Capillary electrophoresis for measuring major and trace anions in thermal water and condensed-steam samples from hydrothermal springs and fumaroles

A new application of capillary electrophoresis for measuring major and trace anions in thermal water and condensed-steam samples is presented. Ten fluid samples were collected from hydrothermal springs and fumaroles located in a volcanic zone of Deception Island, Antarctica. Anion separation was achieved in less than 6 min using indirect UV detection at 254 nm with a negative power supply (−15 kV). The electrolyte consisted of 4.7 mM sodium chromate, 4.0 mM electroosmotic flow modifier (OFM) hydroxide, 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid and 0.1 mM calcium gluconate (pH 9.1). Major anions (Cl⁻, SO₄², PO₄H²⁻, and CO₃H⁻) were measured using hydrostatic injection (10 cm for 30 s) at 25°C. Trace amounts of anions (F⁻, Br⁻, and NO₃⁻) were better determined by electromigration injection (4 kV, 10 s) at 15°C. Good reproducibility of the migration times (<0.72% RSD), a satisfactory linear response and accuracy as well as acceptable detection limits were successfully obtained.     

Dynamic control and indirect absorption detection for high-speed capillary electrophoretic separation of organic acids

Dynamic control and indirect absorption detection have been combined for the separation of eight small aliphatic organic acids in less than 4 min. Electroosmotic flow (EOF) coefficients in 5 mM 8-hydroxyquinoline-5-sulfonic acid (8-HQSA) (pH 3.00) and 3 mM 1,2,4,5-benzenetetracarboxylic acid (BTA) solutions are 4.35 and 1.65·10⁻⁴ cm²/V s, respectively. In the BTA system, relatively large amounts of sodium ions adsorbed into the capillary wall are the most probable reason for the small EOF, in turn causing problems for the separation of all acids. In contrast to BTA, 8-HQSA could be used for the separation of all eight organic acids. Limits of detection of analytes are at the level of several tens of μM at pH 3.00 in the 8-HQSA system. This new technique provides several features such as high speed, reasonable resolution and sensitivity, and ease of operation.     

Analysis of water extracts from airborne dust samples by capillary isotachophoresis

Application of capillary isotachophoresis (CITP) for the analysis of water extracts of the dust samples collected in different periods in air-filtration devices in Prague car traffic tunnels and in Parisian metro station is presented. The extracts were analyzed in cationic mode with a leading electrolyte (LE) of 10 mM KOH, 25 mM acetic acid, pH 4.4, and a terminating electrolyte (TE) of 10 mM β-alanine, adjusted to pH 4.4 with acetic acid, and in anionic mode with LE 10 mM HCl, 20 mM histidine, pH 5.8 and TE 10 mM 2-(N-morpholino)ethanesulphonic acid, pH 3.7. Extracted amounts of UV-absorbing substances, including pollen allergens and organic pollutants, the number of the found components and concentrations of some inorganic ions (e.g. Cl⁻, K⁺, Na⁺, Ca²⁺) in the dust samples were determined. It was found that the extracted amounts of anionic components and their number were much higher than those of cationic components. Significant differences have been found in the analyses of the extracts of different origin. Much more material and more components were present in the extracts of samples from the pollen-rich period than from the pollen-free period, especially in anionic CITP mode.     

Determination of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography.

The present paper describes the development of a micellar electrokinetic chromatographic (MEKC) method for simultaneous determination of andrographolide, deoxyandrographolide and neoandrographolide in ethanol extracts of Andrographis paniculata. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation was achieved using a 20 mM borate buffer, containing 20 mM sodium dodecyl sulphate and 10 mM sodium cholate, adjusted to pH 8.3 at an operating voltage of 25 kV, temperature of 35°C and a hydrodynamic injection of 5 s. The method was validated with good correlation coefficients obtained (0.9986–0.9989) while relative standard deviation (RSD) of migration time was between 1.14 and 2.42. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive diterpenoids in andrographis herb and its derived products.     

Development and optimization of organic acid analysis in tobacco with ion chromatography and suppressed conductivity detection

With the aid of a central composite face-centered design, an ion chromatographic method was developed and optimized for analyzing organic acids in tobacco. A Dionex-100 ion chromatograph with an ion suppressor and a conductivity detector, and a Bio-Rad Aminex HPX-87H column were employed. Only 13 analyses were required to optimize two factors: column temperature and eluent strength. Two sets of optimal conditions for separating nine acids were found: 1.8 mM HFBA eluent and 42 °C column temperature, and 0.8 mM HFBA eluent and 50 °C column temperature. The flow-rate was 0.6 ml min⁻¹ and the analysis time was 18 min or less. A sample preparation procedure included extraction of 2 g ground tobacco with 100 ml of 5 mM sulfuric acid solution for 3 h, filtration of the extract, and dilution of the filtrate 10-fold with deionized water.     

Separation and detection of common mono- and divalent cations by ion chromatography with an ODS column and conductivity/UV detection

The retention and detection behavior of common mono- and divalent cations (M⁺, alkali metal (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺) and ammonium ions (NH₄⁺); M²⁺, alkaline earth metal ions (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) was examined using an ODS column (150×4.6 mm I.D.) and conductivity (CD)/UV detection. The results obtained were as follows: (1) for M⁺, the mobile phase, 0.1 mM sodium dodecyl sulphate (SDS)+10 mM HNO₃ and indirect CD detection were effective. (2) Addition of Ce(III) in the mobile phase accelerated the elution of both M⁺ and M²⁺. The separation of above 10 cations on an ODS column was achieved for the first time without any coelution of cations and disturbance by system peak. Addition of higher SDS resulted in good separation of M⁺ and M²⁺ with longer retention times. CD detection was possible for M⁺ and M²⁺ and UV detection for M²⁺. (3) For M²⁺, the mobile phase, 0.8 mM Ce(III)+0.1 mM SDS+1 mM HNO₃ and indirect UV detection were effective. The IC methods were applied to real samples.     

Optimisation of selectivity in the separation of metallo-cyanide complexes by ion-interaction liquid chromatography

A study into the optimisation and selectivity of a reversed-phase ion-interaction liquid chromatographic method for the separation of metallo-cyanide complexes is described. A stable ion-interaction system was developed in which a C₁₈ stationary phase was equilibrated with a 60 mM solution of tetrabutylammonium hydroxide ion-interaction reagent in order to saturate the stationary phase and to minimise retention changes caused by adsorption and desorption of this material. The effects on retention of the metallo-cyanide complexes caused by changes in pH and ionic strength were minimised through the addition of a high concentration of a phosphate buffer (150 mM)to the mobile phase. Perchlorate (0.32–5.62 mM) was then added to the mobile phase as a competing anion and its effect upon the capacity factors of each complex determined. A linear relationship between the logarithm of capacity factor and the logarithm of the concentration of perchlorate was observed, although the slopes of these plots were not accordance with those predicted by a simple ion-exchange model. However, the linearity of the data allowed a simple optimisation procedure to be applied and the concentration of perchlorate could be used to manipulate the separation selectivity of the system. Three differing elution orders of metallo-cyanide complexes were achieved by varying the concentration of perchlorate in the mobile phase within the range 0.94–5.62 mM.     

Rapid determination of drugs in biofluids by capillary electrophoresis Measurement of antipyrine in saliva for pharmacokinetic studies

A micellar electrokinetic capillary chromatography method was developed that permitted the resolution of antipyrine from endogenous compounds and its quantitation in neat saliva in as little as 1 min. Final conditions were: SpectraPhoresis 1000, 30(23) cm × 50 μm silica capillary, 50 mM sodium phosphate pH 9.6, 50 mM SDS, 10 s hydrodynamic load, detection scanning 200–300 nm or 260 nm, run 25 kV. To overcome the effects of Joule heating the capillary was cooled to 15°C. Sensitivity was <10 μM and linearity extended to 350 μM. Comparison with an HPLC assay demonstrated that hydrodynamic injection gave a loading bias unless samples and standards were of equal viscosity. For 75 samples from five subjects the correlation of CE vs. HPLC was then r = 0.99.     

Development of a capillary zone electrophoresis method for caseinoglycomacropeptide determination

Caseinoglycomacropeptide (CGMP) is a polypeptide of 64 amino acid residues, derived from the C-terminal part of bovine κ-casein. A sensitive and selective capillary zone electrophoresis method has been developed and validated for the analysis and quantitation of CGMP. Separation is carried out at 30 kV, using an uncoated fused-silica capillary and 20 mM sodium citrate buffer at acidic pH 3.5. The described method allows the separation of various CGMP subcomponents. The validation data proves that the method has the requisite selectivity, sensitivity, reproducibility and linearity for CGMP assay and for quality control during CGMP manufacturing (batch-to-batch reproducibility).     

Detection of traces of oxidized and reduced sulfur compounds in small samples by combination of different high-performance liquid chromatography methods

Depending on the sulfur species, picomoles of different inorganic sulfur compounds can be detected and separated by HPLC in one arrangement in a sample volume less than 50 μl. The combination of fluorescence labelling of reduced inorganic sulfur compounds such as sulfide (S²⁻), sulfite(SO₃²⁻ and thiosulfate (S₂O₃²⁻) with monobromobimane followed by an extraction of elemental sulfur (S°) by chloroform treatment enables the detection of all mentioned sulfur compounds as well as sulfate (remaining aqueous phase) in the same sample. While the derivatized sulfur compounds could be detected by their fluorescence emission at 480 nm, elemental sulfur is identified by its UV absorption at 263 nm. Sulfate in the remaining aqueous phase is detected by HPLC with indirect UV detection at 254 nm. Detection ranges for the different sulfur compounds examined are as follows: sulfide (5 μM to 1.5 mM), sulfite (5 μM to 1.0 mM), thiosulfate (1 μM to 1.5 mM), elemental sulfur (2 μM to 32 mM) and sulfate (5 μM to >1 mM).     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Development and validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine

A capillary zone electrophoresis (CZE) method is proposed for the quantitative determination of anthocyanins in wine as an alternative to high-performance liquid chromatography. The CZE separation was carried out using a 46 cm (effective length)×75 μm I.D. fused-silica capillary at 10 °C and a 50 mM sodium tetraborate buffer at pH 8.4 with 15% of methanol as modifier. A voltage of 25 kV and a hydrodynamic injection of 300 mbar s were used. The electropherograms were recorded at 599 nm. It was found that SO₂ (antibacterial and antioxidant agent added to wine during its production) increased the absorbance of anthocyanins at 599 nm in a basic medium. Therefore, a concentration of 250 mg/l of SO₂ was added to the samples and the calibration solution before the analysis in order to avoid errors by this matrix effect. The analytical response was linear (R=0.998) between 10 and 700 μg/ml of malvidin-3-O-glucoside. The limit of detection and the reproducibility (as a relative standard deviation, n=11) were 1 μg/ml and 1.5%, respectively. Finally, the CZE method was validated by the analysis of synthetic wine samples (errors less than 8%) and by the comparison of the results obtained in the analysis of different monovarietal wines by CZE with those obtained by the standard HPLC method. In this comparison, a good correlation (R=0.998) with a slope of 1.005±0.044 and an intercept of −0.752±6.690 was obtained for malvidin-3-O-glucoside.     

Single-column method of chelation ion chromatography for the analysis of trace metals in complex samples

A single-column chelation ion chromatographic system for the preconcentration and separation of trace transition metals is described. The system includes standard chromatographic equipment with a post-column reagent system based on the reaction with 4-(2-pyridylazo)resorcinol followed by photometric detection at 495 nm. Iminodiacetic acid bonded to 5 μm silica (Diasorb IDA) was used as a chelating stationary phase. The strong complexing ability in combination with good kinetics of complexation and ion-exchange selectivity of iminodiacetic functional groups allow both preconcentration of Mn, Co, Cd, Zn, Ni and Cu from waters of high salinity and efficient separation with the same column. The retention characteristics of alkaline-earth and transition metal ions on Diasorb IDA silica (250×4 mm I.D.) column was investigated for a variety of eluents including nitric acid, maleic, malonic, citric, dipicolinic, picolinic, tartaric and oxalic acids. The influence of ionic strength on retention of metal ions involving high nitrate and chloride concentrations was also evaluated. The baseline separation of preconcentrated metals was achieved using a three-step gradient elution scheme which involved first, flushing of the column loaded with the sample with 0.5 M KCl−0.5mMHNO₃ for 10 min, followed by 80 mM tartaric acid for 20 min and finally 10 mM picolinic acid for 20 min.     

Separation of coumarins by micellar electrokinetic capillary chromatography

Nine coumarins were successfully separated simultaneously using micellar electrokinetic capillary chromatography with 4-hydroxybenzoic acid as an internal standard. A carrier composed of buffer solution (20 mM sodium dodecyl sulfate-15 mM sodium borate-15 mM sodium dihydrogenphosphate)-acetonitrile (24:1) was found to be the most suitable electrolyte for this separation. The analysis time (22 min) was shorter than that using high-performance liquid chromatography (47 min). Contents of coumarins in the crude drug of Angelicae Tuhou Radix could be easily determined by the proposed method.     

Micellar electrokinetic chromatography of charged and neutral drugs in acidic running buffers containing a zwitterionic surfactant, sulfonic acids or sodium dodecyl sulphate separation of heroin, basic by-products and adulterants

A procedure for the separation of charged and neutral solutes in acidic micellar running buffers has been developed. The procedure is based on a zwitterionic surfactant 3-N,N-dimethylmyristylammoniopropanesulfonate (MAPS) and alkylsulfonates added to the running buffer at pH 4.0. The alkylsulfonates increase the migration-time window for elution of neutral substances by increasing electro-osmotic mobility and by creating a negative micellar electrophoretic mobility. The test solution contained opiates and adulterants found in heroin seizures. Both the test solution and actual heroin seizures were successfully separated using a running buffer containing 50 mM 6-aminocaproic acid adjusted to pH 4.0 with 50 mM MAPS, 5 mM 1-heptanesulfonic acid and 10% acetonitrile. The procedure offers an alternative to micellar electrokinetic chromatographic separations based on charged surfactants in alkaline buffers.     

Retention behavior of alkali, alkaline earth and transition metal cations in ion chromatography with an unmodified silica gel column

An unmodified silica gel (Develosil 30-5) column (150×4.6 mm I.D.) has been applied to the ion chromatographic separation of alkali, alkaline earth and transition metal cations. The retention behavior of the above cations on the bare substrate was investigated using a number of weak inorganic and organic acid eluents. During this investigation, several separations were achieved and the most suitable eluent conditions were identified. It was concluded that: (a) 1.5 mM HNO₃-0.5mM pyridine-2,6-dicar☐ylic acid eluent was the most effective for the simultaneous separation of common alkali and alkaline earth metal cations, (b) 1.5 mM oxalic acid eluent resulted in the best separation of alkali, alkaline earth, and transition metal cations, (c) 0.5 mM CuSO₄ eluent could be used for the separation of alkali metal cations alone and (d) 0.5 mM ethylenediamine-oxalic acid eluent at pH 5.5 resulted in themost efficient separation of both alkaline earth and transition metal cations.     

Determination of histamine and some other amines by high-performance capillary electrophoresis with on-line mode in-capillary derivatization

We have developed a method for the determination of histamine (His), tyramine (Tyr) and cadaverine (Cad) using high-performance capillary electrophoresis with fluorescence detection and an on-line mode in-capillary derivatization with o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as derivatization reagent. HPCE separation of His, Tyr, Cad and Spermidine (Spd) was influenced by sodium dodecyl sulfate (SDS) and phosphate–borate buffer (pH 10) concentration. After optimization of the method, a 4-component amine solution containing His, Tyr, Cad and Spd could be separated and detected by using 2 mM OPA/NAC–20 mM SDS–20 mM phosphate–borate buffer (pH 10) as a run buffer at an applied voltage of 25 kV, with detection at 340 nm. Although a practical sensitivity level can be obtained by using fluorescence detection (λ_ex=340 nm, λ_em=450 nm) instead of ultraviolet–visible detection, Spd was not detected at all. The precision (relative standard deviation; n=15) of this method for within- and between-days is less than 2.9% (peak area) and 1.3% (migration time), respectively. Linearity for these analytes, except for Spd, was established over a concentration range of 0.02 to 1.00 μmol/ml and detection limits (S/N=3) range from 1 nmol/ml for His and Tyr to 5 nmol/ml for Cad. The determination of His and some amines in aging raw fish meat samples (room temperature, 48 h) was carried out using the described method with fluorescence detection.     

Capillary electrophoretic and thin-layer chromatographic characterization of rhenium complexation with 1-hydroxyethylidenediphosphonic acid

1-Hydroxyethylidenediphosphonic acid (HEDP) labeled by short-lived radionuclides with the nuclear properties suitable for the therapeutical purposes (¹⁸⁶Re, ¹⁸⁸Re, ¹⁶⁶Ho) is similar to the other phosphonates widely applied in the radiopharmaceutical field for the treatment of palliative bone metastases. One of the important steps for the preparation of compounds of radiopharmaceutical interest is the quality control comprehending the radiochemical and chemical purity determination. Chromatographic methods as TLC and HPLC are mostly used for this purpose. Our experiments were focused on the application of capillary electrophoresis with UV detection to the study of rhenium complexation with HEDP. The influence of pH, concentration of the ligand and the reaction time were determined. Taking in account our previous results, the Re:SnCl₂ molar ratio 1:500 (for 0.1 mM Re) was applied to reduce perrhenate to lower oxidation states which enables the Re–HEDP complexation. Different background electrolytes were tested. The mixture of 40 mM Na₂HPO₄ with 15 mM HEDP adjusted to pH 8 was selected as the most suitable system because it enabled the separation of different forms of Re–HEDP complexes. The results obtained in this study were compared to those obtained by thin-layer chromatography with radiometric detection.     

Ion chromatography of organic-rich natural waters from peatlands II. Na, NH4, K, Mg and Ca

Organic rich natural waters from peat bogs in continental (Switzerland) and maritime (Shetland Islands, Scotland) environments were analysed for Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺ using ion chromatography. These cations were determined simultaneously in surface and pore water samples from the continental bogs using a 250-μl injection loop in an isocratic separation. Using this loop, detection limits of the order of 1 ng/g were achieved. An organics-removal cartridge (Dionex OnGuard P) was used to remove humic materials. Analyses of deionized water filtered through these cartridges showed acceptably low blank values (e.g., ca. 5 ng/g) and appeared to have no significant effect on the measured cation concentration. For the maritime bog waters, the low concentrations of NH₄⁺ (ca. 1 μg/g) compared with Na⁺ (ca. 100 μg/g) required improved peak separation. This was accomplished using a gradient separation beginning with 40 mM HCl—1 mM , -2,3-diaminopropionic acid monochloride (DAP) and switching to 40 mM HCl-12 mM DAP after 2 min. Using a 25 μl injection loop, Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺ were determined simultaneously in less than 25 min. In this instance, even with Na⁺/NH₄⁺ > 100, there was no interference from Na⁺ in the determination of NH₄⁺ (baseline separated).     

Simultaneous determination of L-693,612, a topical carbonic anhydrase inhibitor, and two potential metabolites in human whole blood by ion-pair high-performance liquid chromatography

A method for the simultaneous determination of a topical carbonic anhydrase inhibitor, L-693,612, and two of its potential metabolites in human whole blood is described. The analytes are isolated from the matrix via liquid-liquid extraction with a mixture of toluene, ethyl acetate and isopropanol (49:50:1, v/v/v). The analytes are then back extracted into dilute phosphoric acid prior to injection into the HPLC system. A cyano column (Zorbax SB-CN, 150 × 4.6 mm) with a mobile phase of phosphoric acid(0.085%)-acetonitrile (73.5:26.5) containing 10 mM sodium decane sulfonate and adjusted to pH 3 is used for the analysis. Detection is based on UV absorbance at 252 nm. The assay was found to be linear in the concentration range of 5–500 ng/ml for each analyte when 1-ml aliquots of whole blood were extracted.