在线高效液相色谱-毛细管气相色谱联用方法的建立

建立了一种以保留间隙柱技术和阀切换以及定量管样品转移为接口并具有早期溶剂蒸气出口的在线液相色谱与毛细管气相色谱联用方法。考察了主要实验条件,如溶剂蒸发温度、载气压力等对联机系统性能的影响,并用萘和联苯对该系统的线性范围进行了测定。利用联机系统对一种轻柴油样品进行了分析。     

C18柱串联冠醚手性柱高效分离3种芳香族氨基酸对映体

将C18柱与手性冠醚柱串联,建立了一种反相高效液相色谱法用于3种芳香族氨基酸对映体同时拆分的方法.考察了反相色谱流动相的组成、pH值、柱温、流速对对映体拆分的影响.实验结果表明,当流动相为HClO4-乙睛溶液(86:14,V/V,pH 2.0)、柱温20℃、流速0.4 mL/min时,3种氨基酸对映体可获得基线分离.进一步对比了C18柱、冠醚手性柱和串联顺序不同的4种分离模式,结果表明,C18柱不能拆分氨基酸对映体,仅能分离不同种类氨基酸;冠醚手性柱可分离氨基酸映体,但不同种类氨基酸色谱峰出现重叠;串联模式能实现3种氨基酸对映体的基线分离,实现双柱优势互补,而串联顺序对分离影响不大,仅影响色谱峰的峰形.     

Statistics aided optimization for high-performance liquid chromatographic analysis of organic acids in tobacco

HPLC analysis for organic acids in tobacco was optimized with the aid of statistical experimental design, a central composite face-centered design. In the design, only thirteen HPLC analyses were needed for identifying two optimal separation parameters. A Bio-Rad Aminex HPX-87H column was used for the analyses. An optimal separation for seven acids in tobacco was found at a temperature of 57 degrees C and a mobile phase of 0.032 N sulfuric acid solution, or at a temperature of 70 degrees C and a mobile phase of 0.024 N sulfuric acid solution, with a flow rate of 0.6 ml min(-1).     

On-chip temperature gradient interaction chromatography

This paper reports the first integrated microelectromechanical system (MEMS) HPLC chip that consists of a parylene high-pressure LC column, an electrochemical sensor, a resistive heater and a thermal-isolation structure for on-chip temperature gradient interaction chromatography application. The separation column was 8 mm long, 100 microm wide, 25 microm high and was packed with 5 microm sized, C18-coated beads using conventional slurry-packing technique. A novel parylene-enhanced, air-gap thermal isolation technology was used to reduce heater power consumption by 58% and to reduce temperature rise in the off-column area by 67%. The fabricated chip consumed 400 mW when operated at 100 degrees C. To test the chromatography performance of the fabricated system, a mixture of derivatized amino acids was chosen for separation. A temporal temperature gradient scanning from 25 to 65 degrees C with a ramping rate of 3.6 degrees C/min was applied to the column during separation. Successful chromatographic separation of derivatized amino acids was carried out using our chip. Compared with conventional temperature gradient HPLC system which incorporates "macro oven" to generate temporal temperature gradient on the column, our chip's thermal performance, i.e., power consumption and thermal response, is greatly improved without sacrificing chromatography quality.     

HPLC separation of triacylglycerol positional isomers on a polymeric ODS column

A polymeric ODS column was applied to the resolution of triacylglycerol positional isomers (TAG-PI), i.e. 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) and 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), with a recycle HPLC system. To investigate the ODS column species and the column temperatures for the resolution of a TAG-PI pair, a mixture of OPO and OOP was subjected to an HPLC system equipped with a non-endcapped polymeric, endcapped monomeric, endcapped intermediate, or non-endcapped monomeric ODS column at three different column temperatures (40, 25, or 10 degrees C). Only the non-endcapped polymeric ODS column achieved the separation of OPO and OOP, and the lowest column temperature (10 degrees C) showed the best resolution for them. The other pair of TAG-PI, a mixture of 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) and 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (PPO) was also subjected to the system equipped with a non-endcapped polymeric or monomeric ODS column at five different column temperatures (40, 32, 25, 17, and 10 degrees C). Thus, POP and PPO were also separated on only the non-endcapped polymeric ODS column at 25 degrees C. However, no clear peak appeared at 10 degrees C. These results would indicate that the polymeric ODS stationary phase has an ability to recognize the structural differences between TAG-PI pairs. Also, the column temperature is a very important factor for separating the TAG-PI pair, and the optimal temperature would relate to the solubility of TAG-PI in the mobile phase. Furthermore, the recycle HPLC system provided measurements for the separation and analysis of TAG-PI pairs.     

Enantiomeric separation of amino acids and nonprotein amino acids using a particle-loaded monolithic column

A solution is prepared of 5 microm silica particles modified with (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine (particle 1) or (S)-N-3,5-dinitrophenylaminocarbonyl-valine (particle 2) suspended in liquid tetraethylorthosilicate, ethanol, and aqueous hydrochloric acid. This solution is injected under pressure into a 30 cm long, 75 microm inner diameter capillary column and heated for 1 h at 120 degrees C after which the modified particles are embedded in a monolithic column of sol gel. The packed column measures approximately 15 cm from the inlet to the window used to view the laser-induced fluorescence. Thirteen different amino acids and three nonprotein amino acids are derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) before injection onto the column for capillary electrochromatographic separation. The enantiomeric separation of the monolithic column packed with particle 1 results in a resolution ranging from 1.14 to 4.45, whereas that packed with particle 2 results in a resolution ranging from 0.79 to 1.17. On the basis of resolution and amount of chiral packing material the enantiomeric separation obtained by capillary electrochromatography is judged to be superior to that obtained previously with high performance liquid chromatography (HPLC).     

Measurement of the axial and radial temperature profiles of a chromatographic column. Influence of thermal insulation on column efficiency

The temperatures of the metal wall along a chromatographic column (longitudinal temperature gradients) and of the liquid phase across the outlet section of the column (radial temperature gradients) were measured at different flow rates with the same chromatographic column (250 mm x 4.6 mm). The column was packed with 5 microm C18-bonded silica particles. The measurements were carried out with surface and immersion thermocouples (all junction Type T, +/-0.1 K) that measure the local temperature. The column was either left in a still-air bath (ambient temperature, T(ext) = 295-296 K) or insulated in a packing foam to avoid air convection around its surface. The temperature profiles were measured at several values of the inlet pressure (approximately = 100, 200, 300 and 350 bar) and with two mobile phases, pure methanol and a 2.5:97.5 (v/v, %) methanol:water solution. The experimental results show that the longitudinal temperature gradients never exceeded 8 K for a pressure drop of 350 bars. In the presence of the insulating foam, the longitudinal temperature gradients become quasi-linear and the column temperature increases by +1 and +3 K with a water-rich (heat conductivity approximately = 0.6 W/m/K) and pure methanol (heat conductivity approximately = 0.2 W/m/K), respectively. The radial temperature gradients are maximum with methanol (+1.5 K at 290 bar inlet pressure) and minimum with water (+0.8 K at 290 bar), as predicted by the solution of the heat transfer balance in a chromatographic column. The profile remains parabolic all along the column. Combining the results of these measurements (determination of the boundary conditions on the wall, at column inlet and at column outlet) with calculations using a realistic model of heat dispersion in a porous medium, the temperature inside the column could be assessed for any radial and axial position.     

A Simple Design to Realize Micro-column Separation by Conventional Analytical HPLC

The conventional analytical HPLC was successfully developed for micro‐column separation by using a simple eluate splitting system, self‐preparation of packing column and on‐capillary column detector in our laboratory. Porous inlet frit in fused silica capillary was rapidly prepared by sintering stainless steel powders under 500 meshes for about 20 s. The use of such frits or metal meshes in capillary to retain C₁₈ particles of chromatographic packing was demonstrated to be stable and specially robust with continuous packing and long chromatographic runs. Furthermore, the chromatographic behavior was detailedly evaluated by changing the flow rate and the percentage of mobile phase using the prepared capillary column. Under the optimal experimental conditions, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a high column efficiency near 70000N (plates/m) by the developed capillary‐HPLC.     

Optimizing the gas chromatographic separation and detection of polychlorinated biphenyls by use of electronic pressure programming and experimental design

The applicability of electronic pressure control and Taguchi L₂₇ experimental design to the optimization of the gas chromatographic separation and detection of polychlorinated biphenyls has been evaluated. The influence of several experimental variables, column temperature program, carrier gas pressure program, on-column injector temperature program, and make-up gas pressure program, was studied using analysis of variance. Simultaneous optimization of sample introduction, column efficiency, and detector performance could be achieved without compromising system performance. The relationships between system performance and experimental variables were established using regression analysis. Agreement between the simulated and experimental results obtained using suggested optimum conditions demonstrated the applicability of the technique developed in this study. The improvement achieved in the chromatographic separation of PCBs is presented.     

Temperature gradients in HPLC columns due to viscous heat dissipation

Summary Temperature effects in HPLC columns due to viscous heat dissipation are examined. For the case when the thermostatted column wall and mobile phase at the column inlet are at the same temperature an explicit solution of the heat transport equation is given. The predicted temperature profile is parabolic at large distances from the column entrance; the magnitude of the effect is proportional to the square of the mobile phase velocity, and is of the order of a few degrees centigrade. At the upper end of the column a relaxation occurs over a length of a few centimers. Experimental results confirm the validity of the predictions made and indicate that the various assumptions and approximations are justified. Plate height curves obtained with two mobile phases with differing viscosities show a much smaller efficiency for the less viscous mobile phase. The curves show an upward curvature at high reduced velocities. Both phenomena can be related to thermal effects. It is concluded that viscous heat dissipation constitutes an obstacle to obtaining higher speed and efficiency in HPLC by the use of smaller particles. Possible remedies, such as the use of smaller bore columns or special thermostatting devices, look troublesome from the experimental point of view.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

替考拉宁键合手性固定相高效液相色谱法直接拆分泮托拉唑钠对映体

以替考拉宁为手性选择剂制备了大环抗生素类手性固定相替考拉宁键合手性固定相(T-CSP),建立了T-CSP反相液相色谱直接拆分泮托拉唑钠对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温以及流动相流速对拆分泮托拉唑钠对映体的影响。研究发现,用甲醇作有机改性剂比乙腈更有利于对映体的分离;在研究的温度范围内,随着柱温的升高,对映体的保留时间缩短,同时分离因子和分离度降低;在一定范围内降低流速有利于对映体的分离。采用T-CSP色谱柱(150 mm×4.6 mm i.d.,5 μm),以甲醇-水(体积比为35∶65)为流动相,在流速0.6 mL/min、检测波长290 nm、柱温20 ℃的条件下,泮托拉唑钠对映体获得了近于基线的分离,所建立的方法具有简便快速及重复性好等优点。     

Effect of temperature on enantiomer separation of oxzepam and lorazepam by high-performance liquid chromatography on a beta-cyclodextrin derivatized bonded chiral stationary phase

A reversed-phase high-performance liquid chromatography (HPLC) method with beta-cyclodextrin (beta-CD) derivatized as chiral stationary phase is used to directly separate oxazepam (Oxa) and lorazepam (Lor) enantiomers. The effect of temperature on the direct HPLC separation of Oxa and Lor enantiomers is studied for the commercially available beta-CD derivatized bonded chiral stationary phase. Chromatographic peak coalescence, appearing as a plateau between the resolved peaks, is observed at column temperatures of above 13 degrees C. Peak coalescence on the beta-CD derivatized bonded column is attributable to racemization of the Oxa enantiomer. By reducing the column temperature to 13 degrees C, the enantiomeric composition of Oxa and Lor could be determined on the chiral column. This method is expected to be useful for the resolution of 3-hydroxybenzodiazepines. At the same time, the separation mechanism is studied by calculating the thermodynamic parameters. The results reveal that the separation of Oxa and Lor enantiomer is a case of enthalpy-controlled separation, inclusion mechanism does not control the separation. The interaction between Oxa and beta-CD is an additionally strong pi-pi interaction or hydrogen bonding, but that between Lor or beta-CD derivatized is a weak pi-pi interaction or hydrogen bonding.     

高效液相色谱法测定淀粉及淀粉制品中的顺丁烯二酸与顺丁烯二酸酐总含量

建立了基于高效液相色谱(HPLC)测定淀粉及其制品中顺丁烯二酸和顺丁烯二酸酐总含量的方法。通过优化得到最佳样品前处理条件为乙醇体积分数5%,超声时间10 min。色谱分离检测的最佳分析条件为:流动相:甲醇-1‰磷酸(2∶98),色谱柱:Plastisil ODS C18(250 mm×4.6 mm,5μm),检测波长214 nm,流速1.0 mL/min,柱温30℃。该方法对顺丁烯二酸的定量下限为5.0 mg/kg,线性范围为0.25～100 mg/L,相关系数为0.999 7,平均加标回收率为88%～89%,相对标准偏差(n=5)小于2%,能够满足实际检测需要。     

Use of partially porous column as second dimension in comprehensive two-dimensional system for analysis of polyphenolic antioxidants

In the present work, a comprehensive LC system using a microbore HPLC column in the first dimension and a partially porous column in the second dimension was developed and applied to the separation of polyphenolic components in a red wine sample. The performance of the partially porous short column (3.0 cm) was compared to that of a monolithic column, of comparable dimensions. The results obtained demonstrated the possibility to use partially porous columns to obtain fast analyses, using high flow rates, under repetitive gradient conditions and with very brief reconditioning times. A conventional HPLC system was used since the backpressure generated by the shell-packed column, even at very high flow rates, was well within the operational limits. The use of an increased column temperature (60 degrees C) allowed a further pressure-drop decrease, with no stationary phase degradation, or loss in column performance.     

Effects of thermal heterogeneity in hydrophobic interaction chromatography

Manipulating temperature and salt concentration can have a powerful effect on the separation effectiveness in hydrophobic interaction chromatography (HIC). However, use of temperature as an operating variable in large-scale applications may involve undesirable consequences such as radial heterogeneity of the column temperature. In this study non-ideal effects of heat transfer in HIC columns were analyzed. The radial temperature gradients were measured by thermocouples immersed in a bed packed into a preparative column. The column wall was either thermostatted by a water jacket or left under ambient conditions. The influence of ineffective column thermostatting and of heat losses on the radial temperature profiles was demonstrated and predicted by a model of heat dispersion in a packed bed. To analyze possible positive or negative effects of thermal heterogeneity on band propagation, non-isothermal chromatographic elution of a model protein (α-chymotrypsinogen A) was recorded under salt gradient conditions as well as at constant salt concentration. To predict temperature and concentration profiles a model of the column dynamics was used. The model accounted for kinetics of mass and heat transfer. A good agreement between experimental and simulated profiles was achieved. It was shown that by proper selection of the process conditions undesirable temperature effects can be avoided or controlled.     

Development and optimization of an HPLC analysis of citalopram and its four nonchiral impurities using experimental design methodology

In this study, the RP-HPLC method was investigated for the separation of citalopram and its four impurities by use of statistical experimental design. Initially, the influence of different experimental conditions (buffer pH, flow rate, and column temperature) on the chromatographic behavior of citalopram and its four impurities was investigated by use of partial least squares regression (PLSR) and multilayer perceptron (MLP) artificial neural networks (ANNs) trained by back-propagation. The developed models and the corresponding response surface plots were used to select the optimal HPLC conditions, buffer pH 7.0, flow rate 1.0 mL/ min, and column temperature 25 degrees C, for an efficient separation of citalopram and its four impurities. The elaborated HPLC method was found to be linear, specific, sensitive, precise, accurate, and robust. Retention times of citalopram and its impurities, obtained with the developed HPLC method, and the computed molecular parameters of the examined compounds were used in a quantitative structure retention relationship (QSRR) study. The PLSR and ANN algorithms were applied for the development of the QSRR methods. The MLP-two layers-ANN-QSRR model with root mean square error of prediction 0.105 and r(2) (observed versus predicted) 0.978 was selected. Since many different reaction conditions are applied for the synthesis of citalopram, different impurities and degradation products can be formed. Therefore, the developed QSRR model can be extended to the prediction of the retention times with the other citalopram impurities, degradation products, and metabolites.     

Impact of a pressure drop on a monolithic capillary column on the efficiency and separation ability of the column

The impact of inlet and outlet column pressures on column separation properties was investigated for monolithic capillary column in gas chromatography. It was demonstrated that the classical Van Deemter equation does not allow us to make a clear choice of the optimal separation conditions. More relevant data can be obtained from the dependence of the height equivalent to a theoretical plate (HETP) on the inlet and outlet column pressures. The dependence ensures that the minimum HETP value can be achieved at high values of inlet and outlet column pressures, but the ratio of the pressures must approach 1. The efficiency of the column under these optimal conditions can exceed by 25–35% the column efficiency under the optimal conditions found using the classical Van Deemter plot. It was shown that a decrease in inlet and outlet column pressures even at a relative pressure close to 1 leads to an increase in HETP and the loss of column separation ability.     

Improvement of the chromatographic separation of several 1,4-dihydropyridines calcium channel antagonist drugs by experimental design

A high-performance liquid chromatographic method with diode array detection has been developed and optimized for the separation of five calcium channel blockers belonging to the 1,4-dihydropyridine subgroup (nifedipine and related drugs). The possibility of the simultaneous drug analysis allows a decrease of time during the assay as well as a saving of reagents and solvents. In this work, the effect of four experimental parameters (organic modifier percentage, pH value, concentration of the buffer in the mobile phase, and column temperature) on the chromatographic resolution are investigated by experimental design in order to optimize the chromatographic separation of five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine, and lercanidipine). Fractional factorial design, central composite design, and finally the Multisimplex program are used to establish the optimal conditions in terms of resolution and minimum analysis time. Optimal separation of the five compounds under study is achieved in less than 12 min using a Sulpecosil LC-ABZ+Plus C18 column, a composition of mobile phase of acetonitrile-10mM acetic acid acetate buffer pH 5 (72:28, v/v) at a flow rate of 1 mL/min, a column temperature of 30 degrees C +/- 0.1 degrees C, and a detection wavelength of 238 nm.     

黄烷酮对映体的高效液相色谱手性拆分及含量测定

采用环糊精为手性固定相,建立了黄烷酮对映体的高效液相色谱(HPLC)手性拆分方法。考察了流动相组成、流动相比例、流速及柱温对黄烷酮对映体拆分的影响。结果表明,以CD-CSP2手性色谱柱分离,采用乙腈-水(体积比30∶70)为流动相,在流速为1.0mL/min,温度30℃,检测波长254nm下,黄烷酮对映体能达到基线分离,且具有较好的重复性和稳定性,可用于对映体的拆分及质量控制。且R-黄烷酮与固定相的作用弱于S-黄烷酮,在色谱柱中首先被洗脱。以面积归一化法计算可知黄烷酮样品中,R-黄烷酮含量为53.94%,S-黄烷酮含量为46.06%。