Microdialysis hollow fiber as a macromolecule trap for on-line coupling of solid phase microextraction and capillary electrophoresis

On-line coupling of solid phase microextraction (SPME) and capillary electrophoresis (CE) is highly desirable due to the apparent advantages of the two techniques particularly in the context of microanalysis. However, the hyphenation is a significant challenge, because of band broadening and analyte carryover caused by the slow kinetics of analyte desorption in liquid phase. A novel strategy was presented in this study to overcome these problems. Analytes desorbed from an SPME fiber, which was held by an adapter, were first transferred by electrophoretic migration into a short piece of microdialysis hollow fiber, which was located at the inlet of a CE system. Analytes with molecular weights greater than the molecular weight cut-off of the microdialysis material were trapped in the microdialysis hollow fiber due to the dialysis effect. Then, under another electric field with different electrode polarity, the analytes trapped in the microdialysis hollow fiber migrated into the separation capillary and were separated. In the coupling approach, the microdialysis hollow fiber functioned as a macromolecule trap and a sample pre-concentrator as well. Band broadening was eliminated because the initial sample volume was very small (at nL level). Meanwhile, analyte carryover was eliminated because the desorption time could be as long as needed. Coupling of SPME with CE including two modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), was successfully demonstrated with proteins as test analytes. High efficiency and high resolution were obtained. The detection limits with UV absorbance whole-column imaging detection were 3.0 x 10(-7) and 3.0 x 10(-8) M (S/N = 3) for beta-lactoglobulin A and ovalbumin, respectively.     

Development of a simple in-vial liquid-phase microextraction device for drug analysis compatible with capillary gas chromatography, capillary electrophoresis and high-performance liquid chromatography

A simple, inexpensive and disposable device for liquid-phase microextraction (LPME) is presented for use in combination with capillary gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). 1-4 ml samples of human urine or plasma were filled into conventional 4-ml vials, whereafter 15-25 microl of the extraction medium (acceptor solution) was filled into a short piece of a porous hollow fiber and placed into the sample vial. The drugs of interest were extracted from the sample solutions and into the small volumes of acceptor solution based on high partition coefficients and were preconcentrated by a factor of 30-125. For LPME in combination with GC, the porous hollow fiber was filled with 15 microl n-octanol as the acceptor solution. Following 30 min of extraction, the organic acceptor solution was injected directly into the GC system. For LPME in combination with CE and HPLC, n-octanol was immobilized within the pores of the hollow fiber, while the internal volume of the fiber was filled with either 25 microl of 0.1 M HCl (for extraction of basic compounds) or 25 microl 0.02 M NaOH (for acidic compounds). Following 45 min extraction, the aqueous acceptor solution was injected directly into the CE or HPLC system. Owing to the low cost, the extraction devices were disposed after a single extraction which eliminated the possibility of carry over effects. In addition, because no expensive instrumentation was required for LPME, 10-30 samples were extracted in parallel to provide a high number of samples per unit time capacity.     

The fabrication and evaluation of inline coupling of microdialysis with capillary electrophoresis and its application in the determination of blood glucose

A novel method of inline coupling of microdialysis (MD) with CE has been investigated. A polysulfone MD membrane was in situ prepared at the inlet end of the separation capillary, which endued the capillary with an additional function of clean-up sampling and made the pretreatment of complex matrices simple. The properties of the membrane and its influence on electrophoresis were evaluated. The membrane cut off macromolecules and particles efficiently and persistently, and changing the components of membrane-casting solution could control the molecular weight cutoff of the membrane. Using the capillary having the MD membrane in electrophoresis, column efficiency with little reduction was achieved. The mixture of chlorpheniramine (CPA) and BSA was used to verify the analytical characteristics. Only the peak of CPA appears in the electropherograms and the RSDs of migration times and peak areas of CPA were 0.8 and 4.6%, respectively. The method was applied for the determination of glucose in human blood using an electrochemical detector. Whole blood containing glucose and macromolecules was electrokinetically injected directly into the capillary without pretreatment, and the concentration of glucose in human blood was 5.5 mmol/L. Equilibrium dialysis injection is also investigated in this paper. The results show that the method may be further used for in vivo sampling to monitor the endogenous or exogenous small molecules and their metabolites.     

Extraction of lead ions by electromembrane isolation

A new microextraction approach namely electromembrane isolation (EMI) coupled with capillary electrophoresis (CE) and ultraviolet (UV) detection for determining lead (Pb) ions was developed. The EMI technique involved the use of a polypropylene hollow fiber whose channel was filled with a sodium dihydrogen phosphate/sodium tetraborate buffer solution of pH 8.1, immersed in 10 mL of an aqueous sample solution. A voltage of 300 V was applied across the hollow fiber wall for 15 min to extract Pb ions. The extract was then complexed with ethylenediamminetetracetic acid (20 mM EDTA solution, at pH 3.4) for CE analysis. Satisfactory linear dynamic ranges (0.1-10 mg L(-1)), limits of detection (0.019 mg L(-1)) and good repeatability (ranging from 4.9 to 15.6%, n=3) were obtained. EMI exhibits good linearity with a correlation coefficient of 0.9935. The optimized EMI procedure was applied to determine the concentration of Pb(2+) in various matrices, such as amniotic fluid, blood serum, lipstick and urine samples.     

Isoelectric focusing sample injection for capillary electrophoresis of proteins

Carrier ampholyte-free isoelectric focusing (IEF) sample injection (concentration) for capillary electrophoresis (CE) is realized in a single capillary. A short section of porous capillary wall was made near the injection end of a capillary by HF etching. In the etching process, an electric voltage was applied across the etching capillary wall and electric current was monitored. When an electric current through the etching capillary was observed, the capillary wall became porous. The etched part was fixed in a vial, where NaOH solution with a certain concentration was added during the sample injection. The whole capillary was filled with pH 3.0 running buffer. The inlet end vial was filled with protein sample dissolved in the running buffer. An electric voltage was applied across the inlet end vial and etched porous wall. A neutralization reaction occurs at the boundary (interface) of the fronts of H+ and OH-. A pH step or sharp pH gradient exists across the boundary. When positive protein ions electromigrate to the boundary from the sample vial, they are isoelectricelly focused at points corresponding to their pH. After a certain period of concentration, a high voltage is applied across the whole capillary and a conventional CE is followed. An over 100-fold concentration factor has been easily obtained for three model proteins (bovine serum albumin, lysozyme, ribonuclease A). Furthermore, the IEF sample concentration and its dynamics have been visually observed with the whole-column imaging technique. Its merits and remaining problem have been discussed, too.     

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

A CE method based on a dual‐enzyme co‐immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co‐immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z′ factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two‐enzyme inhibition by the present method.     

Capillary electrophoresis of trace metals in highly saline physiological sample matrices

Trace metal ions in highly saline samples such as urine were determined with capillary electrophoresis (CE) without desalting or off-line preconcentration. By mixing with a dye, 4-(2-pyridylazo) resorcinol (PAR), the metal ions were converted into anionic complexes having strong absorbance near 500 nm. A large volume of the metal-PAR complex sample solution injected into a coated capillary was stacked isotachophoretically and separated under a reverse potential. The salt anion (chloride) and PAR in the sample matrix acted as the leading and terminating electrolytes, respectively. In a sample containing a 250 mM NaCl matrix, more than 400-fold enhancement in the absorbance detector response was realized compared to the normal CE injection mode. Combination of the dye complexation and isotachophoretic stacking provided excellent detection limits (S/N = 3) for three trace metal ions in the low ppb range (Fe(2+), 0.7 ppb, Ni(2+), 0.4 ppb; Zn(2+), 1.2 ppb) with absorbance detection. The migration time reproducibility was excellent (relative standard deviations: standard samples < 1%, urine samples approximately 1%). The proposed method is convenient and fast, and the sample analysis can be completed within 20 min.     

Polymer-coated hollow fiber microextraction combined with on-column stacking in capillary electrophoresis

In this work, a novel microextraction method termed polymer-coated hollow fiber microextraction (PC-HFME) was developed in combination with capillary electrophoresis (CE). Polar dihydroxylated polymethylmethacrylate polymer was coated onto a porous propylene hollow fiber membrane and used as an adsorbent and that was placed in a stirred aqueous sample solution. Tumbling of the extraction device within the sample solution facilitated extraction. The amino alcohols (2-amino-1-phenylethanol, norephedrine, alprenolol and atenolol which are beta-blocker drugs), were used as model compounds to investigate the extraction performance. No organic solvent was used in this procedure. The extract was then further concentrated through on-column stacking (normal stacking mode) during CE analysis. The detection limits ranged from 0.9 to 7 ng ml(-1). Relative standard deviations (n=6) ranged from 4 to 6%. The extraction of the amino alcohols in spiked wastewater effluent (representing a complex matrix) was evaluated using the developed procedure.     

Sensitive determination of phenylarsenic compounds based on a dual preconcentration method with capillary electrophoresis/UV detection

A novel method based on off-line hollow fiber based liquid liquid liquid microextraction (HF-LLLME) combined with on-column anion selective exhaustive injection (ASEI)-capillary electrophoresis/ultraviolet (CE/UV) detection was proposed for the speciation of five phenylarsenic compounds including phenylarsonic acid (PAA), 4-aminophenylarsonic acid (4-APAA), 4-hydroxyphenylarsonic acid (4-HPAA), 4-nitrophenylarsonic acid (4-NPAA) and 3-nitro-4-hydroxyphenylarsonic acid (NHPAA) in this paper. In HF-LLLME, the target analytes were extracted from 5 mL aqueous samples (donor solution pH 2.15) through a thin phase of tributyl phosphate (TBP) inside the pores of a polypropylene hollow fiber and finally into an 18 μL 0.8 mmol/L Tris acceptor solution inside the lumen of the hollow fiber. Following HF-LLLME, the acceptor solutions were directly analyzed by ASEI-CE/UV. For ASEI, a large plug of water (91% length of total capillary) was introduced into the separation capillary before sample injection in order to prolong the sample injection time, and thus enhance the stacking efficiency. Under the optimized ASEI conditions, up to 236-fold of enrichment factor (EF) was obtained for the ASEI-CE/UV determination of target phenylarsenic compounds. By combining HF-LLLME with ASEI-CE/UV, EFs ranging from 155 to 1780-fold were achieved and the limits of detection (LODs) (at a signal-to-noise ratio of 3) were in the range of 0.68-6.90 μg/L for five phenylarsenic compounds; the relative standard deviations (RSDs) of corrected peak area were 5.6-11.8%. The proposed HF-LLLME-ASEI-CE/UV method was applied for the determination of five target phenylarsenic compounds in pig feed from a local pig farm, and storage pig litter, soil in agricultural field and lake water collected near this pig farm, the recoveries for the spiked samples were in the range of 85.7-104.5%, 66.7-96.2%, 28.9-46.9% and 86.9-107.8% for pig feed, pig litter, soil and lake water, respectively.     

在柱透析-毛细管电泳联用分析--一种带有透析采样功能的毛细管电泳的实现与初步应用

在石英毛细管的进样端端口内采用相转移法制作聚砜膜,使该毛细管同时具有采样、样品净化与分离功能.建立了一种透析-毛细管电泳在柱联用方法.膜能有效地拦截大分子和颗粒物,性能稳定,12h内连续使用,膜的透过率和对大分子的拦截性能无显著变化.该方法避免了大分子和颗粒物的干扰,可直接对复杂样品中的小分子进行分析.将其应用于咖啡牛奶中游离咖啡因的含量测定,测得其值为0.68mmol/L;用于药物与蛋白的相互作用研究,获得了盐酸异丙嗪与牛血清白蛋白的结合常数,其值为1.47×104L/mol.     

Single-drop microextraction as a powerful pretreatment tool for capillary electrophoresis: A review

Single drop microextraction (SDME) is a convenient and powerful preconcentration and sample cleanup method for capillary electrophoresis (CE). In SDME, analytes are typically extracted from a sample donor solution into an acceptor drop hanging at the inlet tip of a capillary. The enriched drop is then introduced to the capillary for CE analysis. Since the volume of the acceptor drop can be as small as a few nanoliters, the consumption of solvents can be minimized and the preconcentration effect is enhanced. In addition, by covering the acceptor phase with an organic layer or by using an organic acceptor phase, inorganic ions such as salts in the sample solution can be blocked from entering the acceptor phase, providing desalting effects. Here, we describe the basic principles and instrumentation for SDME and its coupling with CE. We also review recent developments and applications of SDME-CE.     

Stroboscopic sampling in comprehensive high-performance liquid chromatography-capillary electrophoresis via a pneumatic sampler

A new approach is presented to solve the problem of a long separation time in the second dimension of comprehensive two-dimensional chromatography. The need for a rapid separation in the second column is overcome by repeating analysis of a sample many times. In each of these individual analysis cases the sample is injected into the first dimension column and after a delay a low amount of the effluent at the end of the first column is sampled to the second-dimensional column. The time interval between the samplings from the first column to the second column is constantly increased. Thus, the system enables a comprehensive analysis of the effluent emerging from the first into the second column. This approach, which we call stroboscopic sampling, is tested for coupling high-performance liquid chromatography (HPLC) to capillary electrophoresis (CE) by an interface which operates on the principle of transporting the effluent from the HPLC column to the capillary inlet by small pressure pulses (0.5 MPa). The performance of the interface for accomplishing the comprehensive HPLC-CE analysis was demonstrated for an on-line connection of a short ion-exchange column and an ion-exclusion column to the CE capillary.     

System peaks in capillary zone electrophoresis of anions with negative voltage polarity and counter-electroosmotic flow

The system peaks that often appear on electropherograms in anion separation by CE with indirect spectrophotometric detection, negative voltage polarity and cathodic EOF are studied. The system peaks are shown to correspond to the zones with the changed concentration of the BGE constituents; they appear while the zone of each analyte anion passes through the outlet end of the capillary and are transported to the detector by EOF. An equation is suggested for predicting migration times of the system peaks with an error of 1%. The ratios of the system peak area to the analyte peak area are found to amount to 20%. It is shown that it is possible to avoid overlapping of the system peaks and analyte peaks by controlling the EOF velocity owing to hydrodynamic pressure. Using the mathematical simulation of CE shows that the system peaks and baseline shift can result from changing the transference numbers of the BGE ions and analyte ions at the capillary edge. The cases when the system peak may be incorrectly identified as the peak of analyte ion are considered. In order to avoid such errors, some practical recommendations are given.     

In-capillary preconcentration of proteins for capillary electrophoresis using a cellulose acetate-coated porous joint

This work describes the in-capillary preconcentration of proteins using a cellulose acetate-coated porous joint. The capillary wall near the inlet end of a capillary was made porous by HF etching. During the etching process, a voltage was applied across the capillary wall and the electric current across it was monitored. As the current passed through the capillary wall, it became porous. A solution of cellulose acetate in acetone was added to the etched porous joint. After the acetone was evaporated off, a cellulose acetate-coated porous joint was formed. To preconcentrate the protein ions, an electric voltage was applied between the inlet end of the capillary and the coated porous joint; the protein ions electromigrated to the porous joint but could not pass through it, while the buffer ions could pass easily through the joint. After allowing a certain amount of time for protein preconcentration, a separation voltage was applied across the two ends of the capillary, and normal capillary electrophoresis was carried out. The preconcentration factors for cytochrome c, lysozyme, ribonuclease, and chymotrypsinogen were 65, 155, 705, and 800, respectively. The cellulose acetate-coated porous joint was shown to be strong and stable over time, and was used to analyze trace proteins and macromolecules in biological samples.     

Liquid-phase microextraction with porous hollow fibers, a miniaturized and highly flexible format for liquid-liquid extraction

Since 1999, substantial research has been devoted to the development of liquid-phase microextraction (LPME) based on porous hollow fibers. With this technology, target analytes are extracted from aqueous samples, through a thin supported liquid membrane (SLM) sustained in the pores in the wall of a porous hollow fiber, and further into a microL volume of acceptor solution placed inside the lumen of the hollow fiber. After extraction, the acceptor solution is directly subjected to a final chemical analysis by liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), or mass spectrometry (MS). In this review, LPME will be discussed with focus on extraction principles, historical development, fundamental theory, and performance. Also, major applications have been compiled, and recent forefront developments will be discussed.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire that is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N = 3) was achieved for detection of myoglobin utilizing a 75-µm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550–1300 Da. The advantages of this new design include (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.     

In-column fiber-optic laser-induced fluorescence detection for CE

A highly sensitive in-column fiber-optic LIF detector for CE has been constructed and evaluated. In this detection system, a 457-nm diode-pumped solid-state blue laser was used as the excitation light source and an optical fiber (40 mum od) was used to transmit the excitation light. One end of the optical fiber was inserted into the separation capillary and was in situ positioned at the detection window. The other end of the fiber was protruded from the capillary to capture the excitation light beam from the blue laser. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a yellow color filter before reaching the photomultiplier tube. The present CE-fluorescence detection is a simple and compact optical system. It reduces the laser scattering effect from the capillary and fiber as compared to the conventional LIF detection for CE. Its utility was successfully demonstrated by the separation and determination of D-penicillamine labeled with naphthalene-2,3-dicarboxaldehyde. The detection limit was 0.8 nM (S/N = 3). The present detection scheme has been proven to be attractive for sensitive fluorescence detection for CE.     

Direct determination of small cations in proteinaceous samples using a flow injection-capillary electrophoresis system.

A method is described for the direct determination of small inorganic cations in samples containing large amounts of proteins, such as milk or blood plasma. The method is based on electrokinetic injection in a flow injection analysis-capillary electrophoresis (CE) system. The selected CE-electrolyte, containing 5 mM 4-aminopyridine and 7 microM cetyltrimethylammonium bromide at pH 4.5, prevents detrimental protein adsorption on the capillary walls. Therefore, no sample pretreatment, except for dilution, is required. Up to 30 repeated injections in one electrophoretic run can be performed, yielding RSD values of the migration time of less than 1 and 2.5% (n=30) for milk and blood plasma samples, respectively.     

Interinstrumental transfer of a fast short‐end injection capillary electrophoresis method: Application to the separation of niobium,tantalum, and their substituted ions

The interinstrumental transfer of a short‐end CE method was studied. A model separation of the hexameric forms of niobium, tantalum, and their substituted ions (Nb_6−xTa_x with 0 ≤ x  ≤ 6) was selected as test case. The method was first optimized on a Beckman instrument and in a second step transferred to an Agilent instrument. The transfer needed updated guidelines that tackled differences in effective capillary length, 8.5 (Agilent) versus 10 cm (Beckman), because of instrumental different capillary cartridges. Differences in effective length lead to migration time and separation efficiency inequalities, illustrated by a decrease in resolution between the substituted ions. The difference in effective length was overcome by adapting the lift offset parameter of the Agilent instrument. The lift offset default setting is 4 mm and by increasing this parameter both the inlet and outlet lifts are lowered and thus the detection window can be displaced and consequently the effective length was increased. The decrease in effective length difference and the effect on the separation efficiency was investigated and led finally to a restored separation of the substituted ions. The adaptation of the lift offset parameter during short‐end injection methods was added to earlier developed guidelines to facilitate interinstrumental method transfer of CE methods.