Methods for studying reaction kinetics in gas chromatography, exemplified by using the 1-chloro-2,2-dimethylaziridine interconversion reaction.

In this paper, methods are described that are used for studying first-order reaction kinetics by gas chromatography. Basic theory is summarized and illustrated using the interconversion of 1-chloro-2,2-dimethylaziridine enantiomers as a representative example. For the determination of the kinetic and thermodynamic activation data of interconversion the following methods are reviewed: (i) classical kinetic methods where samples of batch-wise kinetic studies are analyzed by enantioselective gas chromatography, (ii) stopped-flow methods performed on one chiral column, (iii) stopped-flow methods performed on an achiral column or empty capillary coupled in series with two chiral columns, (iv) on-flow method performed on an achiral column coupled in series with two chiral columns, and (v) reaction gas chromatography, known as a dynamic gas chromatography, where the interconversion is performed on chiral column during the separation process. The determination of kinetic and thermodynamic activation data by methods (i) through (iv) is straightforward as the experimental data needed for the evaluation (particularly the concentration of reaction constituents) are accessible from the chromatograms. The evaluation of experiments from reaction chromatography method (v) is complex as the concentration bands of reaction constituents are overlapped. The following procedures have been developed to determination peak areas of reaction constituents in such complex chromatograms: (i) methods based on computer-assisted simulations of chromatograms where the kinetic activation parameters for the interconversion of enantiomers are obtained by iterative comparison of experimental and simulated chromatograms, (ii) stochastic methods based on the simulation of Gaussian distribution functions and using a time-dependent probability density function, (iii) approximation function and unified equation, (iv) computer-assisted peak deconvolution methods. Evaluation of the experimental data permits the calculation of apparent rate constants for both the interconversion of the first eluted (k (A-->B)(app)) as well as the second eluted (k(B-->A)(app)) enantiomer. The mean value for all the rate constants (from all the reviewed methods) was found for 1-chloro-2,2-dimethylaziridine A-->B enantiomer interconversion at 100 degrees C: k (A-->B)(app)=21.2 x 10(-4)s(-1) with a standard deviation sigma=10.7 x 10(-4). Evaluating data for reaction chromatography at 100 degrees C {k (app)=k(A-->B)(app)=k(B-->A)(app)=13.9 x 10(-4)s(-1), sigma=3.0 x 10(-4)s(-1)} shows that differences between k(A-->B)(app) and k(B-->A)(app) are the same within experimental error. It was shown both theoretically and experimentally that the Arrhenius activation energy (E(a)) calculated from Arrhenius plots (lnk(app) versus 1/T) is proportional to the enthalpy of activation {E(a)=DeltaH+RT}. Statistical treatment of Gibbs activation energy values gave: DeltaG (app)=110.5kJmol(-1), sigma=2.4kJmol(-1), DeltaG (A-->B)(app)=110.5kJmol(-1), sigma=2.2kJmol(-1), DeltaG (B-->A)(app)=110.3kJmol(-1), sigma=2.8kJmol(-1). This shows that the apparent Gibbs energy barriers for the interconversion of 1-chloro-2,2-dimethylaziridine enantiomers are equal DeltaG (app)=DeltaG(A-->B)(app)=DeltaG(B-->A)(app) and within the given precision of measurement independent of the experimental method used.

Gas chromatographic determination of the interconversion energy barrier for dialkyl 2,3-pentadienedioate enantiomers.

The enantiomers of dialkyl 2,3-pentadienedioate undergo interconversion during gas chromatographic separation on chiral stationary phases. In this paper the on-column apparent interconversion kinetic and thermodynamic activation data were determined for dimethyl, diethyl, propylbutyl and dibutyl 2,3-pentadienedioate enantiomers by gas chromatographic separation of the racemic mixtures on a capillary column containing a polydimethylsiloxane stationary phase coupled to 2,3-di-O-methyl-6-O-tertbutyldimethylsilyl-beta-cyclodextrin. A deconvolution method was used to determine the individual enantiomer peak areas and retention times that are needed to calculate the interconversion rate constants and the energy barriers. The apparent rate constants and interconversion energy barriers decrease slightly with an increase in the alkyl chain length of the dialkyl 2,3-pentadienedioate esters. The optimum conformation of the dialkyl 2,3-pentadienedioate molecules, their separation selectivity factors and apparent interconversion enthalpy and entropy data changes with the alkyl chain length. The dependence of the apparent interconversion energy barrier (deltaG(app)(a-->b), deltaG(app)(b-->a)) on temperature was used to determine the apparent activation enthalpy (deltaH(app)(a-->b), deltaH(app)(b-->a)) and apparent entropy (deltaS(app)(a-->b), deltaS(app)(a-->b)) (where a denotes the first and b second eluted enantiomer). The comparison of the activation enthalpy and entropy (deltaS(app)(a-->b), deltaS(app)(a-->b)) indicated that the interconversion of dialkyl 2,3-pentadienedioate enantiomers on the HP-5+Chiraldex B-DM column series is an entropy driven process at 160 degrees C. Data obtained for dimethyl 2,3-pentadienedioate enantiomers on the HP-5+Chiraldex B-DM column series at 120 degrees C (deltaG(app)(a-->b) = 123.3 and deltaG(app)(b-->a) = 124.4 kJ mol(-1)) corresponds (at the 95% confidence interval) with the value of deltaG(#) = 128+/-1 kJ mol(-1) found at this temperature by gas chromatography using a two-dimensional stop flow technique on an empty capillary column [V. Schurig, F. Keller, S. Reich, M. Fluck, Tetrahedron: Asymmetry 8 (1997) 3475].

On the use of computer assisted resolution of non-separable peaks in a congener specific polybrominated diphenyl ether capillary gas chromatographic analysis.

The use of computer assisted deconvolution for chromatographically not separated peaks in the analysis of polybrominated diphenyl ethers (PBDEs) by capillary gas chromatography (CGC) was studied. Twenty-two not separated clusters containing 48 overlapped PBDEs were registered in the separation of a sample containing 122 PBDE congeners on a semipolar poly(8%-phenyl-92%-dimethyl)siloxane column. There were only two clusters in which overlapped PBDEs differ in the number of bromine atoms {PBDE 126(5Br) co-elutes with 154(6Br) and PBDE 105(5Br) co-elutes with 144(6Br)} and therefore their mass spectra could be successfully used for deconvolution purposes. In 22 other clusters 46 isomeric PBDEs with identical mass spectra overlapped and for their resolution a computer assisted deconvolution procedure using a commercial available program was used. A published procedure for the estimation of minimum number of peaks in a peak cluster for which the data found by deconvolution are reliable, has been adapted. Using this procedure, for eight overlapped PBDE full peak data (single peak retention times and peak areas) were extracted.

Computerized separation of chromatographically unresolved peaks.

A computerized peak deconvolution software and mass spectra were successfully applied for the deconvolution of overlapped peak cluster in the chromatogram obtained separating the complex mixture of pesticides by retention time locking gas chromatography-mass spectroscopy. The method based on the unique fragment ions in the spectra can be used for deconvolution of peak clusters if mass spectra of overlapped peaks differ. This method allows determining actual retention times of overlapped peaks. Peak areas found by this method however, cannot be used naturally for the quantitative purposes as the abundance of fragment ions used for this deconvolution procedure can dramatically differ. Computer assisted deconvolution of peaks in the peak clusters gives more realistic peak area ratios as at this method it is supposed equal response for all peaks overlapped in a cluster.

Chemometric studies of retention in capillary gas chromatographic separation of hydrocarbons in coupled columns.

This paper describes how different multivariate analysis and classification methods can be used, to characterize the gas chromatographic separation of complex hydrocarbon mixtures in three columns coupled in series. Principal component analysis (PCA), correspondence factor analysis (CFA), and hierarchical ascending classification (HAC) were used as potential tools for evaluating the experiments on single columns and on column series. It has been demonstrated that: (1) multivariate analysis with PCA and CFA offers a powerful strategy to search for the main factors influencing the separation of hydrocarbons without a priori knowledge of the key factors of the separation. (2) With CFA the contribution of retention due to vapour pressure can be minimized. The use of retention indices, which use the n-alkanes as reference compounds, also helps to decrease the dominant focus on vapour pressure in favor of the more selectivity-based interaction forces. (3) CFA helps to analyze the degree of relevance of the chosen experimental design to the most important factors, controlling chromatographic selectivity.

Selectivity tuning of serially coupled (S,S) Whelk-O 1 and (R,R) Whelk-O 1 columns in HPLC.

The selectivity tuning of two columns coupled in series is investigated in chiral high-performance liquid chromatography. Two columns with reversal enantioselectivities [(R,R) Whelk-O 1 and (S,S) Whelk-O 1] are coupled in series via a T connector. Selectivity of such a column series is tuned by varying the mobile phase flows in the individual columns. The flow ratio necessary for the required selectivity is calculated on the basis of retention factors measured on the individual columns. The performance of this method for adjusting the required selectivity is studied by the separation of enantiomers of alkoxy substituted esters of phenylcarbamic acid. It is demonstrated that the change of the mobile phase flows in the individual columns enables change in the elution order of enantiomers.

On-flow gas chromatographic method for the determination of the enantiomer interconversion energy barrier.

A novel on-flow gas chromatographic (GC) method is developed for the determination of the kinetic rate constants and interconversion energy barrier of thermally labile enantiomers. The validity of the developed method is approved by the study of interconversion of 1-chloro-2,2-dimethylaziridine enantiomers on an achiral column. The overall experiments are performed in a series of three columns placed in two independently heated GC ovens. The racemate of the 1-chloro-2,2-dimethylaziridine is injected and separated in the first chiral column at 60 degrees C in which the interconversion of enantiomers is suppressed. Separated enantiomers are then transferred into the achiral column, where the enantiomers are interconverted at a selected temperature under the current carrier gas flow. Effluent from this column is transferred into the second chiral column, where the native enantiomers and those originated by the on-flow interconversion on an achiral column are again separated at 60 degrees C. Chromatograms obtained by monitoring the effluents from the second chiral column are used to determine the peak areas of the original and the newly interconverted enantiomers. The corresponding peak areas and the interconversion times are used to calculate the interconversion rate constants and energy barriers of the 1-chloro-2,2-dimethylaziridine enantiomers. The apparent energy barriers of the enantiomers of 1-chloro-2,2-dimethylaziridine are equal for both enantiomers within a 95% confidence interval and independent of the polarity of the stationary phase of the column in which the interconversion of enantiomers occur.

Determination of the interconversion energy barrier of enantiomers by separation methods.

Separation methods have become versatile tools for the determination of kinetic activation parameters and energy barriers to interconversion of isomers and enantiomers in the last 20 years. New computer-aided evaluation systems allow the on-line determination of these data after separating minute amount of pure compounds or mixture of isomers or enantiomers, respectively. Both dynamic interconversion during the separation process as well as static stopped-flow techniques have been applied to determine the kinetic activation parameters and interconversion energy barriers by separation methods. The use of (1) combinations of batchwise kinetic studies with enantioselective separations, (2) a continuous flow model, (3) a comparison of real chromatograms with simulated ones, (4) stopped-flow techniques, (5) stochastic methods, (6) approximation functions and (7) deconvolution methods, for the determination of interconversion energy barriers by separation methods is summarized in detail.

Selectivity tuning in chiral dual column gas chromatography.

The enantioselective tuning of two columns coupled in series is investigated in chiral high-resolution gas chromatography. Two columns with opposite enantioselectivities (Chirasil-L-Val and Chirasil-D-Val) are coupled in series via a T connector, and the relative retention of enantiomers chromatographed on the system is changed by varying the individual carrier gas flow rates in the coupled columns. The flow-rate ratio necessary for the required selectivity is calculated on the basis of the measured retention factors on the individual columns. The performance of this method for adjusting selectivity is studied by the separation of enantiomers of the N-TFA-O-methyl esters of six amino acids. It is demonstrated that the change of the coupling point carrier gas pressure, at the constant inlet and outlet pressures, may change the enantioselectivity of the given column series to such an extent that the enantiomer elution order may be reversed.

Evaluation of reversible and irreversible models for the determination of the enantiomerization energy barrier for N-(p-methoxybenzyl)-1,3,2-benzodithiazol-1-oxide by supercritical fluid chromatography.

It has been found that the interconversion of enantiomers on a chromatographic column during the separation process can be studied by the first-order kinetic equations derived both for reversible and irreversible reactions in a stationary system if the extent of interconversion is not too high. The equation derived for irreversible reactions gives, however, results also for higher degrees of enantiomerization while that derived for reversible interconversion failed. The irreversible equation was used to determine the enantiomerization barrier of N-(p-methoxybenzyl)-l,3,2-benzodithiazol-l-oxide enantiomers by supercritical fluid chromatography. The racemate of N-(p-methoxybenzyl)-l,3,2-benzodithiazol-l-oxide was separated by supercritical fluid chromatography on the (R,R)-Whelk-Ol column with supercritical carbon dioxide containing 20% methanol as a mobile phase. Peak areas of enantiomers prior to and after the separation used for the calculation of the enantiomerization barrier were determined by computer-assisted peak deconvolution of peak clusters registered on chromatograms using commercial software.

On the calculation of Gibbs energy corresponding to enantioselective interactions at a direct HRGC separation of enantiomers.

A novel procedure is proposed for the calculation of Gibbs energy corresponding to enantiospecific interactions of 2-(2, 4-dinitrophenoxy)-, 2-phenoxy-, and 2-halogen-n-pentane enantiomers with a beta-cyclodextrin (ChirasilDex) stationary phase under gas chromatographic conditions. This energy is calculated from retention data as a difference between the Gibbs energy of an enantiomer and its corresponding achiral congener. The procedure for the determination of 2-(2,4-dinitrophenoxy)-, 2-phenoxy- and 2-halogen- n-pentane achiral congener retention data is discussed in detail.

Degradation of Delor 103, a technical mixture of polychlorinated biphenyls, by selected bacteria.

Ability to utilize a technical mixture of polychlorinated biphenyls (PCB), Delor 103, as the sole carbon source, has been tested in 14 bacterial strains. For the five best growing strains (Alcaligenes latus, Alcalgenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas putida), the dependence of the degradation of individual PCB congeners on the number of chlorine substituents is discussed.

Analysis of polychlorinated biphenyls by high-performance liquid chromatography and capillary gas-liquid chromatography.

Seventy fractions were obtained by vacuum distillation of a PCB mixture containing 42% of chlorine. Four of these fractions were chosen, the composition of which covered the whole of the observed region, and analysed by means of high-performance liquid chromatography (HPLC) on silica gel using n-pentane as the mobile phase and capillary gas-liquid chromatography (GLC) using OV-101 and Carbowax 20M as stationary phases at 200 degrees C. The chosen distillation fractions were further prepared by HPLC, each yielding 10-14 samples. Individual PCB standards and all samples were analysed by HPLC and capillary GLC. These procedures permitted the identification of those compounds which are eluted simultaneously under the conditions used in either HPLC or capillary GLC alone.

Capillary gas chromatography of s-triazines.

In order to achieve better resolution and lower detection limits for s-triazines, glass capillary columns made of soft soda-lime glass and etched with gaseous hydrogen chloride have been introduced. Columns with non-polar (OV-101, SE-30) mixed (Carbowax 20M + SE-30) and polar stationary liquids (Carbowax 20M) were used. The thickness of the ultra-thin film columns was determined by weighing the capillaries before and after coating of the capillary with stationary phase and conditioning, and varied from 0.05 to 0.5 mum. From the capacity ratios of s-triazines and n-alkanes measured in sections of an originally long dynamically coated column after it had been cut into several equal parts, the constancy of the film thickness along the whole column was judged. The film thickness was not uniform and increased along the whole column. It was found that glass capillary columns with non-polar stationary phase are not very suitable for the analysis of s-triazine herbicides, as tailing occurs with some of them. Symmetrical peaks and the separation of 17 from 18 analysed s-triazines were achieved on glass capillary columns coated with Carbowax 20M.

Possibilities and limitations of capillary gas chromatography and mass spectrometry in the analysis of polychlorinated biphenyls.

The possibilities and limitations of analyses of polychlorinated biphenyls (PCBPs) by capillary gas chromatography and capillary gas chromatography-mass spectrometry have been investigated. Metal capillary columns (WCOT) coated with Apiezon L and OV-101 were not suitable for PCB analyses. Good results were obtained in the separation of model mixtures of PCBs and of Aroclor 1242 on a glass capillary column coated with OV-101. Sources of error are indicated that may be encountered in the characterization of PCB components in Aroclor 1242 by standard additions, Kováts' retention indices and mass spectrometry. The direct coupling of a capillary column (WCOT) to a mass spectrometer produced spectra of the main PCB components, many of which could be used in the identification of isomeric PCBs.

Analytical aspects of capillary gas chromatography of lower fatty acids [up to C18].

The principal aspects influencing analytical capillary gas chromatography of fatty acids up to C18 have been evaluated. Selected fundamental problems of interlaboratory exchange of retention data were problems of defined temperature in commercial thermostated air baths, of capillary tubing, and of stationary phases. A modification of commercial thermostats has been proposed in order to secure a defined temperature for glass capillary columns. It has been found that retention data of fatty-acid methyl esters can be measured under standard conditions with the same accuracy as retention data of hydrocarbons on squalane. Metal capillary columns coated with Apiezon L were found to be unsuitable for the analysis of fatty-acid methyl esters when compared with the results of their quantitative analysis in packed Apiezon L and polar capillary columns. Possibilities of a nontraditional statistical evaluation of the results of measurements are suggested. A program in FORTRAN IV language is given for the calculation of Kovats' retention indices for fatty-acid methyl esters.