Elsevier

Analytica Chimica Acta

Volume 1252, 29 April 2023, 341040
Analytica Chimica Acta

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

https://doi.org/10.1016/j.aca.2023.341040Get rights and content

Highlights

  • Optimization strategy for active solvent modulation in sHILICxRPLC

  • Optimization of transfer and dilution parameters investigated in depth.

  • General recommendations for similar methods are made.

Abstract

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.

Introduction

Despite significant efforts that have been made to improve the performance of one-dimensional liquid chromatography (1D-LC), such as the introduction of small particle packed columns operated at ultra-high pressure (UHPLC) [1], highly efficient core–shell particles [2], monolithic stationary phases [3], and operation at elevated temperatures [4], complex samples generally still result in (fully or partially) overlapping peaks, even for the most efficient (and lengthy) 1D-LC separations [5]. By using two-dimensional LC (2D-LC), which combines two LC separation systems into one method, higher resolving power and peak capacities are achievable, especially when complementary separation systems (operated via different retention mechanisms) are used [[6], [7], [8], [9], [10], [11]]. For this reason, and with the availability of reliable, commercial 2D-LC instrumentation and improved data analysis software nowadays, 2D-LC is increasingly used for the separation of complex samples, as encountered in environmental analysis [12], food analysis [[13], [14], [15]], (bio-)pharmaceutical analysis [[16], [17], [18]], polymer analysis [19], proteomics [20], and other applications [21,22].

In 2D-LC, analyte-containing fractions of the effluent of the first-dimension (1D)-column are transferred to a second-dimension (2D)-column for further separation [5]. In on-line 2D-LC, this transfer takes place in an automated manner, by means of switching valves equipped with multiple sample loops, which leads to a better reproducibility, reduced sample loss and contamination, and faster overall analysis time, compared to off-line 2D-LC [9,10]. Despite these advantages, online hyphenation is constrained by additional considerations, such as the temporal and physical connection between the two dimensions, which brings additional complexity to the method development [9,[23], [24], [25]].

To maximize the resolving power of 2D-LC methods, the selectivity of the two combined LC modes must be sufficiently complementary. This complementarity is usually achieved by selecting suitable mobile phases and stationary phases and by using markedly different retention mechanisms in the two dimensions. One of the main difficulties encountered when combining complementary chromatographic modes is to deal with their mobile phase incompatibility [26,27]. This is typically the case when combining hydrophilic interaction liquid chromatography (HILIC) with reversed-phase LC (RPLC), a combination that is quite attractive for the analysis of samples containing compounds with a broad range of polarities, as encountered in environmental analysis, natural product analysis, drug discovery, metabolomics, and proteomics [[28], [29], [30], [31], [32], [33]]. This specific combination is challenging due to the opposite elution strengths of the mobile phases used in both dimensions [32]. The mobile phase in HILIC typically contains a high percentage of acetonitrile (ACN), which is a strong eluent in RPLC. The transfer of ACN-rich fractions from 1D-HILIC, combined with the effect of a much larger injection volume compared to 1D-LC, can negatively impact the peak shapes in 2D-RPLC [26,29,34]. Typical effects include band broadening, peak distortion, and/or analyte breakthrough, which affects the 2D-separation by decreasing both peak capacity and peak intensity [26,29].

To deal with this mobile phase incompatibility problem, different strategies have been explored over the years [[35], [36], [37]]. These include approaches based on modifying the composition and/or the volume of the fractions transferred from the first to the second dimension using either flow splitting [[38], [39], [40], [41]], in-line mixing [42], on-line dilution (with trapping columns [29,43,44] or sample loops/capillaries [32,[45], [46], [47]]), or solvent evaporation [48,49].

Another strategy to address the mobile phase incompatibility problem, called active solvent modulation (ASM), was recently developed by Stoll et al. [50]. This valve-based approach enables the on-line dilution of the 1D-effluent with a weak (dilution) solvent (with respect to the 2D-column) prior to transfer to the 2D-column, by using restriction capillaries. With ASM, the dilution solvent is provided by the same pump as used for the 2D-separation and unlike other on-line dilution strategies, no additional pump is required for fraction dilution [51] or transfer [52]. The total 2D-cylce time includes an ASM phase, during which an isocratic hold takes place before the start of the 2D-gradient to allow the dilution of the transferred fractions [26]. This can result in peak focusing on the 2D-column head and improvements in peak shape and sensitivity in the second dimension [29,50,53]. With the currently available ASM hardware, the transferred fractions can be diluted up to a maximum ratio of 5.1 [35]. More details regarding the working mechanism of ASM are provided in the experimental and results sections. ASM has been applied in 2D-LC for a variety of applications including the characterization of therapeutic antibodies [18,54] and synthetic oligonucleotides [55], the determination of small molecule target compounds in polymeric matrices [53], the study of monomer incorporation in copolymer dispersants [56], and the analysis of polar and non-polar vitamins [57]. However, clear recommendations or guidelines regarding the optimization of the ASM conditions, such as the dilution factor (DF), the unloading configuration of the sample loops, the duration of the ASM phase, and the filling percentage of the sample loops used at the interface to collect the 1D-fractions, are not readily available.

Therefore, this study investigates the effect of possible optimization parameters for ASM for the combination of two highly orthogonal separation mechanisms with incompatible mobile phases (HILIC and RPLC), to suggest guidelines for future users. In the current work, the study is conducted in the selective comprehensive 2D-LC (sLC x LC) mode, also called high-resolution sampling (HiRes), wherein targeted regions of the 1D-chromatogram are quantitatively transferred into the 2D by a series of relatively small consecutive cuts [58]. Using a simplified setup to mimic HILIC conditions combined with a RPLC-separation in 2D, and a representative sample containing six typical organic micropollutants (OMPs) with a large variety in polarity, the different parameters are investigated and optimized, in order to maximize both the peak capacity and the peak intensity in the second dimension. To evaluate the impact of each of these parameters, peak shapes, peak widths, peak intensities, and recoveries are determined and compared for each studied configuration. To illustrate the improvement in 2D-peak shapes after this optimization, a real sHILIC x RPLC experiment is carried out for a more complex sample consisting of 45 OMPs.

Section snippets

Chemicals

Caffeine, progesterone, estrone, gabapentin, pipamperone, hydrochlorthiazide, ethinylestradiol, bezafibrate, testosterone, atenolol, β-estradiol, metolachlor, (4)-nonylphenol and ketoprofen were obtained from Sigma-Aldrich (Diegem, Belgium), sulfamethoxazole from Roche (Mannheim, Germany), guanylurea, theophylline, metformin, and naproxen from Thermo Fisher Scientific (Geel, Belgium), ibuprofen and lidocaine from Certa (Braine-l’Alleud, Belgium), lorazepam from Fagron (Waregem, Belgium),

Interface without ASM

In 2D-LC, the process of cutting fractions of 1D-effluent and transferring them to the 2D-column is referred to as “modulation”. The number and volume of the collected fractions are important determinants for the quality of the 2D-LC separation. For all modes of 2D-LC separation in use today, it is most common to transfer the 1D-effluent to the 2D-column using a simple open tubular capillary (sample loop). First, the 1D-effluent flows from the 1D-column outlet into the capillary during a

Conclusion

Considering the high degree of orthogonality between HILIC and RPLC, 2D-LC methods combining these two separation methods are of interest to analyze complex samples containing both polar and non-polar compounds, as encountered for example in environmental analysis. ASM is a possible method to deal with the mobile phase incompatibility between HILIC and RPLC. However, clear recommendations or guidelines regarding the optimization of the 2D-LC interface and ASM conditions are not available.

CRediT authorship contribution statement

Marie Pardon: Data curation, Formal analysis, Investigation, Visualization, Validation, Writing – original draft. Soraya Chapel: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Visualization, Validation, Writing – original draft, Writing – review & editing. Peter de Witte: Funding acquisition, Project administration, Supervision, Writing – review & editing. Deirdre Cabooter: Conceptualization, Formal analysis, Funding acquisition, Investigation,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Marie Pardon is funded by the Research Foundation Flanders (FWO) (SBO research project “SmartDetox”, project ID: 3E200795). Soraya Chapel is funded by the joint-initiative of the Research Foundation Flanders (FWO) and the Walloon Fund for Scientific Research (FNRS) (EOS – research project “Chimic” (EOS ID: 30897864)).

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