Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

Abstract

In HPLC analysis of glycoprotein glycans, oligosaccharides released from glycoproteins are often derivatized with fluorescent tags for enhancing sensitivity and selectivity. However, existing analysis methods require multiple steps for removing excess reagents and are time consuming and sometime spoil the reliability in quantitative determination. Here, we developed an online sample cleanup procedure for the analysis of 7-amino-4-methylcoumarin (AMC)-labeled oligosaccharides. An NH 2 -bonded silica packed short column (1 mm I.D. x 15 mm) and a Waters HLB TM -packed column (4 mm I.D. x 5 cm) were used for online cleanup HPLC analysis with Amide80 and ODS columns, respectively. Optimized conditions enabled the direct injection of the diluted solution of the reaction mixture onto the chromatographic system without any prior removal of the excess labeling reagents. The method was successfully applied to the analysis of N-linked glycans released from some glycoprotein samples.

Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

“…The analytes in a sample solution entrapped in an extraction column are delivered to an analytical column by changing valve positions and the analytes are quantitatively recovered by eluting with extractable solvent. Thus, band broadening of the elution process could be minimized by a back flush elution technique [21,22].…”

Three-dimensional Representation Method Using Pressure, Time, and Number of Theoretical Plates to Analyze Separation Conditions in HPLC Columns

“…[1][2][3][4][5][6][7][8][9][10][11] However, it is difficult to select the optimal packing support, and to find suitable separation conditions, given a specific pressure in an HPLC system; and peripheral studies on the theory have thus far been limited. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] In 2005, Desmet et al invented kinetic performance methods 28 by expanding both Bristow and Knox's, 29 and Poppe's 30 methods.…”