Carbohydrate Analysis by HPLC I, An Introduction

Xun Yan
Analytical Sciences, Amway R&D
Ada, Michigan 49355

Introduction

Carbohydrates are widely present in biological systems in their free states, such as starch and cellulose, as well as in conjugated
forms, such as proteoglycans, glycoproteins, glycolipids and glycosides. Besides the energy storage and structural support functions,
carbohydrates participate in many biological processes including cell recognition, interaction, inflammation and development. The
analysis of carbohydrates has been a challenging area due to their complex structure and heterogeneity.

Carbohydrates
Monosaccharides

Sample Preparation

Polysaccharides (Glycans)

Extraction
Hexane, CHCl3: removes fat or extract glycolipids
Alcohol/water: extracts low molecular weight
(LMW) carbohydrates, or
remove acids, peptides.
Water (hot):
extracts high molecular weight
carbohydrate. Often followed with
alcohol precipitation.
Protease:
breaks down proteins
Amylase:
breaks down starch

Glycoproteins
Carbohydrate modified protein via N- or Oglycosylation . Known to participate in cell growth,
inflammation and immune response.

Proteoglycans
Glycosaminoglycan (GAG) modified proteins. Found
in extracellular matrices and cell walls. Help with cell
to cell adhesion, cell signaling and communication.

Glycolipids.
Reside in outer surface of cell membrane. Important
in cell recognition.

Release carbohydrates
Glycoamidase: releases N- linked
oligosaccharides
Exoglycosidase: releases O- linked
oligosaccharides
Hydrazinolysis: effective for both N- and Olinked oligosaccharides
elimination: release O- linked saccharides
but also degrade glycan
TFA hydrolysis: effective to release all types but nonspecific, also breaks down
carbohydrates
Break down HMW carbohydrates
Acidic hydrolysis: TFA (2~6M), HCl (2~8 M), 100oC
Polysaccharides lyases: analyte specific.
e.g. chondroitinase for chondroitin,
lichenase to hydrolyze beta
(1,3)/(1,4) glucan

Summary

HPLC techniques in carbohydrate analysis are summarized in this poster. Different sample preparation techniques can be used to extract, release
and label carbohydrates for sensitive and selective detection. Reversed phase (RP), normal phase (NP), hydrophilic interaction (HILIC), anion
exchange (AE) and size exclusive chromatography (SEC), can be used for carbohydrate separation. Common carbohydrate detection methods
includes refractive index (RID), UV-Vis, fluorescence (FLD), light scattering (ELS or LLS) and electrochemical detection.

Derivatization

Precolumn techniques
Carbonyl (ketones, aldehydes): reductive amination
using aminopyridine, aminobenzene. UV, FLD
detection

Aldehyde: PMP (phenyl methyl pyrazolone)

condensation, UV 245 nm

Aminosaccharides: FMOC (fluorenyl methyl

carbamate), CBQCA (carboxybenzoyl quinoline
carboxyaldehyde), UV or FLD

Post column techniques

Sulfuric acid/ anthrone for general use
DMAB (dimethyl aminobenzyl aldehyde) for sialic
acid, amino sugars
Copper bicinchoninate for reducing sugars

Chromatography Separation
Reverse phase (RP)
stationary phase: Alkyl or aryl modified surface
mobile phase: water/alcohol /acetonitrile/THF
application: derivatized carbohydrates
Hydrophobic interaction (HIC)
stationary phase: spaced hydrophobic ligands on
hydrophilic surface
mobile phase: water with alcohols and salts
application: glycoproteins
Normal phase:
stationary phase: polar surface, silica, diol,
cyano/amino
mobile phase: hexane/ethyl acetate/ chloroform
with propanol, methanol and water
application: glycolipids
Hydrophilic interaction (HILIC)
stationary phase: polar surface, ion exchange
mobile phase: water acetonitrile
application: mono-, oligo- saccharides
Anion exchange (AEC)
stationary phase: anion exchange
mobile phase: strong base (pH 12)
applications: acidic or neutral saccharides
Size exclusive (SEC)
stationary phase: porous
mobile phase: electrolyte solution, high solubility but
not changing the chains conformation
applications: high molecular weight polysaccharides

Detection Methods
Refractive index (RID)

Ultraviolet-visible (UV-VIS)
Fluorescence (FLD)

Evaporative light scattering (ELSD)

Laser light scattering (LLS)

Electrochemical (EC)
Pulsed amperometric (PAD)

Reference

HPLC for Carbohydrate Analysis, in High

Performance Liquid Chromatography: Principles,
Practices and Procedures (Ed.), Nova Publishers: NY,
in press.
Contact:
Xun Yan, Amway 50-2D, 7575 Fulton, Ada, MI