Tag: Two-dimensional liquid chromatography

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Parallel Gradients 2DLC-HRMS of complex protein digest

2D-LC System
2D-LC System

Investigating the proteins in biological samples can help us understand and identify diseases and improve the effectiveness of medication. To study proteins in these samples, they are typically digested into peptides and subsequently analyzed by liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS).

Comprehensive two-dimensional LC (LC×LC) offers increased separation power over traditional LC methods. However, most common gradient designs require re-equilibration of every second-dimension run, resulting in high flow rate operations to limit the empty separation space. This also limits MS sensitivity as flow splitting is required to handle such flow rates.

In this work, we developed an LC×LC method using a so-called parallel-gradient design, which omits the need for column re-equilibration and enables the use of the entire separation space. Moreover, this allows for lower flow rates and maintains the sensitivity for low-abundant analytes. The parallel-gradient design achieved higher surface coverages and sensitivity at lower effective peak capacities. Most importantly, both methods were applied to analyze a Human IMR90 lung fibroblast cell line digest to assess its applicability to real complex samples. The parallel-gradient method was able to identify significantly more proteins than the current state-of-the-art methods while using the same analysis time and at a lower solvent consumption. The applicability of the parallel-gradient design could be improved even further by shortening the modulation times, as it was not limited by column re-equilibration.

The study is a collaborative work done thanks for the contribution of many colleagues and students. The link to the publication is reported below.

https://doi.org/10.1021/acs.analchem.4c02172

 

 

 

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2D-LC in industry: technological innovations reviewed

2D-LC in industry: technological innovations reviewed
2D-LC in industry: technological innovations reviewed

CAST scientist Rick van den Hurk wrote a review on recent developments in 2D-LC and the use of 2D-LC in industry. He did this under the supervision of Bob Pirok and in collaboration with Matthias Pursh (Dow) and Dwight Stoll (Gustavus Adolphus College).

Two-dimensional liquid chromatography (2D-LC) greatly advances the separation powered of analytical separation sciences through a better peak capacity as well as offering more-tailored selectivity combinations.

However, the field of 2D-LC is, in particular in contrast to 2D-GC, still very immature and under significant development. In 2019, Bob Pirok, Dwight Stoll and Peter Schoenmakers published a review in which they examined the latest trends from 2015 until 2019 [1].

In this recent installment [2], Rick van den Hurk reviewed the recent innovations between 2019 and 2023. In addition the review also devotes significant focus to the implementation of the technique in industry. The review was co-written by Bob Pirok, Dwight Stoll (Gustavus Adolphus College) and Matthias Pursch (Dow).

The authors examined over 200 articles and also compared these with the articles published prior to 2019. In their review, the authors concluded that mobile-phase mismatch continues to be an important focus area for the field, and several modulation strategies and new variants were discussed.

Van den Hurk and co-workers also noticed that a third of the publications had at least one author affiliated with industry. Application fields that particularly demonstrated involvement were the polymer characterization, metabolomics, and pharmaceutical and biopharmaceutical analysis. Furthermore, industrial applications favored the use of heart-cut 2D-LC and largely employed on-line hyphenation. The authors did note that the database was likely to be missing out on a number of industrial works that are not published for confidentiality reasons.

Other important developments were the increased popularity of computer-aided strategies, alternative gradient-elution methods to facilitate modulation, as well as multi-stage, multi-dimensional separations, the latter of which were applied to the characterization of protein therapeutics.

2D-LC in industry: technological innovations reviewed
Figure. Number of applications per application area distributed by non-comprehensive (light blue) and comprehensive (dark blue) applications between 2019 and 2023. Reproduced with permission from [2].

Two-dimensional liquid chromatography is of paramount importance to the PARADISE project of CAST scientist Bob Pirok in which multi-dimensional separation technology is used to achieve separation of highly complex samples. In addition, the project aims to characterize the correlation of different sample properties within a single analytical solution. The outcomes of this recent review are thus of value to the ongoing progress in the PARADISE project. Rick van den Hurk is a PhD candidate in the PARADISE project, which stands for Propelling Analysts by Removing Analytical-, Data-, Instrument- and Sample-related Encumbrances and receives funding from the Dutch Research Council (NWO), as well as a number of public and private organisations. Read more about the PARADISE project here.

In addition, the review was part of Pirok’s UPSTAIRS project, which aims to improve the accessibility of advanced separation technology by developing computational methods to leverage chromatographic theory in an unsupervised workflow. This project also receives funding from NWO.

The work was published in TrAC Trends in Analytical Chemistry as open access, and can thus be accessed for free here.

References

  1. Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications, B.W.J. Pirok, D.R. Stoll and P.J. Schoenmakers, Anal. Chem., 2019, 91(1), 240-263, DOI: 10.1021/acs.analchem.8b04841
  2. Recent trends in two-dimensional liquid chromatography, R.S. van den Hurk, M. Pursch, D.R. Stoll, B.W.J. Pirok, TrAC Trends in Analytical Chemistry, 2023, 166, 117166, DOI: 1016/j.trac.2023.117166
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Parallel gradients as alternative to shifted gradients in 2D-LC

In pursuit of full usage of the two-dimensional separation space as prescribed by Giddings [1], the LC×LC chromatographic community is continuously scouting for new methods that yield fully orthogonal separations. With the rich and diverse LC toolkit of available retention mechanisms, chromatographers mainly focus on improving the compatibility of orthogonal – yet incompatible – separation methods. This has spurred the development of active-modulation techniques such as stationary-phase-assisted modulation [2] and active-solvent modulation (ASM) [3]. Ultimately, this angle of innovation is mainly driven by the selection of stationary -and mobile phases, as well as their underlying retention mechanisms.

Rather than fine-tuning selectivity, another branch focuses on tweaking retention factors. For LC×LC separations, this has led to the introduction of shifted gradients. Here, the second-dimension gradient is altered and adapted as a function of the first-dimension gradient program [4]. While extremely effective, the optimization of shifted-gradient assemblies introduces additional complexity to the already more-complex method development process for comprehensive 2D-LC.

The situation of LC×LC contrasts heavily with that of GC×GC. For GC×GC, orthogonal separations are extremely difficult if not impossible due to analyte volatility. As a consequence, wrap-around effects are frequently generated, yet this is rather seen as advantage than disadvantage.

Prof. Tadeusz Gorecki (University of Waterloo, Canada) thus set to investigate what would happen if the same approach would be applied in LC×LC [5]. The project was an international collaboration with Alshymaa Aly (Minia University, Egypt, and University of Waterloo, Canada), Prof. Andre de Villiers and Magriet Muller from Stellenbosch University in South Africa, and Bob Pirok from the CAST team at the University of Amsterdam in the Netherlands.

RPLC×RPLC separations were simulated based on experimental data using the MOREPEAKS framework (formerly PIOTR [6]). Predicted separations using shifted gradients and parallel gradients were compared. The results suggested that parallel gradients indeed may advantageous. To verify this assessment, optimized experimental methods were executed and the resulting separations compared.

Supported by both experimental data and theoretical simulations, the authors concluded that non-orthogonal separation mechanisms could still yield good separation methods in LC×LC.

References

[1] Two-dimensional separations: concept and promise. J.C. Giddings, Anal. Chem. 1984, 56(12), 1258A–1270A, DOI: 10.1021/ac00276a003

[2] Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications. B.W.J. Pirok, D.R. Stoll and P.J. Schoenmakers, Anal. Chem., 2019, 91(1), 240-263, DOI: 10.1021/acs.analchem.8b04841

[3] Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography. D.R. Stoll, K. Shoykhet, P. Petersson, and S. Buckenmaier, Anal. Chem. 2017, 89(17), 9260–9267, DOI: 10.1021/acs.analchem.7b02046

[4] Optimizing separations in online comprehensive two-dimensional liquid chromatography
B.W.J. Pirok, A.F.G. Gargano and P.J. Schoenmakers, J. Sep. Sci., 2018, 41(1), 68–98, DOI: 10.1002/jssc.201700863

[5] Parallel gradients in comprehensive multidimensional liquid chromatography enhance utilization of the separation space and the degree of orthogonality when the separation mechanisms are correlated. A.A. Aly, M. Muller, A. de Villiers, B.W.J. Pirok, T. Górecki, J. Chromatogr. A, 1628, 2020, 461452, DOI: 10.1016/j.chroma.2020.461452

[6] Program for the interpretive optimization of two-dimensional resolution. B.W.J. Pirok, S. Pous-Torres, C. Ortiz-Bolsico, G. Vivó-Truyols and P.J. Schoenmakers, J. Chromatogr. A, 2016, 1450, 29–37, DOI: 10.1016/j.chroma.2016.04.061