The LudgerSep R1 4.6x150mm HPLC column has been developed for the analysis of sialic acids labeled with DMB using the LudgerTag DMB Sialic Acid Labeling Kit.
LudgerSep R1 columns can be used with any HPLC pumping system capable of delivering accurate gradients at a flow rate of 0.3 to 2.0 ml/min. In general, systems that mix solvents at high pressure (after the pump head) have lower dead volumes and supply more accurate gradients that are appropriate at the flow rate needed for LudgerSep columns. We recommend keeping sample injection volumes at or below 25 μL, ideally use full loop injection. Although an example chromatogram is shown in the product guide, retention times will vary dependent on the HPLC system used.
Overview of the Procedue
Release of Sialic Acids
The release of sialic acids from the glycoprotein occurs by a mild acid hydrolysis. Samples and controls are incubated with 2M acetic acid at 80°C for 2 hours.
Sialic Acid Labeling Reaction
The released silica acids are fluorescently labelled with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB). The acid hydrolysed samples and process controls plus Ludger sialic acid standards: Neu5Ac, Neu5Gc, sialic acid reference panel, and Neu5,9Ac2 if using, are incubated with DMB at 50°C for 3 hours.
Analysis via HPLC or UHPLC
A standard curve is calculated using the Neu5Ac and Neu5Gc standards. The sialic acid reference panel and Neu5,9Ac2 are used for sialic acid identification. Analysis can be performed using the LudgerSep R1x150mm column for HPLC or via UHPLC using a LudgerSep uR2x100mm column.
Chromatogram of DMB Labeled Sialic Acid Reference Panel (CM-SRP-01), run on the LudgerSep R1 HPLC column.
Peaks: 1 = Neu5Gc; 2 = Neu5Ac; 3 = Neu5,7Ac2; 4 = Neu5Gc,9Ac; 5 = Neu5,8Ac2; 6 = Neu5,9Ac2; 7 = Neu5,x,xAc3 (where x is an unknown acetyl position); * = Reagent
R1 4.6x150mm HPLC Column Specifications
Particles: 3 μm silica derivatized with octadecylsiliane coating. 120 Angstrom pore size.
Column dimensions: 4.6mm x 150mm
Column volume: 2.49 mls
Typical flow rate: 0.3 – 2.0 ml/min
Pressure limit: 2000 psi
Column tube: Stainless steel, Valco compatible end fittings
pH compatibility: pH 2 to pH 8