Biomolecule analyses in an open-tubular capillary chromatography using ternary mixed carrier solvents with chemiluminescence detection

We examined the elution behavior of isoluminol isothiocyanate (ILITC)-labeled biomolecules (α-amino acids, peptides, and proteins) in an open-tubular capillary chromatography system using an untreated fused-silica capillary tube and a water-acetonitrile-ethyl acetate mixture carrier solution. Such an open-tubular capillary chromatography is called "tube radial distribution chromatography (TRDC)" for convenience. A mixture of ILITC and ILITC-labeled biomolecules was analyzed using TRDC with chemiluminescence detection that provided simple instrument without a light source and complex optical devises. The ILITC and the labeled twenty α-amino acids were separated, in this order or the reverse order, or not separated with an organic solvent-rich and water-rich carrier solution. Their elution behavior was considered to be of hydrophilic or hydrophobic nature of ILITC and the labeled α-amino acids. The ILITC and the labeled protein, alcohol dehydrogenase and bovine serum albumin, were separated in this order with an organic solvent-rich carrier solution, while they were eluted in the reverse order with a water-rich carrier solution, based on the TRDC separation performance. The TRDC system worked with the untreated open-tubular capillary tube not using any specific capillary tubes, such as coated, packed, or monolithic.     

Fluorescence observation supporting capillary chromatography based on tube radial distribution of carrier solvents under laminar flow conditions

We developed a capillary chromatography system by using an open capillary tube made of fused-silica, polyethylene, or polytetrafluoroethylene, and a water-hydrophilic/hydrophobic organic mixture carrier solution, called tube radial distribution chromatography (TRDC) system. By comparing with chromatograms obtained via the TRDC system, fluorescence photographs and profiles of the fluorescent dyes dissolved in the carrier solvents in capillary tubes were observed under laminar flow conditions. The chromatograms were obtained for a model mixture analyte consisting of 1-naphthol and 2,6-naphthalenedisulfonic acid with the TRDC system, by using a fused-silica capillary tube and a water-acetonitrile-ethyl acetate carrier solution. By altering the carrier flow rates, we examined the fluorescence photographs and profiles of the dyes, perylene and Eosin Y, dissolved in the carrier solvents in the capillary tube by using a fluorescence microscope equipped with a CCD camera. As confirmed by fluorescence observations, the major inner and minor outer phases generated in the capillary tube were based on the tube's radial distribution of the carrier solvents. We designed and manufactured a microreactor incorporating microchannels in which three narrow channels combined to form one wide channel. When the carrier solvents containing the dyes were fed into the channels, the inner and outer phase generations were also observed in the narrow and wide channels, strongly supporting the conclusions concerning the tube radial distribution phenomenon of the solvents.     

Tentative Comparison of Tube Radial Distribution Chromatography and CZE

Tube radical distribution chromatography (TRDC) uses an untreated open tubular capillary tube and a ternary mixture of solvents (water and hydrophilic/hydrophobic organic solvents) as a carrier solution. A model analyte mixture comprising 1-naphthol, 1-naphthoic acid, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was examined by the TRDC and capillary zone electrophoresis (CZE) systems that comprised mainly a capillary tube and a detector. In the TRDC system the elution order of analytes could be changed by altering the component ratios of the solvents, whereas in the CZE system the elution order was changed by altering the electroosmotic flow direction. The experimental data obtained provide clues about the features and utility of TRDC as a new separation method.     

Use of tube radial distribution of ternary mixed carrier solvents for introduction of absorption reagent for metal ion separation and online detection into capillary

When ternary mixed solvents consisting of water-hydrophilic/hydrophobic organic solvents are fed into a micro-space under laminar flow conditions, the solvent molecules are radially distributed in the micro-space. The specific fluidic behavior of the solvents is called the "tube radial distribution phenomenon (TRDP)". A novel capillary chromatography method was developed based on the TRDP that creates the inner major and outer minor phases in a tube, where the outer phase acts as a pseudo-stationary phase. This is called "tube radial distribution chromatography (TRDC)". In this study, Chrome Azurol S as an absorption reagent was introduced into the TRDC system for metal ion separation and online detection. The fused-silica capillary tube (75 μm id and 110 cm length) and water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) including 20 mM Chrome Azurol S as a carrier solution were used. Metal ions, i.e. Co(II), Cu(II), Ni(II), Al(III), and Fe(III), as models were injected into the present TRDC system. Characteristic individual absorption characteristics and elution times were obtained as the result of complex formation between the metal ions and Chrome Azurol S in the water-acetonitrile-ethyl acetate mixture solution. The elution times of the metal ions were examined based on their absorption behavior; Co(II), Ni(II), Al(III), Fe(III), and Cu(II) were eluted in this order over the elution times of 4.7-6.8 min. The elution orders were determined from the molar ratios of metal ion to Chrome Azurol S and Irving-Williams series for bivalent metal ions.     

Capillary chromatography based on tube radial distribution of aqueous-organic mixture carrier solvents

A capillary chromatography system was developed using open capillary tubes made of fused-silica, polyethylene, or poly(tetrafluoroethylene), and an aqueous-organic mixture (water-acetonitrile-ethyl acetate mixture) as a carrier solution. Model analyte mixture solutions, such as 2,6-naphthalenedisulfonic acid and 1-naphthol, Eosin Y and perylene, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein and 1,1’-bi-2-naphthol, and 2,7-naphthalenedisulfonic acid and p-nitroaniline, were injected into the capillary tube by a gravity method. The analyte solutions were subsequently delivered through the capillary tube with the carrier solution by a micro-syringe pump. The system worked under laminar flow conditions. The analytes were separated through the capillary tube and detected on-capillary by an absorption detector. For example, 2,6-naphthalenedisulfonic acid and 1-naphthol were detected in this order with a carrier solution of water-acetonitrile-ethyl acetate (volume ratio 15:3:2), while they were detected in the reverse order with a carrier solution of water-acetonitrile-ethyl acetate (volume ratio 2:9:4). The other analyte solutions were similarly separated by the system. The elution times of the analytes could be easily reversed by changing the component ratio of the solvents in the carrier solution.     

Tube radial distribution phenomenon observed in an aqueous micellar solution of non-ionic surfactant fed into a microspace and an attempt of capillary chromatographic application

A new type of tube radial distribution phenomenon was observed in an aqueous micellar solution of non-ionic surfactant that was fed into a microspace. A homogeneous aqueous solution containing 2 wt % Triton X-100 and 2.0 M sodium chloride was fed into a microchannel (40 μm in depth and 200 μm in width) in a microchip at a flow rate of 4.0 μL/min, where the microchip was maintained at a temperature of 34°C. The homogeneous aqueous solution changed to a heterogeneous solution with two phases in the microchannel; the surfactant-rich phase was generated around the middle of the channel, while the aqueous phase containing little surfactant was formed near the wall. The radial distribution of the surfactant was observed through Rhodamine B dissolved in the aqueous micellar solution with a bright-field microscope — CCD camera system. An open-tubular capillary chromatographic system was also tried to develop using the fusedsilica capillary tube (75 μm inner diameter and 120 cm length) as a separation column and the aqueous micellar solution as a carrier.     

Michrochip chromatography using an open‐tubular microchannel and a ternary water–ACN–ethyl acetate mixture carrier solution

A capillary chromatography system has been developed using a ternary mixed‐solvents solution, i.e. water–hydrophilic/hydrophobic organic solvent mixture as a carrier solution. Here, we tried to carry out the chromatographic system on a microchip incorporating the open‐tubular microchannels. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC‐labeled biomolecule was injected to the double T‐junction part on the microchip. The analyte solution was delivered in the separation microchannel (40 μm deep, 100 μm wide, and 22 cm long) with the ternary water–ACN–ethyl acetate mixture carrier solution (3:8:4 volume ratio, the organic solvent rich or 15:3:2 volume ratio, the water‐rich). The analyte, free‐ILITC and labeled BSA mixture, was separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. The outer phase acts as a pseudo‐stationary phase under laminar flow conditions in the system. The ILITC and the labeled BSA were eluted and detected with chemiluminescence reaction.     

Metal speciation using capillary electrophoresis--inductively coupled plasma atomic emission spectrometry and polytetrafluoroethylene capillaries

A capillary electrophoresis--inductively coupled plasma atomic-emission spectrometry (CE-ICP-AES) system using a polytetrafluoroethylene (PTFE) capillary has been developed. The CE-ICP interface was a modified concentric nebulizer. The PTFE capillary (50 microm internal diameter) was used as the central capillary of the nebulizer. Using the PTFE capillaries, the solution flow rate induced by the carrier gas flow was smaller than that of glass capillary. Solution flow was mainly induced by the CE electric field. Baseline separation of Ba2+/Mg2+ ion pair using simple buffer solution of 0.014 M sodium acetate was reported. Separation and correlation of metal species in metallothioneins (MT-1 and MT-2 in MT) of rabbit liver using the CE-ICP system were also discussed.     

Flow-injection extraction and gas-chromatographic determination of terodiline in blood serum

Adsorption losses of terodiline (N-t-butyl-1-methyl-3,3-diphenylpropylamine) from aqueous and organic solutions in the different parts of a flow-injection extraction system are described. Terodiline base adsorbs strongly on PTFE and polypropylene tubing from aqueous solution; 60–80% is lost from low concentration samples during its passage through the tubing (2 m long, 0.7 mm i.d.). For nickel, stainless steel and glass, the adsorption losses were slight. Terodiline in organic solution did not adsorb on any of the tested materials. Based on these results, an extraction manifold was designed for mechanized work-up of human blood serum. The samples were injected from a valve with a steel loop (0.5 ml) at a rate of 30 h^?1 into n-heptane + 2% n-pentanol, made alkaline, segmented with the organic phase and extracted. After phase separation, portions of the extract stream were collected in vials and analyzed for terodiline by using capillary gas chromatography with a nitrogen-selective detector.     

Ultrasonic-Assisted Drop-to-Drop Solvent Microextraction in a Capillary Tube for Analyzing Trace Benzene, Toluene, Xylene in One Drop of a Water Sample

A novel analytical technique termed ultrasonic-assisted drop-to-drop solvent microextraction (USA-DDSME) in a capillary tube was developed to determine trace benzene, toluene, xylene in one drop of a water sample, which was combined with gas chromatography–flame ionization detection (GC–FID). The advantages of this method are rapidity, convenience, ease of operation, simplicity of the device, and extremely little solvent and sample consumption. Extraction conditions including the type of extraction solvent, the volume of extraction solvent, the volume of sample, extraction time and effect of salt concentration were optimized. The best optimum parameters for extraction were achieved with 3 μL of extraction solvent. Chloroform was divided into four equal divisions in 20 μL water sample (without salt addition) in a capillary tube and ultrasonicated for 10 min, centrifugated at 2,500 rpm for 5 min to let the extraction solvent settle at the bottom of the capillary tube, then 1 μL of the separated extraction solvent was injected into the GC–FID for analysis. Linearity of the method was determined by analyzing spiked water samples over a concentration range of 0.1–50 μg mL^?1. Correspondingly, the LOD values were 0.01 μg mL^?1. All calibration curves were found to have good linearity with correlation coefficients (r ²) > 0.995. The precision (RSD) of the system, measured by six repeated determinations of the analytes at 1 μg mL^?1 were in the range of 1.6–3.5%.     

Enantiospecific HPLC and CE Methods for Separation and Determination of S-Darifenacin in Pharmaceutical Formulations

Two separation techniques were developed for the determination of S-(−)darifenacin (DAR) in the presence of its R-(+) isomer: The first method is high performance liquid chromatography (HPLC) and the second is capillary electrophoresis (CE). Chiral separation for chromatographic HPLC method development was carried out for S-DAR on Daicel CROWNPAK CR (+) (5 μm, 4.0 × 150 mm) column which contains (3,3-diphenyl-1,1-binaphthyl)-crown-6 coated onto a 5.5 μm silica support. The mobile phase system was aqueous acidic 70 % HClO₄ (pH 2.5): methanol in the proportion of 90:10 v/v. This current mobile phase was delivered at flow rate 0.8 mL min⁻¹ using UV detector adjusted at 286 nm. In CE method, the enantiomers were separated using 50 μm inner diameter fused-silica capillary cut to total lengths of 31.2 cm using 50 mM phosphate buffer as background electrolyte adjusted to pH 2.5 by triethanolamine. A wide range of cyclodextrins (CDs) were used such as highly sulfated α, γ CDs, hydroxyl propyl-β-CD and sulfobutyl ether-β-CD as chiral selectors. The effects of chiral additives regarding its concentration and content of organic modifier on the enantioseparation were investigated. Linear concentration ranges were from 2.5 to 50 and 40 to 300 μg mL⁻¹ with detection limits 0.67 and 12.28 μg mL⁻¹ for chromatographic HPLC and electrophoretic CE methods, respectively. The two methods were validated according to ICH guidelines with respect to linearity, accuracy, precision, LOQ, LOD and robustness. The suggested methods are suitable for separation and quantitation of S-DAR in tablets.

     

Sample decomposition in an electrically heated cold-trap inlet system for high speed gas chromatography

Thermal decomposition of some hydrocarbon and chlorinated hydrocarbon compounds in metal capillary tubes used in an inlet system for high speed gas chromatography has been investigated. The metal tube is cooled to about ?75°C by a flow of cold nitrogen gas in order to focus a vapor sample cryogenically. A capacitive discharge power supply is then used to heat the metal tube resistively in order to revaporize the sample and introduce it to the separation column as a plug 5-10 ms wide. The effects of tube temperature, tube material, sample vapor residence time, and type of carrier gas on thermal cracking are described. Use of a copper-nickel alloy tube resulted in less cracking than either pure platinum or pure nickel. Cracking is more significant with hydrogen as carrier gas than with helium. Cracking also increases with increasing sample residence time in the hot tube. Quantitative sample injection with minimum decomposition can be obtained for a variety of aliphatic and aromatic hydrocarbons and chlorinated hydrocarbon compounds.     

微流蒸发光散射检测器的研制及评价

构建了适用于纳升级到微升级流量的毛细管分离体系的微流蒸发光散射检测器(μELSD),实现了其与毛细管液相色谱(eLC)的联用.对雾化器孔径和雾化毛细管内径、蒸发管内径和长度、光散射池尺寸、雾化毛细管位置和辅助载气流量等参数进行了优化.在最优条件下,微流蒸发光散射检测器检出限为直接进样葡萄糖1 ng(S/N＞ 10),线性范围0.01～1.0 μg,重复性好,峰面积RSD(n=6)为0.4％,峰高RSD(n=6)为0.3％.本检测器已成功应用cLC-μELSD平台,使用C18毛细管色谱柱(内径250 μm),0.1％甲酸铵溶液(pH 4.5)-甲醇(60∶40,V/V)为流动相,分离检测了3种常用甜味剂,表明本研究构建的系统可以应用于实际分离检测中,具有分析时间快、溶剂消耗量少、样品需求量小的优点.     

Separation and determination of six catechins in tea by pressurized capillary electrochromatography

An efficient pressurized capillary electrochromatography (pCEC) method has been successfully developed for the determination of six catechins in tea. The separation was performed on a reversed-phase EP-100-20/45-3-C18 capillary column (total length of 45?cm, effective length of 20?cm, diameter of 100?μm, ODS packing inside for 3?μm). The mobile phase ratio of organic phase, the concentration of phosphate buffer and sodium heptanesulfonate, separation voltage, and other experimental conditions were investigated and optimized. The mobile phase was 15?mM NaH₂PO₄ and 12?mM sodium heptanesulfonate (pH 3.0)/methanol (64:36) at a flow rate of 0.04?mL/min. Under optimal conditions including applied voltage of ?4?kV and a UV detection wavelength of 230?nm, the six catechins in the tea were well separated. The calibration curves for the analytes had good linearity in the range of 8.02?μg/mL–202.13?μg/mL with a correlation coefficient of 0.9928–0.9997. The limits of detection (LOD) for the six catechins were 4.62?μg/mL–11.63?μg/mL (S/N?=?3). The recoveries of the six catechins were 96.2%–108.4% with a relative standard deviation (RSD) between 0.78% and 4.51%. The method has been used for the determination of six catechins in tea samples with good results.     

Miniaturization of batch- and flow-type chemiluminescence detectors in capillary electrophoresis

Glass and PTFE tubes as detection cells were put in small light-tight boxes to achieve miniaturization of batch-and flow-type chemiluminescence detectors for capillary electrophoresis. These light-tight boxes which included a detection cell and a photosensor module were successfully designed. In the batch-type detector using a glass tube as a detection cell, the influences of a repeated injection of sample and a reagent volume of the detection cell on chemiluminescence intensity were examined in detail. By using 3.8 mm I.D. glass tube including 400 microl chemiluminescence reagent solution, the chemiluminescence peaks were reproducibly observed for the repeated injection experiment up to the eight injection with each run time of 3.0 min. Dansyl-Trp was determined over the range 3 x 10(-8)-1 x 10(-5) M with the detection limit of 0.43 fmol (S/N=3). In the flow-type detector using a PTFE tube as a detection cell, both ends of the PTFE tube were connected to three-way joints; a chemiluminescence reagent solution was delivered into the cell and a capillary was inserted through one of the joints while an electrode was inserted through the other one. Dansyl-Trp was determined over the range 1 x 10(-7)-1 x 10(-5) M with the detection limit of 1.3 fmol (S/N=3). By using the compact flow-type detector, a mixture of dansyl-amino acids was separated and detected in micellar electrokinetic chromatography mode.     

Application of a Square‐Wave Potential Program for Time‐Dependent Amperometric Detection in Capillary Electrophoresis

Use of a square‐wave potential program for time‐dependent amperometric detection of analyte zones in capillary electrophoresis (CE) is described. Electrochemical detection for CE requires that the separation field be isolated from that of the electrochemical detection. This is generally done by physically separating the CE separation field from that of the detection. By applying a time variant potential program to the detection electrode, the detector current has a time dependence that can be used to help isolate the electrochemical detection current from that of the separation. When using a 20 μm inner‐diameter capillary, we find that a square‐wave potential program decreases the RMS baseline current from 4.5×10^?10 A, found with a constant potential amperometric detection, to 1.1×10^?10 A when using a square‐wave potential program. With a 75 μm inner‐diameter capillary, the improvement is even more dramatic, from 2.3×10^?9 A with amperometric detection to 2.06×10^?10 A when using a 1 Hz square‐wave potential program. When not using the time‐dependent detection with the 75 μm capillary, the analyte zones were beneath the S/N for the system and not detected. With the square‐wave potential program and time‐dependent detection, however, the analyte zones for an electrokinetic injection of 200 μM solution of 2,3‐dihydroxybenzoic acid were observed with the 75 μm inner‐diameter capillary. The improvement in the ability to discriminate the analytical signal from the background found experimentally is consistent with modeling studies.     

Simple capillary flow porometer for characterization of capillary columns containing packed and monolithic beds

A simple capillary flow porometer (CFP) was assembled for through-pore structure characterization of monolithic capillary liquid chromatography columns in their original chromatographic forms. Determination of differential pressures and flow rates through dry and wet short capillary segments provided necessary information to determine the mean diameters and size distributions of the through-pores. The mean through-pore diameters of three capillary columns packed with 3, 5, and 7 μm spherical silica particles were determined to be 0.5, 1.0 and 1.4 μm, with distributions ranging from 0.1 to 0.7, 0.3 to 1.1 and 0.4 to 2.6 μm, respectively. Similarly, the mean through-pore diameters and size distributions of silica monoliths fabricated via phase separation by polymerization of tetramethoxysilane (TMOS) in the presence of poly(ethylene glycol) (PEG) verified that a greater number of through-pores with small diameters were prepared in columns with higher PEG content in the prepolymer mixture. The CFP system was also used to study the effects of column inner diameter and length on through-pore properties of polymeric monolithic columns. Typical monoliths based on butyl methacrylate (BMA) and poly(ethylene glycol) diacrylate (PEGDA) in capillary columns with different inner diameters (i.e., 50–250 μm) and lengths (i.e., 1.5–3.0 cm) were characterized. The results indicate that varying the inner diameter and/or the length of the column had little effect on the through-pore properties. Therefore, the through-pores are highly interconnected and their determination by CFP is independent of capillary length.     

Gas chromatography with ballistic heating and ultrafast cooling of column

An overview of the existing methods for minimization of the analysis time in gas chromatography (GC) is presented and a new system for fast temperature programming and very fast cooling down is evaluated. In this study, a system of coaxial tubes, a heating/cooling module (HC-M), was developed and studied with a capillary column placed inside the HC-M. The module itself was heated by a GC oven and cooled down by an external cooling medium. The HC-M was heated at rates of up to 330 °C min⁻¹ and cooled at the rate of 6000 °C min⁻¹. The GC system was prepared for the next run within a few seconds. The HC-M permits good separation reproducibility, comparable with that of a conventional GC, expressed in terms of relative retention times and peak areas of analytes reproducibilities. The HC-M can be used within any commercial gas chromatograph.     

Ethyl Chloroformate as a Derivatizing Reagent for Capillary GC Determination of Dopamine, Adrenaline, Putrescine, and Histamine

Putrescine (Pu), histamine (HA), phenylhydrazine (PHZ), octopamine (OA), dopamine (DA), adrenaline (AD), and noradrenaline (NA) as the ethyl chloroformate (ECF) derivatives have been analyzed by capillary gas chromatography. The derivatives were separated on a 30 m × 0.32 mm i.d. HP-5 column by temperature programming from 100 °C (held for 1 min) to 250 °C at 10 min^?1. The total run time was 16 min. Nitrogen was used as carrier gas at a flow rate of 4 mL min^?1 and detection was by FID. PHZ was used as internal standard. The split ratio was 10:1 (v/v). The calibration curves were linear in the range 4–60 ng injected (1 μL injection) with detection limits 1.3–4.0 ng per injection (1 μL). When the method was used for determination of DA and AD in pharmaceutical preparations the relative standard deviation (RSD) was in the range 1–2.5%. When the effect of several additives was tested these did not affect the analyses. Pu and HA were estimated in fish samples with RSD 0.9–1.1 and 0.9–1.2%, respectively.     

On-line monitoring of cyanide concentration via a gas membrane system in extractive metallurgical processes

The principle of molecular diffusion of gaseous HCN across a tubular microporous hydrophobic PTFE membrane directly immersed in an alkaline solution or in an aqueous mineral suspension has been applied to the design of an on-line sensing system for cyanide. It offers an efficient detection of “available” cyanide and does not require acidification and solution sampling in the reactor. Gaseous HCN diffused through the membrane is dissolved by a sodium hydroxide carder solution which is then submitted to a spectrophotometric analysis by the pyridine-barbituric acid method. The problems related to filtering of sludges are therefore overcome, and many chemical interferences can be eliminated. Complexation reactions of cyanide by metallic cations and the cyanide-consuming properties of sulphide minerals and ores were studied. This method allows the determination of concentrations down to ca. 6 × 10^?7 mol l^?1 HCN + CN^? in the reactor at Ph 10 with a 120-cm membrane tube and a carrier solution flow-rate of 8 cm³ min^?1.