Staffan Folestad

Separation of Amino Acid Enantiomers Using Precolumn Derivatization with o-Phthalaldehyde and 2,3,4,6-Tetra-O-acetyl-1-thio-β-glucopyranoside

Abstract A method is described for resolution of amino acid enantiomers. The D- and L-amino acids were reacted with o-phthalaldehyde (OPA) and the optically active 2,3,4,6-tetra-O-acetyl-1-thio-β-glucopyranoside (TATG). The reaction was complete in a few minutes at room temperature and the derivatives were quite stable. The formed diastereomers were separated by reversed phase chromatography and the selectivity was generally good, except for lysine and ornithine. A mean separation factor (α) of 1.27 was obtained with acetonitrile as an organic modifier. The fluorescence excitation and emission maxima were at 342 nm and 410 nm respectively, and the electrochemical half-wave potential E½ ≡ 0.65–0.75 V. The detection limits (for L-leucine) were 23 pmol and 1 pmol (S/N 3:1) in the respective detection modes. With laser-induced fluorescence detection (He-Cd laser, 325 nm) and microcolumns, a detection limit in the fmol range is obtainable.

Increased Intra‐ and Extracellular Concentrations of γ‐Glutamylglutamate and Related Dipeptides in the Ischemic Rat Striatum: Involvement of γ‐Glutamyl Transpeptidase

Abstract: The present work relates to the possibility that the ATP‐independent enzyme γ‐glutamyl transpeptidase (EC 2.3.2.2), which has been postulated to be part of an amino acid uptake system, is active during cerebral ischemia. This was evaluated in the ischemic rat striatum by determination of intra‐ and extracellular concentrations of γ‐glutamyl dipeptides (the products of the transpeptidation) and glutathione (the physiological γ‐glutamyl donor). An ischemic period (0–30 and 31–60 min) resulted in prominent increases in the respective concentration of extracellular γ‐glutamylglutamate (24‐ and 67‐fold), γ‐glutamyltaurine + γ‐glutamylglycine (5.8‐ and 19‐fold), and γ‐glutamylglutamine (2.6‐ and 6.8‐fold) as revealed using in vivo microdialysis. The changes coincided with increased respective extracellular concentrations of glutamate (83‐ and 115‐fold), taurine (17‐ and 25‐fold), glycine (4.6‐ and 6.1‐fold), and glutamine (1.7‐ and 2.1‐fold). Furthermore, under anoxic conditions in vitro (0–30 and 0–60 min), respective striatal tissue concentrations were increased for γ‐glutamylglutamate (20‐ and 17‐fold), γ‐glutamyltaurine (6.7‐ and 11‐fold), γ‐glutamylglutamine (1.7‐ and 1.2‐fold), and γ‐glutamylglycine (14‐ and 18‐fold), whereas glutathione levels were, on an average, decreased by ∼350 µM. In summary, γ‐glutamyl transpeptidase is involved in de novo dipeptide synthesis in the mammalian brain during anoxic conditions, indicating transport of amino acids such as glutamate.

Performance and preparation of immobilized polysiloxane stationary phases in 5–55 μm I.D. open-tubular fused silica columns for liquid chromatography☆

Cross-linked non-polar polysiloxanes were evaluated as stationary phases in open-tubular column reversed-phase liquid chromatography. Coating of 5–55 μm I.D. fused-silica capillaries with stationary phase films of a well defined thickness in the range 0.03–1.96 μm is described for the two polysiloxane gum phases PS-255 (methylvinyl silicone) and SE-54 (methylphenylvinyl silicone). The chromatographic properties of these columns were investigated using split injection and on-column laser-induced fluorescence detection. Gas chromatography was used complementarily in the evaluation of column stability, retention and inertness. A retentive layer thickness to column diameter ratio up to 1:27 could be prepared. A retentive layer thickness to column diameter ratio up to 1:27 could be prepared, and a linear relationship was observed between the retention and the stationary to mobile phase volume ratio. The selectivity was related to the polysiloxane structure and was constant for films thicker than 0.25 μm. The column band-broadening was studied regarding the contribution from stationary phase diffusion, and compared with theory. Depending on film thickness, the stationary phase diffusion coefficient Ds was in the range 10−8-10−6 cm2/s. The highest efficiency, 351 000 plates (k′ = 0.16), was obtained with a 1.97 m × 11.7 μm I.D. open-tubular-column. An application to the gradient separation of fluorescence labelled amino acids is presented. Preliminary results are also reported on a new type of stationary phase, created by swelling the immobilized polysiloxane with n-heptane. A nearly ten-fold increase in retention and a change in selectivity were obtained.

Separation of precolumn-labelled d- and l-amino acids by micellar electrokinetic chromatography with UV and fluorescence detection

Abstract Micellar electrokinetic chromatography (MEKC) was examined for the separation of labelled d - and l -amino acids to permit rapid screening of protein amino acid enantiomers in microchemical analytical work. Precolumn chiral derivatization was performed using o- phthaldialdehyde /2,3,4,6- tetra-O-acetyl -1- thio -β- d -glucopyranose (OPA/TATG) reagent and the diastereomers formed were detected by UV or fluorescence detection. Optimization of separation buffer pH, ionic strength and surfactant concentration was carried out and was focused on the effective separation window available for the resolution of the amino acid derivatives. The effects of added organic modifiers, methanol, acetonitrile and tetrahydrofuran, on the relative retention of the derivatives were characterized for the purpose of fine tuning the separation selectivity. The resolution of the derivatives of the d - and l -forms of each protein amino acid was very high (mean value of Rs = 14.3, range 0.8–28), except for aspartic acid and glutamic acid, whose enantiomers could not be resolved at the alkaline pH studied. A separation of 34, d,l -amino acids in less than 5 min, is demonstrated with only a few peaks co-eluting.

Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of d- and l-amino acids

Abstract Racemization of free amino acids is considerably lower those bound in peptide. In the same experimental conditions, the rate of racemization of free amino acids is only 20%–80% that of peptide-bound amino acids. When using traditional protein hydrolysis, the racemization was 1.2–1.6 times as high as that obtained at high temperatures (160–180 °C), under conditions ensuring total hydrolysis of the protein. This lower degree of racemization may be explained by the fact that, at high temperatures, the protein hydrolyses more rapidly into free amino acids and the racemization of free amino acids is considerably lower than those bound in polypeptides. When hydrolysis is conducted at lower temperatures for longer times, the amino acids bound in the peptide chain are exposed for a longer time to the effects racemization. As a result, we may say that any factor that speeds up hydrolysis will lower the degree of racemization. Racemization was higher for proteins in milk powder than for pure proteins. This may be explained as a result of catalysis of the racemization by the heavy metals present. After 48 h at 110 °C and in the presence of 4 M barium hydroxide, all amino acids (whether free or bound in peptide) were totally racemized. Therefore, the racemization of tryptophan cannot be determined using barium hydroxide promoted protein hydrolysis. High temperature hydrolysis (at 160 °C for 45–60 min, at 170 °C for 30–45 min and 180 °C for 30 min) is recommended for those who would like to hydrolyse the protein for short times and determine the degree of racemization occurring in the polypeptide chain, but do not wish to use enzyme hydrolysis.

Scatter Correction of Transmission Near-Infrared Spectra by Photon Migration Data: Quantitative Analysis of Solids

The scope of this work is a new methodology to correct conventional near-infrared (NIR) data for scattering effects. The technique aims at measuring the absorption coefficient of the samples rather than the total attenuation measured in conventional NIR spectroscopy. The main advantage of this is that the absorption coefficient is independent of the path length of the light inside the sample and therefore independent of the scattering effects. The method is based on time-resolved spectroscopy and modeling of light transport by diffusion theory. This provides an independent measure of the scattering properties of the samples and therefore of the path length of light. This yields a clear advantage over other preprocessing techniques, where scattering effects are estimated and corrected for by using the shape of the measured spectrum only. Partial least squares (PLS) calibration models show that, by using the proposed evaluation scheme, the predictive ability is improved by 50% as compared to a model based on conventional NIR data alone. The method also makes it possible to predict the concentration of active substance in samples with other physical properties than the samples included in the calibration model.

Swollen polysiloxanes as stationary phases in reversed-phase open-tubular column liquid chromatography

Abstract A new liquid stationary phase was created after swelling cross-linked polysiloxanes with non-polar organic solvents in 11–55 μm I.D. fused-silica open-tubular columns. By choosing different polysiloxane/swelling solvent pairs, the stationary phase film thickness, retention and selectivity could be adjusted within a wide range. Using n-heptane as the swelling solvent, the film thickness increased by a factor of 3–4. Mobile to stationary phase volume ratios, β  Vm/Vs, down to 2 were obtained. An increase in retention of 12–16 times after swelling was observed. Improved stability was obtained as compared with conventional liquid—liquid chromatographic systems with mechanically held stationary phases. The system was stable at very high flow-rates (up to 6.3 cm/s) and showed no mechanical loss of the stationary phase.

Chlorine-selective detector for liquid chromatography

Abstract A chlorine-selective detector based on flame emission has been developed for use with high-performance liquid chromatography. The column effluent is first burnt in a hydrogen-rich total consumption burner in the presence of a platinum catalyst. The so-formed hydrogen chloride reacts with indium yielding indium chloride in an upper cool hydrogen flame, thereby emitting a characteristic emission at 360 nm. The detection limit for chloroform in water was 300 pg/sec (signal-to-noise ratio 3:1). The effects of chemical interferences by organic solvents were studied. In the present design, the total column effluent at flow-rates up to 1.5 ml/min is introduced into the detector.

Age determination based on amino acid racemization: a new possibility

SummaryA method has been developed to determine the age of fossil bone samples based on amino acid racemization (AAR). Approximately one hundred fossil bone samples of known age from Hungary were collected and analysed for D- and L-amino acids. As the racemization of amino acids is affected by temperature, pH, metal content of the soil, and time passed since death, these factors were eliminated by comparing the estimated age to age determined by the radiocarbon method. Determining the D- and L-amino acid contents in samples of known age, determining the half life of racemization and plotting the D/L ratio as a function of time, calibration curves were obtained. These curves can be used for the age estimation of samples after determining their D- and L-amino acid content. The D/L ratio for 2 to 3 amino acids was determined for each sample and the mean value of estimated ages based on calibration curves was considered to estimate age of the fossil samples.

Mercaptoethanesulphonic acid as a protecting and hydrolysing agent for the determination of the amino acid composition of proteins using an elevated temperature for protein hydrolysis

Abstract Mercaptoethanesulphonic acid (MES-OH) (3 M) was used for the hydrolysis of different samples (pure proteins, free typtophan and milk powder with a high sugar content). Different temperatures (160, 170 and 180°C) and time periods (15–90 min) were compared under standard conditions to minimize side-reactions in order to obtain the best recovery of the amino acids (especially tryptophan and methionine). The materials used for testing the hydrolysis methods were bovine ribonuclease, lysozyme, cytochrome c , free tryptophan and mares milk powder. Hydrolysis at high temperature was successfully applied for the amino acid analysis of milk powder with high contents of carbohydrate and pure proteins. In some instances, such as in tryptophan and methionine determination at 160–170°C for 15–30 min, the results were better than those obtained by the original MES-OH method. A disadvantage of the MES-OH hydrolysis method is that it reduces cystine to cysteine, which co-elutes with proline from the ion-exchange column used to separate the released amino acids and it may interfere with the determination of proline in high-cystine proteins.