Instrumentation for capillary electrochromatography

One of the reasons for the immense interest in capillary electrochromatography (CEC) is its feature to combine chromatographic selectivity with the high efficiency and the miniaturization potential of capillary electrophoresis (CE). The capability of commercial CE instruments to run CEC has enforced the readiness of users and researchers to work on this separation technique. Nevertheless, to fully exploit the potential of CEC, a routine CE device can certainly not fulfill all requirements. Two different approaches have been made to overcome this problem. The first was to modify commercial CE instruments for various demands. Pressurization of the packed capillary to prevent "air" bubble formation, gradient elution capabilities and thermostating devices allowing a greater flexibility in column designs have been implemented in CE instruments of several manufacturers. A completely different approach is the development of modular laboratory-made instrumentation dedicated to special CEC requirements. In order to increase mobile phase velocity and thus the speed of analysis the availability of voltages higher than 30 kV was accomplished in some of these devices. Gradient elution was achieved by either coupling of gradient LC systems or an electroosmotic generation of the changing eluent composition. When a pressure gradient is applied between both column ends in addition to the voltage gradient, a hybrid between capillary HPLC and CEC results. This chromatographic mode is named pressure-assisted electrochromatography (PEC). Either CE instruments equipped with additional HPLC pumps or modular laboratory-made devices are suitable for PEC. In CEC, sensitivity for UV detection is rather poor due to the short optical path length for on-column detection in capillary separation techniques. A special cell design with enhanced light path is presented and further principles like, e.g., fluorescence detection and coupling to mass spectrometry are discussed.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Characterization of High‐Pressure Liquid Chromatography Columns using Chromatographic Methods

Abstract

A great variety of columns for liquid chromatography (LC) are available in dimensions ranging from industrial scale to micro‐bore, nano‐bore, and capillary size, and on‐chip columns. The columns may be used in various liquid chromatography modes or in capillary electrochromatography, depending on the support materials and stationary phase chemistry. Every year many new column types are introduced on the market, with improved selectivity and efficiency, long lifetime, and mobile phase compatibility, intended for general use, for liquid chromatography/mass spectrometry (LC/MS) applications, proteomic research, or for the analysis of other specific sample types. Considerable improvement in pH, high‐temperature, and high‐pressure stability of new column types, together with advances in the instrumentation, enabled introduction of capillary, high‐temperature, and ultra‐high‐pressure HPLC into routine practice. Even though reversed‐phase mode is still by the most widely used in contemporary LC, applications of other separation modes (such as ion, normal‐phase, or high‐interaction liquid chromatography (HILC)) have become more frequent recently, because of unique separation selectivity for certain sample types.

Characterization of column quality is not a simple task, because a number of factors should be taken into account, that affect the selectivity, efficiency and resolution of sample separation and the reproducibility of chromatographic data. These include the type of the support, the arrangement and density of the stationary phase on the adsorbent surface, the homogeneity of the chromatographic bed, etc. Various physicochemical techniques are used for characterization of the properties of column packings however, most of them are suitable for bulk materials only and cannot be directly applied for commercial columns without damaging them. Not to destroy the columns, often precious and expensive, practicing chromatographers can apply chromatographic methods to characterize columns and evaluate their analytical suitability under real‐life conditions, where the intermolecular interactions between the analytes, the stationary phase, and the mobile phases affect the retention. The present review reports various chromatographic tests and strategies available for column evaluation.     

Pressurized flow electrochromatography with reversed phase capillary columns

Pressurized flow electrochromatography (PEC) is a hybrid of capillary LC and capillary electroendosmotic chromatography (CEC). Both a pressure gradient and an electric field are applied across a packed capillary. The feasability of a simple, easy to handle PEC instrumentation is demonstrated. Home made capillary columns with four different silica-based reversed phase packings have been operated under PEC conditions separating non ionic and ionic low molecular weight analytes. The capillary columns have been characterized with respect to their separation efficiency and selectivity and the results have been compared to those obtained with the purely pressure driven system. An electrochromatographic capacity factor is discussed.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70th birthday     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.     

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.     

Recent developments in microcolumn liquid chromatography.

An overview of the most recent developments in microcolumn liquid chromatography (LC) is presented. A short theoretical discussion on chromatographic dilution and extracolumn bandbroadening is given and also the recent progress and advances in column technology and instrumentation are reviewed. However, the emphasis of this review is on miniaturized sample clean-up, sample introduction techniques and on both established and more recent detection techniques for microcolumn LC. The hyphenation of miniaturized LC columns with other techniques, specifically on multidimensional chromatography and the coupling of microcolumn LC to mass spectrometry is discussed in detail. Both the on-line and automated off-line interfacing to other separation and detection techniques will also be addressed. Finally, a number of typical microcolumn LC applications are presented in order to demonstrate the potential of microcolumn LC methods in a variety of scientific areas.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

Multidimensional LC x LC analysis of phenolic and flavone natural antioxidants with UV-electrochemical coulometric and MS detection

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.     

Gradient elution techniques for capillary electrochromatography

Capillary electrochromatography (CEC) is a rapidly maturing technique, but still in need of further instrumental development and in need of unique applications that are not possible by traditional pressure-driven LC. We review the development of gradient elution schemes for CEC, beginning with pH gradients initially developed for capillary electrophoresis. Step gradients are the most easily instrumentally implemented, but provide less flexibility in separation than continuous gradients. Pressure-assisted CEC is easily adapted to gradient elution schemes, but does not offer the advantages of very high column efficiency provided by totally electro-driven mobile phases. The development of flow-injection interfaces allows a true solvent gradient to be generated by micro-LC pumps, with the mobile phase drawn into the separation capillary by pure electroosmotic flow. While requiring both a CEC instrument and a traditional pump or pumps capable of generating the gradient, this method offers advantages of greatly reduced column handling, prolonging column lifetimes, and allows simple autosampling. We also discuss voltage gradients, which provide a mobile phase velocity gradient.     

色谱累加进样分离实验条件的研究

在采用反相液相色谱或亲和色谱完成蛋白质等大分子分离时,根据溶质保留值随溶剂梯度变化曲线上突变点的差别,可以通过累加进样分离法进行样品制备或直接柱内富集分析,但这一方法并非在任意条件下、对任何样品都适用。该文根据不同形式的保留值方程从理论上探讨了样品保留值与进样时间差、梯度洗脱速率等实验条件之间的关系;结果表明：两次进样的出峰时间差与进样时间间隔成正比关系,也与其在等度情况下的容量因子有关。样品中的两种组分在间隔进样时的流出时间差主要由两组分的容量因子决定,当样品中存在两种以上保留性能相近组分时,若保证指定的分离度,进样时间间隔存在一极大值,超出该范围,分离条件将不能满足     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

分子印迹手性整体柱的制备及对非对映异构体的分离

 采用原位分子印迹技术 ,单步制备了一种辛可宁印迹的手性整体柱。为了提高柱效和选择性 ,选择了相对低极性的甲苯 /十二醇复合致孔体系。在等度及梯度洗脱条件下 ,非对映异构体辛可宁与辛可尼丁被完全分离。等度洗脱中相对较宽的峰可以在梯度洗脱中得到改善。同时考察了流动相中醋酸浓度、流速以及温度对分离的影响。由于柱中存在大的流通孔 ,大大降低了分离过程中的柱压降 ,从而使这种柱能够在相对高的流速下使用。提高温度可以提高分离因子 ,在 60℃获得最大分离因子 5 40。     

Chiral high‐performance liquid and supercritical fluid chromatographic enantioseparations of limonene‐based bicyclic aminoalcohols and aminodiols on polysaccharide‐based chiral stationary phases

Enantioseparation of limonene‐based bicyclic 1,3‐aminoalcohols and 1,3,5‐ and 1,3,6‐aminodiols was performed by normal‐phase high‐performance liquid chromatographic and supercritical fluid chromatographic (SFC) methods on polysaccharide‐based chiral stationary phases. The effects of the composition of the mobile phase, the column temperature and the structures of the analytes and chiral selectors on retention and selectivity were investigated by normal‐phase LC and SFC technique. Thermodynamic parameters derived from selectivity–temperature‐dependence studies were found to be dependent on the chromatographic method applied, the nature of the chiral selector and the structural details of the analytes. Enantiorecognition in most cases was enthalpically driven but an unusual temperature behavior was also observed: decreased retention times were accompanied by improved separation factors with increasing temperature, i.e. some entropically driven separations were also observed. The elution sequence was determined in all cases. The separation of the stereoisomers was optimized in both chromatographic modalities.     

Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.     

Analytical potential of mid-infrared detection in capillary electrophoresis and liquid chromatography: a review

Literature published in the last decade concerning the use of mid-infrared spectrometry as a detection system in separation techniques employing a liquid mobile phase is reviewed. In addition to the continued use of isocratic liquid chromatographic (LC) techniques, advances in chemometric data evaluation techniques now allow the use of gradient techniques on a routine basis, thus significantly broadening the range of possible applications of LC-IR. The general trend towards miniaturized separation systems was also followed for mid-IR detection where two key developments are of special importance. Firstly, concerning on-line detection the advent of micro-fabricated flow-cells with inner volumes of only a few nL for transmission as well as attenuated total reflection measurements enabled on-line mid-IR detection in capillary LC and opened the path for the first successful realization of on-line mid-IR detection in capillary zone electrophoresis as well as micellar electrokinetic chromatography. Secondly, concerning off-line detection the use of micro-flow through dispensers now enables to concentrate eluting analytes on dried spots sized a few tens of micrometers, thus matching the dimensions for sensitive detection by mid-IR microscopy. Finally in an attempt to increase detection sensitivity of on-line mid-IR detection, mid-IR quantum cascade lasers have been used. Applications cover the field of food analysis, environmental analysis and the characterization of explosives among others. Best detection sensitivities for on-line and off-line detection have been achieved in miniaturized systems and are in the order of 50 ng and 2 ng on column, respectively.     

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 min with a flow rate of 200 μL/min. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 7 0min using a flow rate of 100 μL/min.     

The effect and potential of using a temperature controlled separation column with ultra-high pressure microcapillary liquid chromatography/tandem mass spectrometry on proteomic analysis

The microcapillary liquid chromatography (μLC)/tandem mass spectrometry (MS/MS) system has become a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteome-wide identification of peptides and proteins rely heavily on producing an accurate and reproducible solvent-composition gradient throughout microcapillary separation columns to improve LC separation. With the recent advent of targeted proteomic approaches utilizing the LC retention time as a physicochemical parameter for peptides, high reproducibility of LC separation additionally becomes an important factor. In this study, column temperature elevation is utilized to improve reproducibility and separation efficiency of the μLC-MS/MS system. The simple incorporation of a semi-rigid gas line heater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up to 60 °C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlled temperature elevation on the peptide separation efficiency and reproducibility. In addition to the increased reproducibility in peptide elution time due to the controlled column temperature, the temperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowed a higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements in the performance of μLC systems.     

Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

The application of small porous particles, high temperatures, and high pressures to generate very high resolution LC and LC/MS separations

The effect of combining sub-2 microm porous particles with elevated operating temperatures on chromatographic performance has been investigated in terms of chromatographic efficiency, productivity, peak elution order, and observed operating pressure. The use of elevated temperature in LC does not increase the obtainable performance but allows the same performance to be obtained in less time. Increasing the column temperature did allow the use of longer columns, generating column efficiencies in excess of 100,000 plates and gradient peak capacities approaching 1000. Raising the temperature increased the optimal mobile phase linear velocity, negating somewhat the pressure benefits observed by reducing the solvent viscosity. When operating at higher temperature the analyte retention is not only reduced, but the order of elution will also often change. High temperature separations allowed exotic organic modifiers such as isopropanol to be exploited for alternative selectivity and faster analysis. Finally, care must be taken when using high temperature separations to ensure that the narrow peak widths produced do not compromise the quality of data obtained from detectors such as high resolution mass spectrometers.