Theory and Practice of Two‐Dimensional Liquid Chromatography Separations Involving the HILIC Mode of Separation

Groskreutz, Stephen R.; Stoll, Dwight R.

doi:10.1002/9781118495247.ch9

Cited by 2 publications

(1 citation statement)

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“…Hydrophilic interaction liquid chromatography (HILIC) has become very popular for the separation and determination of polar compounds such as proteins, peptides, amino acids, nucleotides, and carbohydrates. Applications of HILIC began to increase especially after 2003, and many can now be found in the literature [15,16]. The term HILIC or “aqueous normal phase chromatography” should reflect the fact that, unlike RP-HPLC, the stationary phase is polar; moreover, the mobile phase contains an excess of organic solvent, typically more than 80% ( v / v ) of acetonitrile.…”

Section: Introductionmentioning

confidence: 99%

Feasibility of Fraction Collection in HPLC Systems with Evaporative Light Scattering Detector: Analysis of Pectinatella magnifica

Pazourek

Šmejkal

2016

Molecules

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Abstract:The use of a liquid chromatography (LC) splitter inserted between an HPLC column and an evaporative light scattering detector (ELSD) is described. This paper aims to show the feasibility of using the splitter in an HPLC-ELSD system to fractionate a model mixture of analytes, namely salicin (2-(hydroxymethyl)-phenyl-β-D-glucopyranoside) and glucose. The retention factors and efficiency of the separation were studied under various temperatures and water contents in the mobile phase in order to clarify the mechanism of polyols separation on a diol column under the conditions of hydrophilic liquid chromatography (HILIC). Finally, the system was applied to a biological sample (a lyophilized colony gel of Pectinatella magnifica), where the presence of fructose and glucose was confirmed.

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Section: Introductionmentioning

confidence: 99%

Feasibility of Fraction Collection in HPLC Systems with Evaporative Light Scattering Detector: Analysis of Pectinatella magnifica

Pazourek

Šmejkal

2016

Molecules

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Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies

et al. 2018

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Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post-translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC × RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC × RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX × RP separations of the same molecules, we find that chromatograms from the HILIC × RP separations are richer and reveal separation of some glycoforms that coelute in the CEX × RP separations.

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Theory and Practice of Two‐Dimensional Liquid Chromatography Separations Involving the HILIC Mode of Separation

Cited by 2 publications

References 105 publications

Feasibility of Fraction Collection in HPLC Systems with Evaporative Light Scattering Detector: Analysis of Pectinatella magnifica

Feasibility of Fraction Collection in HPLC Systems with Evaporative Light Scattering Detector: Analysis of Pectinatella magnifica

Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies

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