Multidimensional liquid chromatography system with an innovative solvent evaporation interface

An orthogonal two-dimensional liquid chromatographic (2D-LC) system was developed by using a vacuum-evaporation loop-type valve interface. Normal-phase liquid chromatography (NPLC) with a bonded CN phase column was used as the first dimension, and reversed-phase liquid chromatography (RPLC) with a C(18) column was used as the second dimension. All the solvents in the loop of the interface were evaporated at 90 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loop. The mobile phase of the second dimension dissolved the analytes in the loop and injected them onto the secondary column, allowing an on-line solvent exchange of a selected fraction from the first dimension to the second dimension. The chromatographic resolution of analytes on the two dimensions was maintained at their optimal condition. Sample loss due to evaporation in the interface was observed that depended on the boiling point of the compound. Separation of sixteen polycyclic aromatic hydrocarbon mixtures and a traditional Chinese medicine Angelica dahurica was demonstrated.     

A vacuum assisted dynamic evaporation interface for two-dimensional normal phase/reverse phase liquid chromatography

A vacuum assisted dynamic solvent evaporation interface for coupling of two-dimensional normal phase/reverse phase liquid chromatography was developed and evaluated. A normal-phase liquid chromatographic (NPLC) column of a 250 mm × 4.6 mm I.D. 5 μm CN phase was used as the first dimension, and a reversed-phase liquid chromatographic (RPLC) column of 250 mm × 4.6 mm I.D. 5 μm C₁₈ phase was used as the second dimension. The eluent from the first dimension flowed into a fraction loop, and the solvent in the eluent was dynamically evaporated and removed by vacuum as it was entering the fraction loop of the interface. The non-evaporable analytes was retained and enriched in about 5–25 μL solution within the loop. Up to 1 mL/min of mobile phase from the first dimension can be evaporated and removed dynamically by the interface. The mobile phase from the second dimension then entered the loop, and dissolved the concentrated analytes retained inside the loop, and carried them onto the second dimension column for further separation. The operation conditions of the two dimensions were independent from each other, and both dimensions were operated at their optimal chromatographic conditions. We evaluated the interface by controlling the loop temperature in a water bath at normal temperature, and investigated the sample losses by using standard samples with different boiling points. It was found that the sample loss due to evaporation in the interface was negligible for non-volatile samples or for components with boiling point above 340 °C. The interface realizes fast solvent removal of mL volume of fraction and concentration of the fraction into tenth of μL volume, and injection of the concentrated fraction on the secondary column. The chromatographic performance of the two-dimensional LC system was enhanced without compromise of separation efficiency and selectivity on each dimension.     

Two-dimensional reversed-phase liquid chromatography using two monolithic silica C18 columns and different mobile phase modifiers in the two dimensions

Comprehensive two-dimensional (2D) HPLC in the reversed-phase liquid chromatography (RPLC) mode using C18 silica monolith columns at first dimension (1st-D) (10 cm x 4.6mm I.D.) and second dimension (2nd-D) (5 cm x 4.6mm I.D.) was carried out successfully. A mixture of water and tetrahydrofuran (THF) was used as a mobile phase in the 1st-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation. Sample fractions from 1st-D column were directly loaded into an injection loop of the 2nd-D HPLC equipped with two injector valves for one column. The fractionation time at the 1st-D that was equal to the separation time at the 2nd-D was 45 or 60s. Total peak capacity up to 900 was obtained in about 60 min for the isocratic mode separation of aromatic compounds in this system. Gradient elution mode applied to both 1st-D and 2nd-D separations resulted in shorter separation time and better separation efficiencies than the isocratic mode. It was demonstrated that 2D-HPLC systems employing popular C18 stationary phases with different organic modifiers in mobile phases for each dimension could produce large peak capacity. The different selectivities were provided by the difference in polar interactions between a solute and the organic modifier existing in the stationary phase.     

Application of packed column supercritical fluid chromatography to the simultaneous determination of seven anticonvulsant drugs

Studies of speed, resolution, and selectivity have shown that packed column supercritical fluid chromatography (PCSFC) is a viable technique for the isocratic, isothermal and isobaric separation of seven anticonvulsants, viz., phenobarbitone, phenytoin sodium, phethenylate sodium, nitrazepam, clonazepam, carbamazepine, and primidone, and their simultaneous estimation. The drugs were eluted from a JASCO, RP-C₁₈ (250×4.6 mm) 10 μ packed column with a binary mobile phase of carbon dioxide and methanol, using ibuprofen as the internal standard. The effect of pressure, temperature, modifier concentration, and the rate of flow of CO₂ on retention and selectivity of all the analytes were studied and the parameters optimised. Without methanol in the mobile phase none of the solutes eluted. Changing modifier concentration was the most effective physical parameter for changing retention and selectivity. The analytes were detected using a UV detector at 215 nm. An arbitrary mixture of eight components was baseline resolved in 7 min. The study includes a successful attempt at quantification of the drugs. Chromatographic and analytical figures of merit have been listed. The present work holds promise for a possible replacement of HPLC with SFC for the separation and assay of drugs of different families.     

High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.     

Analysis of sulthiame in serum by narrow-bore high-performance liquid chromatography. Comparison of direct sample injection with pre-column switching and extrelut extraction.

Two high-performance liquid chromatographic methods for the analysis of sulthiame in serum are described. In the first method direct injection of serum samples onto a 4 x 4 mm I.D. (C18, 25 microns) precolumn in a column-switching device was used. After a purge step, the adsorbed analytes were eluted onto a 250 x 3 mm I.D. (C18, 5 microns) narrow-bore column for chromatographic separation. In the second method the sample pretreatment was an Extrelut extraction with dichloromethane-propanol-2 (95:5). After evaporation of the solvents, the residue was dissolved in methanol. The chromatographic separation was carried out on the same analytical column as used in the column-switching method. Both sample pretreatment methods were compared with respect to their suitability of routine analysis of sera from patients also receiving other antiepileptic drugs.     

离线二维液相色谱法分离巴天酸模根的化学成分

建立了离线二维反相/反相液相色谱分离体系（2D-RPLC/RPLC）,对巴天酸模中的化学成分进行分离。通过比较巴天酸模乙酸乙酯萃取液在环氧四氮唑和Unitary C18色谱柱上的高效液相色谱图,确定以环氧四氮唑色谱柱为第一维色谱柱,以Unitary C18色谱柱为第二维色谱柱。流动相均采用0.1%（v/v）甲酸水溶液和甲醇,梯度洗脱。经一维色谱分离后,共收集18个流分,采用二维色谱对这18个流分进行了进一步的分离分析。实验结果表明,该二维色谱分离方法高效、可行,为巴天酸模药材的微量组分的分离以及活性化合物的筛选提供了分离方法。     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

填充柱超临界流体色谱系统中的溶剂效应

 考察了填充柱超临界流体色谱法 (SFC)中的样品溶剂及连续进样等因素对化合物保留行为变化的影响规律。以超临界 CO2 或含低体积分数甲醇的 CO2 为流动相时 ,氨基柱上组分的保留时间随着样品溶剂的极性增大而增大 ,而溶剂对 C1 8柱上组分的保留时间影响不大 ;在 C1 8柱上 ,溶剂对连续进样的后续效应不强 ;而在氨基柱上 ,甲醇溶液的后续效应比丙酮、氯仿溶液的后续效应强。当甲醇的体积分数大于 1 .0 %时 ,溶剂的效应明显减弱。这种变化规律对填充柱 SFC的合理进样并获得重现性良好的色谱数据具有实际意义。     

Use of reversed-phase high-performance liquid chromatography-diode array detection for complete separation of 2,4,6-trinitrotoluene metabolites and EPA Method 8330 explosives: influence of temperature and an ion-pair reagent

Explosives such as 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) are widely distributed environmental contaminants. Complete chromatographic separation is necessary in order to accurately determine and quantify explosives and their degradation products in environmental samples and in (bio)transformation studies. The present study describes a RP-HPLC method with diode array detection using a LC-8 guard column, a Supelcosil LC-8 chromatographic column, and a gradient elution system. This gradient method is capable of baseline separating the most commonly observed explosives and TNT transformation metabolites including 2,4,6-triaminotoluene (TAT) in a single run. The TNT metabolites separated were 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, 2,4-dihydroxylamino-6-nitrotoluene, 4,4',6,6'-tetranitro-2,2'-azoxytoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, 4,4',6,6'-tetranitro-2,2'-azotoluene, 2,2',6,6'-tetranitro-4,4'-azotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, 2,4-diamino-6-nitrotoluene, and TAT. The same gradient method at a different column temperature can also be used to baseline separate the explosives targeted in the Environmental Protection Agency (EPA) Method 8330 with approximately 22% reduction in total run time and 48% decrease in solvent consumption compared to previously published methods. Good separation was also obtained when all TNT metabolites and EPA Method 8330 compounds (a total of 23 compounds) were analyzed together; only 2,6-DANT and HMX co-eluted in this case. The influence of temperature (35-55 degrees C) and the use of an ion-pair reagent on the chromatographic resolution and retention were investigated. Temperature was identified as the key parameter for optimal baseline separation. Increased temperature resulted in shorter retention times and better peak resolution especially for the aminoaromatics investigated. The use of an ion-pair reagent (octanesulfonic acid) generally resulted in longer retention times for compounds containing amine functional groups, more baseline noise, and decreased peak resolution.     

高效液相色谱法分离测定人参中的6种人参皂甙

 采用梯度法,以乙腈-水溶液为流动相,用高效液相色谱法分离人参中的6种主要人参皂甙,并采用紫外检测器检测,在203 nm处测定4种人参样品。该方法在25 mg/L～300 mg/L的范围内有良好的线性关系,回收率高于80%。     

反相高效液相色谱法分离测定烟草中的多酚类化合物

对植物中9种多酚类化合物的色谱分离条件进行了优化,分别探讨了流动相组成、流动相中醋酸浓度、醋酸溶液与甲醇的比例对保留时间的影响,确定了梯度分离条件,并对9种天然多酚类化合物进行了定量分析。该方法的检测限为13.26~59.29 mg/kg (S/N=3)。在3.0～100.0 mg/L 范围内呈良好的线性关系,相关系数r2为 0.9979~0.9999。9种待测化合物的加标回收率为96.8%~108%,相对标准偏差(RSD)小于3.8% (n=3)。用80%甲醇超声提取烟草样品,并通过优化的色谱条件对其进行分析,测定了实际烟草样品中芸香苷和绿原酸的含量。结果表明,该方法具有一定的实用价值。     

Comparison of the performance of different reversed-phase columns for liquid chromatography separation of 11 pollutant phenols

A systematic optimization of the HPLC separation of a mixture containing 11 pollutant phenols (PPs) using a Hypersil ODS (250 mm x 4.6 mm, 5 microm) column and UV-DAD detection has been carried out. The binary mobile phases used were obtained by mixing 50 mM phosphate (pH = 3.0) and methanol, ACN, or THF as organic modifiers. After selecting ACN as an organic modifier, the effects of pH and temperature on PPs separation were studied. A mobile phase of 50 mM acetate (pH = 5.0)-ACN (60:40 v/v) at 50 degrees C allowed the separation of 11 phenols but not to baseline in 17 min. To improve the performance of this separation, the following RP columns were tested: Luna C18 (2), Purospher C18, Synergi C12, Synergi Fusion C18, Gemini C18, Luna Cyano, Lichrospher C8, and Envirosep-PP (polymeric). In all the cases, the performance (analysis time, retention, selectivity, resolution, asymmetry factors, and efficiency) was evaluated. A further reoptimization of the mobile phase was carried out for all the columns by studying the ACN content and pH, with the aim of improving the above-mentioned separations and selecting the most suitable one for PPs analysis.     

Comprehensive two-dimensional liquid chromatography (NPLCxRPLC) with vacuum-evaporation interface

A comprehensive two-dimensional liquid chromatographic system incorporating a vacuum-evaporation interface was developed. Normal-phase liquid chromatography with a CN microcolumn was used as the first dimension (1(st)-D), and reversed-phase liquid chromatography with a C(18) monolithic column was used as the second dimension (2(nd)-D). An electronically controlled dual-position, ten-port valve with two identical storage loops served as the interface and the analysis time in the 2(nd)-D was 1.5 min. The solvent in the loops of the interface was evaporated at 25 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loops. The mobile phase of the 2(nd)-D dissolved the analytes in the loop and injected them onto the second column, allowing an on-line solvent exchange of the fractions from the 1(st)-D to the 2(nd)-D. The chromatographic resolution of analytes on the two dimensions was evaluated. Sample loss due to evaporation in the interface was investigated with standard samples having different boiling points. The usefulness of the comprehensive 2-DLC system was demonstrated in the analysis of a traditional Chinese medicine Radix salviae miltiorrhiza bage extract.     

高效液相色谱—化学发光法研究异烟肼和利福平

基于异烟肼和利福平在碱性介质中能与K3Fe(CN)6反应产生强的化学发光，因 此设计了一个经高效液相色谱（HPLC）分离柱后同时检测一线抗结构病药物异烟肼 、利福平的化学发光检测器。研究并优化了流动相、流速以及化学发光检测的条件 。该方法测定异烟肼、利福平的线性范围分别为0.05～6.0mg/L，0.08～20.0mg/L ，其检出限：异烟肼为2×10^-2mg/L，利福平为4×10^-2mg/L，测定的相对标准偏 差分别为1.9，2.9。该方法已成功地用于同时测定复方利福平片中利福平和异烟肼 的含量。     

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。     

高效液相色谱法分析生物样品中的敌鼠

 研究用阴离子交换、氰基及硅胶柱固相萃取技术分离提取血、尿、肝及肾中的敌鼠。以香豆素作内标 ,用高效液相色谱 二极管阵列检测器方法进行分离鉴定。色谱条件 :分析柱为HypersilBDSC18(5 μm ,15 0mm× 4 6mmi.d .) ,保护柱为PhenomenexODS(4mm× 3 0mmi.d .) ;流动相 :A为 0 5 %离子对A水溶液 ,B为 0 5 %离子对A甲醇溶液 ,以梯度淋洗程序分离 ;检测波长为 2 86nm。当敌鼠的质量浓度在 1mg/L～ 10 0mg/L范围时 ,其浓度同其峰面积与内标物的峰面积之比有良好的线性关系 (r=0 9999) ,最小检出限量为 5ng(按S/N =3计 )。     

Evaluation of an International Pharmacopoeia method for the analysis of nelfinavir mesilate by liquid chromatography

A gradient LC method for the determination of related substances in nelfinavir mesilate (NFVM) has been recently published in the International Pharmacopoeia. The method uses a base deactivated reversed phase C18 column (25 cm x 4.6 mm I.D.), 5 microm kept at a temperature of 35 degrees C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 225 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards NFVM components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm I.D.), 5 microm. A two level fractional factorial design was applied to examine the robustness of the method. The method shows good selectivity, precision, linearity and sensitivity. Seven commercial samples were examined using this method.     

Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 μm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 μm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 μm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 μm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 μm average particle size materials for clinical sample analysis.     

Low-Pressure Chromatographic Separation of p-Hydroxybenzoates Using Sequential Injection with Lab-on-Valve System and Miniature Monolithic Column

The use of a small guard column (5-mm length) as a miniature analytical column in low-pressure liquid chromatography based on a sequential injection with lab-on-valve system was demonstrated. A strong initial mobile phase was used to rapidly deliver the sample zone to the column prior to simultaneous separation of analytes with weaker mobile phase. Separation of methyl-, ethyl-, propyl-, and butyl-4-hydroxybenzoates (MP, EP, PP, and BP) was achieved within about 120 s with sufficient peak resolution (all higher than 1.3) using 2,500 µL of mobile phase (composed of 1,087 µL of MeOH) per run. Detection limits were found to be 6.5, 8.0, 0.5, and 0.6 μmol L^?1 for MP, EP, PP, and BP, respectively. The system was tested with common commercially available skin lotions and wet wipe products. The analysis results were in good agreement with those obtained from the high-performance liquid chromatography with a 10-cm-length packed column.