Prediction,optimization of separation,and identification of unknown compounds in capillary gas chromatography

A procedure for the identification and separation of unknown compounds by capillary gas chromatography is described. The procedure involves a live retention time database and optimization of the separation. After initial chromatography of the sample, a rough search lists all the possible compounds it might contain and the analyst then uses his experience to discard those compounds in the list which are unlikely to be present. The multi-component separation is then optimized over the whole range of defined starting temperatures and programming rates, in order to produce the best possible separation of the sample components, and the chromatography repeated using the results obtained from the optimization procedure. Further search operations within a given search window will then report the compound names, and related information, for each peak. Since the identification operation is performed at least twice during the procedure, and the optimization of the separation assists the identification by separating possibly overlapped peaks, the confidence of the qualitative analysis is higher than may be obtained using standards alone. If the reproducibility of isothermal indices measured on columns could be guaranteed, this procedure could be used instead of performing chromatography on standards of the compounds contained in the database, regardless of changes in column dimensions, phase ratio, and operating conditions in temperature programmed analysis.     

Factors affecting the reproducibility and reliability of retention simulation in any form of temperature programmed capillary GC

Conversion of Kováts retention indices on a given stationary phase into the thermodynamic parameters of compounds on a given column leads to a simplified method for retention simulation in isothermal, linear, and multi-ramp temperature programmed capillary gas chromatography. The influence of numerical methods used in the computation, the temperature coefficient of Kováts indices, and the experimental factors such as isothermal temperatures selected in the measurement of n-alkanes, column characterization and sample overloading, on the reproducibility and accuracy of simulation were discussed and examined. When the column used is properly characterized, the error between the simulated values and the experimental data is within ± 0.5 index unit or less than ± 1% of retention time.     

Data for the gas-liquid chromatographic analysis of essential oils. Determination of the composition of the essential oil of Marjoram

Summary By identification of the essential oil component ‘cis-sabinene hydrate acetate’ of marjoram we demonstrate that the mass spectrum—furnish to the chemical composition and molecular configuration of compounds—can lead to incorrect qualitative identification if retention data are not taken into consideration. We studied also the temperature-dependence of the retention data of different compounds, and our measurements showed that because of the different temperature-dependence, a change in the temperature of the analysis results in a change in order of elution. The use of this phenomenon, originating from the change of the retention data, both increases the safety of qualitative identification and aids determination of the optimum measurement conditions in both isothermal and temperature-programmed modes of operation. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

An alternative approach for the estimation of equivalent temperature in gas chromatography

The temperature at which the isothermal retention time (tRiso) is equal to the temperature-programmed retention time (tRTPGC) or the isothermal retention index (Iiso) is equal to the temperature-programmed retention index (ITPGC) is defined as the equivalent temperature (Teq). The Teq of one-, two-, three-, and four-step temperature-programmed gas chromatography (TPGC) of unsaturated fatty acid methyl esters (FAMEs) from Chinese mustard seed oil is calculated. All of the tR values obtained at the Teq (tRTeq) are very close to the tRTPGC. The highest difference for each chromatogram is less than 0.5%. The slight deviation may partly arrive from the difficulty in setting the desired carrier gas flow rate. Also, good agreement among equivalent chain length values determined by different methods is observed, including the graphical method at the Teq. Therefore, it is speculated that the proposed method may facilitate the GC identification of FAMEs as well as other organic compounds in TPGC by using the available isothermal retention index database.     

色谱与色谱/质谱法相结合分析热裂解汽油C9馏分

采用毛细管气相色谱-氢火焰离子化检测器（CGC-FID）和气相色谱-质谱法(GC/MS)分析了热裂解汽油C9 馏分的组成。实验使用PONA毛细管气相色谱柱(100 m×0.25 mm i.d.×0.5 μm),根据烃类化合物在PONA柱上的保留规律,以正构烷烃标样保留值作为碳数分布依据,定量分析了裂解汽油C9 馏分中烃类化合物的碳数分布和单体烃含量;用GC/MS联用技术和CGC保留值定性法相结合对裂解汽油C9 馏分中相对含量大于0.2%的39种化合物进行了定性。     

Lipid profile of fish species by liquid chromatography coupled to mass spectrometry and a novel linear retention index database

In a previous article, Rigano et al. established a new linear retention index system for the identification of triacylglycerols by liquid chromatography methods only on the basis of the retention behavior and independently from many experimental parameters. In that work, a database of 209 compounds was built, but only 54 of them, typical of vegetable oils, were confirmed by mass spectrometry. The aim of the present research is to extend the applicability of the novel approach to more complex samples, such as fish lipid extracts, and assess the complementarity between mass spectromtery and retention information to achieve univocal identification. With this purpose, a new software was implemented to make the identification process easy and automatic as in gas chromatography‐mass spectrometry where the retention index filter is added in the spectral search to discriminate between compounds with similar mass spectrometry spectra. A total of 69 species were identified and, thanks to their baseline separation obtained by an ultra high performance liquid chromatography method, a semiquantification was also performed. The species under investigation were Dicentrarchus labrax, coming from aquaculture and the wild. Some differences in their native lipid composition were observed, probably related to a different diet. A major number of samples would be necessary to confirm such a preliminary finding.     

Use of Retention Data as the First Step in the Identification of Cyclic Organic Peroxides in Temperature-Programmed Gas Chromatography

Temperature-programmed retention indices for eleven cyclic organic peroxides were determined by gas chromatography on slightly polar 5% biphenyl 95% dimethylpolysiloxane columns (DB-5 and Rtx-5MS) at three heating rates (5, 10, and 20° min⁻¹) from 60 to 300°C, using different chromatographs. Cyclic organic diperoxides and triperoxides had nearly constant retention indices when different heating rates and a short isothermal hold time (5 min) before the programmed increase in temperature were used. The usefulness of temperature-programmed retention indices was shown by using the data to predict the retention times and structures of unknown diperoxides or triperoxides derived from ketones. This is the first step in the identification of unknown cyclic organic peroxides, a process would otherwise require the availability of reference compounds. Revised: 7 and 17 November 2005     

GC behaviour of symmetrical n-dialkyl sulphides under isothermal and temperature programming conditions

Retention indices were determined for a homologous series of n-dialkyl sulphides on three stationary phases (SE-30, OV-17 and XE-60) under isothermal and linear temperature programming conditions. Under these two different GC conditions, equations were derived for each of the three stationary phases which showed the dependence of retention index on the number of carbon atoms and the boiling points for a homologous series of n-dialkyl sulphides. The equation for the correlation isothermal retention index was shown to be applicable to the identification of n-dialkyl sulphides using linear temperature programming. It was found that the GC behaviour of n-dialkyl sulphides makes these compounds suitable for use as a standard series instead of n-alkanes for the calculation of retention indices in GC analysis in which detectors insensitive to n-alkanes are employed. The use of the homologous series of n-dialkyl sulphides for the calculation of sulphide retention indices can be great practical importance in the microanalysis of natural compounds. We have used this method successfully in the analysis of pesticides containing S-atoms.     

Determination of retention indices in constant inlet pressure mode and conversion among different column temperature conditions in comprehensive two-dimensional gas chromatography

A method to determine the second dimensional real retention time, dead times on both dimensions and retention indices in constant inlet pressure mode was developed in comprehensive two-dimensional gas chromatography. At the same time, the conversion of GC x GC retention indices among different column temperature conditions were also conducted based on some thermodynamics parameters. The calculation accuracies are better than 1.0 retention index unit. Furthermore, a retention index database was developed and used to identify the compounds in a cigarette essential oil sample. It showed that identification by the database was of close agreement with by time-of-flight mass spectrometry, and some isomers could also be distinguished based on the retention index database.     

Temperature programmed retention indices: Calculation from isothermal data. Part 2: Results with nonpolar columns

The procedure for calculating linear temperature programmed indices as described in part 1 has been evaluated using five different nonpolar columns, with OV-1 as the stationary phase. For fourty-three different solutes covering five different classes of components, including n-alkanes and alkyl-aromatic compounds, both isothermal and temperature programmed indices were determined. The isothermal information was used to calculate temperature programmed indices. For several linear programmed conditions accuracies better than 0.51^T-units were usually obtained. The results are compared with published procedures. It is demonstrated that isothermal retention information obtained on one column can be transferred to another column with the same stationary phase but different column dimensions and/or phase ratio. The temperature programmed indices calculated in this way also have an accuracy better than 0.51^T-u. The temperature accuracy and precision of the GC-instrumentation used was of the order of 0.1°C. All calculations can be run with a Basic-programmed microcomputer.     

Use of Large Retention Index Database for Filtering of GC–MS False Positive Identifications of Compounds

The use of the large retention index database for identification and filtering of false positive hits in GC–MS analysis of the ylang-ylang essential oil is illustrated. Differences between experimental retention indices and database values of retention indices of candidate compounds provide additional constraints on the list of candidates for a target compound. Over 100 components of ylang-ylang essential oil (total grade) were identified. The main components, with concentrations more than 4 %, are β-caryophyllene, germacrene D, benzyl benzoate, linalool, geranyl acetate, α-(E,E)-farnesene and isobornyl acetate.

     

Estimation of Kováts retention indices using group contributions

We have constructed a group contribution method for estimating Kováts retention indices by using observed data from a set of diverse organic compounds. Our database contains observed retention indices for over 35,000 different molecules. These were measured on capillary or packed columns with polar and nonpolar (or slightly polar) stationary phases under isothermal or nonisothermal conditions. We neglected any dependence of index values on these factors by averaging observations. Using 84 groups, we determined two sets of increment values, one for nonpolar and the other for polar column data. For nonpolar column data, the median absolute prediction error was 46 (3.2%). For data on polar columns, the median absolute error was 65 (3.9%). While accuracy is insufficient for identification based solely on retention, it is suitable for the rejection of certain classes of false identifications made by gas chromatography/mass spectrometry.     

Prediction of retention times and efficiency in linear gradient programmed pressure analysis on capillary columns

A method for the prediction of the retention time and the resolution of chromatographic peaks in different experimental conditions by starting from few experimental data measured in isothermal and isobaric analyses was published previously. In this paper, the same mathematical model was implemented for calculating the retention times and the column efficiency in programmed pressure runs. Some models originated from the Golay equation and reported in the literature are compared, and a new modified equation for the calculation of the peak width at half height is proposed. The procedure for the prediction of the retention time and the peak width at half height at programmed pressure of the carrier gas and different column temperature and linear gradient by using retention data of different compounds obtained in few isobaric runs is described. The prediction of the retention time and the separation efficiency of compounds with different polarity gave good results for the programmed pressure runs with linear gradient. The effect of the variation of the initial parameters of the experimental analyses and of the mathematical model on the accuracy of the prediction has been evaluated.     

Multidimensional separation of isomeric species of chlorinated hydrocarbons such as PCB,PCDD, and PCDF

A multidimensional GC-system containing two capillary columns of different polarities, operated within a double oven instrument, was used for the optimized separation of complex isomeric mixtures of various types of chlorinated aromatic compounds such as PCB, PCDD, or PCDF. Electron capture was applied parallel to flame ionization detection to obtain sensitive signals of the PCB or PCDD and the n-alkane standards required for identification by Kovats indices, respectively. Electron capture detection within a valvelessly operated MDGC system forced changes in the usual instrumental set-up, the live-switching coupling piece being located within the second oven of the double oven instrument. This oven is operated isothermally to ensure optimal and interference-free detection, for stable flow conditions within the coupling piece and for the determination of the isothermal retention indices (Kovats), which are much more suitable for identification by retention than the so-called “linear” indices.     

Exact-mass library for pesticides using a molecular-feature database

An automated molecular-feature database (MFD) consisting of the exact monoisotopic mass of 100 compounds, at least one exact mass product ion for each compound, and chromatographic retention time were used to identify pesticides in food and water samples. The MFD software compiles a list of accurate mass ions, excludes noise, and compares them with the monoisotopic exact masses in the database. The screening criteria consisted of +/-5 ppm accurate mass window, +/-0.2 min retention time window, and a minimum 1000 counts (signal-to-noise (S/N) ratio of approximately 10:1). The limit of detection for 100 tested compounds varied from <0.01 mg/kg for 72% of the compounds to <0.1 mg/kg for 95% of the compounds. The MFD search was useful for rapid screening and identification of pesticides in food and water, as shown in actual samples. The combined use of accurate mass and chromatographic retention time eliminated false positives in the automated analysis. The major weakness of the MFD is matrix interferences and loss of mass accuracy. Strengths of the MFD include rapid screening of 100 compounds at sensitive levels compared with a manual approach and the ease of use of the library for any accurate mass spectrometer instrumentation capable of routine sub-5-ppm mass accuracy.     

MassBank: a public repository for sharing mass spectral data for life sciences

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron‐ionization mass spectrometry(EI‐MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)‐MSⁿ data of 2337 authentic compounds of metabolites, 11 545 EI‐MS and 834 other‐MS data of 10 286 volatile natural and synthetic compounds, and 3045 ESI‐MS² data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI‐MS² data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass‐to‐charge ratio are optimized to the ESI‐MS² data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI‐MS² data on an identical compound under different collision‐induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21–23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data. Copyright © 2010 John Wiley & Sons, Ltd.     

Evaluation of the Linear-Elution-Strength Model for the Prediction of Retention and Selectivity in Isothermal Gas Chromatography

Linear-elution strength theory and temperature-programmed gas chromatography is evaluated as a rapid method for predicting isothermal retention factors and column selectivity. Retention times for a wide range of compounds are determined at the program rates of 3 and 12 °C/min for the temperature range 60 to 160 °C on three open-tubular columns (DB-1701, DB-210 and EC-Wax) and used to predict isothermal retention factors for each column over the temperature range 60 to 140 °C. The temperature-program predicted isothermal retention factors are compared with experimental values using linear regression and the solvation parameter model. It is shown that isothermal retention factors predicted by the linear-elution-strength model only approximately represents the experimental data. The model fails to predict the slight curvature that exists in most plots of the experimental retention factor (log k) as a function of temperature. In addition, regression of the temperature-program predicted isothermal retention factors against the experimental values indicates that the slopes and intercepts deviate significantly from their target values of one and zero, respectively, in a manner which is temperature dependent. The temperature-program predicted isothermal retention factors result in system constants for the solvation parameter model that are different to those obtained from the experimental retention factors. These results are interpreted as indicating that linear-elution-strength theory predicts retention factors that fail to accurately model stationary phase interactions over a wide temperature range. It is concluded that temperature-program methods using linear-elution-strength theory are unsuitable for constructing system maps for isothermal separations.     

Determination of the Enthalpies of Vaporization of Cyclic Organic Peroxides by Correlation of Changes in Gas Chromatographic Retention Times

Liquid and solid cyclic peroxides derived from aliphatic ketones are explosive materials so their enthalpies of vaporization and other thermodynamic or condensed-phase properties cannot be measured directly. In this work the enthalpies of vaporization of peroxides at 298.15 K were estimated simply from gas chromatographic retention times measured at different temperatures. The technique correlates changes in the retention times of compounds whose enthalpies of vaporization are known (called the reference series), with those of the compounds of interest. If t _R′ is the adjusted retention time (retention time of each compound minus the retention time of unretained diethyl ether, used as solvent) a plot of ln t _R′ against 1/T for each compound (reference compounds and cyclic peroxides) results in a straight line (r ² > 0.99 for all compounds). The enthalpy of transfer from solution to the vapor state (Δ_sol^g H _m) can be obtained by multiplying the slope by the gas constant (R). A second plot correlates the enthalpies of transfer from solution to the vapor state (Δ_sol^g H _m), as measured by gas–liquid chromatography (GLC), with enthalpies of vaporization of reference materials (Δ_vap H _m at 298.15 K) available in the literature. C₉–C₁₅ fatty acid methyl esters and hydrocarbons were used as reference compounds. The enthalpies of vaporization of the cyclic organic peroxides were calculated from the equation of the line obtained in this second correlation, the slope of which was Δ_vap H _m (at 298.15 K)/Δ^g _sol H _m. The experiments were performed under isothermal conditions with a DB-5 capillary column, flame-ionization detection (FID), and nitrogen as carrier gas. The column temperature was varied over a range of at least 30–70 K between 403 and 473 K, with chromatograms being acquired at 10 K intervals. Enthalpies of vaporization of cyclic organic peroxides are not available in the literature, and the values given in this paper, obtained by gas chromatography, are the first to be reported.     

Multivariate Optimization Approach for Chiral Resolution of Chlorophenoxy Acid Herbicides Using Teicoplanin as Chiral Selector in Capillary LC

In this paper, the selectivity and resolution of enantiomeric separation by capillary liquid chromatography (cLC) of racemates of phenoxy acid herbicides are modelled. The compounds studied were 2-(±)-(2,4,5-trichlorophenoxy)propanoic acid (2,4,5-TP), 2-(±)-(2,4-dichlorophenoxy)propanoic acid (2,4-DP), 2-(±)-(4-chloro-2-methyl)phenoxypropanoic acid (MCPP) and 2-(±)-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid] (diclofop acid), using a capillary column packed with silica particles modified with teicoplanin as chiral selector. Several mixtures of methanol (MeOH), water and triethylamine acetate (TEAA) buffer at different pHs have been tested as mobile phases, and experimental parameters such as resolution (Rs), retention factor (k) and enantioselectivity factor (α) have been measured in all tested conditions. The chemometric methods used to explore and to model the data were principal component analysis (PCA), stepwise multiple linear regression (stepwise-MLR) and response surface analysis (RSA). The results show that it is possible to quantitatively predict the quality of enantiomeric separations of related compounds in a given chromatographic system.     

Decoding of complex isothermal chromatograms recovered from space missions. Identification of molecular structure

A chemometric approach, based on the study of the autocovariance function, is described to study isothermal GC chromatograms of multicomponent mixtures: isothermal GC analysis is the method of choice in space missions since it is, to date, the only method compatible with flight constraints. Isothermal GC chromatograms look inhomogeneous and disordered with peak density decreasing at higher retention times: a time axis transformation is proposed to make retention an homogeneous process so that CH2 addition in terms of an homologous series yields a constant retention increment. The time axis is transformed into a new scale based on the retention times of n-alkanes, as they are the basis of the universal Kovats indices procedure. The order introduced into the chromatogram by retention time linearization can be simply singled out by the experimental autocorrelation function (EACF) plot: if constant inter-distances are repeated in different regions of the chromatogram, well-shaped peaks are evident in the EACF plot. By comparison, with a standard mixture it is possible to identify peaks diagnostic of specific molecular structures: study of the EACF plot provides information on sample chemical composition. The procedure was applied to standard mixtures containing compounds representative of the planetary atmospheres that will be investigated in the near future: in particular, those related to Titan's atmosphere (Cassini-Huygens mission) and cometary's nucleus (Rosetta mission). The employed experimental conditions simulated those applied to GC instruments installed on space probes and landers in space missions. The method was applied to two specific investigations related to space research, i.e., a comparison of retention selectivity of different GC columns and identification of the chemical composition of an unknown mixture.