Hartmut Frank

Enantiomer labelling, a method for the quantitative analysis of amino acids

Enantiomer labelling a method for the quntitative analysis of optically active natural compounds by gas chromatography, involves the use of the unnatural enantiomer as an internal standard. With Chirasil-Val, a chiral stationary phase that is thermally stable up to up to 240 degrees, the enantiomers of amino acids and a variety of other compounds can be separated and quantitated. Incomplete recovery from the sample, incomplete derivatization, hydrolysis and thermal decomposition of the derivative and shifting response factors can be compensated for by adding the unnatural enantiomer. The accuracy of amino acid analysis by enantiomer labelling is equal or superior to that of hitherto known methods. The procedure affords a complete analysis of peptides with respect to both amino acid composition and the optical purity of each amino acid.

Monitoring lipid peroxidation by breath analysis: endogenous hydrocarbons and their metabolic elimination.

Abstract Monitoring of ethane and pentane in breath as a noninvasive method to measure lipid peroxidation is performed by an increasing number of laboratories. The alkanes are generated through peroxidative breakdown of polyunsaturated fatty acids. Fasted Sprague-Dawley rats exhale these hydrocarbons at a rate of approximately 1.7 nmol/kg·hr. Through an improved analytical procedure other volatile hydrocarbons could be detected in the breath of the animals, i.e., ethene (1.1 nmol/kg·hr), propane (0.7 nmol/kg·hr), n -butane (0.7 nmol/kg·hr), iso-pentane, and iso-butene. The exhaled hydrocarbons accumulate in the atmosphere if an animal is confined in a closed system. However, the increase is not linear but a steady-state concentration is approached, presumably due to reuptake and subsequent hepatic metabolism. Hydrocarbons are oxidized by the hepatic monooxygenases, at rates increasing with their molecular masses. The same has been found for the elimination of ethane, propane, butane, pentane, and iso-butene by a rat from closed system. Metabolism is inhibited by a variety of substances, e.g., dithiocarb, ethanol, and tetrahydrofurane. Therefore, the increased release of hydrocarbons into the gas phase, after treatment with compounds suspected to induce lipid peroxidation, may merely result from decreased metabolism. Especially if the hydrocarbon exhalation is only moderately elevated as after ethanol administration, this aspect must be taken into account. Similarly, peroxidizing microsomes show elevated hydrocarbon output when treated with carbon monoxide in order to block metabolism. This study provides a procedure for discrimination of inhibitory and peroxidative action which is of particular importance if the increase over control levels is only moderate. In addition, it may serve as monitor for the metabolic capacity of a laboratory animal.

Gas chromatographic—mass spectrometric analysis of optically active metabolites and drugs on a novel chiral stationary phase

Chirasil-Val, a novel chiral polysiloxane-type stationary phase is capable of separating the enantiomers of optically active drugs and metabolites of several compound classes; alpha-amino acides, alpha-amino alcohols, glycols, aromatic and aliphatic alpha-hydroxy carboxylic acids and amines. Due to their high thermal stability, columns coated with Chirasil-Val may be coupled to a mass spectrometer. Potential applications of the new stationary phase include analysis of the optical purity of enantiomeric drugs, determination of the configuration of metabolites, and quantitation of optically active drugs and metabolites using the unnatural enantiometer as internal standard. Direct separation of enantimoers on Chirasil-Val is especially useful if only minute amounts of the optically active compounds are availalbe for analysis.

Haloacetic acids, phytotoxic secondary air pollutants

Haloacetic acids are atmospheric oxidation products of airborne C2-halocarbons which are important solvents and propellants. Levels of trichloroacetate (TCA) in conifer needles from mountain ranges in Germany (Black Forest, Erzgebirge) and from two sites in Finland are compared; TCA is present in conifer needles at concentrations up to 0.7 μmol/kg, MCA up to 0.2 μmol/kg. At the Finnish sites, TCA-concentrations and branch degeneration symptoms of Scots pine are correlated. Monochloroacetate (MCA) has been determined in needle samples from Southern Germany in concentrations exceeding its phytotoxicity threshold towards photoautotrophic organisms. Data on atmospheric chloroacetate levels in Germany are also given; ambient air levels of chloroacetic acids range from about 2 pmol/m3 (TCA) to 390 pmol/m3 (MCA). TCA and dichloroacetic acid (DCA) arise from atmospheric oxidation of airborne C2-chlorocarbons, while the source of MCA is not yet known; several tentative pathways are suggested.

A new gas chromatographic method for determination of amino acid levels in human serum.

Amino acid levels of biological fluids are usually determined by the classical ion exchange procedure. Although the gas chromatographic method offers the advantage of higher sensitivity and speed of analysis, apparent difficulties in achieving accuracy and precision similar to the ion exchange procedure prevented its acceptance as a routine method. The main problems associated with gas chromatographic amino acid analysis were overcome by application of a novel approach called enantiomer-labelling: the optical antipode to each L-amino acid is added to the sample prior to the clean-up and serves as an internal standard having identical chemical properties. The enantiomers of all amino acids are separated by gas chromatography on capillaries coated with a thermally stable, chiral stationary phase. The shortcomings previously inherent in the gas chromatographic method are thus eliminated. Accuracy and precision of the new procedure are equal or better than of the classical ion exchange method.

Derivatives for separation of amino acid enantiomers

SummaryAn optimum gas chromatographic separation of all protein amino acids in one run on capillaries coated with Chirasil-Val is difficult to achieve. Overlap of enantiomers of different amino acids may occur because the relative retention times depend upon the overall polarity of the stationary phase, the film thickness and the actual temperature programm. Employment of different derivatives formed by esterification with isopropanol, n-propanol, isobutanol and n-butanol and by acylation with trifluoroacetic, pentafluoropropionic and heptafluorobutyric anhydrides yields patterns of relative elution of all amino acid enantiomers. Thus, even critical pairs of amino acid enantiomers can be separated or shifted in their relative retention times. All amino acid enantiomers can be separated and quantitatively estimated.

A modified procedure for acid-catalyzed esterification with isopropanol

SummaryAcid-catalyzed esterification of carboxyl groups with acetyl chloride in isopropanol is investigated. Yields are equivalent to those obtained with reagent prepared conventionally by bubbling gaseous hydrogen chloride into isopropanol. The preparation of the former reagent is less time consuming and no special equipment is needed. The procedure is applicable for other alcohols as well.

Gas chromatographic resolution of 2-hydroxycarboxylic acid enantiomers

Abstract Several types of derivative can be employed for gas chromatographic resolution of optical isomers of 2-hydroxycarboxylic acids, viz. 2-acyloxycarboxamides, 2-trimethylsiloxycarboxamides, 2-carbamoyloxycarboxylic esters, 2-carbamoyloxy-carboxamides and 2-hydroxycarboxylic esters. In all cases hydrogen bonding is the main interaction which leads to chiral recognition. Each type of derivative offers specific advantages whilst exhibiting certain limitations. The choice of derivative will depend primarily upon the sample, the respective hydroxy acid and the relative amounts of isomers.

Trichloroacetic acid as a phytotoxic air pollutant and the dose-response relationship for defoliation of Scots pine

Various ubiquitous volatile organic air pollutants (VOCs), especially C2-halocarbons, may be converted to secondary air pollutants that are phytotoxic and known as herbicides. One of these is trichloroacetic acid (TCA), found in concentrations ranging from 10 to 130 ng/g in the foliage of forest trees in northern Finland. TCA has been used as a herbicide against monocotyledonous weeds. It has formative effects, inhibits growth, and induces chlorosis and necrosis of light-exposed leaves, including those of woody plant species. Twenty Scots pine trees in an experimental stand 50 km southeast of Rovaniemi were sampled for correlation of TCA levels and needle loss. The trees located at the northwesterly edge of the stand could be divided into two groups, one more resistant to the phytotoxicant TCA than the second. The range of TCA concentrations in needles was 8–65 ng/g. The extent of defoliation (range 25–90%) was lower in the TCA-resistant group, with a gradient of 0.32% defoliation per unit TCA concentration; in the sensitive group, the correlation line had a steeper slope of 0.78% defoliation per unit TCA concentration. These two groups serve as a basis for further studies on morphological, anatomical, and biochemical parameters.

The micro-trap: An alternative to cryofocussing in capillary gas chromatography

SummaryFor routine analyses involving numerous samples or for field operation of on-site instruments, cryogenic traps are inconvenient: their operation is timeconsuming and automation is technically demanding. complete trapping and efficient band-focussing may be achieved at ambient temperature with micro-traps packed with suitable sorbents. Two approaches have been evaluated: if the boiling points of the analytes are within a range of about 100°C, a single-sorbent trap and direct coupling to the column are recommended; for samples containing components wih widely different volatilities, a trap composed of two or more segments with complementary affinties and coupling to the chromatographic column under carrier-gas flow-reversion is preferable. The procedure has been evaluated for airborne C1 and C2-halocarbons; it is also applicable to trace analysis of other volatile organic air pollutants.