ABEE derivatization
Materials
ABEE reagent (p-Aminobenzoic acid ethyl ester 35 mg, NaBH3CN 3.5 mg, Acetic acid 41ul, Methanol 350 ul)
Test tube with screw cap
Pre-Sep C18 cartridge column (Senetek PLC)
Heating Block
Method
HPLC Analysis
- Acidic Oligosaccharides
Anion exchange-HPLC
column: TSK gel DEAE-5PW (0.75 cm x 7.5 cm, Tosoh Co., Ltd.)
gradient: 10 mM NaH2PO4 (10 min) -->250 mM NaH2PO4 (60 min)
flow rate: 0.5 ml / min
temp.: 25 ℃
detection: 304 nm
- Neutral Oligosaccharides
Normal phase-HPLC
column: TSK gel Amide-80 (0.46 cm x 25 cm, Tosho)
solvent A: Acetonitrile : Water, 9 : 1 by vol
solvent B: Acetonitrile : Water, 1 : 9 by vol
gradient: B in A: 20 % ---> 50% (60 min)
flow rate: 0.8 ml / ml
Temp.: 40 ℃
detection: 304 nm
Reverse phase-HPLC
column: Wakosil 5C18-200 (0.4 cm x 25 cm, Wako Pure Chemical Industries)
solvent C: 5 % acetonitril in 100 mM acetic acid
solvent D: 15% acetonitril in 100 mM acetic acid
gradient: D in C: 40 % ---> 60% (60 min)
flow rate: 0.8 ml / min
temp.: 40 ℃
detection: 304 nm
Reference
Matsuura, F.and Imaoka, A.
Chromatographic separation of asparagine-linked oligosaccharides labeled with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester
Glycocojugate J 5, 13-26 (1988)Matsuura, F., Ohta, M., Murakami, K., Hirano, K. and Sweeley, C.C.
The combination of normal phase with reversed phase high performance liquid chromatography for the analysis of asparagine-linked neutral oligosaccharides labelled with p-aminobenzoic ethyl ester
Biomed Chromatogr 6, 77-83 (1992)Ohta, M., Kobatake, M., Matsumura, A. and Matsuura, F.
Separation of Asn-linked sialyloligosaccharides labeled with p- aminobenzoic acid ethyl ester by high-performance liquid chromatography
Agric Biol Chem 54, 1045-7 (1990)
