Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization

With 3-trimethoxysilylpropyl chloride as the spacer, a proline-coated capillary column was prepared for the capillary electrochromatographic (CEC) separation of amino acids by in-column derivatization. Nine standard mixtures, including aspartic acid, glutamic acid, valine, phenylalanine, alanine, isoleucine, leucine, tyrosine, and tryptophan, were injected. o-Phthalaldehyde (OPA), OPA/2-mercaptoethanol (2-ME) and OPA/N-acetylcysteine (NAC) in borate buffer were tested as the derivatizing agent. Among them, OPA (50 mM) in borate buffer (pH 9.5, 50 mM) gave the best performance. The formation of isoindole could be detected by UV detection. The sandwich-type injection was carried out in hydrostatic mode (10 cm) with the program R(10 s)S(10 s) R(10 s)W(10 min) with R, S, and W being the reagent, sample, and waiting times. Mesityl oxide, benzyl alcohol, and acetone showed some interaction with the column. A current monitoring method was used instead of the determination of the electroosmotic flow (EOF). The direction of EOF was from anode to cathode even under acidic condition lower than the pI value (6.31) of the bonded group due to some unreacted silanol groups. Some parameters including pH, nature, and concentration of the mobile phase and the effect of organic modifier with regard to the CEC separation were investigated. With the proline-coated column (75 (50) cm x 75 microm ID) the best separation was performed in phosphate buffer (pH 4.00, 100 mM) with an applied voltage of -15 kV. The established method was also compared with those precolumn derivatized prior to the separation with proline-coated column as well as with in-capillary derivatization and separation with a bare fused-silica column.     

Post-column derivatization capillary electrochromatography for detection of biogenic amines in tuna-meat

A system to perform post-column derivatization capillary electrochromatography (CEC) was developed for the first time. The system mainly included a 4-microm (O.D.) silica packed column (200 mm effective length x 0.1 mm inner diameter I.D.) with micro-magnetic particles (MMPs) frits, a T-junction connector, an in-line fluorescence detector and a high-voltage power supply. The system was evaluated by using histamine (HA) as a standard biogenic amine for this study. A 5 microM HA solution was loaded at the anodic site of the capillary column by applying 3 kV for 5s. Then, HA was electrophoretically eluted with a 20mM phosphate buffer (pH 7) by applying 3 kV, and was derivatized with 3mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in 100 mM borate (pH 10), which was continuously delivered through the reagent-loading capillary tube by gravity into the T-junction connector. HA derivative was finally detected with the in-line fluorescence detector (lambda(Ex)=340 nm, lambda(Em)=450 nm) at 9.7 min after sample loading. To test the utility of this system, it was next employed for its ability to detect the presence of HA and other kinds of biogenic amines, including cadaverine (Cad), spermidine (Spm) and tyramine (Tyr) in tuna-meat, once the validity of the method had been confirmed.     

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.     

Capillary electrophoresis method for determination of D-serine and its application for monitoring of serine racemase activity

Serine racemase (SR) is an enzyme responsible for the biosynthesis of D-serine, the coagonist of the N-methyl-D-aspartate receptor, in the brain. Therefore, it has been suggested as a possible therapeutic target for the treatment of various neurodegenerative diseases. To develop a potent inhibitor of SR, a simple, sensitive, fast, and robust assay is needed. In this paper, a new CE method for the determination of D-serine is described. Serine enantiomers are resolved in the form of o-phthaldialdehyde (OPA)/2-mercaptoethanol (2-ME) derivatives in an alkaline BGE composed of 50 mM sodium tetraborate, pH 9.7, and containing 40 mM 2-hydroxypropyl-gamma-CD as a chiral selector. The problem of time-limited stability of OPA/2-ME derivatives has been overcome by employing in-capillary derivatization of the sample, i.e., the derivatization reaction was carried out directly in the separation capillary in the first phase of the CE run. UV-absorption detection at 230 nm allowed concentration detection limit of 3 microM. Baseline resolution of D- and L-serine derivatives was achieved in less than 10 min. This fact, together with the simple sample pretreatment, allowed application of the method to medium-throughput monitoring of SR activity, such as the screening of potential SR inhibitors. A good agreement was achieved between the developed CE method and the previously established HPLC method for determination of the inhibition constant, K(i), of a new SR inhibitor, L-erythro-3-hydroxyaspartate.     

Facile separation and determination of Aconitine alkaloids in traditional Chinese medicines by CE with tris(2,2'-bipyridyl) ruthenium(II)-based electrochemiluminescence detection

A facile CE method coupled with tris(2,2'-bipyridyl) ruthenium(II)-based electrochemiluminescence [Ru(bpy)(3) (2+)] detection was developed for simultaneous determination of Aconitum alkaloids, i.e., hypaconitine (HA), aconitine (AC), and mesaconitine (MA) in baseline separation. The optimal separation of these Aconitum alkaloids was achieved in a fused-silica capillary column (50 cm x 25 microm id) with 30 mM phosphate solution (pH 8.40) as running buffer at 12 kV applied voltage. The three alkaloids can be determined within 10 min by a single run. The calibration curves showed a linear range from 2.0 x 10(-7) to 2.0 x 10(-5) M for HA, 3.4 x 10(-7) to 1.7 x 10(-5) M for AC, and 3.8 x 10(-7) to 1.9 x 10(-5) M for MA. The RSDs for all analytes were below 3.01%. Good linear relationships were found with correlation coefficients for all analytes exceeding 0.993. The detection limits were 2.0 x 10(-8) M for HA, 1.7 x 10(-7) M for AC, and 1.9 x 10(-7) M for MA under optimal conditions. This method was successfully applied to determine the three alkaloids in Aconitum plants.     

Capillary electrochromatographic separation of proteins on a column coated with titanium dioxide nanoparticles

A TiO2 nanoparticle (TiO2 NP)-coated open-tubular column for the capillary electrochromatographic separation of proteins is described. The surface chemistry of the TiO2 NPs on the inner wall of the fused silica was significantly affected by the running buffer. By varying of the phosphate buffer pH, only cathodic EOF was indicated. The results showed that TiO2 NPs are existed as a complexed form with the buffer ligand. Good separation of conalbumin (ConA), apo-transferrin (apoTf), ovalbumin (OVA), and BSA could be achieved with phosphate buffer (40 mM, pH 8.0) and an applied voltage of 15 kV. Five peaks of glycoisoforms of OVA were observed under these conditions. In comparison with the retention behavior of the analytes on the bare fused-silica column, the new column's high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the TiO2 NPs. The method was also used to separate egg-white proteins. Both acidic and basic proteins in egg white were separated in a single run. The microheterogeneities of OVA could also be found in it. The separation efficiency for the main peak of OVA in egg white was around 10,000 plates/m.     

Method development and validation for the separation and determination of omeprazole enantiomers in pharmaceutical preparations by capillary electrophoresis

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.     

CE-electrochemiluminescence with ionic liquid for the facile separation and determination of diester-diterpenoid aconitum alkaloids in traditional Chinese herbal medicine

A CE‐electrochemiluminescence(CE‐ECL) detection system, CE/tris(2,2′‐bipyridyl) ruthenium(II)ECL with ionic liquid, was established for the determination of diester‐diterpenoid aconitum alkaloids (aconitine (AC), mesaconitine (MA) and hypaconitine (HA)) in traditional Chinese herbal medicine. Running buffer containing 25 mM borax‐20 mM 1‐ethyl‐3‐methylimidazolium tetrafluoroborate at pH 9.15 was used, which resulted in significant changes in separation and obvious enhancement in ECL intensity for AC, MA and HA with similar structures. End‐column detection was achieved in 50 mM phosphate buffer with 5 mM (pH 9.15) at applied detection voltage of 1.20 V when the distance between the Pt working electrode and outlet of capillary (50 cm×25 μm id) was set at 150 μm. One single quantitative analysis of three alkaloids was achieved at a separation voltage of 15 kV within 10 min. Moreover, two extraction processes (ethanol extraction and ethyl ether extraction after basification) were investigated. The result showed that ethanol extraction process has higher extraction efficiency than ethyl ether extraction process. Under the optimized conditions, the detection limits of AC, MA and HA were 5.62×10^?8, 2.78×10^?8 and 3.50×10^?9 mol/L (S/N=3), respectively. The method was successfully applied to determine the amounts of AC, MA and HA in the aconitum herbal samples.     

Determination of chlorophenoxy acid herbicides by capillary electrophoresis with integrated potential gradient detection

This report describes separation and detection of chlorophenoxy acid herbicides spiked in drinking water by the technique combining solid-phase extraction, field-amplified sample stacking, capillary electrophoresis, and potential gradient detection. The herbicide solution (400 mL) was concentrated to 0.1 mL by the solid-phase extraction procedure. The buffer containing 3 mM ammonia and 0.3 mM hydroxypropyl-beta-cyclodextrin was adjusted to pH 9.0 with ammonia. The sample solution was injected into the capillary to 30% of the whole length, and -9 kV and 9 kV were employed for field-amplified sample stacking and separation, respectively. The herbicides were baseline separated and the detection limits with the above combined techniques were in the range of 1-4 x 10(-2) ng/mL.     

Experimental design optimization of the capillary electrophoresis separation of leucine enkephalin and its immune complex

To optimize the capillary electrophoretic separation conditions for leucine enkephalin (LE) and the immune complex of the LE and anti-LE reaction, an analysis using a three-level, three-factorial Box-Behnken design was performed. Three separation parameters, buffer pH (X(1)), buffer concentration (X(2)), and applied voltage (X(3)), were chosen to observe the effect on separation responses. The responses were theoretical plate number, migration time of the LE peak, and resolution between the peaks. The optimum conditions and process validation were determined using statistical regression analysis and surface plot diagrams. The capillary electrophoresis optimum separation conditions were established to be 75 mM phosphate buffer at pH 7.00 with an applied separation voltage of 15 kV. By using the analysis technique, the prediction of responses was satisfactory and process verification yielded values within the +/-5% range of the predicted efficiency.     

Separation and Determination of Five Anthraquinones in Rheum and Rheum-Containing Preparations by Capillary Zone Electrophoresis

A capillary zone electrophoresis method modified by β-CD was, for the first time, developed for the simultaneous determination of five anthraquinones (physcion, chrysophanol, aloe-emodin, emodin and rhein) in rheum and rheum-containing preparations. The factors relevant to the run buffer media and their effects on the separation were studied. A buffer solution composed of 15 mM sodium borate, 30 mM β-CD, 20% acetonitrile and 1.0% ethanediol was found to be the most suitable electrolyte for separation, whereby the contents of the five anthraquinones in rheum and rheum-containing preparations could be easily determined in 20 min at 20 kV. The relative standard deviation for the determination of the five constituents in the samples varied form 0.16–5.47%, and the recovery ranged between 92.0 and 107.3%. Moreover the inclusion constants of the target compounds, except for rhein with β-CD, were also determined.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.     

Quantitation of caffeine by capillary zone electrophoresis with end-column amperometric detection at a carbon microdisk array electrode

Capillary zone electrophoresis is employed for the determination of caffeine using end-column amperometric detection with a carbon fiber microdisk array electrode at a constant potential of 1.45 V versus a saturated calomel electrode. The optimum conditions of separation and detection are 0.1 52mM NaH2PO4-0.648mM Na2HPO4 for the buffer solution, 20 kV for the separation voltage, 5 kV for the injection voltage, and 10s for the injection time. The limit of detection is 2.9 x 10(-4)mM or 1.2 fmol (signal-to-noise ratio = 2). The relative standard deviation is 0.68% for the migration time and 2.3% for the electrophoretic peak current. The method is applied to determining caffeine in human serum and a cola drink.     

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.     

Silanizing agent for semipermanent wall coating in micellar electrokinetic capillary chromatography

In this paper, the long-chained, silanizing agent chloro(dodecyl)dimethylsilane (CDDS) was investigated as a semipermanent coating in micellar electrokinetic capillary chromatography (MEKC). CDDS coating had great stability due to the formation of covalent bonding with the silanol groups on the surface of fused-silica capillary and remained stable for over 100 min after removal of the rinse step of CDDS solution. Anionic surfactant sodium dodecyl sulfate (SDS) could aggregate at this CDDS coating by the hydrophobic group and formed a SDS layer which could increase the electroosmotic flow (EOF). The separation was performed with the running buffer composed of 60mM sodium tetraborate, 12 mM SDS at pH 9.9, with the applied voltage of 20 kV and capillary temperature 25 degrees C. The effect of the coating agent was investigated by the analysis of amino acids. Compared with previous no-coating method, the EOF increases from 4.34 x 10(-4)cm(2)V(-1)s(-1) to 7.02 x 10(-4)cm(2)V(-1)s(-1). Migration time reproducibility was less than 0.97% R.S.D. from run to run and less than 1.56% R.S.D. from day to day.     

Development of a perfusion ion-exchange chromatography method for the separation of soybean proteins and its application to cultivar characterization

A perfusion ion-exchange chromatography method has been designed, for the first time, for the separation of soybean proteins and its application to the characterization of soybean cultivars. For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized. The method consisted of a two-step gradient (0% B for 2 min and from 0 to 50% B in 10 min) being mobile phase A a 2 0mM borate buffer (pH 9) and mobile phase B a 20 mM borate buffer (pH 9) containing 1M sodium chloride. The procedure used for the preparation of sample solutions was significantly simpler than that proposed by other authors and basically consisted of dissolving in water. This method enabled the separation of soybean proteins from a soybean protein isolate in 11 peaks in about 9 min. The method was used to separate soybean proteins in different commercial soybeans. In general, the 11 peaks yielded by the soybean protein isolate were also observed in the chromatograms of all soybeans. However, the area percentages of every peak in every soybean enabled the differentiation between soybeans. Moreover, the method was also used to separate soybean proteins in the proteic fractions obtained from every soybean. Multivariate methods were used for patterns recognition and the classification of samples.     

Development of a capillary electrophoresis method for the characterization of "palo azul" (Eysenhardtia polystachya)

The tree Eysenhardtia polystachya (Ortega) Sarg. has quite a wide popular use within the traditional Mexican medicine as herbal remedy. Popular practices constitute a relevant enough basis to design optimum analytical methods in order to determine basic principles of diverse medicinal plants. This has become one of the essentials needed to characterize such products, for which it is fundamentally important to develop an efficient and reliable separation method. This work presents the results concerning the development and optimization of a novel CE method for the separation of components from water/etanol (1:1) extracts of E. polystachya, using the following conditions, considered the best obtained: phosphate buffer 10 mM, 20 kV voltage, and pH 8.1 at 214 nm and 50 mM, 12.5 kV voltage with pH 8.1 at 426 nm. The optimization takes into account the parameters associated in the resulting electropherograms, such as number of peaks, migration times, and the Deltat(m) of the neighboring peaks. Under optimal conditions the separation intended was attained within 15 and 20 min for 214 and 426 nm, respectively. The characterization method developed was applied to the analysis of diverse extracts of E. polystachya.