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Solving the dilution problem in LC×LC via exploitation of the solute refocusing effect in TRLC×RPLC

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Abstract
Achievable sensitivity of online 2D-LC systems is still hampered compared to conventional (U)HPLC, as sensitivity is often traded off to increase peak capacity. This limitation is mostly imposed by the modulation problem. To maximize orthogonality, less compatible separation modes are combined. Thus, solvents of high elution-strength are transferred between the two dimensions. A possible solution is the use of narrow columns in the 1D, maintained at low flow rates to naturally decrease the transferred volumes, but consequently, sample-loading capacities are also lowered. To minimize these effects and allow for fast gradients, the second dimension (2D) column then often must be rather broad, operated at high flow rates. The combination of a 1 mm column in the 1D paired with a 4.6 mm column in the 2D, however, leads to a >20-fold loss in sensitivity for concentration-sensitive detectors due to the dilution of the sample in the 2D flow when assuming comparable flow velocities. The latter is in reality even further exacerbated in LC×LC due to the shorter residence times in the fast flow operated second dimension. RPLC×RPLC allows for some more freedom in column choices, nowadays more often a 2 mm ID column is combined with a 3 mm ID column, also lowering detection sensitivity. Rarely, 2 mm columns are implemented in the 2D, as this is mostly only applicable if aqueous mobile phases are used in the 1D, e.g in IEX×RPLC. Nevertheless, it was shown that 2D-LC is promising, also for the determination of low abundant impurities at a 0.05% level in pharmaceutical quality control if methodical parameters are optimized for high sensitivity. Here, temperature-responsive liquid chromatography (TRLC) is paired with RPLC. Such stationary phases allow for separation in a purely aqueous mobile phase, which solves some of the methodical complexities discussed above. Because full solute refocusing at the 2D column head is obtained in TRLC×RPLC, the transferred volume of 1D effluent is not a determining factor. Thus, the dilution problem experienced with all concentration-sensitive detectors in comprehensive 2D-LC offers the prospect to be overcome, as columns of the same diameter in both dimensions can be used (2.1×2.1 mm I.D.). Optimally, the detrimental dilution problem can even be inversed towards a refocusing based sensitivity enhancement, by combining a broader 1D column with a narrower 2D column (2.1×1 mm I.D.). In this work these aspects are demonstrated through comparative LOD determinations for various column I.D. combinations in comprehensive TRLC×RPLC-UV. Addiontally, split-less transfer of the 2D effluent to the MS from the 1 mm column is introduced. This strategy allows more facile and more sensitive 2D-LC-MS/UV implementations.

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MLA
Wicht, Kristina, et al. “Solving the Dilution Problem in LC×LC via Exploitation of the Solute Refocusing Effect in TRLC×RPLC.” 17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts, 2022.
APA
Wicht, K., Baert, M., Schipperges, S., von Doehren, N., de Villiers, A., & Lynen, F. (2022). Solving the dilution problem in LC×LC via exploitation of the solute refocusing effect in TRLC×RPLC. In 17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts. Ghent, Belgium.
Chicago author-date
Wicht, Kristina, Mathijs Baert, Sonja Schipperges, Norwin von Doehren, André de Villiers, and Frederic Lynen. 2022. “Solving the Dilution Problem in LC×LC via Exploitation of the Solute Refocusing Effect in TRLC×RPLC.” In 17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts.
Chicago author-date (all authors)
Wicht, Kristina, Mathijs Baert, Sonja Schipperges, Norwin von Doehren, André de Villiers, and Frederic Lynen. 2022. “Solving the Dilution Problem in LC×LC via Exploitation of the Solute Refocusing Effect in TRLC×RPLC.” In 17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts.
Vancouver
1.
Wicht K, Baert M, Schipperges S, von Doehren N, de Villiers A, Lynen F. Solving the dilution problem in LC×LC via exploitation of the solute refocusing effect in TRLC×RPLC. In: 17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts. 2022.
IEEE
[1]
K. Wicht, M. Baert, S. Schipperges, N. von Doehren, A. de Villiers, and F. Lynen, “Solving the dilution problem in LC×LC via exploitation of the solute refocusing effect in TRLC×RPLC,” in 17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts, Ghent, Belgium, 2022.
@inproceedings{8725919,
  abstract     = {{Achievable sensitivity of online 2D-LC systems is still hampered compared to conventional (U)HPLC, as sensitivity is often traded off to increase peak capacity. This limitation is mostly imposed by the modulation problem. To maximize orthogonality, less compatible separation modes are combined. Thus, solvents of high elution-strength are transferred between the two dimensions. A possible solution is the use of narrow columns in the 1D, maintained at low flow rates to naturally decrease the transferred volumes, but consequently, sample-loading capacities are also lowered. To minimize these effects and allow for fast gradients, the second dimension (2D) column then often must be rather broad, operated at high flow rates. The combination of a 1 mm column in the 1D paired with a 4.6 mm column in the 2D, however, leads to a >20-fold loss in sensitivity for concentration-sensitive detectors due to the dilution of the sample in the 2D flow when assuming comparable flow velocities. The latter is in reality even further exacerbated in LC×LC due to the shorter residence times in the fast flow operated second dimension.
RPLC×RPLC allows for some more freedom in column choices, nowadays more often a 2 mm ID column is combined with a 3 mm ID column, also lowering detection sensitivity. Rarely, 2 mm columns are implemented in the 2D, as this is mostly only applicable if aqueous mobile phases are used in the 1D, e.g in IEX×RPLC. Nevertheless, it was shown that 2D-LC is promising, also for the determination of low abundant impurities at a 0.05% level in pharmaceutical quality control if methodical parameters are optimized for high sensitivity.  Here, temperature-responsive liquid chromatography (TRLC) is paired with RPLC. Such stationary phases allow for separation in a purely aqueous mobile phase, which solves some of the methodical complexities discussed above. Because full solute refocusing at the 2D column head is obtained in TRLC×RPLC, the transferred volume of 1D effluent is not a determining factor. Thus, the dilution problem experienced with all concentration-sensitive detectors in comprehensive 2D-LC offers the prospect to be overcome, as columns of the same diameter in both dimensions can be used (2.1×2.1 mm I.D.). Optimally, the detrimental dilution problem can even be inversed towards a refocusing based sensitivity enhancement, by combining a broader 1D column with a narrower 2D column (2.1×1 mm I.D.). In this work these aspects are demonstrated through comparative LOD determinations for various column I.D. combinations in comprehensive TRLC×RPLC-UV. Addiontally, split-less transfer of the 2D effluent to the MS from the 1 mm column is introduced. This strategy allows more facile and more sensitive 2D-LC-MS/UV implementations.}},
  author       = {{Wicht, Kristina and Baert, Mathijs and Schipperges, Sonja and von Doehren, Norwin and de Villiers, André and Lynen, Frederic}},
  booktitle    = {{17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-17), Abstracts}},
  language     = {{eng}},
  location     = {{Ghent, Belgium}},
  title        = {{Solving the dilution problem in LC×LC via exploitation of the solute refocusing effect in TRLC×RPLC}},
  year         = {{2022}},
}