Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis.

The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.

Dechlorination of polychlorinated biphenyls, dibenzo-p-dioxins and dibenzofurans on fly ash.

Dechlorination of commercial mixtures of polychlorinated biphenyls (PCB) as well as polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) on extracted and non-extracted fly ash obtained from municipal waste incinerator (MWI) was studied in closed systems under nitrogen atmosphere at temperatures of 260 degrees C and 340 degrees C. Decomposition results (given as the difference between PCB or PCDD/F molar amounts before and after the experiment (in %) due predominantly to dechlorination reactions) and detoxification data (expressed similarly but related to toxic PCB and PCDD/F congeners only and given in I-TEQ units) are reported. Detoxification of Delor 105/80T at 260 degrees C and 340 degrees C at a loading of 0.65 wt%, was 99.48% and 100%, respectively. The decomposition of Delor 103 at 340 degrees C and for the loading of 0.75 wt%, corresponded to 99.99%. The detoxification capability of PCDD/Fs on extracted and non-extracted fly ash for loading of 130 and 264 ng/0.4 g of fly ash at 340 degrees C made 96 and 98%, respectively.

Validation of a method for determination of phospholipase A2 and melittin in bee venom preparations by capillary electrophoresis.

A method was validated for the determination of the 2 main components of bee venom, phospholipase A2 and melittin, by capillary electrophesis (CE). Optimum resolution and selectivity were attained with a running electrolyte of 150 mM phosphoric acid, pH 1.8. The repeatability and day-to-day reproducibility of the migration times were better than 0.36 and 2.8%, respectively. The repeatability and day-to-day reproducibility of the normalized peak areas were better than 1.3 and 2.6%, respectively. The response of the UV detector at 190 nm was linear over < 2 concentration decades, from 0.05 to 1.5 mg/mL, with correlation coefficients of 0.9994 for phospholipase A2 and 0.9997 for melittin. The limits of detection and quantitation were 4.5 and 15 microg/mL, respectively, for phospholipase A2 and 1.6 and 6 microg/mL, respectively, for melittin. The reproducibility of the measurements with 2 different CE instruments was satisfactory; the mean concentration and relative standard deviation (RSD) values for phospholipase A2 and melittin were 14.4% (RSD, 1.3%) and 51.4% (RSD, 1.1%), respectively, with instrument I; the corresponding values with instrument II were 14.5% (RSD, 2.8%) and 52.3% (RSD, 2.2%). The accuracy was estimated by comparison with a liquid chromatographic (LC) method. Differences between the CE and LC measurements were attributed to irreversible adsorption of the analytes on the LC column. The recoveries of phospholipase A2 and melittin with the CE method were 98.8 and 101.7%, respectively.

A study of the distribution of lead, cadmium and copper between water and kaolin, bentonite and a river sediment.

Experiments were carried out to monitor the equilibrium distribution of lead, cadmium and copper between an aqueous phase modelling natural water and a solid phase modelling natural sediment, under varying conditions. The aqueous phase was analysed using ETAAS and differential pulse anodic stripping voltammetry (DPASV), whereas XRD and FTIR were used to study the solid phase. Sorption isotherms at constant pH were measured. Conditional distribution constants were calculated as functions of the pH, the time of equilibration and the amount of solid material. The results obtained stress the need for standardization of the approaches to the study of water-sediment interactions in order to be able to evaluate and compare the extensive data from field measurements and to predict these interactions.

High-performance liquid chromatographic determination of some anthraquinone and naphthoquinone dyes occurring in historical textiles.

A reversed-phase HPLC method has been developed for identification and quantitation of nine natural quinone dyes and applied to historical textile fibres. A Purospher RP18e column was used with a convex gradient of methanol in a mobile phase of 0.1 M aqueous citrate buffer (pH 2.5) and spectrophotometric diode-array detection at 270 nm. For identification of alizarin, purpurin and xanthopurpurin, occurring together in the madder plant, an isocratic method was used with a methanol-0.2 M acetate buffer (pH 4.3) (75:25) as the mobile phase. After an acid extraction of textile fibres and the analysis of the extracts, alizarin and purpurin were identified and quantitated in three fibres.

High-performance separations in isolation and characterization of allergens.

The present state of the use of separation techniques in the identification and characterization of allergens and in the monitoring of the quality of allergenic preparations is critically surveyed. After a brief summary of the range of problems encountered in obtaining and in the application of allergenic preparations and of the principal physico-chemical properties of allergens, chromatographic and electromigration methods of separation of components of these systems and their combinations with immunochemical procedures are discussed, with selected examples of application to real materials. Emphasis is placed on evaluation of the most important analytical parameters, such as reliability of the results, separation efficiency and resolution, and on the most recent results in the field.

Capillary electrophoresis of cytokinins and cytokinin ribosides.

A CE separation of cytokinins and cytokinin ribosides and some other purine and pyrimidine bases has been developed. Two electrolyte systems have been tested: 150 mM phosphoric acid, pH 1.8 and 50 mM sodium dodecylsulphate + 20 mM borate, pH 9.2. The migration times were measured and the effects of the solute structures were discussed. Preliminary experiments with plant extracts have been performed to identify the cytokinins and their ribosides. Both the systems can be used, but 150 mM phosphoric acid is better suited for identification of cytokinins in plant extracts, as the electropherograms are subject to fewer interferences.

A simple method for the trace determination of methanol, ethanol, acetone and pentane in human breath and in the ambient air by preconcentration on solid sorbents followed by gas chromatography.

A simple technique has been developed for preconcentration of gaseous trace organic compounds on solid sorbents, followed by gas chromatography. The sorbent is packed in a cartridge from a syringe needle placed in the gas chromatographic injector and the analytes previously adsorbed are thermally desorbed at the injector temperature and then directly swept by the carrier gas into the column. The system has been tested for a charcoal-based adsorbent and silica gel, with pentane, methanol, ethanol and acetone as the model analytes. The procedure is rapid, the detection limits vary from a few nmol l(-1) to values below 0.1 nmol l(-1) (i.e., a few ppb), the linear dynamic range amounts to at least five concentration decades and a typical relative standard deviation is 10% at the nmol l(-1) concentrations. It has been shown that the method is readily applicable to determination of instantaneous concentrations of the analytes in natural and industrial atmosphere and to their monitoring in human breath which is important for medical and hygienic practice. In general, the procedure is applicable to low-molecular volatile organic compounds.

Size-exclusion liquid chromatography and capillary electrophoresis of pollen allergens.

Allergens from the pollen of Phleum pratense, Dactylis glomerata. Arrhenatherum elatius, Secale cereale, Lolium perrene and Festuca sp. were analysed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). SEC was used for the determination of the molecular masses of main allergens. A CE method, using either 150 mmol/l phosphoric acid (pH 1.8) or a micellar system consisting of 50 mmol/l sodium dodecyl sulphate-20 mmol/l borate (pH 9.35), was developed as a rapid and efficient alternative to SEC, especially for process control of allergenic preparations. The results obtained by the two methods confirmed similarities in the structures of the studied pollen allergens.

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography.

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C18 stationary phase and mobile phases of 20-40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4-5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50 mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the D- and L-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM beta-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.

Quantitative structure-chromatographic retention relationship study of six underivatized equine estrogens.

Underivatized estrone (ES), equilin (EQ), equilenin (EQN) and their corresponding 17 alpha-diols 17 alpha-estradiol (ESD), 17 alpha-dihydroequilin (DHEQ) and 17 alpha-dihydroequilenin (DHEQN) were separated by TLC, RP-HPLC and capillary GC. Their dipole moments (mu) and Randic's connectivity indices ((1)chi) were determined as parameters of importance for the separation. The number of H atoms was taken as an additive structural parameter of importance for the quantitative structure-chromatographic retention relationship study (QSRR). Principal component analysis (PCA) was applied in order to find similarities and dissimilarities between 9 TLC and 10 RP-HPLC systems. PCA indicated that proton donor-proton acceptor interactions play the most important role for the TLC and RP-HPLC separation. The two-dimensional non-linear map of PC variables showed that the keto-estrogens (ES, EQ and EQN) and the corresponding diols (ESD, DHEQ and DHEQN) form two separate clusters. The relationship between GC retention of equine estrogens characterized by Kováts indices (KI), their (1)chi and mu was expressed by the equation KI/100 = al(1)chi+ b/mu(2) + c. The biological activity of the estrogens was related to log 1/mu(2).

High-performance liquid chromatographic determination of creatinine in serum, and a correlation of the results with those of the Jaffé and enzymic methods.

A high-performance liquid chromatographic method for the determination of creatinine in control sera is reported, based on its separation from deproteinated serum components on the ion-exchange material HEMA Bio 1000 SB and ultraviolet detection at 230 nm. Groups of eleven to fourteen samples of human serum and several control materials were simultaneously analysed by the Jaffé, enzymic ultraviolet and enzymic peroxidase aminophenazone methods. Another group (52-115 sera) was analysed for correlations with spectrophotometric methods. The precision of the chromatographic method ranges between 2.0 and 1.0% (relative standard deviation) for serum creatinine concentrations of 115.1 to 471 mumol/l, respectively. A very good accuracy was found in analyses of reference materials Kontrollogen-L and -LP. Some results of analyses of the other control sera were higher and the other lower than those obtained by the Jaffé and enzymic methods, because both interferences and enzyme inhibitors were encountered. Correlations between the chromatographic and spectrophotometric methods were good.

An ion-exchange separation of metal cations on a C-18 column coated with dodecylsulphate.

The retention mechanism was studied for the cations of the alkaline earth metals and Zn(2+) Ni(2+), Co(2+), Cd(2+) and Bi(3+) on a C(18) column permanently coated with sodium dodecylsulphate, with aqueous mobile phases containing cupric chloride or sulphate, or cerous nitrate. The dependencies of the logarithm or the capacity ratio on the logarithm of the eluent concentration were linear, demonstrating that ion-exchange was the predominating separation mode; the slopes of these dependencies were in good agreement with the values predicted from the ion-exchange theory. Indirect UV photometric detection yielded limits of detection (LOD) of 21, 44, 120 and 275 ng in the volume injected, 20 mul, for Mg(2+), Ca(2+), Sr(2+) and Ba(2+), respectively, with the 10(-2)M copper(II) chloride mobile phase; the respective LOD values decreased to 0.8, 1.6, 3.0 and 6.7 ng with the 5 x 10(-4)M cerium(III) nitrate eluent. The method was found to be primarily suitable for determination of the alkaline earths and was applied to analyses of surface and mineral waters.

An ion-exchange separation of Cu(2+), Cd(2+), Pb(2+) and Tl(+) on silica gel with polarographic detection.

The HPLC separation of heavy metal cations was studied with a column packed with Separon SGX silica gel. The retention of the cations is controlled by an ion-exchange mechanism. The ion-exchange capacity is primarily dependent on the mobile phase pH. The analyte retention is further affected by the type and concentration of the completing agent present and of the counterion. The effect of acetate, tartrate and alpha-hydroxyisobutyrate as complexing agents and that of methanol as the organic modifier were studied in detail and the results were compared with the theoretical model of ion-exchange separation. Simple mixtures of metals can be rapidly separated on a short column (30 x 3.3 mm i.d.), e.g., with a mobile phase containing 10(-2)M tartrate at pH 6.0. The metals separated can be detected by dc amperometry at a hanging mercury drop electrode. The limits of detection at an electrode potential of -0.95 V (Ag/AgCl) are in the units-tens of ng range with 20-mul samples with satisfactory precision (RSD values of 2-6%). The main advantages of the method are rapidly and simplicity because derivatization of the analytes is not required.

High-performance liquid chromatographic determination of cholesteryl esters in the blood of obese children.

The serum of obese children and adolescents was analyzed for cholesteryl esters. The test substances were first separated from the sample matrix by solvent extraction and thin-layer chromatography and then resolved in a reversed-phase high-performance liquid chromatographic system involving a Separon SGX C18 column and a mobile phase of 2-propanol-acetonitrile (40:60, v/v), with ultraviolet detection at 206 nm. Cholesterol and 10-cholesteryl esters could be separated and determined within ca. 25 min at a flow-rate of 1 ml/min. The method was applied to a study of the effect of external conditions (physical stress, diet) on the content of cholesteryl esters in a test group of obese boys and girls aged from 13 to 16 years. The analyses have demonstrated that the above conditions do not affect the concentrations of the individual cholesteryl esters, although the total cholesterol concentration decreased significantly after spa treatment.

Gas chromatography-mass spectrometry and high-performance liquid chromatographic analyses of thermal degradation products of common plastics.

The thermo-oxidation of five commonly used materials, namely low-density polyethylene, retarded polyethylene, paper with a polyethylene foil, a milk package and filled polypropylene, was studied. Capillary gas chromatography and gas chromatography-mass spectrometry were used to analyze the volatile degradation products, while high-performance liquid chromatography was employed to measure polycyclic aromatic hydrocarbons. The results are discussed from the point of view of toxicity of the products.

Determination of narciclasine in serum by reversed-phase high-performance liquid chromatography: comparison of amperometric, ultraviolet photometric and fluorescence detection.

Narciclasine was determined in the blood of mice by reversed-phase high-performance liquid chromatography, using a C18 stationary phase and a mobile phase of methanol-0.025 M potassium dihydrogen phosphate (50:50, v/v) of pH 5.5. Amperometric detection at a carbon fibre array working electrode held at +1.8 V (Ag/AgCl) permitted determination down to concentrations of 10 and 15.4 ng ml-1 (at a signal-to-noise ratio of 2) in aqueous solution and in serum, respectively. Fluorescence detection (excitation and emission wavelengths of 360 and 480 nm, respectively) exhibited somewhat poorer sensitivities for aqueous and serum samples: the respective limits of detection were 25 and 32 ng ml-1 at a signal-to-noise ratio of 2. Both the amperometric and the fluorescence detection were free from interference from blood components, but the fluorescence measurement required a post-column pH adjustment. UV photometric detection at 254 nm exhibited detection limits of 15 and 65 ng ml-1 in aqueous samples and in serum, respectively, and suffered from interferences from blood components that strongly absorbed in the ultraviolet region. All three detection techniques exhibited good linearity and precision.

High-performance liquid chromatographic determination of some polar phospholipids in serum.

The experimental conditions have been optimized for high-performance liquid chromatographic determination of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM) in serum. The phospholipids are separated on a silica gel column, using a mobile phase of acetonitrile-methanol-water (100:10:18, v/v), with ultraviolet photometric detection at 200 nm. The limit of detection was 0.2 micrograms (in 20 microliters) for natural phospholipids and 2.5 micrograms for synthetic phospholipids; the relative standard deviation was ca. 5%. An alternative detection is tensammetry at a mercury electrode, at a potential of -1.8 V, with an a.c. current frequency of 60 Hz and an amplitude of 20 mV. The tensammetric detection has an advantage in its independence of the structure of the phospholipids. In measurements without a column (flow-injection analysis), the tensammetric detection also yields a somewhat lower limit of detection than photometry (0.15 micrograms per 20 microliters), but this value increases more than ten times in chromatographic detection. The precision is poorer and is more susceptible to interferences. The method was applied to the determination of the above substances in the blood of obese children, as a function of physical stress and spa treatment. It was shown that physical exercise causes a decrease in the contents of PE and PC in the patients. On the other hand, the spa treatment has no pronounced effect on the phospholipid content in the blood.