Table 2.
An overview of glycan separation by different liquid chromatography approach. Columns, column dimensions, column manufacturers, analytes, mobile phase compositions and references are summarized
| Column, column dimensions and manufacturer | Analyte | Mobile phase composition | References | ||
|---|---|---|---|---|---|
| Reversed phase chromatography |
Acclaim PepMap™ 100 C18 0.075 × 150 mm; Thermo Fisher Scientific |
Permethylated glycans of human milk |
80% ACN with 0.1% FA/ 2% ACN with 0.1% FA in 1 mM sodium acetate |
[117] | |
|
Acclaim PepMap™ C18 0.075 × 150 mm; 2 µm; Thermo Fisher Scientific |
Permethylated glycans of bovine RNase B, fetuin, mouse brain extracts, recombinant HIV gp120 |
Proteomic buffer: 80% ACN with 0.1% FA/0.1% FA Low lithium buffer: 80% ACN with 0.1% AA containing 0.1 mM lithium acetate/0.1% AA containing 0.1 mM lithium acetate High lithium buffer: 80% ACN with 0.02% AA containing 1 mM lithium acetate/0.02% AA containing 1 mM lithium acetate |
[118] | ||
|
Acclaim PepMap™ C18 0.075 × 150 mm; 2.0 µm; Thermo Fisher Scientific |
Permethylated glycans of cerebrospinal fluid from Alzheimer’s disease patients | ACN with 0.1% FA/0.1% FA | [119] | ||
|
Acclaim PepMap™ C18 0.075 × 150 mm; 2.0 µm; Thermo Fisher Scientific |
Permethylated N- and O-glycans of RNase B and fetuin | ACN with 0.1% FA/2% ACN with 0.1% FA | [121] | ||
|
C18 no information given |
Permethylated glycans of RNase B and fetuin, human blood serum and breast cancer cell lines | ACN with 0.1% FA/2% ACN with 0.1% FA | [120] | ||
|
μPAC columns 5 μm pillar diameter, 2.5 µm inter pillar distance, 18 µm pillar height, 50 or 200 cm bed length; PharmaFluidics |
Permethylated N- and O-glycans of RNase B and fetuin |
ACN with 0.1% FA/2% ACN with 0.1% FA or 80% ACN with 0.1% FA/2% ACN with 0.1% |
[121] | ||
|
InertSustain AQ-C18 2.1 × 150 mm; GL Sciences |
Human A1GP, transferrin and bovine fetuin tagged with 2-AB | 20% ACN with 0.2% FA/0.2% FA | [123] | ||
|
Magic C18 AQ 0.075 × 150 mm; 5 µm; Tosoh Bioscience |
Native and P2PGN derivatized glycans from human plasma | 98% ACN with 0.2% FA/2% ACN with 0.2% FA | [125] | ||
|
T3 C18 2.1 × 150 mm; 1.7 µm; Waters Corporation |
2-AB labeled N-glycans of IgG derived from mammalian species | ACN with 0.1% FA/ 0.1% FA | [133] | ||
| Reversed phase chromatography |
Acquity UPLC Glycan BEH Amide 2.1 × 100 mm; 1.7 µm; Waters Corporation |
2-AA labeled N-glycans of recombinant therapeutic glycoproteins | ACN/150 mM ammonium formate (pH 4.5) | [124] | |
|
Acquity UPLC Glycan BEH amide 2.1 × 50 or 150 mm; 1.7 µm; Waters Corporation |
RFMS labeled glycans of anticitrinin murine monoclonal IgG1, pooled human IgG, and bovine fetuin |
ACN/50 mM ammonium formate (pH 4.4) |
[128] | ||
| Hydrophilic interaction liquid chromatography |
Acquity UPLC Glycan BEH amide 2.1 × 50, 100 or 150 mm; 1.7 µm; Waters Corporation |
2-AB labeled glycans of RNase B, fetuin, and IgG | ACN/100 mM ammonium formate (pH 4.5) | [129] | |
|
Acquity UPLC Glycan BEH amide 2.1 × 50 mm; 1.7 µm; Waters Corporation |
N-glycans of infliximab labeled with RFMS |
ACN/50 mM ammonium formate (pH 4.4) |
[130] | ||
|
Acquity UPLC Glycan BEH amide 2.1 × 150 mm; 1.7 µm; Waters Corporation |
2-AB labeled N-glycans of IgG derived from mammalian species |
ACN/50 mM ammonium formate (pH 4.4) |
[133] | ||
|
TSKgel Amide-80 2.0 × 250 mm; Tosoh Biosciences |
2-AA labeled human plasma |
ACN/50 mM ammonium formate (pH 4.4) |
[127] | ||
|
TSKgel Amide-80 4.6 × 250 mm; 5.0 µm; Tosoh Biosciences |
Native and derivatized glycans of mAbs and RNAse B |
ACN/250 mM ammonium formate (pH 4.4) | [131] | ||
|
In house packed Amide column 0.075 × 250; 5 µm; Prozyme |
DMT-MM derivatized haptoglobin and human plasma | ACN with 0.1% FA/0.1% FA | [132] | ||
|
SeQuant ZIC-HILIC 2.1 × 150 mm; 3.5 µm; Merck |
Native and derivatized glycans of (mAbs) and RNAse B |
ACN/34 mM AA (pH 3.0) | [131] | ||
|
SeQuant ZIC-HILIC 2.1 × 150 mm; 3.5 µm; Merck |
2-PA derivatized glycans of A1GP and IgG from human serum | ACN/ACN/ammonium acetate (90%/50%/ 20, 5 or 100 mM) | [33, 34] | ||
|
SeQuant ZIC-HILIC 2.1 × 150 mm; 3.0 µm; Merck |
2-AB labeled and reduced glycans of mAbs | ACN/0.1% AA | [136] | ||
|
Halo® Penta-HILIC column 2.1 × 150 mm; 2.7 µm; Advanced Materials Technology |
ProA labeled N-glycan of bovine fetuin |
ACN/5% ACN with 50 mM ammonium formate (pH 4.4) | [22] | ||
| Porous graphitized carbon |
PGC 0.075 × 43 mm; 5.0 µm |
Native N-glycans of human serum | 90% ACN with 0.1% FA/3% ACN with 0.1% FA | [135] | |
|
PGC 0.075 × 43 mm; 5.0 µm; Agilent Technologies |
N-glycans derived from RNase B, fetuin, thyroglobulin, A1GP, and IgG (both from human serum) |
10% ACN with 0.1% FA/0.1% FA or ACN/2% ACN containing 7.5 mM ammonium acetate (pH 8.4) |
[102] | ||
|
PGC 0.32 × 100 mm; Thermo Fisher Scientific |
Borohydride-reduced glycans of human IgG, commercial mAb expressed in CHO cells, anti-HIV antibody 4E10 | 80% ACN in 65 mM ammonium formate/65 mM ammonium formate (pH 3.0) | [138] | ||
|
PGC 0.15 × 100 mm; 5 µm; Thermo Fisher Scientific |
O-glycans of porcin gastric mucin | ACN in 10 mM ammonium bicarbonate/water | [142] | ||
|
HyperCarb 0.075 × 150 mm; 5 µm; Thermo Fisher Scientific |
Permethylated glycans of RNase B and fetuin, human blood serum, and breast cancer cell lines | ACN with 0.1% FA/2% ACN with 0.1% FA | [120] | ||
|
HyperCarb 2.1 × 150 mm; 5 µm; Thermo Fisher Scientific |
2-AB labeled and reduced glycans of mAbs | ACN/0.1% AA | [136] | ||
|
HyperCarb 0.18 × 100 mm; Thermo Fisher Scientific |
Reduced N-glycans of fibrinogen (derived from different species) and transferrin |
ACN/65 mM ammonium formate (pH 3.0) |
[137] | ||
|
HyperCarb 0.15 × 150 mm or 0.1 × 200 mm; 3.0 µm; Thermo Fisher Scientific |
Native glycans of RNase B and fetuin | ACN with 0.1% FA/0.1% FA | [140] | ||
|
HyperCarb 0.075 × 100 mm; 5.0 µm; Thermo Fisher Scientific |
Permethylated glycans of RNase B, fetuin, A1GP, human blood serum, and breast cancer cells | ACN with 0.1% FA/2% ACN with 0.1% FA | [141] | ||
|
HyperCarb 0.18 × 200 mm; 5.0 µm; Thermo Fisher Scientific |
O-glycans of MUC1 | ACN with 8 mM ammonium bicarbonate/water | [143] | ||
|
ProteCol HyperCarb 0.3 × 100 mm or 0.15 × 100 mm; SGE Analytical Science |
Reduced O- and N-linked glycans isolated from mucosal surfaces and ovarian tissues | 80% ACN in 10 mM ammonium bicarbonate/10 mM ammonium bicarbonate | [139] | ||
| Multi-dimensional chromatography |
AEX × HILIC (TSKgel DEAE-5PW × TSKgel Amide-80 or SeQuant ZIC-HILIC AEX: 2.0 × 50 mm; 5.0 µm; Tosoh Biosciences Amide-80: 2.0 × 150 mm; 5.0 µm; Tosoh Biosciences ZIC-HILIC: 2.1 × 150 mm; 5.0 µm; Merck |
2- PA derivatives of N-glycans of human serum | ACN/water/0.5 M ammonium acetate | [144] | |
|
HILIC × PGC (Click-Maltose × HyperCarb) HILIC: 4.6 × 250 mm; 5.0 μm; Acchrom HyperCarb: 4.6 × 150 mm; 5.0 μm; Thermo Fisher Scientific |
N-glycans of ovalbumin |
HILIC: ACN with 0.1% FA/0.1% FA PGC: ACN/water |
[145] | ||
|
HILIC × RP (Acquity UPLC Glycoprotein amide × RP PLRP-S) HILIC: 2.1 × 150 mm; 1.7 µm; Waters Corporation RP: 2.1 × 30 mm; 5.0 µm; Agilent Technologies |
mAbs |
HILIC: ACN/0.3% TFA RP: ACN/0.1% FA |
[146] | ||
RNase A and B ribonuclease A and B, A1GP α1-acid glycoprotein, MUC1 mucin 1, CHO Chinese hamster ovary, mAbs monoclonal antibodies, 2-AB 2-aminobenzamide, 2-PA 2-aminopyridine, AEX anion-exchange, ACN acetonitrile, FA formic acid, AA acetic acid, TFA trifluoroacetic acid, IgG immunoglobulin G, RNase A and B ribonuclease A and B, A1GP α1-acid glycoprotein, 2-AB 2-aminobenzamide, P2PGN 4-phenethyl-benzohydrazide, ACN acetonitrile, IgG immunoglobulin G, RNase A and B ribonuclease A and B, A1GP α1-acid glycoprotein, mAbs monoclonal antibodies, RFMS RapiFluor-MS, 2-AA 2-aminobenzoic acid, 2-AB 2-aminobenzamide, 2-PA 2-aminopyridine, DMT-MM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, P2PGN 4-phenethyl-benzohydrazide, ProA procainamide, ACN acetonitrile