Separation of di- and trisaccharide mixtures by comprehensive two-dimensional liquid chromatography. Application to prebiotic oligosaccharides

Abstract

Carbohydrates are one of the most important ingredients in foods. They are normally present as complex mixtures with different glycosidic linkages, monomeric units and degrees of polymerization. This structural heterogeneity impairs their comprehensive characterization and requires the use of analytical techniques with high resolving power and sensitivity. The use of chromatographic techniques, especially liquid chromatography (LC), has been extremely helpful for the analysis of carbohydrates. However, in many cases, the use of monodimensional LC is not enough to resolve these complex mixtures; then, the use of techniques with a higher resolving power, as multidimensional LC, could be a good alternative. To the best of our knowledge, our findings are pioneer in applying online LC×LC for the analysis of carbohydrate mixtures. For this purpose, different conditions such as stationary phases (BEH amide, C18 and PGC columns) and chromatographic conditions for the separation of di-and trisaccharide mixtures were optimized. The BEH amide × C18 combination was selected for the LC×LC analysis of carbohydrate standards with different degree of polymerization, linkages and monomeric units. In order to allow their proper UV detection, carbohydrates were previously derivatized using p-aminobenzoic ethyl ester. This method also resulted to be successful for the separation of commercial prebiotic mixtures of galacto-oligosaccharides and gentio-oligosaccharides. This is the first time that LC×LC has been applied for the separation of bioactive carbohydrate mixtures and it could be considered as a powerful analytical technique for the characterization of other oligosaccharide complex mixtures.

Structure and function of non-digestible carbohydrates in the gut microbiome

“…Interestingly, these authors reported MS fragmentation rules to distinguish reducing GOS isomers with a mono-and disubstituted terminal glucose. Likewise, in a pioneering work, the BEH amide stationary phase in the first dimension was coupled to a reversed-phased column in the second dimension for the first application of on-line comprehensive two-dimensional LC (LC × LC) to the successful separation of previously derivatised commercial prebiotic mixtures of GOS and gentio-oligosaccharides (GEOS) (Martín-Ortiz et al, 2019). Carevic et al (2016) have recently reported a complete elucidation of GOS structures from DP 2 to DP 4 based uniquely on IMMS-MS/MS.…”

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

“…The first peak is approximately not retained (marked by an asterisk) while the second one is eluted at the expected retention time. This phenomenon is explained by the fact that a significant part of the sample (diluted in a strong solvent) travels through the column without much interaction with the stationary Increasing 2 C F HIC × RPLC Antibody-drug conjugates [16,17] IEC × RPLC Proteins [64] IEC × RPLC Wine and Chinese medicine [65] IEC × RPLC Monoclonal antibody digests [66] IEC × RPLC Monoclonal antibodies [67,68] Aqueous-SEC × RPLC Peptides [69] RPLC × RPLC Peptides [8] Decreasing column id ratio HILIC × RPLC Fatty alcohol derivatives [70] HILIC × RPLC Phenolics [71][72][73][74] HILIC × RPLC Procyanidins Large loop volume [27] HILIC × RPLC Phenolic and flavonoids [75] HILIC × RPLC Phlorotannins [23,76] HILIC × RPLC Surfactants [77] HILIC × RPLC Liquorice metabolites [24] HILIC × RPLC Red wine sample [78] HILIC × RPLC Liquorice metabolites Large loop volume [28] HILIC × RPLC Lipids [79] HILIC × RPLC Oligosaccharides Large loop volume [80] RPLC × HILIC Peptides [25] Silver ion LC × RPLC Triacylglycerides [81] RPLC × RPLC Biomass by-product standards [29] RPLC × RPLC Bio-oil extract [26,82,83] RPLC × RPLC Phenolics [73,84] RPLC × RPLC Hop cone and pellet extracts [85] RPLC × RPLC Proteins [86] RPLC × RPLC Plant metabolites [87] LC × organic-SEC Polymers [88] RPLC × SFC Bio-oil extract …”

“…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…”

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

“…A typical example of this duality is the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC). In the past decade, HILIC x RPLC has received a substantial increase in interest for the on-line LC x LC separation of polar and ionisable compounds [9][10][11][12]. This growing popularity is driven by the complementarity of the two separation mechanisms which gives rise to high coverage of the two-dimensional retention space [2,13].…”