Development of an At-Column Dilution Modulator for Flexible and Precise Control of Dilution Factors to Overcome Mobile Phase Incompatibility in Comprehensive Two-Dimensional Liquid Chromatography

Abstract

With the combination of different mechanisms, two-dimensional liquid chromatography has brought revolutionary changes compared to the traditional one-dimensional separation, which dramatically improve the peak capacity in separation and meet the ever-increasing demand for the analysis of complex samples in different research fields, such as chemistry, medicine, etc. However, the incompatibilities between two columns due to the transport of the large sample volume and the solvent effect always limit the wide use of two-dimensional liquid chromatography. In order to resolve this problem an at-column dilution (ACD) modulator was established to overcome the solvent incompatibility in the orthogonal combination within the comprehensive two-dimensional liquid chromatography. This interface is modified from normal two-dimensional interfaces by an additional transfer pump, which realize the at-column dilution without a flow split during the transportation. Moreover, with the control of the transfer flow and the second-dimensional gradient flow, it is able to precisely regulate the at-column dilution factor and conveniently optimize the separation conditions in both dimensions. In this work, a systematic research has been done between the setups with/without the at-column interface in the combination of reversed-phase liquid chromatography and hydrophilic interaction chromatography (RPLC × HILIC) and HILIC × RPLC, which proved that the at-column interface is able to resolve the solvent conflict problem very well. Furthermore, red ginseng was chosen as a real sample to investigate the applicability of the at-column dilution modulator for comprehensive two-dimensional chromatography with high orthogonality.

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

“…HILIC × RPLC Phenolic acids Make-up flow [58] HILIC × RPLC Phenolics Make-up flow [95][96][97] HILIC × RPLC Oligosaccharides Make-up flow [80] HILIC × RPLC Standard mix At-column dilution [41] RPLC × HILIC Red ginseng extract At-column dilution [41] RPLC × HILIC Herbal medicine At-column dilution [42] Organic-SEC × RPLC Polymers Active solvent modulation [98,99] RPLC × RPLC Monoclonal antibody digests Active solvent modulation [100] HILIC × RPLC Small molecule probes Active solvent modulation [44] HILIC × RPLC Monoclonal antibodies Active solvent modulation [45] Diluting with a weak solvent (trapping columns) SFC × RPLC Pharmaceuticals [101] SFC × RPLC Citrus oil extract [101] SFC × RPLC Fatty acids [102] SFC × RPLC Triacylglycerols [103] SFC × RPLC Carotenoids [104] HILIC × RPLC Oligonucleotides [54] HILIC × RPLC Surfactants [55] HILIC × RPLC Phenolics [59,105] HILIC × RPLC Licorice metabolites [28] HILIC × RPLC Phenolic acids [58] HILIC × RPLC Lipids [106] HILIC × RPLC Cocoa bean metabolites [107] HILIC × RPLC Histone proteoforms [56] HILIC × RPLC Ginsenosides [57,108] HILIC × RPLC Oligosaccharides [80] HILIC × RPLC Microalgae digests [109] RPLC × HILIC Ginseng extract [110] IEC × RPLC Peptides [50] IEC × RPLC Dye extracts [111] HDC × RPLC Nanoparticles [112] RPLC × RPLC Vacuum gas oil [113] RPLC × RPLC Flavonoids [105] RPLC × RPLC Anabolic steroids…”

“…The sample is then diluted by the 2 D-pump with a weak initial mobile phase. This method was used in both RPLC × HILIC and HILIC × RPLC by Chen et al [41,42] for the analysis of red ginseng extract and herbal medicine.…”

Trendbericht Analytische Chemie II: Trenntechniken und Elektroanalytik

“…Durch neuere Entwicklungen bei der Modulation lässt sich nun die Elutionskraft des Eluats der 1-D-für die 2-D-LC herabsetzen. 13,14) Abb. 2.…”

Application of the new at-column dilution (ACD) modulator for the two-dimensional RP×HILIC analysis of Buddleja davidii

“…As a further improvement, a novel modulator, called atcolumn dilution (ACD) modulator, was proposed in our previous work to achieve free adjustment of the online dilution factor [10]. The RPLCxHILIC configuration equipped with an ACD modulator is shown in Fig.…”