2D-LC Separations Research Articles

Eyes on the Prize: Overcoming Uncertainty to Realize the Power of 2D-LC Separations

Two-dimensional liquid chromatography (2D-LC) is a technique that extends the separation capabilities of conventional liquid chromatography by adding a second separation step to resolve compounds that are coeluted from a first column. This approach holds tremendous potential to solve difficult separation challenges in fields ranging from pharmaceutical analysis to biofuel characterization. Currently, method development is a significant bottleneck impeding more widespread implementation of 2D-LC methods, particularly for new users who are uncertain about how to proceed. In this month’s column, I highlight some of the primary considerations we face in method development and point to resources that can help users overcome uncertainty and develop highly effective 2D-LC methods.

A Strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part II: Development of second-dimension gradient conditions

The importance of therapeutic peptides continues to increase in the marketplace for treating a range of diseases including diabetes and obesity. Quality control analyses for these pharmaceutical ingredients usually depends on reversed-phase liquid chromatography, and it is critically important to ensure that no impurities coelute with the target peptide at levels that would compromise the safety or effectiveness of the drug products. This can be challenging due to the broad range of properties of impurities that can be present on one hand (e.g., amino acid substitutions, chain cleavages, etc.), and the similarity of other impurities on the other hand (e.g., d-/l-isomers). Two-dimensional liquid chromatography (2D-LC) is a powerful analytical tool that is well suited to address this particular problem; the first dimension can be used to detect impurities over a broad range in properties, while the second dimension can be used to focus specifically on those species that might coelute with the target peptide in the first dimension. While hundreds of papers have been published on the use of 2D-LC for proteomics applications, there are very few papers that have focused on its use for characterisation of therapeutic peptides. This paper is the second in a two-part series. In Part I of the series, we studied several different column / mobile phase combinations that could be useful in 2D-LC separations of therapeutic peptides, with a focus on selectivity, peak shape, and complementarity to other combinations, particularly for isomeric peptides under mass spectrometry-friendly conditions (i.e., volatile buffers). In this second part in the series, we describe a strategy to derive second-dimension (2D) gradient conditions that both, ensure elution from the 2D column, and increase the likelihood of resolving peptides with very similar properties. We find that a two-step process yields conditions that place the target peptide in the middle of the 2D chromatogram. This process begins with two scouting gradient elution conditions in the second dimension of a 2D-LC system, followed by building and refining a retention model for the target peptide using a third separation. The process is shown to be generically useful by developing methods for four model peptides, and application to a sample of degraded model peptide to demonstrate its utility for resolving impurities in a real sample.

Flow Dynamics and Analyte Transfer in a Microfluidic Device for Spatial Two-Dimensional Separations

In the last decade, chip-based separations have become a major area of interest in the field of separation science, especially for the development of “spatial” two-dimensional liquid chromatography (xLC × xLC). In xLC × xLC, the analytes are first separated by migration to different positions in a first-dimension (1D) channel and subsequently transferred with the aid of a flow distributor in a perpendicular direction to undergo a second-dimension (2D) separation. In this study, several designs for 2D separations are explored with the aid of computational fluid dynamics simulations. There were several aims of this work, viz. (1) to investigate the possible anomalies arising from the location of analyte bands in the first-dimension channel before transfer to the second dimension induced by the flow distributor, (2) to study the distribution ratio of the analytes across the different outlets of the 1D channel, and (3) to study the flow behaviour confinement in the flow distributor. In all designs, the simulated absolute flow velocity was not equal in all regions of the 1D channel. The extreme segments showed higher velocities compared to the central zones. This will eventually influence the migration times (first moments) and the variances (second moments), as confirmed by CFD results. The study has contributed to the understanding of the effects of the peak locations and, ultimately, to progress in spatial 2D-LC separations.

Dealing with Wandering First-Dimension Peaks in 2D-LC Separations

The retention of large molecules, such as peptides and proteins, is strongly dependent on mobile-phase composition and temperature. Consequently, the retention times (tR) of these molecules often fluctuate from one separation to the next. In two-dimensional liquid chromatography (2D-LC), this fluctuation can be challenging to deal with, especially if only one or a few specific peaks in the first dimension (1D) separation are targeted for further separation. In this installment, we describe instrumental concepts that are useful for coping with this challenge.

Mapping the Separation Landscape in Two-Dimensional Liquid Chromatography: Blueprints for Efficient Analysis and Purification of Pharmaceuticals Enabled by Computer-Assisted Modeling.

Recent developments in two-dimensional liquid chromatography (2D-LC) now make separation and analysis of very complex mixtures achievable. Despite being such a powerful chromatographic tool, current 2D-LC technology requires a series of arduous method development activities poorly suited for a fast-paced industrial environment. Recent introductions of new technologies including active solvent modulation and a support for multicolumn 2D-LC are helping to overcome this stigma. However, many chromatography practitioners believe that the lack of a systematic way to effectively optimize 2D-LC separations is a missing link in securing the viability of 2D-LC as a mainstay for industrial applications. In this work, a computer-assisted modeling approach that dramatically simplifies both offline and online 2D-LC method developments is introduced. Our methodology is based on mapping the separation landscape of pharmaceutically relevant mixtures across both first (1D) and second (2D) dimensions using LC Simulator (ACD/Labs) software. Retention models for 1D and 2D conditions were built using a minimal number of multifactorial modeling experiments (2 × 2 or 3 × 3 parameters: gradient slope, column temperature, and different column and mobile phase combinations). The approach was first applied to online 2D-LC analysis involving achiral and chiral separations of complex mixtures of enantiomeric species. In these experiments, the retention models proved to be quite accurate for both the 1D and 2D separations, with retention time differences between experiments and simulations of less than 3.5%. This software-based concept was also demonstrated for offline 2D-LC purification of drug substances.

High resolution two-dimensional liquid chromatography coupled with mass spectrometry for robust and sensitive characterization of therapeutic antibodies at the peptide level

Separations of complex peptide mixtures have been a common target application for two-dimensional liquid chromatography over the last few decades. These separations have most frequently been carried out at the capillary scale, with columns on the order of 75 µm i.d. and flow rates on the order of 500 nL/min. Recently, however, several groups have worked to optimize comprehensive 2D-LC (LC × LC) separations of peptides at the analytical scale (i.e., 2 mm i.d. columns, and ca. 1 mL/min flow rates) and demonstrated peak capacities on the order of 5000 in analysis times of a few hours, using reversed-phase separations in both dimensions. In this paper we aim to advance the performance of such separations in two primary ways. First, we demonstrate that active solvent modulation (ASM) can be used to improve the 2D peak capacity by both enabling use of long and highly efficient first dimension (1D) columns, and by mitigating the deleterious effects of injecting large fractions of 1D effluent into the small columns that are required for fast and highly sensitive second dimension (2D) separations. Taken together these two benefits enable the realization of a peak capacity of 10,000 in an analysis time of four hours. This comes at the cost of increased instrument complexity compared to 1D-LC separations, but the 2D-LC approach is unquestionably the most efficient way to improve upon the resolving power of existing 1D-LC. Second, we have systematically studied the compromise between the peak capacity of each 2D separation and the operating pressure required to achieve that peak capacity. Understanding this compromise will be important to the development of LC × LC methods that both produce high peak capacities, and are sufficiently robust to operate for days at a time without significant losses in separation performance. Based on the results of this study we chose conditions for subsequent separations that required less than 400 bar operating pressure in the second dimension, but yielded a 2D peak capacity of about 3500 in 2 h. After 160 h of continuous operation of the LC × LC separation under these conditions (and about 20,000 injections into the 2D column) the 2D column had only lost about 18% of its initial isocratic efficiency. These results should motivate further development and implementation of such high performing and robust separations for the identification and quantification of peptides in a variety of application areas, including digests of therapeutic proteins such as monoclonal antibodies.

A study of the re-equilibration of hydrophilic interaction columns with a focus on viability for use in two-dimensional liquid chromatography

In recent years there has been increasing interest in the use of HILIC separations in two-dimensional liquid chromatography (2D-LC), mainly because the selectivity of HILIC separations complement that of reversed-phase separations for a variety of molecules. Historically, the re-equilibration of HILIC phases following gradient elution has been perceived as too slow to be useful in the second dimension of 2D-LC separations in particular. Recent studies of re-equilibration of HILIC phases by McCalley and coworkers using a limited set of conditions showed that highly repeatable gradient separations could be obtained with re-equilibration times as short as 4.3 min [1,2]. In this study we aimed to study re-equilibration of HILIC phases under a broader set of conditions, and at much shorter re-equilibration times, in the interest of determining whether or not HILIC separations can be generally considered as a viable option for use in the second dimension of 2D-LC separations. To this end we studied the effects of mobile phase pH, buffer concentration, and preparation method, flow rate, analyte and stationary phase chemistry, column length, and re-equilibration time on retention of a variety of small molecule probe solutes following gradient elution. In general, we have found that excellent separation repeatability can be obtained with quite short (≪10 min) re-equilibration times, even when progress toward full equilibration of the column is quite slow (≫10 min). In other words, even if the stationary phase is not fully equilibrated, as long as it is partially equilibrated in a highly precise manner, highly repeatable retention times can be obtained. Higher flow rate has a positive effect on both the rate of progress toward full equilibration and the repeatability of separation. No significant, consistent effects of eluent pH or buffer concentration on repeatability of gradient separation were observed for the stationary phases studied. Excellent gradient separation repeatability was obtained with shorter columns (30 mm length) with re-equilibration times as short as 3 s. A proof-of-concept 2D-LC separation of several small molecule probes using HILIC separations in both dimensions was performed to illustrate that re-equilibration of these columns can be fast enough for HILIC columns to be considered as a viable option for the second dimension of comprehensive 2D-LC separations.

Evaluation of active solvent modulation to enhance two-dimensional liquid chromatography for target analysis in polymeric matrices

A new methodology is presented for two-dimensional liquid chromatography (2D-LC) separations of polymers. Active solvent modulation (ASM) was evaluated in its effectiveness to enhance solvent compatibility for both separation dimensions. As an example the determination of target compounds in epoxy resins was used. Ultra-high pressure size-exclusion chromatography was applied in the first dimension using THF as the solvent. The second dimension separation was operated in reversed-phase mode using an acetonitrile/water gradient. ASM prevents sample breakthrough in the second dimension and produces chromatograms that are of great peak shape and high resolution. It enables very sensitive determination of target components down to the low ppm level. The resulting high-speed 2D-LC method (10 min analysis time) showed good linearity (R2 > 0.9995) and reproducibility (as low as 0.3–0.7% peak area RSD). ASM was also applied in comprehensive 2D-LC (SECxLC) mode for characterization of molecular weight and chemical composition distribution of a polymer blend consisting of epoxy novolac and phenol novolac. The SECxLC separation was executed at short run times (20 min). ASM technology can markedly enhance productivity in 2D-LC analysis for many complex sample matrices.

Two-Dimensional Liquid Chromatography Analysis of Polystyrene/Polybutadiene Block Copolymers.

A detailed characterization of a commercial polystyrene/polybutadiene block copolymer material (Styrolux) was carried out using two-dimensional liquid chromatography (2D-LC). The Styrolux is prepared by statistical linking reaction of two different polystyrene- block-polybutadienyl anion precursors with a multivalent linking agent. Therefore, it is a mixture of a number of branched block copolymers different in molecular weight, composition, and chain architecture. While individual LC analysis, including size exclusion chromatography, interaction chromatography, or liquid chromatography at critical condition, is not good enough to resolve all the polymer species, 2D-LC separations coupling two chromatography methods were able to resolve all polymer species present in the sample; at least 13 block copolymer species and a homopolystyrene blended. Four different 2D-LC analyses combining a different pair of two LC methods provide their characteristic separation results. The separation characteristics of the 2D-LC separations are compared to elucidate the elution characteristics of the block copolymer species.

Simulation of elution profiles in liquid chromatography − II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography

An important research direction in the continued development of two-dimensional liquid chromatography (2D-LC) is to improve the detection sensitivity of the method. This is especially important in applications where injection of large volumes of effluent from the first dimension (1D) column into the second dimension (2D) column leads to severe 2D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the 1D mobile phase overwhelms the 2D column with each injection of 1D effluent, leading to low resolution in the second dimension. In a previous study we validated a simulation approach based on the Craig distribution model and adapted from the work of Czok and Guiochon [1] that enabled accurate simulation of simple isocratic and gradient separations with very small injection volumes, and isocratic separations with mismatched injection and mobile phase solvents [2]. In the present study we have extended this simulation approach to simulate separations relevant to 2D-LC. Specifically, we have focused on simulating 2D separations where gradient elution conditions are used, there is mismatch between the sample solvent and the starting point in the gradient elution program, injection volumes approach or even exceed the dead volume of the 2D column, and the extent of sample loop filling is varied. To validate this simulation we have compared results from simulations and experiments for 101 different conditions, including variation in injection volume (0.4–80μL), loop filling level (25–100%), and degree of mismatch between sample organic solvent and the starting point in the gradient elution program (−20 to +20% ACN). We find that that the simulation is accurate enough (median errors in retention time and peak width of −1.0 and −4.9%, without corrections for extra-column dispersion) to be useful in guiding optimization of 2D-LC separations. However, this requires that real injection profiles obtained from 2D-LC interface valves are used to simulate the introduction of samples into the 2D column. These profiles are highly asymmetric − simulation using simple rectangular pulses leads to peak widths that are far too narrow under many conditions. We believe the simulation approach developed here will be useful for addressing practical questions in the development of 2D-LC methods.

Solvent minimization in two-dimensional liquid chromatography

An algorithm was developed for the minimization of consumption of organic solvent in comprehensive two-dimensional liquid chromatography (2DLC). It was shown that one can reach higher peak capacities only by using more eluent. The equilibration volume of the second dimension, however, did not affect the solvent consumption significantly. Calculations confirmed that the same target peak capacity could be achieved by consuming significantly different volume of organic modifier depending on the number of fractions analyzed in the second dimension suggesting that 2D separations can be optimized for eluent consumption. It was shown that minimization of eluent usage requires the use of small and high efficient columns in the second dimension. A simple equation was derived for the calculation of the optimal number of collected fractions from the first dimension that allowed the minimization of eluent usage, cost and environmental impact of comprehensive 2DLC separations.

New zwitterionic polymethacrylate monolithic columns for one‐ and two‐dimensional microliquid chromatography

We prepared 0.53 and 0.32 mm id monolithic microcolumns by in situ copolymerization of a zwitterionic sulfobetaine functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) and dioxyethylene dimetacrylate crosslinkers. The columns show a dual retention mechanism (hydrophilic-interaction mode) in acetonitrile-rich mobile phases and RP in highly aqueous mobile phases. The new 0.53 mm id columns provided excellent reproducibility, retention, and separation selectivity for phenolic acids and flavonoids. The new zwitterionic monolithic columns are highly orthogonal, with respect to alkyl silica stationary phases, not only in the hydrophilic-interaction mode but also in the RP mode. The optimized monolithic zwitterionic microcolumn of 0.53 mm id was employed in the first dimension, either in the aqueous normal-phase or in the RP mode, coupled with a short nonpolar core-shell column in the second dimension, for comprehensive 2D LC separations of phenolic and flavonoid compounds. When the 2D setup with the sulfobetaine-BIGDMA column was used for repeated sample analysis, with alternating gradients of decreasing (hydrophilic-interaction mode), and increasing (RP mode) concentration of acetonitrile on the sulfobetaine-BIGDMA column in the first dimension, useful complementary information on the sample could be obtained.

Characterization of functionalized polyolefins by high-temperature two-dimensional liquid chromatography

High-temperature two-dimensional liquid chromatography (HT 2D-LC) was developed for the separation and characterization of functional polyolefins. Therefore, the key experimental parameters, namely the injection volume, the mobile phase composition, the flow rate in SEC and the time and phase of sampling into the second dimension, were systematically varied and their influence on the resolution of separation were studied. The HPLC separation of ethylene-vinylacetate waxes was realized using silica gel as stationary phase and a solvent gradient decalin→cyclohexanone, while SEC separations were realized in the chromatographic system polystyrene divinyl benzene column/1,2,4-trichlorobenzene. By choosing suitable experimental parameters, the run time needed for one complete 2D-LC analysis of a polymer sample was shortened from about 200min to 100min. However, the developed method failed to adsorb polypropylene and ethylene/1-butene copolymers grafted with 13 or 3mol.% of methyl methacrylate respectively. Using porous graphite as a stationary phase and a solvent gradient 1-decanol→1,2,4-trichlorobenzene as mobile phase 2D-LC separations of both grafted polyolefins were realized.

Recent progress in online, comprehensive two-dimensional high-performance liquid chromatography for non-proteomic applications

Comprehensive two-dimensional HPLC (2DLC) has been used very successfully in proteomics applications for over a decade. Increasingly we are seeing online, comprehensive 2DLC used for non-proteomic applications. This article gives an overview of the state of the art of this technique, with emphasis on current trends in theory and practice that are exciting and hold the most promise for advancing the performance of 2DLC. Specifically, the recently commercialized small, superficially porous packing materials are very well suited to use in the second dimension of online 2DLC where analysis speed is critical and largely dictates the performance of the 2DLC system. Further, the recent development of optimization schemes and associated software that support the use of different second-dimension elution modes in a single 2DLC analysis will improve the flexibility and effectiveness of 2DLC separations. Excluding separations of peptides, proteins, and polymers, 2DLC systems utilizing reversed-phase separation in one dimension, and either normal phase or reversed-phase in the other dimension continue to be the most popular. Although these systems have been applied mostly to complex biological materials, we are beginning to see applications in the analysis of pharmaceutical materials for ingredient purity and degradation profiling. The general lack of robust, easy-to-use commercially available software is arguably the greatest impediment to wider application of 2DLC methods. This situation is improving slowly, however, and at least two commercially available software packages have been described in the peer-reviewed literature. This is an exciting time in the development of online 2DLC.

Detection issues in two-dimensional on-line chromatography

The dilution of analytes during their migration through two-dimensional liquid chromatographic systems was investigated. Simplified equations for the calculation of the dilution factors in the first and second dimension columns were derived considering the variance of the injected sample, the flow rates, the split ratio, the volume of the intermediate sample loop and the sampling time of the first-dimension effluent. The net dilution factor is the product of the dilution factors in the two single dimensions. It is between 200 and 300 in typical two-dimensional liquid chromatographic (2D-LC) separations. These values are less pessimistic than those reported previously [1]. It was shown that the fraction of organic modifier or stronger eluent component in the injected sample has a significant effect on the widths and heights of the eluted peaks. Analytes with high retention factors and high molecular weights are less sensitive toward this effect than those having small retention factors. These results suggest that the optimization of the experimental conditions of two dimension separations must be made from the points of view of both the retention factor and the detector response factor. The frequency of sampling of the eluent of the first-dimension separation does not have a significant effect on the detection limit of a 2D system if the split ratio is adjusted in the ratio of the frequency of sample collection.

1 mm ID poly(styrene‐co‐divinylbenzene) monolithic columns for high‐peak capacity one‐ and two‐dimensional liquid chromatographic separations of intact proteins

The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.

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 -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.

Equation for Peak Capacity Estimation in Two-Dimensional Liquid Chromatography

Two-dimensional liquid chromatography (2DLC) is a very powerful way to greatly increase the resolving power and overall peak capacity of liquid chromatography. The traditional "product rule" for peak capacity usually overestimates the true resolving power due to neglect of the often quite severe under-sampling effect and thus provides poor guidance for optimizing the separation and biases comparisons to optimized one-dimensional gradient liquid chromatography. Here we derive a simple yet accurate equation for the effective two-dimensional peak capacity that incorporates a correction for under-sampling of the first dimension. The results show that not only is the speed of the second dimension separation important for reducing the overall analysis time, but it plays a vital role in determining the overall peak capacity when the first dimension is under-sampled. A surprising subsidiary finding is that for relatively short 2DLC separations (much less than a couple of hours), the first dimension peak capacity is far less important than is commonly believed and need not be highly optimized, for example, through use of long columns or very small particles.

Comparison of the Practical Resolving Power of One- and Two-Dimensional High-Performance Liquid Chromatography Analysis of Metabolomic Samples

Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to its higher peak capacity compared to one-dimensional LC (1DLC). Because of the long analysis times typically associated with 2DLC (tens of hours) and its use primarily in proteomics applications, 2DLC in the context of general HPLC has been regarded as a niche technique for use in analysis of mixtures containing hundreds to thousands of components compared to the far more common techniques of isocratic and gradient elution 1DLC. A significant next step in the analytical development of 2DLC is to consider using its higher resolving power to reduce the analysis time of rather "simple" mixtures, in the range of only tens to hundreds of chemical constituents. The chief objective of this paper is to provide guidance to practitioners who need to decide whether 1DLC or 2DLC gives the superior separation in a given analysis time. Conditional peak capacities are predicted for fully optimized 1DLC and practical 2DLC separations of the low molecular weight constituents of an extract of corn seed at several analysis times using a model based on the chromatographic properties of compounds that are representative of real mixtures of lower molecular weight species. Two important corrections to the ideal 2DLC peak capacity are made to account for both incomplete usage of the separation space and the serious effect of first-dimension undersampling; this allows, we believe for the first time, a fair comparison of the resolving power of 1D- and 2DLC under realistic conditions. The predicted optimum conditions are then used to carry out experimental separations of low molecular weight corn seed extract, and peaks are counted in each 1D- and 2DLC chromatogram. Based on comparisons of both the predicted peak capacities and number of peaks observed in experimental chromatograms, we believe that practical 2DLC will be superior to fully optimized gradient 1DLC for separations lasting more than only about 10 min. This crossover time is much shorter than intuitively expected, and we believe this finding will inevitably have a major impact on the practice of 2DLC in liquid-phase separations in general.