Accucore 150 amide hilic lc column

Labeled N-glycans were separated with Thermo Scientific Accucore 150 Amide HILIC LC Column (2.6 μm, 150 A, 2.1 × 150 mm) at 50°C with Thermo Scientific Vanquish Horizon UHPLC System equipped with 2 μl flow cell FLD at 455 nm and 500 nm excitation and emission wavelength, respectively, at high power lamp mode and 10 Hz of data collection rate. Separation was performed with 15 μl injection volume at a 45 min gradient of B from 32% to 45%, 45.5–47 min at 60%, and 47.5–50 min at 32% at constant flow rate of 0.45 ml/min, with mobile phase B being 100 mM ammonium formate (pH 4.4) and mobile phase A being 100% acetonitrile.

SimplyBlue™ SafeStain from Novex by Life Technologies (Cat# LC6065), NuPAGE MOPS SDS running buffer (P/N# NP0001), NuPAGE LDS sample buffer (4x) (Cat# NP0008), NuPAGE Bis-Tris Mini Gels (Cat# IM-8042), GlycanAssure HyPerformance APTS kit (Cat# A33953), 1 M DTT (P/N# P2325), formic acid, 99.5+%, Optima™ LC/MS grade (P/N#A117-50) from Thermo Fisher Scientific, HPLC grade 100% acetonitrile (P/N#AC610010040) from Fisher Scientific, Aeris™ 3.6 μm WIDEPORE XB-C18 (200 Å, LC Column 150 × 4.6 mm) (P/N 00F-4482-E0) from Phenomenex, Thermo Scientific™ Accucore™ 150 Amide HILIC LC Column (2.6 μm, 150 A, 2.1 × 150 mm), and Thermo Scientific Vanquish™ Horizon UHPLC System, glycoproteins of RNase B and bovine fetuin from Sigma (P/N#J62334, lot R112C021) and QA-Bio (P/N# GCP 500 μg, lot# B1B2-04), IgG1κ monoclonal antibody (NIST mAb #8671 (lot# 14HB-D-001)) in 12.5 mmol/L L-histidine, 12.5 mmol/L L-histidine HCl (pH 6.0) from NIST were used.