Theoretical and practical guidelines for solvent dilution between the two dimensions in online comprehensive two-dimensional liquid chromatography.

Online comprehensive two-dimensional liquid chromatography (online LC x LC) has become increasingly popular. Among the different chromatographic modes that can be combined, hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) are particularly interesting because they offer a high degree of orthogonality. However, this combination remains complex due to the incompatibility of the solvents in the two dimensions. To avoid this problem, it is possible to dilute the first dimension (1D) effluent with (zdilution -1) volumes of a weaker solvent added to one volume of 1D-effluent, where zdilution represents the extent to which the fraction volume has been multiplied. This can be done using either active solvent modulation technology or an additional pump, prior to the second dimension analysis. The objective of this study was to develop theoretical models to predict whether or not dilution can be effective, and, if so, what is the minimum zdilution value required. This approach is based on the calculation of the ratio (called xdilution) between the peak standard deviation due to the injection process and the peak standard deviation in the absence of extra-column dispersion. xdilution was calculated from theoretical relationships and plotted as a function of zdilution, to predict the value required to obtain good peak shapes for the compound of interest. The maximum xdilution value was found to be of the order of 1 for chromatographically acceptable peak shapes. The proposed theoretical approach was experimentally validated on a number of representative small molecules and peptides. Agreement between experimental results and theoretical models was very high, especially for small molecules. Finally, it is shown that this approach helps to predict the most appropriate set of conditions in HILIC x RPLC, depending on the compounds to be separated.

Comprehensive analysis of the effective and intra-particle diffusion of weakly retained compounds in silica hydrophilic interaction liquid chromatography columns.

A detailed analysis of intra-particle volumes and layer thicknesses and their effect on the diffusion of solutes in hydrophilic interaction liquid chromatography (HILIC) was made. Pycnometric measurements and the retention volume of deuterated mobile phase constituents (water and acetonitrile) were used to estimate the void volume inside the column, including not only the volume of the mobile phase but also part of the enriched water solvent acting as the stationary phase in HILIC. The mobile phase (hold-up) volume accessible to non-retained components was estimated using a homologous series approach. The joint analysis of the different approaches indicated the formation of enriched water layers on the hydrophobic silica mesopore walls with a thickness varying significantly with mobile phase composition. The maximal thickness of the enriched water layers, which corresponded to the minimum void volume accessible to unretained solutes, marked a transition in the retention behavior of the studied analytes. Discrepancies between deuterated solvent measurements and pycnometry were explained by the existence of an irreplaceable water layer adsorbed on the silica surface. Regarding the diffusion behavior in HILIC, peak parking experiments were used to interpret the influence of the acetonitrile content on the effective diffusion coefficient Deff. A systematic decrease in Deff and molecular diffusion Dm was observed with decreasing acetonitrile concentration, primarily attributed to variations in mobile phase viscosity. Notably, Deff/Dm remained nearly unaffected by variations in mobile phase composition. Finally, the effective medium theory was used to make a comprehensive analysis of Dpart/Dm to study the contribution to band broadening when the solute resides in the mesopores. The obtained data unveiled a curvature with a minimum corresponding to conditions of maximum water-layer thickness and retention. For the weakly retained compounds (k' < 0.5) the Dpart/Dm-values were found to be relatively high (order of 0.35-0.5), which directly reflects the high γsDs/Dm-values that were observed (order 0.35-7).

Detailed comparison of in-house developed and commercially available heart-cutting and selective comprehensive two-dimensional liquid chromatography systems.

Recently, two-dimensional liquid chromatography (2D-LC) has become a popular approach to analyze complex samples. This is partly due to the introduction of commercial 2D-LC systems. In the past, 2D-LC was carried out on in-house developed setups, typically consisting of several switching valves and sample loops as the interface between the two dimensions. Commercial systems usually offer different 2D-LC modes in combination with specialized software to operate the instrument and analyze the data. This makes them highly user-friendly, however, at an increased cost compared to in-house developed setups. This study aims to make a comparison between an in-house developed 2D-LC setup and a commercially available 2D-LC instrument. The comparison is made based on experimental differences, in addition to more general differences, including cost price, flexibility, and ease of operation. Special attention is also paid to the different strategies to deal with the mobile phase incompatibility between the highly orthogonal separation mechanisms considered in this work: hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC). For the commercial 2D-LC instrument, this is done using active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension column before transfer to the second dimension (2D) column. For the in-house developed setup, a combination of restriction capillaries and a trap column is used. Using a sample of 28 compounds with a large polarity range, peak shapes and recoveries of the 2D-chromatograms are compared for both setups. For early eluting compounds, the selective comprehensive approach, currently only possible on the commercial 2D-LC instrument, results in the best peak shapes and recoveries, however, at the cost of an increased analysis time. In general, depending on the analytical goal (single heart-cut versus full-comprehensive 2D-LC), an in-house developed system can be satisfactory for the analysis of specific target compounds/samples. For more complex problems, it can be interesting to use a more specialized commercial 2D-LC instrument. Overall, this comparison study provides advice for analytical scientists, who are considering to use 2D-LC, on the type of equipment to consider, depending on the needs of their particular applications.

Reversed HILIC Gradient: A Powerful Strategy for On-Line Comprehensive 2D-LC.

The aim of the present work is to evaluate the possibilities and limitations of reversed hydrophilic interaction chromatography (revHILIC) mode in liquid chromatography (LC). This chromatographic mode consists of combining a highly polar stationary phase (bare silica) with a gradient varying from very low (1-5%) to high (40%) acetonitrile content (reversed gradient compared to HILIC). The retention behavior of revHILIC was first compared with that of reversed-phase LC (RPLC) and HILIC using representative mixtures of peptides and pharmaceutical compounds. It appears that the achievable selectivity can be ranked in the order RPLC > revHILIC > HILIC with the two different samples. Next, two-dimensional liquid chromatography (2D-LC) conditions were evaluated by combining RPLC, revHILIC, or HILIC with RPLC in an on-line comprehensive (LC × LC) mode. evHILIC × RPLC not only showed impressive performance in terms of peak capacity and sensitivity, but also provided complementary selectivity compared to RPLC × RPLC and HILIC × RPLC. Indeed, both the elution order and the retention time range differ significantly between the three techniques. In conclusion, there is no doubt that revHILIC should be considered as a viable option for 2D-LC analysis of small molecules and also peptides.

Optimizing transfer and dilution processes when using active solvent modulation in on-line two-dimensional liquid chromatography.

Two-dimensional liquid chromatography (2D-LC) is becoming increasingly popular for the analysis of complex samples, which is partly due to the recent introduction of commercial 2D-LC systems. To deal with the mobile phase incompatibility between highly orthogonal retention mechanisms, such as hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC), several strategies have been introduced over the years. One of these strategies is active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension before transfer to the second dimension. This strategy has gained a lot of attention and holds great potential, however, no clear guidelines are currently in place for its use. Therefore, this study aims to investigate how the ASM process can be optimized when using highly incompatible LC combinations, such as HILIC and RPLC, in a simplified selective comprehensive 2D-LC set-up (sHILIC x RPLC) to suggest guidelines for future users. Using a representative sample, the dilution factor (DF), the duration of the ASM phase, the filling percentage of the sample loops, and their unloading configuration are investigated and optimized. It is observed that a DF of 10 with an optimal ASM phase duration, a sample loop filling of maximum 25%, and an unloading configuration in backflush mode, result in the best peak shapes, intensities, and recoveries for early eluting compounds, while keeping the total analysis time minimal. Based on these results, some general recommendations are made that could also be applied in other 2D-LC modes, such as comprehensive 2D-LC (LC x LC), heart-cutting 2D-LC (LC-LC), and other chromatographic combinations with mobile phase incompatibility issues.

Sense and nonsense of shifting gradients in on-line comprehensive reversed-phase LC × reversed-phase LC.

In on-line comprehensive two-dimensional liquid chromatography (LC × LC), the combination of similar chromatographic modes such as reversed-phase liquid chromatography × reversed-phase liquid chromatography (RPLC × RPLC) usually leads to the partial occupation of the available separation space. A possible solution to circumvent this issue may be to dynamically adjust the gradient elution in the second dimension (2D) throughout the LC × LC analysis. This allows the gradient elution to be tailored to the elution conditions of each fraction instead of using a conventional full gradient program in which the same gradient profile is repeated for each 2D-fraction. In this study, an online RPLC × RPLC method is optimized with shifting gradients in 2D. The logic behind implementing such programs in on-line LC × LC is explained. The optimized method consists of a combination of segmented and shifted gradient modes. It is shown that the retention space coverage can be increased by 50 % compared to a conventional full gradient program, leading to a significant increase in peak capacity (about 35 %). However, such an increase comes at the expense of larger peak widths in 2D and thus lower peak intensities. It is shown here that the use of shifting gradients raises another serious issue related to the repeatability of retention times between two successive 2D-separations.

Development of a sub-hour on-line comprehensive cation exchange chromatography x RPLC method hyphenated to HRMS for the characterization of lysine-linked antibody-drug conjugates.

This study details the development of on-line two-dimensional liquid chromatography (2D-LC) methods combining cation-exchange chromatography (CEX) and reversed-phase liquid chromatography (RPLC) for the separation of the charge variants of a lysine-linked antibody-drug conjugate (ADC). This combination gives an excellent example of the potential benefits of 2D-LC approaches for the analysis of such complex protein formats. CEX is considered the reference technique for the separation of protein charge variants but its retention mechanism usually requires the use of a high concentration of non-volatile salts, which impedes its compatibility with MS detection. In this context, the use of an on-line 2D-LC-MS approach not only allows on-line desalting and indirect coupling of CEX with mass spectrometry (MS) detection but it also provides increased and complementary information within a single analysis. The first part of this study was devoted to the choice of stationary phases and the optimization of chromatographic conditions in both dimensions. Based on the results obtained in 1D-CEX with ultraviolet detection (UV) and 1D-RPLC with UV and high-resolution mass spectrometry (HRMS) detections, an on-line comprehensive two-dimensional liquid chromatography method combining CEX and RPLC was developed. The last part of this study was devoted to the identification of the separated species using HRMS detection and in the comparison of three ADC samples exposed to different durations of thermal stress.

Strategies to circumvent the solvent strength mismatch problem in online comprehensive two-dimensional liquid chromatography.

On-line comprehensive two-dimensional liquid chromatography is a powerful technique for the separation of highly complex samples. Due to the addition of the second dimension of separation, impressive peak capacities can be obtained within a reasonable analysis time compared to one-dimensional liquid chromatography. In online comprehensive two-dimensional liquid chromatography, the separation power is maximized by selecting two separation dimensions as orthogonal as possible, which most often requires the combination of different mobile phases and stationary phases. The online transfer of a given solvent from the first dimension to the second dimension may cause severe injection effects in the second dimension, mostly due to solvent strength mismatch. Those injection effects may include peak broadening, peak distortion, peak splitting or breakthrough phenomenon. They are often found to reduce significantly the peak capacity and the peak intensity. To overcome such effects, arising specifically in online comprehensive two-dimensional liquid chromatography, different methods have been developed over the years. In this review, we focused on the most recently reported ones. A critical discussion, supported by a theoretical approach, gives an overview of their advantages and drawbacks.

A comprehensive study on the phenomenon of total breakthrough in liquid chromatography.

Differences in elution strength between the sample solvent and the mobile phase usually give rise to undesirable effects on the chromatographic separation, which may range from slight broadening to severe peak deformation or even splitting. In the most extreme case, the retention factor of the analyte at the head of the column is so small at the time of injection that part of the analyte goes through the column with very little interaction with the stationary phase and hence elutes very close to the column dead time. This phenomenon is known as breakthrough. Usually, during breakthrough, the retained peak displays a wide array of deformations and it is not rare that multiple retained peaks appear for a given injected analyte. However, under certain conditions, it has been demonstrated that these deleterious effects could fully disappear, leaving only one breakthrough peak and one symmetrical peak on the chromatogram. This so-called "total breakthrough" phenomenon was recently highlighted in the specific context of the 2D-LC separation of peptides but has yet to be explained. In the present paper, we describe the results of a comprehensive study aiming to better understand and define the conditions of emergence of both breakthrough and total breakthrough phenomena in liquid chromatography. The effects of a broad range of parameters, including the nature of the solute, the retention mechanism, the injection and elution conditions, the column temperature, and the injected sample concentration on the occurrence of both phenomena were investigated. While breakthrough was found to occur for all studied compounds, it appears that the presence of positive charges on the molecule is a prerequisite for observing a total breakthrough phenomenon. Among all the parameters investigated in this work, only the injection conditions and the analyte retention were found to be impactful on the onset of both phenomena. This finding allowed us to suggest one necessary and sufficient condition, relying on the injection of critical volumes to observe each respective phenomenon. These critical volumes only depend on the column dead volume and the retention factor of the analyte in the injection solvent.

Detailed numerical study of the peak shapes of neutral analytes injected at high solvent strength in short reversed-phase liquid chromatography columns and comparison with experimental observations.

We report on a numerical investigation of the different steps in the development of the spatial concentration profiles developing along the axis of a liquid chromatography column when injecting large relative volumes (>10 to 20% of column volume) of analytes dissolved in a high solvent strength solvent band as can be encountered in the second dimension (2D) column of a two-dimensional liquid chromatography (2D-LC) system. More specifically, we made a detailed study of the different retention and the axial band broadening effects leading to the double-headed peak shapes or strongly fronting peaks that can be experimentally observed under certain conditions in 2D-LC. The establishment of these intricate peak profiles is discussed in all its fine, mechanistic details. The effect of the volume of the column, the volume and the shape of the sample band, the retention properties of the analyte and the band broadening experienced by the analytes and the sample solvent are investigated. A good agreement between the simulations and the experimental observations with caffeine and methylparaben injected in acetonitrile/water (ACN/H2O) mobile phase with different injection volumes is obtained. Save the difference in dwell volume, key features of experimental and simulated chromatograms agree within a few %. The simulations are also validated against a number of simple mathematical rules of thumb that can be established to predict the occurrence of a breakthrough fraction and estimate the amount of breakthrough.

Comparison of existing strategies for keeping symmetrical peaks in on-line Hydrophilic Interaction Liquid Chromatography x Reversed-Phase Liquid Chromatography despite solvent strength mismatch.

In two-dimensional liquid chromatography, the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) is very attractive due to the complementarity of their separation mechanisms. On-line comprehensive HILIC x RPLC is well-known to give rise to a large retention space coverage when dealing with ionisable compounds. However, method development in on-line HILIC x RPLC is challenging due to the reversed solvent strength between both dimensions, which can greatly affect the peak shapes in the second RPLC dimension, and thus the separation quality and the method sensitivity. In the present contribution, we compared four strategies designed to avoid this problem: (1) flow splitting, which consists in reducing the injection volume in the second dimension (2D), (2) on-line dilution with a make-up flow and (3) on-line dilution with Active Solvent Modulation (ASM), which both consist in reducing the solvent strength of the injected fractions, and (4) Total Breakthrough Strategy, which we recently proposed. Unlike the three preceding strategies, this latter one consists in injecting large volumes of strong solvent in 2D. The performance of each strategy was evaluated for sub-hour separations of a tryptic digest in on-line HILIC x RPLC. In this work, we considered the critical case for which the same column internal diameters (i.e. 2.1 mm here) are used in both dimensions. Peak capacity, peak shapes and peak intensities were considered for this evaluation. The highest peak capacity could be achieved with Total Breakthrough Strategy while the lowest one with on-line dilution using ASM. Peak intensities were usually higher with on-line dilution approaches (make-up flow and ASM). However, despite the presence of breakthrough, peak intensities were approximately 7-fold higher with Total Breakthrough Strategy than with flow splitting.

Two-Dimensional Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for the Analysis of ADCs.

From a structural point of view, the complete characterization of ADCs is a challenging task due to their high complexity. ADCs combine the heterogeneity of the initial antibody to the variability associated with the conjugation strategy, the manufacturing process, and the storage. Given the inherent complexity of these biomolecules, online comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive technique to address the challenges associated with ADC characterization. Compared to conventional one-dimensional liquid chromatography techniques (1D-LC), LC × LC combines two different and complementary separation systems. In the context of ADC analysis, LC × LC has been proven to be a rapid and efficient analytical tool: (1) to provide a higher resolving power by increasing the overall peak capacity and thus allowing to gain more information within a single run and (2) to allow mass spectrometry (MS) coupling with some chromatographic techniques that are not MS-compatible and hence to facilitate the structural elucidation of ADCs. In this chapter, we present the coupling of different chromatographic techniques including hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophilic liquid chromatography (HILIC). The interest of HIC × SEC, SEC × SEC, HIC × RPLC, IEX × RPLC, RPLC × RPLC, and HILIC × RPLC, all hyphenated to high-resolution mass spectrometry (HRMS), is discussed in the context of the characterization of ADCs.

Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples.

In the present work, we have investigated the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC) for the separation of peptides in on-line HILIC x RPLC. This combination usually leads to significant solvent strength mismatch, since a weak solvent in HILIC becomes a strong solvent in RPLC. This may result in band broadening, peak distortion, and breakthrough phenomena. Our focus was directed towards the reduction of band broadening and peak distortion. The conditions of the emergence of breakthrough could be investigated with high resolution mass spectrometry (HRMS) detection. The importance of both the injection volume and the difference in composition between injection and elution solvents was highlighted. Reported strategies to avoid bad peak shapes mostly rely either on flow splitting to limit the injection volume, or on on-line dilution. Here, we propose an alternative approach which consists in injecting large volumes in the second dimension. In this case, no flow-splitting nor dilution prior to the second dimension is required. Our results show that above a certain critical injected volume, depending on both the compound and the elution conditions, narrow and symmetrical peaks can be obtained, despite the persistence of breakthrough. As a result, the injected volume in the second dimension must be larger than the largest critical volume. This counter-intuitive approach was applied for the on-line HILIC x RPLC-UV-HRMS analysis of a complex tryptic digest sample. A peak capacity close to 1500 could be achieved in 30 min, which is two-fold higher than in RPLC x RPLC within the same analysis time.