Column selectivity for two-dimensional liquid chromatography

Selectivity of phase system is of primary concern when designing a 2-D separation, as it affects the 2-D system orthogonality and consequently the peak capacity controlling the number of peaks that can be separated in the available 2-D retention space limited by the time of analysis. Possibilities for characterization of LC phase system selectivity with respect to different polar and nonpolar structural units are compared, with special attention to multidimensional samples with various types of repeat groups, such as homopolymers, (co)polymers, fatty acid esters with various acyl lengths and number and position of double bonds, etc. Possibilities of the 2-D LC separations of these and other sample types, including pharmaceuticals, natural phenolic compounds, biopolymers, etc., using various combinations of separation modes are reviewed. Rules for design of comprehensive 2-D LC x LC systems are discussed, with respect to mobile phase compatibility in the two systems and modulation techniques suppressing band broadening connected with the sample fraction transfer from the first to the second dimension. Pitfalls connected with online connection of normal-phase and RP LC systems and their possible practical solutions are addressed and illustrated by practical examples.     

Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection

A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.     

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.     

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.     

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.     

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.     

Optimization strategies for off-line two-dimensional liquid chromatography

A step by step strategy of optimization of comprehensive off-line two-dimensional liquid chromatography (2D-LC) separations is proposed. The goal of an optimization process in the separation sciences is either to achieve a given resolution (a target peak capacity in 2D-LC) within as short a time as possible or to reach the highest possible resolution in a given analysis time. The proposed method takes into account the characteristics of the columns used in the first and the second dimension and the number of fractions of the first dimension eluent that should be collected. The effect of the time spent during the analysis on the second dimension column to carry out necessary tasks that are not the separation itself (called the additional time) on the maximum peak capacity that is achievable was carefully investigated. It was shown that (1) an increase in the peak capacity of the first dimension column combined with the collection of larger volume fractions permits a significant reduction of the time needed to achieve the desired peak capacity; and (2) there is an optimum fraction collection ratio (or number of collected fractions per peak) which yields the target peak capacity in the minimum time. The proposed strategy was used for the optimization of the separation of samples of BSA tryptic digest by an off-line 2D-LC using an SCX⊗RP$\text{SCX}\otimes \text{RP}$-HPLC method. As a result of this optimization, a peak capacity of 4000 could be achieved in about 5 h with the two columns available. The time needed for the optimized analysis was less than two thirds of the analysis time that would have been needed had the conventional rule of thumb of sample collection in comprehensive on-line 2D-LC (4 samples/peak) been followed.     

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.     

The impact of sampling time on peak capacity and analysis speed in on-line comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (2DLC) offers a number of practical advantages over optimized one-dimensional LC in peak capacity and thus in resolving power. The traditional “product rule” for overall peak capacity for a 2DLC system significantly overestimates peak capacity because it neglects under-sampling of the first dimension separation. Here we expand on previous work by more closely examining the effects of the first dimension peak capacity and gradient time, and the second dimension cycle times on the overall peak capacity of the 2DLC system. We also examine the effects of re-equilibration time on under-sampling as measured by the under-sampling factor and the influence of molecular type (peptide vs. small molecule) on peak capacity. We show that in fast 2D separations (less than 1 h), the second dimension is more important than the first dimension in determining overall peak capacity and conclude that extreme measures to enhance the first dimension peak capacity are usually unwarranted. We also examine the influence of sample types (small molecules vs. peptides) on second dimension peak capacity and peak capacity production rates, and how the sample type influences optimum second dimension gradient and re-equilibration times.     

Improved 2D‐HPLC of red wine by incorporating pre‐process signal‐smoothing algorithms

Two multidimensional HPLC separations of an Australian red wine are presented, >70% of the available separation space was used. A porous graphitic carbon (PGC) stationary phase was used as the first dimension in both separations with both RP core–shell and hydrophilic interaction chromatography fully porous columns used separately in the second dimension. To overcome peak analysis problems caused by signal noise and low detection limits, the data were pre‐processed with penalised least‐squares smoothing. The PGC × RP combination separated 85 peaks with a spreading angle of 71° and the PGC × hydrophilic interaction chromatography separated 207 peaks with a spreading angle of 80°. Both 2D‐HPLC steps were completed in 76 min using a comprehensive stop‐and‐go approach. A smoothing step was added to peak‐picking processes and was able to greatly reduce the number of false peaks present due to noise in the chromatograms. The required thresholds were not able to ignore the noise because of the small magnitude of the peaks; 1874 peaks were located in the non‐smoothed PGC × RP separation that reduced to 227 peaks after smoothing was included.     

Resolution prediction and optimization of temperature programme in comprehensive two-dimensional gas chromatography

A model is developed for predicting the resolution of interested component pair and calculating the optimum temperature programming condition in the comprehensive two-dimensional gas chromatography (GC x GC). Based on at least three isothermal runs, retention times and the peak widths at half-height on both dimensions are predicted for any kind of linear temperature-programmed run on the first dimension and isothermal runs on the second dimension. The calculation of the optimum temperature programming condition is based on the prediction of the resolution of "difficult-to-separate components" in a given mixture. The resolution of all the neighboring peaks on the first dimension is obtained by the predicted retention time and peak width on the first dimension, the resolution on the second dimension is calculated only for the adjacent components with un-enough resolution on the first dimension and eluted within a same modulation period on the second dimension. The optimum temperature programming condition is acquired when the resolutions of all components of interest by GC x GC separation meet the analytical requirement and the analysis time is the shortest. The validity of the model has been proven by using it to predict and optimize GC x GC temperature programming condition of an alkylpyridine mixture.     

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.     

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.     

Fast, comprehensive two-dimensional liquid chromatography

The absolute need to improve the separating power of liquid chromatography, especially for multi-constituent biological samples, is becoming increasingly evident. In response, over the past few years, there has been a great deal of interest in the development of two-dimensional liquid chromatography (2DLC). Just as 1DLC is preferred to 1DGC based on its compatibility with biological materials we believe that ultimately 2DLC will be preferred to the much more highly developed 2DGC for such samples. The huge advantage of 2D chromatographic techniques over 1D methods is inherent in the tremendous potential increase in peak capacity (resolving power). This is especially true of comprehensive 2D chromatography wherein it is possible, under ideal conditions, to obtain a total peak capacity equal to the product of the peak capacities of the first and second dimension separations. However, the very long timescale (typically several hours to tens of hours) of comprehensive 2DLC is clearly its chief drawback. Recent advances in the use of higher temperatures to speed up isocratic and gradient elution liquid chromatography have been used to decrease the time needed to do the second dimension LC separation of 2DLC to about 20s for a full gradient elution run. Thus, fast, high temperature LC is becoming a very promising technique. Peak capacities of over 2000 and rates of peak capacity production of nearly 1 peak/s have been achieved. In consequence, many real samples showing more than 200 peaks with signal to noise ratios of better than 10:1 have been run in total times of under 30 min. This report is not intended to be a comprehensive review of 2DLC, but is deliberately focused on the issues involved in doing fast 2DLC by means of elevating the column temperature; however, many issues of broader applicability will be discussed.     

Two‐dimensional HPLC analysis of oligostyrenes: Comprehensive and online heart‐cutting techniques

2‐D HPLC incorporating two reversed phase (RP) environments was employed for the isolation of oligomers and their diastereomers of low molecular weight oligostyrenes. The operation of a comprehensive method of analysis was compared to a heart‐cutting approach. The comprehensive approach employed a high resolution diastereomer separation in the first dimension and a low peak capacity C18, high speed separation according to molecular weight. Because of solvent incompatibility between the dimensions in the comprehensive method, successful separation of the diastereomers of the oligomers was not possible. The heart‐cutting approach used a C18 monolith in the first dimension, which was selective only for molecular weight. Entire molecular weight fractions were then transported to the second dimension in an online heart‐cutting process for the separation of diastereomers. The heart‐cutting process was more successful in that 228 components of the 511 within the sample were recognized. This series of separations was undertaken in less than 6 h.     

A graphical method for understanding the kinetics of peak capacity production in gradient elution liquid chromatography

A novel graphical method for assessing the compromise between conditional peak capacity and separation speed for packed bed columns under gradient conditions has been developed and applied to the separation of peptides. This approach is analogous to and complements the conventional "Poppe plot" used to study plate count in isocratic separations. The use of the new plot can assist the design of appropriate column formats (e.g. particle size and column length) for both dimensions in gradient elution two-dimensional liquid chromatography (2DLC). Particularly for the second dimension of 2DLC, we find that smaller particles provide faster separations even though fast separations based on particles smaller than 2 microm are practically limited by the required miniscule column length. We also find that high temperatures strongly enhance the kinetics of peak capacity production whereas higher pressures help achieve larger absolute peak capacities albeit at the cost of longer analysis time.     

Separation of fatty acid methyl esters by comprehensive two-dimensional supercritical fluid chromatography with packed columns and programming of sampling duration

Fatty acid methyl esters from various fats and oils were separated by comprehensive two-dimensional supercritical fluid chromatography with conventional packed columns and FID detection. The first dimension was a silica gel column and the second dimension was an ODS column. This combination was largely orthogonal for the separation of fatty acid methyl esters. The first dimension separations were primarily based on the number of double bonds while the second dimension separations were based on the chain length. The highly-ordered chromatograms and improved resolution allowed the easy detection and identification of minor components. Although the first dimension separations were performed under isobaric conditions where the peak width increased in proportion to the retention, the programming of the sampling duration allowed us to maintain the optimum re-injection frequency (3–4 times) per peak into the second dimension and so to minimize the total analysis time without deteriorating the resolution.     

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.     

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.     

Comprehensive multidimensional liquid chromatography: theory and applications

Comprehensive two-dimensional (2D) liquid chromatographic (LC x LC) techniques can be considered innovative methods only recently developed and adopted in many configurations. The revolutionary aspect of comprehensive two-dimensional techniques, with respect to classical multidimensional (MD) chromatography, is that the entire sample is subjected to the 2D advantage. The major benefit is that the separation capacities of each dimension are multiplied, offering a high peak capacity to resolve samples of great complexity. The first part of the present review briefly describes the theoretical and practical aspects related to the development of a multidimensional comprehensive liquid chromatographic method. Applicational experiences in comprehensive liquid chromatography are then described, divided into four groups, according to the HPLC modes used in the two dimensions and to the nature of the samples analyzed.