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

Chapel, Soraya; Rouvière, Florent; Heinisch, Sabine

doi:10.1016/j.chroma.2021.462001

Cited by 13 publications

(11 citation statements)

References 35 publications

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“…F I G U R E 9 Hydrophilic interaction chromatography (HILIC) × RPLC separations of a tryptic digest (contour-plots and 3D-plots) highlighting the differences between (A) total breakthrough strategy, (B) flow splitting, and (C) online dilution. Adapted from [47] with permission from Elsevier At first sight, injection effects can be avoided by reducing the volume injected in 2 D, either by reducing the id of the column in 1 D compared to 2 D or by splitting the flow between the two dimensions. However, such strategies always come at the cost of decreased method sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…Very good peak shapes were obtained in 2 D, resulting in an impressive peak capacity of 1500 achieved in 30 min only [63]. Compared to more conventional strategies, such as flow splitting and online solvent dilution, it was shown that this strategy performs better in terms of peak capacity and much better in terms of peak shapes [47], but usually less well in terms of sensitivity than online solvent dilution, due to the presence of a breakthrough peak for all peptides. The HILIC × RPLC separations of a tryptic digest shown in Figure 9 highlight these different features.…”

Section: Total Breakthrough Strategymentioning

confidence: 98%

“…The breakthrough peak is indicated by an asterisk. Adapted from [47] with permission from Elsevier transferred fraction can be diluted up to a maximum ratio of 5.1, which is the current limit of the process. Since the dilution process takes place at the beginning of each modulation, the use of ASM reduces the available time for the separation.…”

Section: Diluting With a Weaker Solvent (Use Of Empty Loops)mentioning

confidence: 99%

“…Similarly, broad and distorted peaks were reported for little retained aromatic compounds in HILIC x RPLC [38] despite online dilution with a countergradient. A recent study on the separation of peptides in HILIC x RPLC [47] showed the occurrence of distorted peaks and breakthrough phenomena with the two dilution approaches based on make-up flow or ASM. As shown in Figure 6, poorly and highly retained peptides were both affected because this effect also depends on the percentage of acetonitrile at elution.…”

Section: Diluting With a Weaker Solvent (Use Of Empty Loops)mentioning

confidence: 99%

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

Chapel

Heinisch

2021

J of Separation Science

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Total Breakthrough Strategymentioning

confidence: 98%

Section: Diluting With a Weaker Solvent (Use Of Empty Loops)mentioning

confidence: 99%

Section: Diluting With a Weaker Solvent (Use Of Empty Loops)mentioning

confidence: 99%

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

Chapel

Heinisch

2021

J of Separation Science

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“…In addition, there are two methodical factors that influence the sensitivity of analysis. This comprises the analyte focusing on the 2 D column head upon fraction transfer, evaluated via the compression factor and the dilution factor (DF) of the method. , The injection compression factor takes the analyte retention on the 2 D column in the 1 D mobile phase and the analyte retention in the 2 D mobile phase into consideration. This relationship describes the additional band broadening due to the injection of 1 D effluent of higher eluotropic strength on the 2 D column, which affects sensitivity and repeatability .…”

Section: Introductionmentioning

confidence: 99%

Enhanced Sensitivity in Comprehensive Liquid Chromatography: Overcoming the Dilution Problem in LC × LC via Temperature-Responsive Liquid Chromatography

et al. 2022

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In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension ( 1 D), which lead to peak broadening in the second dimension ( 2 D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1 D paired with a 3 mm (or broader) column in the 2 D leads to at least a 9-or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2 D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1 D, which is a purely aqueous separation mode. Full solute refocusing at the 2 D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3−2.1−1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2 D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2 D is that it allows split-free coupling of 2 D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.

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Impact of conditioning runs on hydrophilic interaction chromatography repeatability and its application as a second dimension in online comprehensive two‐dimensional liquid chromatography

Gély,

Monneau,

Hologne

et al. 2024

J of Separation Science

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A common separation approach for polar compounds involves coupling reversed‐phase liquid chromatography (RPLC) with hydrophilic interaction chromatography (HILIC) in two‐dimensional chromatography. The higher proportion of acetonitrile used in the HILIC mobile phase, which enhances mass spectrometry detection, encourages its use in the second dimension. Previous studies demonstrated that the HILIC column can be partially equilibrated within very short timeframes without compromising retention time stability, rendering it suitable in online comprehensive two‐dimensional liquid chromatography (LC×LC) setups. In addition, a specific number of conditioning cycles seems necessary to establish stable retention times. Here, the repeatability of HILIC when employed as second dimension in LC×LC was investigated, with a focus on determining the required number of conditioning cycles to achieve repeatable retention times. Various parameters influenced by the LC×LC online modulation system were studied, such as steep gradient slopes up to 8%, and very short equilibration times, less than or equal to dead time, as well as injection volume and solvent, which depend on the first dimension. Finally, the use of HILIC as a second dimension with tailored conditioning runs was applied to the analysis of hyaluronic acid hydrogel digests. The application of an RPLC×HILIC method using five conditioning runs yielded exceptional stability in second‐dimension retention times.

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

Cited by 13 publications

References 35 publications

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

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

Enhanced Sensitivity in Comprehensive Liquid Chromatography: Overcoming the Dilution Problem in LC × LC via Temperature-Responsive Liquid Chromatography

Impact of conditioning runs on hydrophilic interaction chromatography repeatability and its application as a second dimension in online comprehensive two‐dimensional liquid chromatography

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