正相模式/反相模式的二维液相色谱系统的构建与应用

以4.6mm×50 mm i. d.的Hypersil SiO2正相色谱柱为第一维,4.6mm×250 mm i. d.的Kromasil C18反相色谱柱为第二维,通过升高第二维色谱温度的方法增加两维流动相间互溶性的方法构建了定量环-阀切换接口的二维液相色谱系统(NPLC×RPLC)。根据有机溶剂的特征,在第一维正相色谱流动相中加入二氧六环;第二维反相色谱流动相中加入异丙醇,在改善流动相兼容性的同时,有效调整分离选择性。采用此系统对正天丸样品进行分离分析,达到1120的峰容量。     

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography x liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ((2)D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ((1)D) column can be derived. Theory clearly indicates that the choice of the (1)D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8mm for SEC and 4.6mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.     

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.     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

Comprehensive two-dimensional liquid chromatography applying two parallel columns in the second dimension

The design of a new interface for comprehensive two-dimensional liquid chromatography (LC x LC) is described. To the conventionally used LC x LC system with the loop-type interface consisting of a two-position/ten-port switching valve equipped with two loops, an extra two-position/ten-port switching valve, a detector, a pump and a second column placed in parallel with the column in the second dimension, are added. The features of the interface are that the separation space in the second dimension is significantly enlarged and that the number of fractions transferred from the first to the second dimension can be increased, reducing the risk to lose resolution of the primary dimension. The potential of the system in NPLC x 2RPLC is illustrated with the analysis of a standard mixture and a lemon oil extract. For the lemon oil analysis, the effective peak capacity was increased from 437 using a conventional interface to 1095 with the new interface. RPLC x 2RPLC in combination with reduced modulation times was applied to the analysis of steroids and to the detection of impurities at the 0.05% relative concentration level in a sulfonamide drug sample.     

A comprehensive two-dimensional normal-phase × reversed-phase liquid chromatography based on the modification of mobile phases

A comprehensive orthogonal two-dimensional liquid chromatography (2D-LC) based on the modification of mobile phases was developed with a sample loop–valve interface. To improve the compatibility of mobile phases and analysis speed, some special solvents were chosen as the mobile phases, and the column temperature was elevated to decrease the viscosity of mobile phases of reversed-phase liquid chromatography (RPLC). Based on this principle, the first dimension was normal-phase liquid chromatography (NPLC) with a SiO₂ column, and the second dimension was reversed-phase liquid chromatography containing two tandem C18 columns. 1,4-Dioxane was used in the NPLC mobile phase, and isopropyl alcohol was employed in the RPLC mobile phase. Moreover, the elevated column temperature enabled the reduction of the backpressure and using tandem C18 columns to improve the resolving power in RPLC. The new comprehensive 2D-LC system and applied strategy offered a novel idea for construction of 2D-LC system. A traditional Chinese medicine, Zhengtian pill, was used as the test sample to evaluate the constructed 2D-LC system. 876 peaks were detected, and the peak capacity reached 1740.     

全二维液相色谱(NPLC×RPLC)接口及其应用

用内径为0.53 mm的填充毛细管正相液相色谱为第一维, 用4.6 mm(i.d.)×50 mm RP-18e整体柱反相色谱为第二维, 建立了定量环-阀切换接口的全二维液相色谱系统(NPLC×RPLC). 第一维色谱分离洗脱出的组分交替存储在十通阀上的两个定量环中, 同时定量环中前一个组分被转移到第二维进行反相分离. 因为第一维的流动相流量仅是第二维的1/500, 自然解决了流动相兼容问题. 采用芳香族化合物的混合物和中药丹参正己烷提取液对该全二维液相系统的分离能力进行了评价.     

Study of two-dimensional liquid chromatography with high temperature NPLC and room temperature RPLC

To overcome the peak band broadening and to increase the peak capacity and separation efficiency of a two-dimensional liquid chromatographic system, a high-temperature normal phase liquid chromatography (HTNPLC) was used as the first dimension (1^st-D), and a RPLC was used as the second dimension (2^nd-D). The sample was first separated on the 1^st-D CN column and the primary eluent stored in the sampling-loop system alternatively (in HTNPLC×RPLC mode) or selectively (in HTNPLC/RPLC mode) and was then transferred to 2^nd-D C₁₈ column for further separation. The resolution and separation efficiency of the systems were greatly improved. The systems were evaluated by analyzing several polycyclic aromatic hydrocarbons and Glycyrrhiza uralensis extract. __________ Translated from Journal of Instrumental Analysis, 2008, 27(1) (in Chinese)     

Analysis of ginseng root and leaf by multiple columns and detections liquid chromatography

Abstract

A multiple columns and detections liquid chromatography system, including size exclusion chromatography (SEC) and reversed phase liquid chromatography (RPLC), for the analysis of macromolecules and micromolecules in ginseng root and leaf was developed. The columns were connected by two switching valves. Macromolecules were separated on a SEC column (TSK gel SuperMultipore PW-H column, 6?mm× 150?mm, 8?μm) by isocratic elution of 50?mM ammonium acetate aqueous solution, 0.3?mL/min of flow rate and detected by evaporative light scattering detection (ELSD). Micromolecules were analyzed on a Poroshell RP column (Agilent Poroshell 120?SB-Aq column, 4.6?mm × 50?mm, 2.7 µm) with gradient elution of water and acetonitrile, 0.6?mL/min of flow rate and detected by ultraviolet detection (UV). As a result, in the macromolecules chromatogram of ginseng root sample showed two main peaks while only one major peak for ginseng leaf. For micromolecules analysis, 17 compounds (3 nucleosides + 14 saponins) and 17 compounds (3 nucleosides + 1 flavonoid + 13 saponins) were found in ginseng root and leaf, respectively. The developed method is helpful for the quality evaluation of ginseng root and leaf.     

Overloading of the second-dimension column in comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GC x GC) is based on a coupling of two GC columns of different characteristics by means of a device that allows portions of the effluent from the primary column to be injected onto the second dimension column for an additional separation. The time available for the separation in the second-dimension column is very short. Thus, this separation should be very efficient. The vast majority of GC x GC practitioners use very narrow bore columns for the second dimension. While this approach is justified in principle, if peaks in the second dimension overload this column, its peak capacity is severely reduced. A series of second-dimension columns of varying internal diameters, but similar phase ratios, were used to study these effects. The results indicate that 250 microm columns often provide comparable second dimension peak widths to 100 microm columns, while at the same time being less prone to overloading, indicating that they may often be a better choice than smaller diameter columns in the second dimension of GC x GC systems.     

Serial coupling of reversed-phase and hydrophilic interaction liquid chromatography to broaden the elution window for the analysis of pharmaceutical compounds

It is presently a common practice in drug discovery to analyse samples by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). To increase throughput, HILIC was connected in series to RPLC by means of a T-piece with make-up flow. The first column is a 2mm I.D. column having an optimal flow between 0.1 and 0.2mL/min. Via the T-piece, the flow for the second dimension column with an I.D. of 4.6mm is adjusted to 1.5-2.0mL/min with a high acetonitrile content (i.e. >/=80%) mobile phase. Therefore, even in gradient RPLC analysis starting with a mobile phase with high water content, the HILIC column is always operated at high acetonitrile concentration which is required to obtain retention on the HILIC column. The performance of the hyphenated RPLC/HILIC set-up is illustrated with the analysis of two model samples of pharmaceutical interest. Optimization of the conditions in the HILIC dimension is discussed.     

Application of supplementary flow in comprehensive 2D liquid chromatography combining SEC and RPC

A comprehensive two-dimensional HPLC system (SEC x RPC) was evaluated. Various model compounds with differing hydrophobicity (log D: -0.08 to 2.22) and size (MW: 194 to 66.0 x 10(3)) were used. In order to reduce the run time of the second dimension, and thereby optimize the number of runs per unit volume from the first dimension, short RPC columns were used (7.5 mm). This column size demanded a low concentration of methanol in the mobile phase from the first dimension, in order to avoid severe band broadening and solute loss. Secondary interactions on SEC make a high methanol concentration in the mobile phase a necessity. Up to 40% methanol was required to diminish non-ideal SEC behavior. These conditions were non-compatible with trapping of hydrophilic compounds on RPC. The use of a supplementary flow (0.1% TFA) mixed after the first dimension led to better peak shape and trapping of hydrophilic compounds in the second dimension. This demanded flow adjustment in SEC, which in turn caused performance improvement and an increase in analysis time, making more RPC separations possible. These factors contributed to a larger peak capacity.     

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.     

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

Characterization of a novel diol column for high-performance liquid chromatography

For the investigation of a diol phase (Inertsil Diol column) in hydrophilic interaction chromatography, urea, sucrose and glycine were used as test compounds. The chromatographic conditions were investigated for optimal column efficiency. The column temperature used in common reversed-phase liquid chromatography could also be used for the separation and the flow-rate should be adjusted to 0.3-0.5 ml/min to optimize column efficiency. It is suggested that the velocity of the hydrophilic interaction is slower than the hydrophobic interaction in RPLC. The addition of trifluoroacetic acid is effective for the retention of glycine, but ineffective for urea and sucrose. The diol phase exhibited sufficient chemical stability even if exposed to water in high percentage, and could be applied with isocratic elution for the separation/analysis of amino acids and glucose.     

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.     

Use of Two-Dimensional Liquid Chromatography Combined with Diode-Array and Mass Spectrometric Detection for Analysis of Compounds in Flos Lonicera

Comprehensive two-dimensional liquid chromatographic separation by means of a combination of ion-exchange chromatography (IEC) and reversed-phase liquid chromatography (RPLC) has been realized and applied to the analysis of components of a traditional Chinese medicine Flos Lonicera. In the first dimension the components of a Flos Lonicera extract is separated on strong anion-exchange (SAX) column according to the charge-exchange properties of the components. In the second dimension the components are further fractionated by reversed-phase liquid chromatography (on ODS) on the basis of their hydrophobicity. The RPLC effluent is desalted and split and the components are identified on-line by use of both diode-array detection and mass spectrometry with atmospheric pressure chemical ionization (APCI–MS). In this way, the extract of Flos Lonicera can be rapidly separated, desalted, and analyzed, with determination of molecular weight. More than 58 components of an extract of Flos Lonicera were well resolved and six were tentatively identified from their UV and mass spectra.     

Comprehensive on-line RPLC-CZE-MS of peptides

Peptide standards and tryptic digests of ribonuclease B are separated by comprehensive two-dimensional reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) and detected on-line by electrospray mass spectrometry. The RPLC column is coupled to the CZE column by a transverse flow gating interface. A new rugged microelectrospray needle is described that combines high ionization efficiency, low flow rates, and a sheath flow. The result is a system combining the separation capabilities of both RPLC and CZE with on-line mass spectrometric detection, all in about 15 min.     

Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature

In recent years, two-dimensional liquid chromatography (2D-LC) has been used increasingly for the analysis of synthetic polymers. A 2D-LC analysis provides richer information than a single chromatography analysis at the cost of longer analysis time. The time required for a comprehensive 2D-LC analysis is essentially proportional to the analysis time of the second dimension separation. Many of 2D-LC analyses of synthetic polymers have employed size exclusion chromatography (SEC) for the second-dimension analysis due to the relatively short analysis time in addition to the wide use in the polymer analysis. Nonetheless, short SEC columns are often used for 2D-LC analyses to reduce the separation time, which inevitably deteriorates the resolution. In this study, we demonstrated that high temperature SEC can be employed as an efficient second-LC in the 2D-LC separation of synthetic polymers. By virtue of high temperature operation (low solvent viscosity and high diffusivity of the polymer molecules), a normal length SEC column can be used at high flow rate with little loss in resolution.