Evaluation of the two-dimensional reversed-phase-reversed-phase separations of low-molecular mass polystyrenes

The resolving power of four reversed-phase-reversed-phase two-dimensional (2D) chromatographic systems was evaluated using information theory (IT) and a geometric approach to factor analysis. The first separation dimension employed a C18 column, while the second separation dimension employed a carbon clad zirconia column. Mobile phases in each dimension were either methanol or acetonitrile. The sample matrix that was employed in this study contained a mixture of 58 components, comprised of stereoisomers and structural isomers of a systematic variation in molecular weight. Each of the components were oligostyrenes, with between two and five configurational repeating units having either n-butyl, sec-butyl or tert-butyl end groups. In the two-dimensional systems employed in this study, between 46 and 49 of the 58 components could be separated, depending on the mobile phase combinations--with apparent resolution that could not possibly be achieved in a single one-dimensional separation. The results from this study indicate that in order to fully evaluate the resolving power of a 2D system multiple methods of analysis that evaluate the separation potential are most appropriate. This becomes increasingly important when the sample contains components that are very closely related and the retention of solutes displays a high degree of solute crowding.     

毛细管等电聚焦-毛细管区带电泳二维蛋白质分离平台的构建及其性能

通常采用二维聚丙烯酰胺凝胶电泳 ( 2 D- PAGE)分析组织或细胞的全蛋白质 [1] ,但难与质谱 ( MS)直接联用 .用高效液相色谱 ( HPLC)和毛细管电泳 ( CE)分离分析蛋白质和多肽的一维分离模式的分辨率和峰容量有限 .多维柱联用技术比一维分离有更高的分辨率和峰容量 [2 ] ,便于和 MS直接联用 [3,4 ] ,易于实现自动化 .目前 ,有关 2 D- CE的报道相对较少 [5～ 7] ,我们初步实现了将 2 D- PAGE由平板转移到毛细管中[6 ,7] ,但凝胶柱的制作烦琐 ,存在交叉污染 ,不能与 MS直接联用 .本文用微透析中空纤维膜为接口构建了毛细管等电聚焦 ( …     

Comprehensive coupled reversed-phase reversed-phase separations of a complex isomeric mixture

Using predictions based on results obtained from Information Theory and Factor Analysis for the two-dimensional separation of a complex isomeric mixture, a practical experimental comprehensive coupled reversed phase-reversed phase chromatographic system was developed. In total four reversed phase-reversed phase systems were studied, each of which theory predicted would be able to resolve essentially equal numbers of components. However, in practice only one of these coupled systems realised the theoretical potential. This system employed as the first dimension, a C18 stationary phase with methanol as the mobile phase and as the second dimension, carbon clad zirconia as the stationary phase and acetonitrile as the mobile phase. In this system, 27 of the 32 isomers of a mixture of oligostyrenes were resolved. Failure of the remaining coupled systems to achieve the theoretical potential was attributed to high solute crowding, low efficiency of separation space utilisation and long analysis times in the second dimension.     

Separation of oligostyrene isomers in a complex mixture using two-dimensional heart-cutting reversed-phased liquid chromatography

The development of a two-dimensional liquid chromatographic system requires a process of assessment that can yield an optimum performing system with minimal experimental evaluation. Information Theory and a geometric approach to Factor Analysis are two techniques that when used in combination, provide important information on the expected two-dimensional performance. In the present study, we compare the predicted separation performance of two-dimensional systems that have been subjected to analysis by Information Theory and Factor Analysis to that of actual chromatographic separation performance. Our test separation comprised a mixture of 32 oligostyrene structural isomers and stereoisomers. The optimal combination as determined by Information Theory and Factor Analysis consisted of a C18 column with a methanol mobile phase in the first dimension and a carbon clad zirconia column with an acetonitrile mobile phase in the second dimension. This system was also shown to be the most successful practical system when a heart-cutting approach was employed. The practical results were in total agreement with the results from Information Theory and Factor Analysis. The number of isomers resolved using this system was 27. A second system, namely one comprising of a C18 column and methanol mobile phase in the first dimension and a carbon clad zirconia column with a methanol mobile phase in the second dimension was also predicted to be a system with high separation potential. However, practical assessment of this system did not realise the theoretical predictions, largely due to the long separation times required in the second dimension. Furthermore, all combinations that employed a C18 column with an acetonitrile mobile phase in the first dimension failed to realise the theoretical separation potential due to high solute crowding, low orthogonality and a disordered arrangement of bands along the first separation axis. This was also predicted by the theoretical assessment.     

Comprehensive two-dimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.     

Comprehensive two-dimensional separations of complex mixtures using reversed-phase reversed-phase liquid chromatography

A comprehensive two-dimensional reversed-phase reversed-phase liquid chromatographic system for the separation of a complex mixture of oligostyrenes was developed using results from a previous theoretical assessment of the informational similarity, percent synentropy, orthogonality and peak capacity of hypothetically coupled systems. The degree of sample attribute order in the first separation dimension was also used in the development of the experimental two-dimensional system. A C18(methanol)/CCZ(acetonitrile) two-dimensional system was chosen for the comprehensive analysis of the oligostyrene mixtures because this system had the lowest solute crowding, highest orthogonality and was observed to have order with respect to a sample attribute in the first separation dimension. The separations achieved were in full agreement with the results from information theory and (a geometric approach to) factor analysis assessments. High sampling rates in the first liquid chromatographic dimension were shown to be impossible or inefficient when the peak capacity and separation time of the second dimension was high or when the aim of the exercise was to isolate individual sample constituents in high yield.     

High electric field strength two-dimensional peptide separations using a microfluidic device

New instrumentation has been developed to improve the resolution, efficiency, and speed of microfluidic 2D separations using MEKC coupled to high field strength CE. Previously published 2D separation instrumentation [Ramsey, J. D. et al., Anal. Chem. 2003, 75, 3758-3764] from our group was limited to a maximum potential difference of 8.4 kV, resulting in an electric field strength of only approximately 200 V/cm in the first dimension. The circuit described in this report has been designed to couple a higher voltage supply with a rapidly switching, lower voltage supply to utilize the best features of each. Voltages applied in excess of 20 kV lead to high electric field strength separations in both dimensions, increasing the separation resolution, efficiency, and peak capacity while reducing the required analysis time. Detection rates as high as six peptides per second (based on total analysis time) were observed for a model protein tryptic digest separation. Additionally, higher applied voltages used in conjunction with microfluidic chips with longer length channels maintained higher electric field strengths and produced peak capacities of over 4000 for some separations. Total separation time in these longer channel devices was comparable to that obtained in short channels at low field strength; however, resolving power improved approximately threefold.     

Phase system selectivity and two-dimensional separations in liquid column chromatography

Correlations between the separation selectivity in aqueous and non-aqueous reversed-phase systems and in normal-phase LC systems were investigated for samples containing different numbers of two repeat structural elements. Such samples are best separated in "orthogonal" two-dimensional chromatographic systems, showing selectivity for one type of the repeat structural element only in the first dimension and for the other structural element only in the second dimension. The number of resolved compounds improves as the degree of orthogonality of the separation systems increases with decreasing correlation between the selectivities for the sample structural distribution in the two dimensions. Orthogonal systems with non-correlated selectivities for each repeat structural element provide the highest number of separated peaks and regular arrangement of the peaks over the two-dimensional retention space according to the individual structural element distribution and the best use of the available peak capacity. Fully orthogonal systems are difficult to find in practice. Partially orthogonal system with correlated selectivities for one structural type distribution, but with one system non-distinguishing the distribution for the other structural element are still useful for the two-dimensional separations. The correlations between the selectivities for repeat regular structural increments were employed to evaluate the suitability of phase systems for two-dimensional HPLC separations. The selectivity correlation in various reversed-phase and normal-phase systems was evaluated for two sample types: (1) Various RP columns show significantly inversely correlated selectivities for acyl lengths and numbers of double bonds distribution, but the differences in the double bond selectivity can be used for practical separations of triacylglycerols with the same equivalent carbon numbers. (2) Synthetic EO-PO block (co)oligomers with two-dimensional distribution of oxyethylene and oxypropylene monomer units were separated according to the two distribution types using on-line two-dimensional reversed-phase-normal-phase LC with a C18 column in the first dimension and an aminopropyl silica column in the second dimension.     

无死体积蚀刻多孔膜接口的制作及其在二维毛细管电泳分离系统中的应用

介绍了一种在毛细管柱上原位腐蚀而成的多孔膜接口的制作方法,并用该接口构建了一类毛细管电泳二维分离技术平台。柱上原位腐蚀刻成的多孔膜接口具有零死体积、制作过程简易、成本低廉、耐用、柱间切换便捷等优点,特别适合作为基于毛细管柱的二维及多维电泳联用中的接口,是目前二维及多维毛细管柱联用中一类较为新型、实用、理想的接口。以鹿茸冻干粉可溶物样品为例,验证了该接口在二维毛细管电泳联用系统中的可行性和分离效能。实验结果表明：鹿茸冻干粉可溶物整个二维分离分析的时间在1 h内完成,二维分离系统的分辨率和总峰容量都比一维的高。     

Targeted three-dimensional liquid chromatography: a versatile tool for quantitative trace analysis in complex matrices

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.     

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.     

Fast, high-efficiency peptide separations on a 50-microm reversed-phase silica monolith in a nanoLC-MS set-up

Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-microm reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC-MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETP(min)=5-10 microm at 2.4 mm/s). Flow rates up to 1.95 microL/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-microm monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

Multidimensional LC-LC and LC-CE for high-resolution separations of biological molecules

In multidimensional separations, two or more independent separation methods are coupled in an effort to resolve complex mixtures. The displacement mechanisms of each method should be orthogonal, such that little correlation exists between the retention of compounds in each dimension. When multiple orthogonal separation methods are coupled such that all sample components are subjected to complete analysis on all dimensions, the method is considered comprehensive. The primary advantage of comprehensive multidimensional separations over their one-dimensional counterparts is the potential for dramatically enhanced resolution. High resolving power can be achieved because the peak capacity of a comprehensive multidimensional separation is roughly equal to the product of the individual peak capacities of each dimension. In this review, the theory and instrumentation of two-dimensional liquid chromatography (LC-LC) and liquid chromatography-capillary electrophoresis (LC-CE) separations are discussed. Some applications of these techniques to the separation of biological molecules are highlighted. Future directions for the development of multidimensional separations are also considered.     

Concepts and comparisons in multidimensional separation

Multidimensional separation methodology provides a mechanism for a very substantial increase in the resolving power of chromatographic and related systems. After defining the conditions required for multidimensional separation and reviewing the origin of its high resolving power, a comparison is drawn between the two principal kinds of multidimensional systems, represented by continuous two-dimensional operation and coupled column assemblies. It is shown that their roles are complementary. The two-dimensional system gives a broad powerful overview of a sample while the coupled column system can be used flexibly to magnify and resolve selected regions of the sample space.     

Comprehensive chromatographic separations in proteomics

In such a complicated field as proteomic analysis, scientists are more and more challenged in implementing separation systems capable to provide enhanced separation power, as well as sensitivity of detection for adequate identification and, to a lesser extent, quantification of the separated compounds. To address such issues, several combinations of different separation modes have been investigated in comprehensive liquid chromatographic platforms, in which the entire sample eluted from the first dimension is subjected to a secondary chromatographic separation. The different applications exploited for comprehensive LC analysis of intact or digested proteins are the focus of this review, in which advantages and disadvantages of the different columns combinations, interfaces, and operating modes are pointed out. The combination with mass spectrometry as part of the total system is stressed, and illustrated in more detail. Theoretical concerns and practical requirements will be briefly discussed, as well.     

Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation,dual detectors,and multivariate curve resolution

Comprehensive two-dimensional liquid chromatography (LC × LC) is rapidly evolving as the preferred method for the analysis of complex biological samples owing to its much greater resolving power compared to conventional one-dimensional (1D-LC). While its enhanced resolving power makes this method appealing, it has been shown that the precision of quantitation in LC × LC is generally not as good as that obtained with 1D-LC. The poorer quantitative performance of LC × LC is due to several factors including but not limited to the undersampling of the first dimension and the dilution of analytes during transit from the first dimension (¹D) column to the second dimension (²D) column, and the larger relative background signals. A new strategy, 2D assisted liquid chromatography (2DALC), is presented here. 2DALC makes use of a diode array detector placed at the end of each column, producing both multivariate ¹D and two-dimensional (2D) chromatograms. The increased resolution of the analytes provided by the addition of a second dimension of separation enables the determination of analyte absorbance spectra from the ²D detector signal that are relatively pure and can be used to initiate the treatment of data from the first dimension detector using multivariate curve resolution–alternating least squares (MCR–ALS). In this way, the approach leverages the strengths of both separation methods in a single analysis: the ²D detector data is used to provide relatively pure analyte spectra to the MCR–ALS algorithm, and the final quantitative results are obtained from the resolved ¹D chromatograms, which has a much higher sampling rate and lower background signal than obtained in conventional single detector LC × LC, to obtain accurate and precise quantitative results. It is shown that 2DALC is superior to both single detector selective or comprehensive LC × LC and 1D-LC for quantitation of compounds that appear as severely overlapped peaks in the ¹D chromatogram – this is especially true in the case of untargeted analyses. We also anticipate that 2DALC will provide superior quantitation in targeted analyses in which unknown interfering compounds overlap with the targeted compound(s). When peaks are significantly overlapped in the first dimension, 2DALC can decrease the error of quantitation (i.e., improve the accuracy by up to 14-fold compared to 1D-LC and up to 3.8-fold compared to LC × LC with a single multivariate detector). The degree of improvement in performance varies depending upon the degree of peak overlap in each dimension and the selectivities of the spectra with respect to one another and the background, as well as the extent of analyte dilution prior to the ²D column.     

Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples

A new analytical two-dimensional supercritical fluid chromatography/mass spectrometry system (2D SFC/SFC/MS) has been designed and implemented to enhance the efficiency and quality of analytical support in drug discovery. The system consists of a Berger analytical SFC pump and a modifier pump, a Waters ZQ 2000 mass spectrometer, a set of switching valves, and a custom software program. The system integrates achiral and chiral separations into a single run to perform enantiomeric analysis and separation of a racemic compound from a complex mixture without prior clean up. The achiral chromatography in the first dimension separates the racemate from all other impurities, such as un-reacted starting materials and by-products. Mass-triggered fractionation is used to selectively fractionate the targeted racemic compound based on its molecular weight. The purified racemate from the achiral chromatography in the first dimension is then transferred to the chiral column in the second dimension to conduct the enantiomeric separation and analysis. A control software program, we coined SFC2D, was developed and integrated with MassLynx to retrieve acquisition status, current sample information, and real time mass spectrometric data as they are acquired. The SFC2D program also monitors the target ion signal to carry out mass-triggered fractionation by switching the valve to fractionate the desired peak. The 2D SFC/SFC/MS system uses one CO(2) pump and one modifier pump for both first and second dimension chromatographic separations using either gradient or isocratic elution. Similarly, a preparative 2D SFC/SFC/MS system has been constructed by modifying an existing Waters preparative LC/MS system. All components except the back pressure regulator are from the original LC/MS system. Applications of the 2D SFC/SFC/MS methods to the separation and the analysis of racemic pharmaceutical samples in complex mixtures demonstrated that an achiral separation (in first dimension) and a chiral separation (in second dimension) can be successfully combined into a single, streamlined process both in analytical and preparative scale.