HPLC 2025 Preview: Boosting the Separation Power of LC×LC

The article discusses advancements in two-dimensional liquid and gas chromatography (LC×LC and GC×GC) to address the challenges of analyzing complex samples in non-target analysis. Traditional chromatography struggles with ion suppression and mixed spectra, particularly in complex samples, making identification difficult. LC×LC and GC×GC improve separation by using different stationary phases for enhanced resolution. Recent developments, such as multi-2D-LC×LC, combine different phases to optimize separation. However, the method’s complexity and optimization challenges limit its acceptance. Innovations, such as multi-task Bayesian optimization or feature clustering aim to enhance performance and make the technique more acceptable for wider use.

Oliver J. Schmitz and Katharina Wetzel © Image courtesy of authors

Partly—but not exclusively—due to the -omics research of recent years, the demands on analytical measurement have increased enormously. The attempts by industry to establish a circular economy will further increase the demands on analytics in the future. Not only are the samples to be analyzed becoming ever more complex and numerous, but an all-encompassing non-target analysis is also increasingly required. While there is a gold standard for target analysis, namely gas chromatography (GC) or high performance liquid chromatography (HPLC) coupled with a triple-quad mass spectrometer (MS), the situation in non-target analysis is much more difficult. Here, HPLC with an ultrahigh-resolution mass spectrometry technique such as orbital ion trap-MS, high-resolution (quadrupole time-of-flight [QTOF])-MS or a GC coupled with a quadrupoleMS or TOF-MS is currently used the most frequently. However, the complexity of these samples means that baseline separation in upstream one-dimensional chromatography is not possible in the vast majority of cases, which is why the use of atmospheric pressure mass spectrometers (electrospray ionization [ESI], atmospheric pressure chemical ionization [APCI], atmospheric pressure photoionization [APPI]) leads to ion suppression and mixed spectra, which can often make identification more difficult. In GC–MS with electron impact ionization (EI), ion suppression plays a smaller role, but inadequate chromatographic pre-separation can lead to mixed spectra, which, because of the hard ionization and the associated strong fragmentation by EI, leads to unevaluable mixed spectra, so that the otherwise very useful spectral databases cannot make a contribution to the identification of the analytes. Two-dimensional chromatography, both in liquid and gas form, can provide a remedy here. Here, the different interactions of the analytes with the stationary phase (hydrophobic, dipole-dipole, ionic, π-π , or steric interactions, as well as hydrogen bonding) are utilized. Classical two-dimensional LC (LC-LC) and GC (GC-GC), in which individual fractions from the first separation dimension are transferred to a second separation column and further separated there, have been used successfully for many decades.

However, there are two disadvantages with classical two-dimensional chromatography. First, only a few fractions can be transferred from the 1^st to the 2^nd dimension, as a new fraction can only be injected once the previous analysis in the 2^nd dimension has been completed. Second, there is no guarantee that any pre-separation in the 1^st dimension will be retained in the 2^nd dimension, which may also lead to a reduction in separation performance. The latter can be counteracted by transferring only very small fractions (individual peaks) to the second dimension. The number of fractions to be analyzed in one analysis run could be drastically increased by the multiple heart-cutting method presented by Pursch and Buckenmaier (1), in which the individual fractions can be stored in several loops and successively transferred to the second dimension. This method is now used very successfully in many applications, particularly in the pharmaceutical industry. However, heart-cutting techniques are not suitable for an all-encompassing analysis of a complex sample. The most effective chromatographic method for this problem to date is comprehensive two-dimensional gaschromatography (GC×GC) and comprehensive two-dimensional liquid (LC×LC) chromatography. Both methods were first published almost simultaneously, with LC×LC being introduced by Bushey and Jorgenson in 1990 (2) and GC×GC by Liu and Phillips in 1991 (3). Over the last few decades there have been many advances in LC×LC and GC×GC, involving both the modulator and data interpretation; for further reading the reviews by Mondello et al. (4,5) are recommended.

Combinations of two different reversed-phase phases were used in LC×LC in the early years (6); now hydrophilic interaction liquid chromatography (HILIC) phases are increasingly being used in one of the two dimensions. However, these column combinations experience the same problem, namely that the elution force of the eluate from the first dimension is often too strong to focus the analytes at the head of the column in the second dimension. In recent years, this has led to several modifications in modulation and a commercial modulator, the active solvent modulator (ASM), which addresses this problem (7,8). Here, the elution force is reduced in various ways by adding a solvent (water for RP phase and acetonitrile for HILIC phase in the 2^nd dimension). One problem with complex samples comprised of analytes over a wide polarity range is that polar analytes in the second dimension can be separated well with HILIC but poorly with an RP phase, while the reverse is true for non-polar analytes. For this reason, multi-2D LC×LC was recently introduced, in which a six-way valve can be used to select between a HILIC or an RP phase as the 2^nd dimension depending on the analysis time in the first dimension (9), which significantly improves the separation performance (Figure 1).

Figure 1: LC×LC and multidimensional 2D LC×LC (bottom left) analysis of Sambucus nigra.

Recently, various approaches have been pursued to further increase separation performance. For example, attempts are currently being made to generate a comprehensive spatial three-dimensional liquid-phase separation technology platform using 3D-printing, which would be characterized by peak capacities of over 30,000 within one hour (10).

Another way to further increase the separation performance would be to couple the LC×LC system with an ion mobility mass spectrometer instead of a TOF-MS. However, this would mean a four-dimensional separation method (two retention times, one drift time and one mass-to-charge ratio value). To be able to display and reasonably evaluate these complex data, however, the data dimension needs to be reduced by feature clustering, which is currently being developed in my working group.

Despite all the improvements in LC×LC and the increasing use of this high performance separation technology, there is still a major acceptance problem. This may be mainly the result of the complex method optimization, which requires experienced users with a sound knowledge of chromatography. Various working groups have addressed this problem and have attempted to simplify method optimization, for example, by using multi-task Bayesian optimization (11). More innovation can certainly be expected here in the future, which will hopefully further increase the acceptance of LC×LC.

HPLC 2025 Talk: Boosting the Separation Power of LCxLC (KN02)

MO-01 – Multidimensional LC

Monday, June 16, 2025 08:30–10:15 AM

References

(1) Pursch, M.; Buckenmaier, S. Loop-Based Multiple Heart-Cutting Two-Dimensional Liquid Chromatography for Target Analysis in Complex Matrices. Anal. Chem. 2015, 87 (10), 5310–5317. DOI: 0.1021/acs.analchem.5b00492

(2) Bushey, M. M.; Jorgenson, J. W. Automated Instrumentation for Comprehensive Two-Dimensional High-Performance Liquid Chromatography of Proteins. Anal. Chem. 1990, 62, 161–167. DOI: 10.1021/ac00201a015

(3) Liu, Z.; Phillips, J. B. Comprehensive Two-Dimensional Gas Chromatography Using an On-Column Thermal Modulator Interface. J. Chromatogr. Sci. 1991, 29 (6) 227–231. DOI: 10.1093/chromsci/29.6.227

(4) Mondello, L.; Dugo, P.; Donato, P.; et al. Comprehensive Two-dimensional Liquid Chromatography. Nature Reviews Methods Primers 2023, 3, 86. DOI: 10.1038/s43586-023-00269-0

(5) Mondello, L.; Cordero, C.; Janssen H.-G.; et al. Comprehensive Two-dimensional Gas Chromatography-Mass Spectrometry. Nature Reviews Methods Primers 2025, 5, 7. DOI: 10.1038/s43586-024-00379-3

(6) Li, D.; Jakob, C.; Schmitz, O. J. Review: Practical Considerations in Comprehensive Two-dimensional Liquid Chromatography Systems (LCxLC) with Reversed-phases in Both Dimensions. Anal. Bioanal. Chem. 2015, 407, 153–167. DOI: 10.1007/s00216-014-8179-8.

(7) Chen, Y.; Montero, L.; Schmitz, O. J. Advance in On-line Two-dimensional Liquid Chromatography Modulation Technology. TRAC 2019, 120, 115647. DOI: 10.1016/j.trac.2019.115647

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

(9) Montero, L.; Ayala-Cabrera, J. F.; Bristy, F. F.; Schmitz, O. J. Multi-2D LC × LC as a Novel and Powerful Implement for the Maximum Separation of Complex Samples. Anal. Chem. 2023, 95, 3398–3405. DOI: 10.1021/acs.analchem.2c04870

(10) Eeltink, S.; De Vos, J.; Desmet, G. Toward Unrivaled Chromatographic Resolving Power in Proteomics: Design and Development of Comprehensive Spatial Three-Dimensional Liquid-Phase Separation Technology. Annu. Rev. Anal. Chem. 2024, 17, 475–93. DOI: 10.1146/annurev-anchem-061522-044510

(11) Boelrijk, J.; Molenaar, S. R. A; Bos, T. S.; et al. Enhancing LC×LC Separations Through Multi-task Bayesian Optimization. J. Chromatogr. A 2024, 1726, 464941. DOI: 10.1016/j.chroma.2024.464941

Oliver J. Schmitz has been a full professor at the University of Duisburg-Essen, Germany, since 2012, and is the chair of the Institute of Applied Analytical Chemistry. In 2018, he founded, together with Agilent, the Teaching and Research Center for Separation (TRC) at the University of Duisburg-Essen. Schmitz´s main research area is separation science, with a particular focus on non-target analysis of complex samples, the development of ion sources, the use and optimization of multidimensional LC and GC, ion mobility-mass spectrometry, and metabolomics.

Katharina Wetzel is currently a PhD student in the working group Applied Analytical Chemistry of Oliver J. Schmitz at the University of Duisburg-Essen, Germany. The focus of her research is the analysis of complex samples using two-dimensional liquid chromatography in general and in particular of European medicinal plants as well as sustainable extraction techniques.