Some factors affecting separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Some factors influencing the separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection were investigated. These factors include eluent concentration, column temperature, and detection waveform. The selectivity changes in weakly retained amino acids are slight with changing sodium hydroxide eluent concentration. When sodium acetate eluent concentration is changed, the selectivity variations between strongly retained amino acids containing two carboxyl groups and containing only one carboxyl group are obviously different. Significant but slight selectivity changes in weakly retained amino acids can be achieved through changing the column temperature. Sodium hydroxide and sodium acetate eluent concentration affect the detection of amino acids. Detection sensitivity of amino acids can be improved by increasing the concentration of sodium hydroxide and sodium acetate in a certain concentration range. The detections of amino acids at two different detection waveforms were compared. The hydroxyl amino acids can be selectively detected by choosing a modified detection waveform. The optimized gradient elution condition and column temperature for analyzing 19 amino acids were obtained. The time for the gradient elution program was 60 min. The column temperature was 35 degrees C. Under the optimized conditions, detection limits for 19 amino acids were 0.15-4.52 pmol. The calibration graphs of peak area for all the analytes were linear for about three orders of magnitude. The RSDs (n=5) of peak area were 0.6-5.6%. The determination of trace amino acid impurities in valine product is shown as an application example.     

Potential of Flame Ionization Detection Coupled On‐Line with Microcolumn Liquid Chromatography Using Aqueous Eluents and an Eluent‐Jet Interface

The potential of the on‐line coupling of microcolumn liquid chromatography (μLC) using aqueous eluents with a flame ionization detector (FID) was evaluated. An eluent‐jet interface was modified to allow the efficient introduction of the eluent into the FID. The potential of the method is demonstrated by the μLC–FID determination of lower alcohols and bis(2‐hydroxyethylthio)alkanes on porous and non‐porous stationary phases, respectively. Flow injection analysis (FIA)–FID experiments with highly polar, thermolabile, semi‐volatile and non‐volatile compounds like amino acids, organic acids, alkylphosphonic acids, and carbohydrates showed the developed configuration to be a promising approach for the detection of a wide range of analytes. Compared with a nebulization interface, the eluent‐jet interface showed 4–10 times higher peaks for citric acid. Detection limits by FIA for all compounds were in the range of 0.2–5 ng injected. With ribose as test compound, plots of peak height vs. amount injected showed good linearity (r² > 0.999) in the range of 75–12,000 μg/mL. The repeatability showed relative standard deviations of less than 5%.     

Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection

A direct, sensitive, simple and practical method for simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection was developed. The retention behavior of amino acids and carbohydrates on the anion-exchange column and the detection of amino acids and carbohydrates at different integrated pulsed amperometric detection waveforms were investigated. The optimized gradient eluent conditions for analysis of 17 amino acids and nine carbohydrates were obtained. Separation time was 100 min. Detection limits for amino acids and carbohydrates were 5.2-207.1 nM under injection volume of 25 microl. The RSDs of peak area were 1.2-3.3%. The calibration graphs of peak area for the analytes were linear over about three orders of magnitude with a correlation coefficient of 0.9950-0.9999. The method was applied to determine amino acids and carbohydrates in a liquid condiment with satisfactory results.     

阴离子交换-积分脉冲安培检测法分离测定氨基酸注射液中的氨基酸和葡萄糖

 用阴离子交换 积分脉冲安培检测法测定了氨基酸注射液中 1 7种氨基酸和葡萄糖。研究了氨基酸和葡萄糖在阴离子交换中的保留行为。采用了优化的水、NaOH和NaAc三元梯度淋洗条件。在优化的梯度淋洗条件和积分脉冲安培检测条件下 ,氨基酸和葡萄糖的检出限为 0 3pmol～ 1 0 3pmol,线性范围约为 2个数量级。样品加标回收率为 88 3 %～ 1 0 4 6 %。方法简单、灵敏、准确。     

Ion-exclusion chromatography of aliphatic car☐ylic acids on a cation-exchange resin by elution with polyvinyl alcohol

Ion-exclusion chromatography of aliphatic car☐ylic acids of different acidity (pK_a) and hydrophobicity was investigated on a polystyrene-divinylbenzene (PS-DVB) based strongly acidic cation-exchange resin in the H⁺ form and conductivity detection by elution with polyvinyl alcohol (PVA). When water was used as an eluent, the resolution of the car☐ylic acids was very low and the peak accompanied a fronting depending on their hydrophobicities. Therefore, to improve the peak shape and the peak resolution, aqueous eluents containing PVAs (degrees of polymerization, n=500, 1500and2000) with many OH groups were tested for the ion-exclusion chromatographic separation of the car☐ylic acids. When aqueous eluents containing PVA were used, the fronting was decreased dramatically by the effect of increased hydrophilicity of the PS-DVB cation-exchange resin surface due to adsorption of OH group in PVA. The high resolution ion-exclusion chromatographic separation without the fronting and highly sensitive conductimetric detection of the car☐ylic acids was accomplished successfully by elution with a 0.2% PVA (n=1500)-10% methanol-water.     

Gradient HPLC Separation of Alkylphosphonic Acids on a Hypercarb Porous Graphitic Carbon Adsorbent with an Aqueous Formic Acid Solution as the Mobile Phase

An approach to the gradient HPLC separation of alkylphosphonic acids on a Hypercarb porous graphitic carbon adsorbent with an aqueous formic acid solution as a mobile phase is proposed. Analytes were detected using a quadrupole mass spectrometer. To increase the retention of the analytes, the chromatographic column was washed with water before the injection of a sample solution. This procedure results in a three- to fourfold increase in the retention factors of alkylphosphonic acids in comparison with the analogs described in the publications.     

离子色谱法测定高氯、高钠油田回注水中的阴、阳离子及有机酸

建立了高氯、高钠油田回注水中痕量无机阴、阳离子和有机酸的离子色谱分析方法。对高钠基质中痕量阳离子的测定,选用IonPac CS12A分析柱、H2SO4溶液梯度淋洗、电导检测器检测;对高氯基质中阴离子及有机酸的测定,选用对OH-具有高选择性的高容量的IonPac AS11-HC柱、KOH梯度淋洗、电导检测器检测。在优化的梯度淋洗条件下,高氯或高钠的存在不影响痕量阴离子或阳离子的测定。该方法具有良好的线性(r=0.9926~0.9990)和精密度(测定组分峰面积的相对标准偏差(n=7)在8.0%以下),回收率     

Vacancy ion-exclusion chromatography of carboxylic acids on a weakly acidic cation-exchange resin

In this preliminary study, a new approach to ion-exclusion chromatography is proposed to overcome the relatively poor conductivity detection response which occurs in ion-exclusion chromatography when acids are added to the eluent in order to improve peak shape. This approach, termed vacancy ion-exclusion chromatography, requires the sample to be used as eluent and a sample of water to be injected onto a weakly acidic cation-exchange column (TSKgel OApak-A). Vacancy peaks for each of the analytes appear at the retention times of these analytes. Highly sensitive conductivity detection is possible and sharp, well-shaped peaks are produced, leading to efficient separations. Retention times were found to be affected by the concentration of the analytes in the eluent, and also by the presence of an organic modifier such as methanol in the eluent. Detection limits for oxalic, formic, acetic, propionic, butyric and valeric acids were 0.1, 0.2, 0.3, 0.3, 0.4 and 0.5 microM, respectively, and linear ranges for some acids extended over two orders of magnitude. Precision values for retention times were 0.21% and for peak areas were <1.90%. The vacancy ion-exclusion chromatography method was found to give detection responses four to 10 times higher than conventional ion-exclusion chromatography using sulfuric acid eluent and two to five times higher than conventional ion-exclusion chromatography using benzoic acid eluent.     

离子色谱法同时分析董酒中的有机酸与无机阴离子

研究了用离子色谱法同时分析董酒中可离解性有机酸和无机阴离子。首次采用邻苯二甲酸氢钾和邻苯二甲酸的混合水溶液作淋洗剂,改善了分离效果,提高了检测灵敏度。所建立的方法无需进行样品前处理,无干扰,有一定的实用价值。     

离子交换色谱法同时测定啤酒中有机酸和无机阴离子

建立了用亲水性阴离子交换分离柱,KOH为淋洗液等浓度泵作梯度淋洗,电导检测,同时分离和检测16种无机阴离子和低分子量有机酸的离子色谱法。方法对所测无机阴离子和有机酸检出限在9．3～32μg/L之间;线性范围均在2个数量级以上;回收率在90．2％～107．2％之间。方法用于啤酒样品的分析,结果满意,样品的RSD小于5．3％(n=7)。     

Field-amplified sample stacking for the detection of chemical warfare agent degradation products in low-conductivity matrices by capillary electrophoresis-mass spectrometry

Preconcentration of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids) in low-conductivity matrices (purified water, tap water and local river water) by field-amplified sample stacking (FASS) was developed for capillary electrophoresis (CE) coupled to ion trap mass spectrometry. FASS was performed by adding a mixture of HCOONH(4) and NH(4)OH in appropriate concentrations to the sample. This allowed to control the conductivity and the pH of the sample in order to obtain FASS performances that are independent of analyte concentration. The influence of different parameters on FASS (sample to background electrolyte (BGE) conductivity ratio, injection volume and concentration of BGE) was studied to determine the optimal conditions and was rationalized by using the theoretical model developed by Burgi and Chien. A good correlation was obtained between the bulk electroosmotic velocity predicted by this model and the experimental value deduced from the migration time of the electroosmotic flow marker detected by mass spectrometry (MS). This newly developed method was successfully applied to the analysis of tap water and local river water fortified with the analytes and provided a 10-fold sensitivity enhancement in comparison to the signal obtained without preconcentration procedure. The quite satisfactory repeatability and linearity for peak areas obtained in the 0.5-5 microg mL(-1) concentration range allow quantitative analysis to be implemented. Limits of detection of 0.25-0.5 microg mL(-1) for the alkyl alkylphosphonic acids and of 0.35-5 microg mL(-1) for the alkylphosphonic acids were reached in tap water and river water.     

柱后加碱脉冲积分安培检测-离子色谱法测定水产品中十种生物胺

建立了利用淋洗液自动发生梯度淋洗的离子交换色谱法同时测定水产品中酪胺、5-羟色胺、腐胺、尸胺、组胺、庚二胺、苯乙胺、亚精胺、精胺、色胺等10种生物胺的方法。样品经处理后用Ionpac CS17分离柱和Ionpac CG17型保护柱分离,以EG40自动淋洗液发生器生成的5.0～55 mmol/L的MSA为淋洗液梯度洗脱,脉冲积分安培检测器检测。对梯度进行优化,10种生物胺都能基线分离,并且浓度和峰面积在一定范围内呈良好的线性关系。检出限在0.04 mg/kg以下,回收率在91.2%～102.5%之间,样品的RSD(n=6)小于5%。方法可用于水产品的检测。     

Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high performance liquid chromatography with ultraviolet detection

A precolumn derivatization method for the determination of amino acids using 6-aminoquinolyl-N-hyroxy-succinimidyl carbamate (AQC) followed by high-performance liquid chromatography is described. Ultraviolet detection was used for the assay of AQC derivatives of amino acids with the detection wavelength set at 248 nm. The reagent peak interference was minimized by optimizing the pH of the eluent and the gradient elution profile to improve the resolution between the reagent peak and amino acid derivatives. All nineteen amino acids were separated in 35 min with resolutions 1.6. The correlation coefficients of the calibration graphs for seventeen amino acids were fairly good (r 0.9999) at concentrations of 25–500 μM. The detection limits for all common amino acids including cystine and trytophan were at the range of 0.07–0.3 pmol. Good reproducibility and accuracy of the method were demonstrated by the determination of amino acids in three typical kinds of samples (protein, peptide and feed.) The average relative standard deviations for bovine serum albumin (BSA) and neuromedin were 0.86% and 1.36, respectively, and the average relative errors were 3.2% and 2.3%, respectively. The results of the analysis of feed hydrolysates agreed with those obtained by an ion-exchange method and the average recovery of the method for feed hydrolysates was 98%.     

离子色谱法测定水中4种有机酸

建立了水样中甲酸、乙酸、丙烯酸和甲基丙烯酸的离子色谱测定方法,采用IonPac AS11-HC阴离子分离柱,以KOH为淋洗液,采用浓度梯度洗脱,可同时测定上述4种有机酸。方法对实际水样中甲酸、乙酸、丙烯酸和甲基丙烯酸的平均加标回收率在80.2%~103.3%,相对标准偏差在5.8%之内,检出限分别为:0.005,0.003,0.005和0.004mg/L;定量下限分别为:0.020,0.012,0.020和0.016mg/L。方法准确、简便、环保,能够满足实际水样的测定需要,且水样中常见的阴离子不会对目标污染物的测定产生影响。     

阴离子交换色谱-积分脉冲安培法测定鱼粉和玉米粉水解液中氨基酸

将高效阴离子交换色谱．积分脉冲安培直接检测法应用于鱼粉和玉米粉水解液中的氨基酸的分析测定。该方法使用Dionex AminoPac PA10阴离子交换柱为分析柱，控制柱温为35℃，以NaOH和NaAc的强碱性溶液为淋洗液，在合适的梯度条件下，可以实现对17种常见氨基酸的高效分离。在强碱性条件下，获得良好分离的各个氨基酸可在配有金工作电极的电化学检测器上以积分脉冲安培法获得高灵敏度的检测。在优化的测定条件下，该方法对这17种氨基酸的检出限在0．21—3．38pmol之间；峰面积校正曲线的线性范围为2—3个数量级；5次平行测定的峰面积的相对标准偏差RSD在0．32％—4．7％之间。该方法应用于玉米粉和鱼粉水解液中氨基酸含量的测定，结果良好。对玉米粉和鱼粉水解液标准加入的回收率分别在81．9％—108．0％和90．0％～108．0％之间。     

Selective enrichment of the degradation products of organophosphorus nerve agents by zirconia based solid-phase extraction

Selective extraction and enrichment of nerve agent degradation products has been achieved using zirconia based commercial solid-phase extraction cartridges. Target analytes were O-alkyl alkylphosphonic acids and alkylphosphonic acids, the environmental markers of nerve agents such as sarin, soman and VX. Critical extraction parameters such as modifier concentration, nature and volume of washing and eluting solvents were investigated. Amongst other anionic compounds, selectivity in extraction was observed for organophosphorus compounds. Recoveries of analytes were determined by GC-MS which ranged from 80% to 115%. Comparison of zirconia based solid-phase extraction method with anion-exchange solid-phase extraction revealed its selectivity towards phosphonic acids. The limits of detection (LOD) and limit of quantification (LOQ) with selected analytes were achieved down to 4.3 and 8.5 ng mL(-1), respectively, in selected ion monitoring mode.     

Application of Doehlert design in optimizing the determination of degraded products of nerve agents by ion-pair liquid chromatography electrospray ionization tandem mass spectrometry

A qualitative method was developed for the determination of degraded products of nerve agents by using ion-pair liquid chromatography electrospray ionization tandem mass spectrometry (IP-LC-ESI-MS(n)). Generally, alkylphosphonic acids (APAs) and O-alkyl alkylphosphonic acids (AAPAs) give deprotonated molecular ion [M-H](-) in negative mode. Interestingly, first time we obtained the molecular radical anion [M](.-) of phosphonic acids in negative mode by using tri-n-butyl amine as an ion-pairing agent. We interpreted this observation as an indication of electrochemical reduction of phosphonic acids in electrospray needle. Three variables such as sheath gas flow, electrospray needle voltage and pH of the mobile phase were investigated to enhance the molecular radical anion [M](.-) signal of each analyte. The Doehlert design was used to obtain the region in which the optimum value of such variables is simultaneously achieved. Limit of detection achieved was 0.5 microg mL(-1) for AAPAs and 10 microg mL(-1) for APAs. Excellent precision was observed with less than 8.61% RSD. Finally, the method was applied for the detection of ethyl methylphosphonic in aqueous extract of soil sample.     

Direct, sensitive and selective detection of free fatty acids by high-performance liquid chromatography with post-column ion-pair extraction and absorbance detection

Free fatty acids (C8-C18) are separated by reversed-phase liquid chromatography and detected using a simple post-column dynamic extraction system in which the acids are extracted as ion pairs with chloroform from the aqueous acetonitrile (gradient: 79-99% acetonitrile) mobile phase after the post-column addition of aqueous Methylene Blue solution. The chloroform phase containing the ion pairs is monitored with an absorbance detector at 651 nm. The detection limits ranged from 26 to 83 ng, depending upon the acid, with coefficients of variation of 1.2-14%. Application of the method to butter and margarine samples permitted detection of free fatty acids down to 35 ppm and in orange juice, down to 0.5 ppm using only an organic solvent extraction without further sample clean-up for isolation of the fatty acids.     

反相硅胶整体柱离子对色谱法快速测定吡啶离子液体阳离子

建立了整体柱离子对色谱-紫外检测法梯度淋洗快速分离测定4种吡啶离子液体阳离子的方法。分离采用C18反相硅胶整体柱,以离子对试剂(用柠檬酸调节pH值)-乙腈为淋洗液,并采用多级梯度洗脱程序。实验考察了色谱柱、离子对试剂、乙腈浓度、色谱柱温度及流速对吡啶阳离子保留的影响,并讨论了其保留规律。咪唑阳离子的保留符合碳数规律。最佳色谱条件是:在流速3.0 mL/min,柱温30℃下,以1.0 mmol/L庚烷磺酸钠(pH 4.0)(A)+乙腈(B)为淋洗液进行梯度洗脱。淋洗梯度为0～2.0 min,10%B;2.0～2.5 min,10%～15%B;2.5～4.0 min,15%B;4.0～4.5 min,15%～20%B;4.5～10.0 min,20%B。在此条件下,4种吡啶阳离子可在7 min内基线分离。所测阳离子的检出限(S/N=3)为0.05～0.17 mg/L;峰面积的相对标准偏差(n=5)小于0.6%。将本方法用于实验室合成的离子液体样品和污水样品的分析,加标回收率在95.7%～99.0%之间。本方法准确、快速,具有较好的实用性。     

离子转换色谱-紫外检测法测定啤酒中的无机阴离子和有机酸

建立了离子转换色谱与紫外检测器联用检测啤酒中无机阴离子与有机酸的新方法。在传统的离子色谱基础上引入两根离子转换柱,无机阴离子与有机酸经过两步转换,定量转换成有相同紫外响应的碘酸盐,然后用紫外检测器代替电导检测器进行定量分析。在Dionex AS11-HC阴离子色谱柱上,采用KOH梯度淋洗方式实现了12种无机阴离子与有机酸的分离。结果表明,12种无机阴离子和有机酸检出限（S/N=3）与定量限（S/N=10）分别为6.168~29.01 μg/L与20.56~66.30 μg/L;线性关系良好（r在0.9994以上）,回收率为89.0%~117.0%,RSD均小于1.0%,该方法与传统的电导检测器检测结果相当。该方法简单快捷,只需采用少数标准曲线,就可以实现啤酒中无机离子与有机酸的定量测定。