Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation

We report the use of a sheath flow reactor for post-column fluorescence derivatization of proteins. The derivatization reaction employed naphthalene-2,3-dicarboxaldehyde (NDA) and beta-mercaptoethanol, which were added in the sheath buffer. The labeled proteins were detected by laser-induced fluorescence with an argon-ion laser beam at 488 nm. The performance of this detection scheme was evaluated by separation of some protein standards. A column efficiency of 450,000 plates/m was obtained without stacking. The limits of detection for those standard proteins were determined to be from 8 to 32 nM. Excellent linear relationship was obtained with correlation coefficient of 0.9998 for alpha-lactalbumin concentration ranging from 3.91 x 10(-7) to 1.25 x 10(-5) M. Separation of protein standards at low pH was also demonstrated by reversing the electroosmotic flow (EOF) with addition of cetyltrimethylammonium bromide (CTAB) to the running buffer. Different separation selectivity was achieved, but the sensitivity is poorer than that at high pH. This post-column derivatization detection system was applied successfully to analyze the protein extract from HT29 human colon cancer cells as well as tryptic peptides.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

New technique for capillary electrophoresis directly coupled with end-column electrochemiluminescence detection

A new end-column electrochemiluminescence (ECL) detection technique coupling to capillary electrophoresis (CE) is characterized. A 300 microm diameter Pt working electrode was used to directly couple with a 75 microm inner diameter separation capillary without an electric field decoupler. The hydrodynamic cyclic voltammogram (CV) of Ru(bpy) 3 2+ showed that electrophoretic current did not affect the ECL reaction. The presence of high-voltage (HV) field only resulted in the shift of the ECL detection potential. The distance of capillary to electrode was an important parameter for optimizing detection performance as it determined the characteristics of mass transport toward the electrode and the actual concentration of Ru(bpy) 3 2+ in the detection region. The optimum distance of capillary to electrode was decided by the inner diameter of the capillary, too. For a 75 microm capillary, the working electrode should be placed away from the capillary outlet at a distance within the range of 220-260 microm. The effects of pH value of ECL solution and molecular structure of analytes on peak height and theoretical plate numbers were discussed. Using the 75 microm capillary, under the optimum conditions, the method provided a linear range for tripropylamine (TPA) between 1 x 10(-10) and 1 x 10(-5) mol/L with correlation coefficient of 0.998. The detection limit (signal-to-noise ratio S/N = 3) was 5.0 x 10(-11) mol/L. The relative standard deviation in peak height for eight consecutive injections was 5.6%. By this new technique lidocaine spiked in a urine sample was determined. The method exhibited the linear range for lidocaine from 5.0 x 10(-8) to 1.0 x 10(-5) mol/L with correlation efficient of 0.998. The limit of detection (S/N = 3) was 2.0 x 10(-8) mol/L.     

Micellar electrokinetic chromatography of enkephalin-related peptides with laser-induced fluorescence detection

Eight enkephalin-related peptides were derivatized using fluorescein isothiocyanate (FITC), and the derivatization products were separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The optimum molar ratio of FITC and peptide for derivatization was found to be 40:1, and 5 mmol/L sodium borate buffer (pH 9.2) was selected as derivatization media in order to get the high efficiency. Enkephalin-related peptides were completely separated in the pH range of 10.51-10.60 in a running buffer consisting of 100 mmol/L sodium borate and 60 mmol/L sodium dodecyl sulfate (SDS). The detection limit for these eight enkephalins ranged from 0.18 to 2.25 nmol/L, and the linear response range was 1.0 x 10(-6) to 1.0 x 10(-9) mol/L with correlation coefficients between 0.9947-0.9988. A separation efficiency as high as 380000 theoretical plates could be obtained for these analytes.     

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection

Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 microm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal-to-noise ratio. The limit of detection is 1 x 10(-7) mol/L or 26 amol and the linear dynamic range is 2 x 10(-7) to 2 X 10(-5) mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser-induced fluorescence detection.     

Capillary electrophoretic determination of sulfite using the zone-passing technique of in-capillary derivatization.

A new capillary electrophoretic (CE) method was developed for the simple and selective determination of sulfite. The proposed method is based on the in-capillary derivatization of sulfite with iodine using the zone-passing technique and direct UV detection of iodide formed. The optimal conditions for the separation and derivatization reaction were established by varying concentration of iodine, electrolyte pH and applied voltage. The optimised separations were carried out in 20 mmol l(-1) Tris-HCl electrolyte (pH 8.5) using direct UV detection at 214 nm. Experimental results showed that the injection of the iodine zone from anodic end of the capillary gives significantly better precision. Common UV absorbing anions such as Br-, l-, S2O3(2-), NO3-, NO2-, SCN- did not give any interferences. Valid calibration (r2=0.998) is demonstrated in the range 1 x 10(-5) - 8 x 10(-4) mol l(-1) of sulfite. The detection limit (SIN=3) was 2 x 10(-6) mol l(-1). The proposed system was applied to the determination of free sulfite in wines. The recovery tests established for wine samples were within the range 92-103%. The CE results were compared with those obtained by iodometric titration technique.     

Detection of duck hepatitis B virus DNA fragments using on-column intercalating dye labeling with capillary electrophoresis-laser-induced fluorescence.

A rapid on-column DNA labeling technique is used to detect viral restriction DNA fragments by capillary electrophoresis-laser induced fluorescence detection. Intercalating dyes such as POPO3 or ethidium homodimer-2 are incorporated into the detection buffer. The cationic dyes migrate into the capillary during electrophoresis and bind to the oppositely migrating DNA fragments. A post-column sheath-flow fluorescence detector is used in the experiment. Excellent labeling efficiency is achieved at minimal background fluorescence by diluting the dyes to between 1 x 10(-7) M and 5 x 10(-7) M in a buffer with low ionic strength relative to the running buffer within the capillary. This dilute sheath-flow buffer allows stacking of dye molecules inside the capillary when an electric field is applied. Calibration curves using a series of DNA size markers (between 72 and 1353 base pairs) were linear over an order of magnitude in DNA concentration. Sensitivity also increased linearly with fragment length, and detection limits ranged from 4 x 10(-14) M to 5 x 10(-13) M for the size-standards. Analysis of cloned viral DNA using duck hepatitis B virus demonstrated a concentration detection limit of 3.9 x 10(-16) M. Last, the technique produced very high separation efficiency, 14 x 10(6) theoretical plates which is greater than 47 x 10(6) plates m-1, for the duck hepatitis B viral genome.     

Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection

We have developed a compact polytetrafluoroethylene (PTFE) assembly-type capillary electrophoresis with chemiluminescence (CL) detection system. Luminol-microperoxidase-hydrogen peroxide chemiluminescence reaction was adopted. The device is rectangular in shape (60 mm x 40 mm x 30 mm) and includes three reservoirs (sample, migration buffer, and detection reservoirs) with electrodes. The detection reservoir includes an optical fiber to transport light at the capillary tip to a photomultiplier tube. Isoluminol isothiocyanate (ILITC) was analyzed as a model using this device with fused-silica or polytetrafluoroethylene capillary tubes 10 cm in length. We also used the sample reservoir as a reactor for an immune reaction between anti-human serum albumin immobilized on glass beads and isoluminol isothiocyanate-labeled human serum albumin. The present polytetrafluoroethylene assembly with the capillary tube was useful as a palm-sized analysis device for separation and detection, as well as a reactor.     

Fluorescence enhancement for aflatoxins in HPLC by post-column split-flow iodine addition from a solid-phase iodine reservoir

Summary A method for post-column derivatization of the highly carcinogenic aflatoxins with iodine has been developed. It involves splitting of the mobile phase used for the reversed phase HPLC separation. One part flows through the injection valve and the C₁₈ analytical column to achieve the separation. The other part flows through a column packed with solid iodine. The iodine-containing solution is recombined with the flow coming from the analytical column. The derivatization reaction takes place in a knitted open tubular reactor maintained at 60 °C. Detection is done by fluorescence measurement. Due to the low solubility of iodine in the mobile phase, the iodine solid-phase column can be used for very long periods of time before refilling is necessary. The analytical system consists of only one pump and therefore gives the opportunity to carry out low-cost post-column reaction detection. The method yields reproducible results, a linear response over at least two orders of magnitude and detection limits of about 1 ppb, both for standard solutions and for peanut butter samples.     

电泳芯片结构和芯片电泳分离操作参数的模拟、优化和实验验证

选择了L-精氨酸和L-苯丙氨酸为分离样品体系,根据电泳实验提出样品基本参数,通过模拟计算考察了进样管道宽度和进样时间对进样方差的贡献;根据分离度与分离长度拟合曲线确定电泳芯片的有效分离长度;对化学发光柱后衍生管道施加的夹流电压进行了模拟优化,得出氨基酸体系分离分析的电泳芯片设计方案和操作参数为:进样管道宽度为分离管道宽度的1/2,简单进样充样时间应大于5 s,分离管道有效分离长度为30 mm,衍生夹流比1.0~1.6。根据模拟优化结果提出了电泳芯片设计方案,采用整体浇注法制作带有柱后衍生反应器的PDMS电泳芯片,按照模拟计算提出的电压操作参数实现了精氨酸和苯丙氨酸样品体系的准确进样、芯片电泳分离和柱后衍生化学发光检测。电泳过程模拟结果和实验结果相结合,考察了柱后衍生对样品谱带展宽的影响,简单进样过程样品泄露引起的谱峰拖尾现象,并讨论了夹流进样法对减小进样方差和抑制样品泄露的贡献。     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.     

离子色谱-在线柱后电化学衍生-荧光法测定酪氨酸

采用自制电解池作为电化学衍生装置,建立了离子色谱一电化学衍生一荧光法测定饮料中的酪氨酸。在碱性淋洗液作用下,酪氨酸在阴离子交换柱上被分离,到达自制电解池的阳极室,在阳极上被氧化,氧化后的产物因具有较强的荧光而被荧光检测器检测。离子化试剂既可以做色谱分离所需的淋洗液,又可以作为电化学反应优良的支持电解液,因此,离子色谱和电化学衍生具有较好的兼容性。最佳的实验条件为：淋洗液NaOH（10mmol／L）＋乙腈（ACN,1＋9）,流动相流速1．0mL／min,电解池电压1．0V,激发／发射波长320／420NM。在优化的实验条件下,酪氨酸的线性范围为0．01～10mg／L,检出限为1．2μg／L（信噪比S／N=3）。50μg／L的酪氨酸标准溶液进样7次,得到的色谱峰面积相对标准偏差为2．5％。方法具有快速,灵敏和选择性好的特点,并成功用于饮料中酪氨酸的测定。     

Capillary electrophoresis with post-column addition of terbium and sensitized lanthanide-ion luminescence detection for the determination of diflunisal and salicylic acid

A detection scheme based on the Post-column addition of terbium followed by sensitized luminescence detection has been developed. Two model Compounds- saliculic acid and diflunisa- were separated by capillary electophoresis in an alkaline phosphate buffer. A Solution containing terbium and CTAB was added post-column via a “home-built” reactor resulting in the rapid fromation of the strongly luminescent terbium-salicylic acid and terbium-diflunisal complexes in a reaction capillary. Following complexationn in the reaction capillarty, the 325 nm line from a HeCd laser was used to excite the analytes which efficiently transfer their energy to terebium. Resuulant luminescence of terebium was liner of 2 orders of magnitude. This novel and practical system enables the detection of micromolar concentrations of diflunisal and salicylic acid- a 10 fold improvement over UV absorbance detection in the same system. Post-column addition of terbium also eliminates the problems associated with strong adsorption of the lanthanide ion to the silica walls of the separation capillary and the formation of metal-ligand complexes with different coordination numbers.     

Study of the separation and determination of monosaccharides in soluble coffee by capillary zone electrophoresis with electrochemical detection

A simple, fast and reliable method, based on capillary electrophoresis with electrochemical detection, for the separation and determination of six monosaccharides, namely glucose, galactose, arabinose, fructose, xylose and ribose, in soluble coffees was developed. A copper disk electrode was used as the working electrode. The optimum conditions for separation and detection were 50 mmol L-1 sodium hydroxide buffer (pH 12.7), separation voltage 5 kV and detection potential 0.65 V (vs. Ag/AgCl). The linear ranges were from 5.0 x 10(-3) to 0.5 mmol L-1 for all six sugars. All regression coefficients were > 0.99. The detection limits for all the sugars were 1.0 x 10(-3) mmol L-1. The RSD of the peak current was < 4.2% (n = 5). The proposed method was applied directly to the separation and determination of the six sugars without prior derivatization, and the assay results were satisfactory.     

Modification of a Capillary for Electrophoresis by Electrostatic Self‐Assembly of an Enzyme for Selective Determination of the Enzyme Substrate

The integration of a separation capillary for capillary electrophoresis (CE) with an on‐column enzyme reaction for selective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the GOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at the capillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre‐ or post‐separation chemical modification of the analyte. The enzyme reactor is found to have an optimal response to glucose when a 5 : 1 mixture of PSS:GOx is used. Under these conditions the limit of detection for glucose is found to be between 5.0×10^?4 and 1.3×10^?3 M, dependent upon the inner‐diameter of the capillary. The apparent Michaelis‐Menten constant for the enzyme reaction was determined to be 0.047 (±0.001) M and 0.0037 (±0.0007) M for a 50 and 10 μm inner‐diameter capillaries, respectively. These results indicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution. This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.     

NBD-Cl as a Post-Column Reagent for Primary and Secondary Amines after Separation by Ion-Exchange Chromatography

In this study 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is proposed as a post-column derivatization (PCD) reagent for the fluorescence detection of aliphatic primary and secondary amines after HPLC separation. Five primary (methylamine, isoamylamine, 2-phenylethylamine, putrescine, and histamine) and one secondary amine (dimethylamine) were separated isocratically on a cation-exchange column using HNO₃ (5 × 10^?3 mol L^?1) as the mobile phase. Post-column derivatization was based on two steps: 1) the derivatization of amines with NBD-Cl in alkaline medium, and 2) the acidification of the resulted mixture in order to minimize the background signal of the reagent and improve dramatically the sensitivity and determination range. The variables of the post-column reaction (concentration of NBD-Cl, buffer concentration and pH, reaction temperature, concentration of HCl, flow rates of the reagents) were thoroughly investigated. Critical chromatographic parameters such as the concentration of HNO₃, the percentage of organic solvent, and the column temperature were also examined to achieve adequate separation. An internal standard of 1,7-diaminoheptane was used. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LODs 20–100 µg L^?1 with S/N = 3), and it requires less sample preparation. The applicability of the proposed analytical scheme was demonstrated by the determination of histamine (HIS) in tuna fish tissues according to US Food and Drug Administration (FDA) guidelines.     

UV-VIS detection of amino acids in liquid chromatography: online post-column solid-state derivatization with Cu(II) ions

In this work, we describe the introduction of a post-column solid-state reactor in the HPLC system used for the analyses of amino acids. The reactor used was filled with copper(II) oxide. Passage of the analytes through the reactor leads to the formation of Cu(II) complexes. Unlike free amino acids, the Cu-complexes show significant absorbance in the UV region and accordingly sensitivity of UV-VIS detection is increased by two to three orders of magnitude. As a result of this improvement in sensitivity, we have obtained LOD values in micromolar range and good linearity over the studied concentration range (5.0×10(-5) to 2.0×10(-3) mol/L). The method exhibits advantages typical of solid-state reactors, such as negligible loss of efficiency due to the derivatization, simplicity of realization and a long-term durability. The presented system brings an easy and versatile solution for UV-VIS detection of coordinating compounds, which do not normally absorb well in the UV-VIS region.     

Improved method for the determination of manganese in nuclear power plant waters

An improved method for manganese determination in nuclear power plant waters has been developed. This method combines a selective chelation concentration method with a unique analytical separation for manganese from the interfering matrix using a weak acid cation exchange column. The detection sensitivity by conventional post-column derivatization is improved with the combination of chemical eluent suppression and subsequent post-column derivatization. The detection limit for manganese in ammonium matrix is approximately 2 pg/ml and the limit of quantitation 10 pg/ml with 100 ml sample volume.     

柱后衍生HPLC法测定功能饮料中的牛磺酸

建立柱后衍生–高效液相色谱法测定功能性饮料中牛磺酸的含量。功能性饮料中的牛磺酸经水溶提取后与邻苯二甲醛柱后衍生,以柠檬酸三钠溶液(p H 3.2)为流动相,用AMINO–NA色谱柱分离,荧光检测器检测,激发波长为338 nm,发射波长为425 nm,柱后衍生反应温度为55℃,流量为0.4 m L/min。牛磺酸质量浓度在5.0~25.0μg/m L范围内与色谱峰面积线性关系良好,r=0.999 8,检出限(S/N=3)为0.11μg/m L,测定结果的相对标准偏差为0.73%(n=6),加标回收率在99.2%~101.6%之间。该方法灵敏度高、选择性好,可用于市售功能性饮料中牛磺酸的测定。     

Determination of hesperidin and synephrine in Pericarpium Citri Reticulatae by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection (CE-ED) has been developed for the determination of hesperidin (HP) and synephrine (SP) in the Chinese traditional herbal drug, Pericarpium Citri Reticulatae, the dried rind of the ripe fruits of Citrus reticulata Blanco (mandarin orange). The effects of some important factors such as the acidity and concentration of running buffer, separation voltage, and detection potential were investigated to determine the optimum conditions. The working electrode was a 300 microm diameter carbon disc electrode positioned opposite the outlet of the capillary. Both analytes could be well separated within 5 min in a 40 cm long capillary at a separation voltage of 12 kV in 50 mmol L(-1) borate buffer (pH 9.0). Excellent linearity was observed for the dependence of peak current on analyte concentration in the range from 2.5 x 10(-6) to 1.0 x 10(-3) mol L(-1) for SP and from 5.0 x 10(-6) to 1.0 x 10(-3) mol L(-1) for HP. The detection limits (S/N=3) for SP and HP were 4.96 x 10(-7) mol L(-1) and 6.54 x 10(-7) mol L(-1), respectively. This method has been successfully applied for the analysis of real samples, with satisfactory results.