal mixture and the elution orders of some liquid crystal were changed. The better resolution and the change in the elution orders on the new column might be originated from additional π–π interaction between the π-acidic 3,5-dinitrobenzoyl group of the stationary phase and the π-basic aromatic group of liquid crystals.     

Method validation for determination of the 15 European-priority polycyclic aromatic hydrocarbons in primary smoke condensates by gas chromatography/mass spectrometry: interlaboratory study

A collaborative study was conducted to validate an analytical method for quantification of the 15 polycyclic aromatic hydrocarbons (PAHs) regarded in 2002 as a health concern by the former Scientific Committee on Food of the European Commission (SCF) in primary smoke condensates. The method is based on gas chromatography/mass spectrometry of a cyclohexane extract with solid-phase cleanup through silica gel. The analytes were detected in the selected-ion monitoring mode and quantified by using 3 isotopically labeled internal standard compounds. Seventeen laboratories participated in the collaborative validation study, of which 12 reported valid results. The data were subjected to Cochran, single Grubbs, and double Grubbs tests for statistical outliers. A maximum of 2 outliers was eliminated before further statistical evaluation of the method performance characteristics. Depending on the analyte, the results showed relative standard deviations for repeatability between 4.2 and 30% and for reproducibility from 9.9 to 60%. The recoveries varied between about 50 and 85%, except those for cyclopenta[cd]pyrene, dibenzo[a,l]pyrene, and dibenzo[a,h]pyrene. Nevertheless, because Commission Directive 2005/10/EC allows for a recovery range of 50-120% for (BaP) benzo[a]pyrene in various foods, it can be concluded that the method performs appropriately within the analytical range between 5 and 25 microg/kg of primary smoke condensate. For BaP the validated analytical range covered 5-20 microg/kg, and for benzo[a]anthracene (BaA) 10-25 microg/kg. The method is suitable for monitoring BaP and BaA at their respective maximum permitted levels of 10 and 20 microg/kg. Three analytes, benzo[b]-, benzo[j]-, and benzo[k]-fluoranthene could not be separated by all of the participants and were therefore treated as the sum. Nevertheless, with this method the pattern of the respective concentrations of these 15 PAHs can be monitored in primary smoke condensate as suggested by the SCF.     

Comparative chemical analysis of commercial creosotes and solvent refined coal-II materials by high resolution gas chromatography

The chemical composition of a commercially available creosote was compared to a direct coal liquefaction product, i.e., solvent refined coal-II fuel oil blend (SRC-II FOB) using high resolution gas chromatography (HRGC). In addition, hydrogenated products of these materials were studied. Samples were fractionated by chemical class on neutral alumina. Those fractions previously shown to be the most mutagenic and tumorigenic in laboratory bioassays of coal-derived materials were analyzed and compared by HRGC and gas chromatography/mass spectrometry (GC/MS). Individual components were tentatively identified and quantitated. Although similar chemical components were present in the creosote and SRC-II FOB fractions studied, the creosotes had higher concentrations of heavy molecular weight materials and a lower ratio of alkylated to parent polycyclic aromatic compounds than the coal liquefaction products. The creosote samples also had a significantly higher concentration of components which eluted in the polycyclic aromatic hydrocarbon (PAH) chemical class fraction. Amino-substituted PAH were present in both nonhydrogenated coal liquid and creosote materials. The creosote and SRC-II FOB crudes and nitrogen-containing polycyclic aromatic compound (NPAC) chemical class fractions expressed similar microbial mutagenicity. Based on chemical analysis data, the predicted tumorigenic potency of the creosote in laboratory bioassay systems would be equivalent to or greater than the SRC-II FOB.     

Zur Hochdruck-Flüssigkeits-Chromatographie von polycyclischen aromatischen Kohlenwasserstoffen

Two aspects of high-pressure liquid chromatography (HPLC) of polycyclic aromatic hydrocarbons (PAH) have been studied: The application of HPLC to the analysis of very highly condensed PAH, that cannot be analyzed by gas chromatography, and the application of u.v. spectroscopy to the identification of PAH, that have been separated by HPLC. A device, that allows the rapid scanning of u.v. spectra within the HPLC apparatus, is described.     

Two-dimensional liquid chromatography coupled with mass spectrometry for the analysis of Lobelia chinensis Lour. using an ESI/APCI multimode ion source

A comprehensive approach for the separation and identification of components in a traditional Chinese medicine Lobelia chinensis Lour. was developed using 2D-HPLC coupled with an online photodiode array (PDA) detector and a mass spectrometer. The extract of L. chinensis Lour. was separated on a CN column in the first-dimensional HPLC, and then each of the collected fractions was further separated on an ODS column followed by an online PDA detector. After separation in the two different chromatographic modes, the eluents were delivered to a quadrupole mass spectrometer equipped with a multimode ion source of an ESI and an atmospheric pressure chemical ionization (ESI/APCI). At least 536 components in L. chinensis Lour. extract were detected and 6 of them were identified as apigenin 7-O-rutinoside, luteolin, lobetyolinin, lobetyolin, diosmin, and linarin, respectively, according to their UV spectrum and mass spectrum. The results demonstrated the powerful resolution, high peak capacity, as well as the identification capability of the 2D-HPLC combined with PDA and ESI/APCI-MS for the analyses of complex samples.     

Separation of fatty acid methyl esters by comprehensive two-dimensional supercritical fluid chromatography with packed columns and programming of sampling duration

Fatty acid methyl esters from various fats and oils were separated by comprehensive two-dimensional supercritical fluid chromatography with conventional packed columns and FID detection. The first dimension was a silica gel column and the second dimension was an ODS column. This combination was largely orthogonal for the separation of fatty acid methyl esters. The first dimension separations were primarily based on the number of double bonds while the second dimension separations were based on the chain length. The highly-ordered chromatograms and improved resolution allowed the easy detection and identification of minor components. Although the first dimension separations were performed under isobaric conditions where the peak width increased in proportion to the retention, the programming of the sampling duration allowed us to maintain the optimum re-injection frequency (3–4 times) per peak into the second dimension and so to minimize the total analysis time without deteriorating the resolution.     

Isolation and Characterization of Sulfur-Containing Polycyclic Aromatic Compounds (Thia-Arenes) from Complex Environmental Mixtures

Abstract

A multi-dimensional chromatographic method was developed and applied to the separation of thia-arenes and polycyclic aromatic hydrocarbons (PAH) derived from coal tar. A thia-arene-rich fraction, prepared using a combination of alumina chromatography and palladium chloride/silica gel chromatography, was further separated using normal phase HPLC to isolate fractions containing thia-arenes with molecular masses ranging from 184 amu to 284 amu. These fractions were analysed using gas chromatography-mass spectrometry; approximately one-half of the thia-arenes in a coal tar extract were retained and separated by the palladium chloride-silica gel step. This methodology has been successfully applied to coal tar, air particulate material, sediments and biological samples.     

A manual sequence method of peptides and phosphopeptides using 4-(1'-cyanoisoindolyl)phenylisothiocyanate

A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1'-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.     

Nachweis von Dibenzofuranen im Zigarettenrauchkondensat von Virginiatabak mittels Kapillargaschromatographie-Massenspektrometrie nach Wasserdampfdestillation sowie dünnschichtund hocheistungsflüssigkeitschromatographischer Abtrennung

Zusammenfassung Dibenzofuran und die vier isomeren Monomethyldibenzofurane wurden aus dem Zigarettenrauchkondensat von Virginiatabak mittels Wasserdampfdestillation, Dünnschichtchromatographie, Hochleistungsflüssigkeitschromatograhpie und Gaschromatographie abgetrennt. Charakterisierung und Identifizierung erfolgten durch Kapillargaschromatographie-Massenspektrometrie, sowie durch UV-Spektrometrie und durch den Vergleich chromatographischer Daten unter Zuhilfenahme authentischer Vergleichssubstanzen.

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Determination of dibenzofurans in cigarette smoke condensate of virginia tobaccos by means of gaschromatography — Mass spectrometry after separation by steam distillation, thin-layer chromatography and high-performance-liquid chromatograpby

Summary Dibenzofuran and the four isomers of monomethyldibenzofuran were isolated from cigarette smoke condensate of Virginia tobaccos by means of steam distillation, thin-layer chromatography and high performance-liquid chromatography. The characterization and identification were mainly achieved by capillary gaschromatography in combination with mass spectrometry as well as by comparison of Chromatographie retention data applying the respective substances as standards.