See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/301698343

HPLC for Carbohydrate Analysis

Chapter · October 2014

CITATIONS READS

0 1,757

1 author:

Xun Yan
Amway Corp, ADA, MI
13 PUBLICATIONS 172 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

carbohydrates View project

LB film View project

All content following this page was uploaded by Xun Yan on 29 April 2016.

The user has requested enhancement of the downloaded file.

Chapter

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

FOR CARBOHYDRATE ANALYSIS.

Xun Yan*
Analytical Sciences, Research & Development, Amway Corporation.

ABSTRACT
Carbohydrates are widely present in biological systems in both free states (e.g., starch,
cellulose) and conjugated forms (e.g., proteoglycans, glycoproteins, glycolipids),
providing energy storage as well as structural support functions. Carbohydrates
participate in many biological processes including cell recognition, development,
interaction, and inflammation. The analysis of carbohydrates is challenging due to their
complex structure and heterogeneity. This chapter reviews current high performance
liquid chromatography (HPLC) techniques for carbohydrate analysis.
To analyze carbohydrate content by HPLC, the test sample must first be extracted and
purified. Hydrolysis and derivatization techniques are used to release and label
carbohydrates for sensitive and selective detection. There are several operational modes
of HPLC that are widely used for carbohydrate separation including, reversed phase (RP)
and normal phase (NP) chromatography, hydrophobic interaction chromatography (HIC),
hydrophilic interaction liquid chromatography (HILIC), anion exchange chromatography
(AEC), and size exclusion chromatography (SEC). Common detection methods used for
HPLC include refractive index (RI), ultraviolet and visible (UV-Vis) absorbance,
fluorescence, evaporative or laser light scattering (ELS or LLS), and electrochemical
(EC) detection.
To demonstrate an HPLC application for analyzing carbohydrates, we have included a
chitosan analysis method in this chapter. Chitosan, is a copolymer of glucosamine (2-
amido-2-deoxy-D-glucose) and N-acetyl glucosamine via a β(1-4) linkage. It is a widely
used food ingredient and additive. To quantify chitosan, it is first hydrolyzed to
glucosamine with hydrochloric acid (HCl), then undergo derivatization with N-(9-
fluorenylmethoxycarbonyloxy)succinimide (FMOC-OSu). The glucosamine-FMOC
derivatives are then separated and quantified using reversed phase HPLC with UV
detection.

Keywords: Carbohydrates, Polysaccharides, HPLC, Derivatization, Detection, Chitosan,

Glucosamine, FMOC

* Corresponding Author 7575 Fulton Street E., Ada, MI, 49355

Email: xun.yan@amway.com
2 Xun Yan

INTRODUCTION
Carbohydrates are the most abundant and diverse class of natural organic compounds.
They function as energy reserves and as structural material in tissues. Their protein and lipid
conjugates play crucial roles in many biological processes, such as inflammation, immunity,
and cell recognition and communication [Varki and Lowe, 2009]. The analysis of
carbohydrates and their conjugates is one of the most requested analyses at both biochemical
and nutritional analytical laboratories [Yamada and Kakehi, 2011]. In the nutritional
supplements industry, specific and accurate analyses of carbohydrates are required as a result
of nutrition labeling regulations, as well as, due to increased consumer demand for high
quality products.

CARBOHYDRATES AND THEIR CONJUGATES

Carbohydrates

Carbohydrates consist of polyhydroxy aldehydes and ketones and their derivatives

[Robyt, 1998]. Monosaccharides, the simplest carbohydrates, consist of three to nine carbon
atoms with a carbonyl functional group. If the carbonyl group is an aldehyde the
monosaccharide is an aldose, if the functional group is a ketone, a ketose is formed. A linear
monosaccharide can convert to a cyclic configuration through a hemiacetal formation
between one of its hydroxyl groups and the carbonyl group. In its cyclic form, the hemiacetal
group of one monosaccharide can react with the alcohol group of another monosaccharide to
form a glycosidic bond, resulting in a disaccharide (e.g., maltose, sucrose, and lactose).
Three to nine monomeric saccharides linked together through glycosidic bonds are
oligosaccharides. The number of sugar residues is the degree of polymerization. Many
oligosaccharides occur naturally in plants as free compounds, such as raffinose, stachyose,
and fructo-oligosaccharides.
Polysaccharides, or glycans, are linear or branched polymers of monomeric sugars and
their derivatives, and may contain hundreds or even thousands of monosaccharide units. The
well-known polysaccharides include starch, glycogen, cellulose, inulin, and chitin. Due to the
diversity of monosaccharides, polysaccharides are a very heterogeneous chemical group,
varying in charge, shape, and solubility. Even within one type of polysaccharide, there is
considerable variation in molecular weights, linkage positions, and branch number and length.

Glycoproteins and glycopeptides

Monosaccharides or polysaccharides can attach to proteins or polypeptides through

glycosylation, forming glycoproteins and glycopeptides [Marino et al., 2010]. The
carbohydrate groups are covalently bound to the polypeptide backbone through N-
glycosylation of an asparagine residue, or through an O-glycosylation with a serine,
threonine, and, occasionally, a hydroxyproline residue [Geyer and Geyer, 2006; Lennarz,
1980]. The carbohydrate content in glycoproteins can vary between one and eighty percent.
The degree of glycosylation is often critical to the physiological properties of the protein,
 High Performance Liquid Chromatography for Carbohydrate Analysis 3

affecting such parameters as solubility and stability. Glycosylated proteins are reported to
affect a wide range of biological processes including tumor growth, cancer metastasis,
inflammation, and immunological and cardiovascular disorders.

Proteoglycans and peptidoglycans

Proteoglycans and peptidoglycans are a subclass of glycoproteins in which the

carbohydrate units are glycosaminoglycans (GAGs) [Lennarz, 1980]. GAGs, such as
hyaluronan, chondroitin sulfate, keratin sulfate, dermatan sulfate, and heparan sulfate, are
linear polymers of glucosamine or galactosamine alternating with uronic acid or a neutral
sugar.
Proteoglycans are found primarily in extracellular matrices, such as blood vessels,
cartilage, and skin. They bind cations and water to regulate the transfer of molecules through
the cell matrix, and to affect the stability and activity of proteins in cell-cell adhesion, and cell
signaling, migration, and proliferation.
Peptidoglycans are an essential component of bacterial cell walls, helping to maintain cell
structure and counteract the high osmotic pressure within the bacteria [Thiemermann, 2002].
Peptidoglycans regulate the transport of molecules into cells and participate in the regulation
of cell division. Bacteria often release peptidoglycan fragments that function as signals in
cell-cell communication.

Glycolipids

Glycolipids consist of short carbohydrate chains (mono- or oligosaccharides) with

attached lipids [Barraud and Stott, 2008]. Common glycolipids include glycosyl
diacylglycerols, glycosphingolipids, and glucose or sucrose esterified with fatty acids and
sterols. Glycosyl diacylglycerols are the most abundant glycolipids, consisting of mono- or
disaccharides glycosidically linked to diacylglycerol. Glycolipids contain hydrophobic
aliphatic chains and hydrophilic sugar head groups contributing to their amphiphilic
properties. They typically reside at the outer surface of the cell membrane. They participate in
cell physiology through cell recognition and by modulating functional membrane proteins,
such as cellular receptors, transducers, and transporters.

Lipopolysaccharides

Lipopolysaccharides, or lipoglycans, are polysaccharides with covalently attached lipids

[De Castro et al., 2010; Esko et al., 2009; Raetz and Whitfield, 2002]. They are found in the
outer cell membrane of all gram-negative bacteria. The general structure of
lipopolysaccharides consists of three regions, a lipid region, serving as the hydrophobic
membrane anchor, a core oligosaccharide, and an O-polysaccharide region.
Lipopolysaccharides play a critical role in the interactions between the bacterium and its
environment. They are known to act as endotoxins and modulate immune functions.
4 Xun Yan

PREPARATION OF CARBOHYDRATE SAMPLES FOR ANALYSIS

Sample preparations prior to HPLC separation and analysis of carbohydrates generally
include extraction, hydrolysis, and derivatization steps. Depending on the types of
carbohydrates in the test sample, different techniques may be selected [Lee et al., 1989; Mort
and Pierce, 2002].

Extraction and purification

A simple carbohydrate sample typically needs only solvent extraction and filtration prior
to HPLC analysis where, in other cases, especially when analyzing tissue and complex food
products, sample preparation can be more complex [Aoki and Tiemeyer, 2010]. Lipophilic
compounds are typically removed by solvent extraction with chloroform, ether, or hexane.
Proteins may be removed from samples enzymatically with proteases. Treatment with a hot
80% ethanol solution can be used to extract minerals, acids, amino acids, and low molecular
weight sugars and peptides. High molecular weight carbohydrates can be extracted with hot
water and then precipitated with four volumes of ethanol or isopropyl alcohol. Removal of
residual starch can be accomplished by digesting the samples with α-amylase or
amyloglucosidase, followed by centrifugation. Further purification of carbohydrates can be
achieved with solid phase extraction using ion exchange, such as diethylaminoethanol
(DEAE) or HILIC cartridges [Paulsen et al., 2002; Svejcar and Robertson, 1967]. Cellulose
and other water insoluble carbohydrates can be precipitated by centrifugation following hot
water extraction.
To purify glycolipids, total lipids are first extracted from the sample by homogenization
in 20 volumes of chloroform/methanol (v/v, 2/1) or hexane/isopropanol (v/v, 4/1)
[Kesselmeier and Heinz, 1987; Van Die, et al., 2010]. The addition of water to the clarified
organic phase allows partitioning of the polar carbohydrates and liposaccharides into the
aqueous phase. The organic phase contains glycolipids with short oligosaccharide segments
as well as other lipids. These glycolipids can be further separated using thin layer or normal
phase chromatography [Kesselmeier and Heinz, 1987; Moreau et al., 2008].

Release of carbohydrate from its conjugates

To analyze carbohydrate structure and content, it is often necessary to cleave the

carbohydrate from its conjugates. Glycoproteins often contain more than one type of
oligosaccharide, which needs to be released prior to characterization [Anumula, 2006; Geyer
and Geyer, 2006]. There are several enzymatic and chemical methods for the release of
oligosaccharides from glycoproteins. The choice of method depends on the oligosaccharide
structure, their linkages to the protein, and the quantity of glycoprotein.
The N- linked glycans can be released using a glycoamidase, such as peptide -N-
glycosidase F (PNGase F), which cleaves the acid amide linkage in asparagine residues
[Freeze and Kranz, 2006; Rohrer et al., 1993]. Exo- and endoglycosidases can be used to
release O-linked oligosaccharides from their conjugates, yielding smaller oligosaccharide
chains.
Hydrazinolysis and β-elimination are two commonly used chemical methods to release
glycans from glycoproteins [Didraga et al., 2006]. Heating glycoproteins with anhydrous
 High Performance Liquid Chromatography for Carbohydrate Analysis 5

hydrazine results in transamidation of the amide bonds and releases both free N- and O-linked
oligosaccharides from the protein. O-linked oligosaccharides can also be released through β-
elimination using an alkaline/borohydride mixture [Merry et al., 2002]; however, the strong
basic conditions may lead to glycan degradation.
Another method used to extract oligosaccharides from glycoproteins is trifluoroacetic
acid (TFA) hydrolysis. TFA hydrolysis cleaves the amide bonds between the amino acids, the
acetamido linkages, and the N- and O-glycosidic linkages. However, in this method, the
oligosaccharides are also hydrolyzed, leading to a mixture of products [Nilsson and Svensson,
1979]. Oligosaccharides can be extracted from glycolipids by a mild acid hydrolysis using
acetic acid. The lipid fraction, which is not water-soluble, can be removed by centrifugation
[Raetz, 1990].

Hydrolysis of polysaccharides

While direct analysis of polysaccharides by HPLC has been recently reported, it poses a
number of challenges [Kakehi and Honda, 1986]. Polysaccharides can be quite large,
polydisperse, and occasionally highly charged. They can also contain a high degree of
sequence variability due to the uneven distribution of substitution groups. In complex tissue
samples, polysaccharides can be difficult to separate from one another due to their similar
physico-chemical properties. In conventional analyses, they are first broken down into
monosaccarides, disaccharide, or oligosaccharide components, using acidic hydrolysis or
enzymatic depolymerization. The released saccharides are then identified using
chromatographic techniques.
Acidic hydrolysis is a common method used to hydrolyze glycosidic linkages in glycans.
In the presence of a strong acid and heat, the glycosidic bonds are cleaved, consuming one
molecule of water for each bond cleaved. Sulfuric acid, hydrochloric acid, phosphoric acid,
and TFA are commonly used for acid hydrolysis. TFA and hydrochloric acid are preferred
due to their high volatility and ease of removal prior to HPLC analyses [Anumula, 1994;
Clarke, 1993; Fu and O'Neill, 1995]. Since not all glycosidic linkages are cleaved at the same
rate and the released monosaccharides, especially neutral and acidic sugars, are susceptible to
degradation during the hydrolysis process, the reaction can be a challenge if the sample
contains both hydrolysis-resistant and acid and heat labile sugar residues. The hydrolytic
conditions need to be optimized based on the sample type and the focus of the analysis.
Generally, hydrolytic conditions of 2 M TFA at 100°C for 4 hours is optimal for neutral-sugar
analysis, and a stronger acid, such as 4 M HCl, and longer hydrolysis time, approximately 6
hours, for aminoglycan analyses.
When dealing with an unknown polysaccharide, it is best to check its hydrolysis kinetics.
As the hydrolysis proceeds, the quantity of the released sugar should increase until all the
sugar has been released from the polysaccharide. If the hydrolysis proceeds past this point,
degradation of released sugars will become evident as the monosaccharide concentration
decreases.
Enzymatic glycan depolymerization utilizes polysaccharide lyases. These enzymes are
specific for different types of glycans and can break the polysaccharides down to
monosaccharides, disaccharides, or oligosaccharides. Enzymatic depolymerization offers the
advantage of high specificity, high sensitivity, and protection of liable sugar residues. These
enzymes generally act at temperatures ranging from 25 to 37 C in a variety of buffers. GAG
6 Xun Yan

lyases, such as hyaluronidase and chondroitinase, use an elimination mechanism to lyse

hyaluronan and chondroitin into unsaturated uronate dissaccharides from the non-reducing
end [Ji et al., 2007; Kakehi et al., 1993; Vyios et al., 2002]. β-glucan can be hydrolyzed with
lichenase to oligosaccharides which can be further hydrolyzed to glucose with β-glucosidase.

Carbohydrate derivatization

Carbohydrates do not contain selective chromophores, which make them difficult to

detect using traditional analytical techniques. Chemical derivatization converts carbohydrates
into compounds that can be detected with increased sensitivity while also increasing
analytical selectivity [Harazono et al., 2011]. Derivatization methods can be performed before
or after chromatographic separation [Hase, 2002].
The derivatization sites in carbohydrates are the carbonyl groups (aldehydes and ketones)
at the reducing end, hydroxyl groups, free amino groups, and the carboxylic groups in acidic
saccharides [Lamari et al., 2003]. Reductive amination of carbonyl groups is a common
carbohydrate derivatization scheme, in which the carbonyl reacts with an amino group in the
derivatizing label, yielding a Schiff base. The imine derivative is further reduced to a stable
secondary amine with sodium cyanoborohydride or triacetoxyborohydride under mild acidic
conditions [Paulus and Klockow, 1996]. Aminopyridine, aminonaphthalene sulfonic acid, and
aminobenzene are common derivatizing labels and the resulting derivatives can be detected
with either UV or fluorescence detectors. Reducing sugars can also be condensed with phenyl
methyl pyrazolone (PMP) to form saccharide-PMP derivatives that strongly absorb at 245 nm
UV wavelength [Honda et al., 1989; Kakehi et al., 1993; Strydom, 1994]. Aminosugars with
primary amine groups can be derivatized with fluorenylmethyl carbamates (FMOC) and
carboxybenzoyl quinoline, both well-developed techniques used in amino acid and peptide
analysis [Rosenfeld, 2003].
In post-column derivatization, the sample is injected directly into the HPLC, and after
separation, a number of colorimetric methods are used to derivatize the carbohydrate analytes
prior to detection [Honda, 1996]. Since the derivatization occurs online, a quick reaction is
required. When treated with sulfuric acid, carbohydrates can be dehydrated and converted to
furaldehyde derivatives, which can form chromogens with anthrone or phenol. Sialic acid,
amino sugars, and their derivatives can react with dimethylaminobenzaldehyde (DMAB or
Ehrlich's reagent) to form chromogens. Reducing sugars can also be quantified with copper
bicinchoninate reagent due to their capability to reduce copper ions. In this method, a reagent
pump and an online reactor are required for detection, increasing the instrument and operation
complexity.

HPLC SEPARATION OF CARBOHYDRATES

HPLC has been widely used to separate carbohydrates and their conjugates [Kakehi and
Honda, 1986]. HPLC separates compounds in a mixture by using the compounds different
partition properties between a stationary phase and a mobile phase [Snyder et al., 2010]. The
separation can be manipulated by varying the make-up and concentration of the mobile phase,
and the adsorption properties of the stationary phase. A compound will elute faster if it has
 High Performance Liquid Chromatography for Carbohydrate Analysis 7

greater affinity to the mobile phase than the stationary phase; by reducing its relative affinity
to the mobile phase, it will elute slower.
HPLC uses several types of separation mechanisms characterized by the type of
stationary and mobile phases employed. In NP chromatography, the stationary phase is
hydrophilic and the mobile phase is hydrophobic. In RP chromatography, the stationary phase
is hydrophobic. Ionic exchange chromatography uses ion exchange material as its stationary
phase to separate ionic compounds. SEC separates compounds by molecular size; it slows the
elution of small molecules by increasing their pathway through a porous stationary phase, and
increases elution of large molecules by excluding them from the small pore solid support.

Reversed phase (RP) chromatography

RP chromatography employs a non-polar stationary phase, which traditionally consists of

bound alkyl or aryl groups on a micro-silica particle surface. The polar mobile phase consists
of a water and organic solvent mixture containing methanol, propanol, acetonitrile (ACN), or
tetrahydrofuran with an optional buffer to adjust pH and ionic strength.
Direct analysis of free carbohydrates, using RP chromatography, can be difficult due to
short retention times and poor resolution as a result of their high polarity. The carbohydrates
are typically derivatized to increase their affinity to the hydrophobic stationary phase. When
separating ionic saccharides, ion-pairing agents, such as alkyl ammonium salts, can be added
to the mobile phase to increase retention and separation of the analytes. When separating
glycoproteins with RP chromatography, the composition of the mobile phase, besides
achieving high resolution, must cause minimal protein denaturation during the separation
process [El Rassi, 2002].

Hydrophobic interaction chromatography (HIC)

In HIC, the stationary phase is more hydrophilic than in RP chromatography [El Rassi,
2002]. Hydrophobic ligands (e.g., short alkyl, phenyl, polyether, and glycol molecules) are
attached with wide spacing onto a hydrophilic surface. The hydrophilic sites on the surface of
the stationary phase become hydrated when in contact with the aqueous mobile phase. Under
these conditions, glycoproteins interact with both hydrophilic and hydrophobic sites on the
support surface and are effectively separated. The HIC mobile phase does not denature the
protein portion of the glycoproteins because an organic solvent is not required for elution, and
the mildly hydrophobic stationary phase does not cause any substantial change in the
conformation of the protein conjugates. Additives, such as ammonium salts and alcohols may
added to adjust the retention and recovery of the glycoproteins.

Normal phase (NP) chromatography

In NP chromatography, the stationary phase is highly polar and the mobile phase is less
polar or hydrophobic. Silica, or silica particles modified with cyano, diol, or amino groups, is
a common stationary phase for NP applications. Hexane, ethyl acetate, and chloroform can be
used as a mobile phase with propanol, methanol, and water additives. Contrary to RP
separation, more polar compounds are retained longer in NP chromatography. Because of the
8 Xun Yan

hydrophobic nature of the mobile phase, NP chromatography is limited to analysis of

glycolipids and hydrophobic carbohydrate derivatives [Moreau et al., 2008].

Hydrophilic interaction liquid chromatography (HILIC)

HILIC uses a stationary phase similar to NP chromatography and an organic solvent

balanced with water (up to 40%) as its mobile phase. Using the same stationary phase as NP
chromatography, HILIC can be considered an aqueous NP chromatography method. The
organic component of the HILIC mobile phase is often acetonitrile, although ethyl acetate,
acetone, and methanol are also used. In HILIC conditions, water serves as the stronger
solvent and an increase in water content leads to a decrease in analyte retention. Polar and
ionized solutes tend to be more strongly retained in HILIC [Churms, 2002a; Slimestad and
Vagen, 2006].
When using HILIC for carbohydrate analysis, the silica stationary phase may be too polar
to use for unsubstituted sugars so cyano and diol stationary phases are often used. Mixtures of
mono-, di-, and oligosaccharides up to 20 units can be separated using this HILIC system
[D’Amboise et al., 1980]. For chromatographic analysis of oligosaccharides with a degree of
polymerization greater than three, a higher water content mobile phase is required. Amino-
modified silica particles can be used as the stationary phase, but in HILIC it presents one
serious disadvantage, the glycation of reducing sugars causes rapid degradation of the particle
surface. Polymeric ion exchange resins, such as crosslinked polystyrene divinylbenzene
sulphonate and crosslinked polyvinylpyridinium provide a more stable HILIC stationary
phase and offer multiple separation mechanisms [Churms, 2002a].

Anion exchange chromatography (AEC)

AEC is a popular choice for carbohydrate analysis [Bruggink et al., 2005; Clarke, 1993;
Pecina and Bonn, 1984]. It separates negative analyte ions based on their different affinity to
the cationic stationary phase and water-based mobile phase. Sodium hydroxide and sodium
acetate are used in the mobile phase to control pH and ionic strength. Crosslinked
polystyrene, modified with quaternary ammonium, is commonly used as the stationary phase
for high pH AEC.
Acidic saccharides, such as sialic acids, uronic acids, and sulfated saccharides, and
glycoproteins that are negatively charged under neutral or mildly acidic conditions, can be
easily separated using AEC [Zhang and Lee, 2002]. At high pH (12 to 14), the hydroxyl
groups on carbohydrates ionize into oxyanions, which can also be separated by AEC [Hardy
et al., 1988]. Alternatively, borate can be used to complex with carbohydrates to form anions,
which can also be separated with AEC [Malcolm et al., 1964; Zhang and Lee, 2002]. It has
been demonstrated that when combined with sensitive electrochemical detection, high
performance AEC can achieve baseline resolution of an homologous series of
polysaccharides with a degree of polymerization up to 60 (e.g., inulin) [Franck, 2006].

Size exclusive chromatography (SEC)

The most common technique for investigating intact polysaccharides is SEC, also known
as gel permeation chromatography or gel filtration chromatography. It does not rely on
 High Performance Liquid Chromatography for Carbohydrate Analysis 9

chemical differences but on molecule size to separate the polysaccharide molecules. It has
been the primary tool in measuring molecular weight and molecular weight distribution of
starch, fructan, alginate, chitosan, hyaluronic acid, glycoproteins, and proteoglycans [Churms,
2002].
SEC uses a porous material as the stationary phase. The mobile phase flows through
porous stationary phase and has a velocity distribution dependent on the pathway. The flow
around the adsorbent particles is faster than the flow through the pore space. The laminar flow
in the pore has a velocity profile that increases with increasing distance from the pore wall.
Small molecules diffuse into small pores, nearing the pore walls, and slowing their average
migration speed. Larger molecules cannot enter the small pores and, in the pores that they can
enter, they have a center mass allocated closer to the pore center than the pore wall, resulting
in a faster average migration speed. As a result, the larger molecules elute from the column
first, and the smaller ones last. SEC chromatograms can represent molecular weight
distribution in the effluent but to associate a specific elution volume to a known molecular
weight requires calibration with appropriate molecular weight standards. When selecting a
mobile phase for SEC, it needs to dissolve the sample and preferably, avoid significant
swelling of the polymer chains, which will affect its molecular shape. The solvent must also
suppress interactions of the analytes with the stationary phase surface since specific
interactions will change the retention patterns of the analytes. In carbohydrate analysis, an
electrolyte solution of sufficiently high ionic strength is often used as the mobile phase.

DETECTION METHODS FOR CARBOHYDRATE ANALYSIS

Detection methods capture a signal related to the concentration, and physical and
chemical properties of the analytes and effluent [Snyder et al., 2010]. To obtain precise and
accurate quantitative data, a proper detector needs to be selected based on the properties of
both the mobile phase and the carbohydrate analytes. The detector properties are
characterized by detection limit (the minimum detectable concentration), sensitivity (the
change in signal per unit change in concentration), signal to noise ratio (the detector signal
quality), and dynamic range (the concentration range over which the signal is proportional to
analyte concentration).

Refractive index detection (RID)

RID is often used for carbohydrate detection in HPLC analysis since intact carbohydrates
and their conjugates do not have appreciable UV absorbance. The detection principle of RID
is to measure the change in refractive index of effluent passing through the flow-cell relative
to the mobile phase [Bruno and Krattiger, 1995]. The greater the refractive index differences
between the effluent and the mobile phase, the greater the signal. If an eluted component has
the same refractive index as the mobile phase, it will be undetectable. RID is a purely
differential measurement so any change in the composition of the mobile phase will require
recalibrating the detector thus limiting the application of RID to only methods that do not
require a mobile phase gradient. Refractive index is a universal detection method but it has a
high detection limit and poor selectivity. It is also sensitive to fluctuations in temperature.
10 Xun Yan

Ultraviolet-visible detection (UV-Vis)

The most widely used detectors in HPLC are photometers based on ultraviolet and visible
light absorption (UV-Vis). UV-Vis detectors can detect a broad spectrum of compounds that
absorb light between 190 nm and 600 nm wavelengths. They also demonstrate good linearity
and dynamic range. Analyte concentrations in the flow cell are linearly proportional to the
light absorbance as described by Beer’s law.
UV-Vis detection is used to detect acidic saccharides and unsaturated oligosaccharides
produced by GAG lyase depolymerization [Ji et al., 2007]. Since most carbohydrates, as well
as many organic solvents and compounds, adsorb light at a UV range below 200 nm, UV
detection has limited use for un-derivatized carbohydrates. UV-Vis detection is more
commonly used to detect derivatized carbohydrates in which chromophore groups have been
introduced.

Fluorescence detection

In a fluorescence detector, an excitation light of a specific wavelength illuminates the

effluent sample as it passes through the flow cell. The compound absorbs light at its
excitation wavelength and then emits or fluoresces light at its emission wavelength. The
source of the excitation signal is a UV or Xenon lamp where the needed wavelength is
selected through a filter or monochromator. The emitted signal, following wavelength
selection, is monitored and detected by a photo detector. Since fluorescence is emitted in all
directions, it is common to monitor the emitted light at a right angle to the incident light.
Fluorescence detectors are very selective for fluorescent analytes so non-fluorescent
matrices or variations in the mobile phase do not interfer with the signal. Many of the
derivatization methods previously described can be used to prepare fluorescent carbohydrate
derivatives. Fluorescent detection methods often provide improved detection limits and
compound selectivity.

Evaporative light scattering (ELS) detection

ELS detection is a measure of light scattered by non-volatile or semi-volatile analyte

particles after the evaporation of the mobile phase. The column effluent is nebulized in a
stream of nitrogen and evaporated in a heated drift tube leaving the analyte particles
suspended in the carrier gas and mobile phase vapor. A laser light is passed through the
sample stream and the light scattered by the particles is detected by a photo detector mounted
at a fixed angle (90º or 120º) from the incident beam. Considered a universal detector, ELS
detection responds to most compounds though the signal may decrease for highly volatile
analytes. ELS detection response correlates to analyte quantity through a power equation. Its
response is independent of the solvent so ELS can be used with an elution gradient. It is also
insensitive to mobile phase temperature or fluctuations in flow-rate. The mobile phase for
ELS must be volatile and free of non-volatile additives, such as phosphate buffer. ELS
detection has a 1ng to 100 ng analyte on-column detection limit, which is 10– to 100–fold
lower than that seen with RI detection. The detector response is optimal with a constant
carrier gas flow rate and vaporization temperature.
 High Performance Liquid Chromatography for Carbohydrate Analysis 11

ELS detection has proven useful for carbohydrate determination because of the low
volatility of carbohydrates and their derivatives, and because of the ability to use gradient
elution to separate complex carbohydrate mixtures [Lafosse and Herbreteau, 2002].

Laser light scattering (LLS) detection

Where ELS detection measures light scattering in a gas, LLS detection measures particle
light scattering properties in solutions. In LLS detection, laser light is passed through a flow
cell, and the scattered light is measured with a photometer. Using the proper mathematical
model (Rayleigh equation), the signal can be used to determine molecular mass and size of
the analytes. The LLS detector is usually used in conjunction with SEC for the determination
of polymer molecular weights in the range of 103-106 Da [Jumel, 2002].
There are two types of LLS detection, low angle laser light scattering (LALLS) or
multi-angle laser light scattering (MALLS). In MALLS detection, a circular sample cell is
surrounded by detectors, which simultaneously collect the scattered light at different discrete
angles. Fitting the data into a Zimm plot, MALLS detection can be used to determine absolute
molecular weight and its distribution, radius of gyration, and conformational information.
LALLS measures light scattering intensity at a single low angle (< 10). When using low
angle LLS, data fitting and extrapolation are unnecessary, so the calculation is greatly
simplified. However, in doing so, conformational information is no longer available. When
molecular size is small compared to the light wavelength, a right angle scattering analysis is
preferred. Since analyte concentration is needed in molecular weight calculations, LLS
detection is always paired with an RI detector.

Electrochemical (EC) detection

The aldehyde and terminal alcohol moieties of carbohydrate species, including simple
sugars, acidic saccharides, polysaccharides, and their conjugates, can be oxidized and
detected directly by EC detection [Lacourse, 2002]. The oxidation reaction occurs on the
surface of the working electrode at a specific electrical potential, and the current through the
working electrode is measured as a function of applied voltage against a reference electrode.
EC detectors are more accurately termed amperometric devices since current is the measured
signal. The working electrode can be fouled by sample adsorption and oxidation so its surface
requires regular regeneration to provide stable and reproducible results. To regenerate the
electrode surface, it is scanned with brief positive and negative potential pulses, known as
pulsed amperometric detection (PAD).
Gold electrodes and platinum electrodes are widely used in PAD. Gold electrodes are
preferred over platinum due to the interference caused by dissolved oxygen on platinum
electrode surfaces. For carbohydrate analysis on gold electrodes, the mobile phase should be
alkaline (pH>12) and conductive. The high pH can be easily achieved by post-column
addition of alkaline buffer solutions. Alternatively, the mobile phase pH can be adjusted
providing the stationary phase is stable at high pH.
Besides the detection techniques discussed above, the application of mass spectroscopy is
critical to understanding the structure and sequence of carbohydrates and their conjugates
[Harvey, 2001; Stahl et al., 2002]. The different ionization techniques and ion analyzers used
in mass spectrometry offer broad analytical versatility, high sensitivity, and precise results.
12 Xun Yan

More information on the theory, instrumentation, and detailed methodology of mass

spectrometry and its application in carbohydrate structural analysis can be found in several
reviews [Harvey, 2011; Zaia, 2004] and is not discussed in this chapter.

HPLC APPLICATION TO CHITOSAN ANALYSIS

Chitosan is a copolymer of glucosamine and N-acetyl glucosamine, which can be derived
from chitin ([1→4]-2-acetamido-2-deoxy-β-D-glucan), a naturally occurring amino
polysaccharide [Toan et al., 2006]. The use of chitosan can be found in food, dietary
supplements, agricultural, pharmaceutical, and biotechnological industries [Li et al., 1996;
Kumar et al., 2004].
Direct analysis of chitosan can be difficult due to its wide range of molecular weights and
the extent of acetylation. In this report, we demonstrate an indirect chitosan analytical method
in which we hydrolyze chitosan under acidic conditions into glucosamine, derivatize the
liberated glucosamine with N-(9-Fluorenylmethoxycarbonyloxy)succinimide) (FMOC-OSu),
and quantify the concentration of glucosamine derivatives in the resultant mixture using
HPLC [Yan and Evenocheck, 2012; Zhang et al., 1993; Zhou et al., 2005].

Experimental conditions

Materials

All the reagents were used as purchased without further purification. Glucosamine
hydrochloride (C6H13NO5HCl), N-(9-Fluorenylmethoxycarbonyloxy)succinimide) (FMOC -
OSu), ACN, and sodium borate were purchased from Sigma-Aldrich (St. Louis, MO).
Chitosan (95% purity) was obtained from Wilke Resources (Lenexa, KS). Acetic acid, TFA,
and hydrochloric acid (HCl, trace metal grade) were purchased from Fisher Chemical
(Fairlawn, NJ). Different concentrations of hydrochloric acid were prepared by diluting
concentrated HCl with deionized (DI) water.
Chitosan hydrolysis and derivatization
Samples containing 50 mg chitosan were weighed into thick-walled glass digestion tubes,
2 mL of 1% acetic acid solution was added, and the mixture was vortexed until it formed a
consistent gel. Chitosan was hydrolyzed following the addition of 10 mL concentrated HCl
(8, 10, or 12 M) and heat (90 or 105C). After a designated period of hydrolysis (for kinetic
studies, the samples were taken at 30 minute intervals), the digestion mixture was cooled to
room temperature.
Since the efficiency of the FMOC -OSu derivatization is pH sensitive, the hydrolysate
has to be either neutralized or dried. To neutralize the hydrolysate, 1 mL is transferred into a
mixture of sodium borate (3.8 g) and DI water (30 ml), pH adjusted to 7.00 with 10 M HCl,
and the solution volume adjusted to 50 mL using 0.2 M borate buffer, pH 7.0. The
derivatization was carried out by mixing 1 mL of the neutralized hydrolysate with 1 mL of 10
mg/mL FMOC -OSu in acetonitrile and the reaction allowed to progress to completion at
ambient temperature for at least 4 hours without agitation.
Alternatively, 100 μL of the hydrolysate can be transferred into 2 mL deep-well plates
and dried under vacuum at 40 – 50C overnight, the resultant glucosamine film extracted with
100 μL triethylamine/DI water (0.75%), and derivatized with the addition of 400 μL of 10
mg/mL FMOC -OSu in acetonitrile.
 High Performance Liquid Chromatography for Carbohydrate Analysis 13

After derivatization, the samples were diluted with equal amounts of HPLC mobile phase
(0.05% TFA/ACN, 1:1 [v/v]).

HPLC analysis

HPLC analysis was conducted using an Agilent 1100 system equipped with degasser,
quaternary pump, autosampler, and photodiode array detector. Agilent Zorbax SB-C18, 5 μm,
150 mm x 4.6 mm column was used for the chromatography. To analyze glucosamine FMOC
derivatives, the HPLC conditions were as described by Zhou et al. [Zhou et al., 2005]. In
brief, the elution gradient begins at 70% of 0.05% TFA in water (A) and 30% ACN (B). After
6 minutes, B is elevated to 100% ACN in 5 minutes. The elution rate was held at 0.8 mL/min
and the injection volume was 10 μL for both samples and standards. The chromatogram was
recorded at 265 nm UV, integrated, and analyzed with Agilent Chemstation.
Chitosan content was calculated as follows:

mg (mg )
C (%)   [161.2  DA(%)  42  (1  DA(%))  M A ]  100%
215.7  mo (mg )

where, C is the chitosan content (%); mg is the glucosamine content as determined by

HPLC; 215.7 is the glucosamine (HCl) molecular weight; mo is the sample weight for
analysis; 161.2 is the mole weight of glucosamine repeating unit in chitosan; DA is the degree
of acetylation in chitosan; 42 is the mole weight of one acetyl group; MA is the molecular
weight of the counter acid in chitosan.

Results and discussion

Glucosamine separation and quantitative determination

Chitosan and a formulation containing chitosan were hydrolyzed, derivatized, and

analyzed with HPLC. The chromatograms for the hydrolysates were the same as those for the
glucosamine standards. As shown in the chromatogram (Figure 1), glucosamine derivatives
were detected in two peaks at 6 and 7.5 minutes. This is due to the presence of two possible
conformations of free glucosamine (α- and β-glucosamine) in solution. The sum of the two
peak areas were used to calculate total glucosamine content
14 Xun Yan

Figure 1. HPLC chromatogram of FMOC -OSu glucosamine derivatives.

Chitosan hydrolysis

The mechanism of chitosan hydrolysis was studied by Shabrukova et al. and Einbu et al.
[Einbu et al., 2007; Shabrukova et al., 2002]. In strong acid, chitosan could be fully
hydrolyzed to acetic acid and glucosamine. When the hydrolysis is incomplete, acetyl
glucosamine is observed. When weak acid is used at low temperature, oligosaccharides are
dominant in the hydrolysate products.
Chitosan was first dissolved in acetic acid to aid its dispersion in the concentrated HCl
solutions. The dispersed chitosan was then hydrolyzed at two different temperatures. The
glucosamine content in the hydrolysate solution was analyzed and plotted against hydrolysis
time. Figure 2 shows the glucosamine yield during the hydrolysis process with respect to
hydrolysis time at various acid concentrations and temperatures.

100 a b
100
Glucosamine Conversion (%)

Glucosamine Conversion (%)

80 80

60 60

40 40
o
105 C and 12 M HCl
o
105 C and 10 M HCl
20 o 20
105 C and 8 M HCl o
90 C and 12M HCl
o
90 C and 10M HCl
o
90 C and 8M HCl
0 0
0 2 4 6 8 10
0 2 4 6 8 10
Time (Hours) Time (Hours)
 High Performance Liquid Chromatography for Carbohydrate Analysis 15

Figure 2. Effects of acid concentration and temperature on glucosamine yield in chitosan

hydrolysis. (a) Percent glucosamine conversion following chitosan hydrolysis with 8 M, 10
M, and 12 M HCl at 105˚C. The glucosamine conversion is calculated based on HPLC
measurement and theoretical content. Data is presented as best fit curves. (b) Similar to a,
except the hydrolysis temperature is 90˚C.

As shown in Figure 2, using 8 M HCl, after 8 hours of hydrolysis, chitosan is not fully
hydrolyzed into glucosamine, and less than 40% glucosamine yield is detected at 90 ˚C. The
glucosamine yield reaches 98% using a digestion condition of 10 M HCl and 105 ˚C in 6
hours. We also observed similar recovery using 12 M HCl and 90 ˚C in 6 hours. Based on
these results, hydrolysis conditions of 10 M HCl, 105ºC, and a six-hour duration were
selected and used for method validation. The hydrolysis conditions of 12 M HCl and 105˚C
result in complete glucosamine conversion in two hours, but the endpoint is difficult to
control because glucosamine decomposes rapidly under these conditions.

Effects of acid concentration and temperature on glucosamine decomposition

During acid hydrolysis of chitosan, the end product stability is a critical concern for
analysis accuracy. Since glucosamine is known to be deaminated by acid hydrolysis, it is
necessary to minimize the decomposition to achieve an accurate result for chitosan
quantitation [Einbu et al., 2007; Shabrukova et al., 2002]. To determine the degree of
decomposition, glucosamine samples were weighed into digestion tubes and treated with the
selected hydrolysis condition (105˚C, 10 M HCl). At the end of each hour interval, a sample
was taken and analyzed for glucosamine. The results were recorded and are shown in Figure
3.

100
Glucosamine conversion (%)

95

90

85

80

75
0 2 4 6 8 10
Time (hours)
Figure 3. Glucosamine decomposition at selected digestion conditions (10 M HCl,
105˚C) with first-order decomposition model fitting.
16 Xun Yan

The degradation of glucosamine fits a first-order decomposition model. After 6 hours of

hydrolysis, we observed approximately a 20% loss of glucosamine. However, the
decomposition kinetics during the chitosan hydrolysis process are complicated by the
processes of chitosan depolymerization and deacetylation, and glucosamine generation. The
glucosamine decomposition during chitosan hydrolysis does not appear to have a significant
impact on chitosan recovery. The glucosamine content of the chitosan plain material is 93.6
% (w/w) which is experimentally close to the nominal chitosan content of the plain material
(95%). The recovery of glucosamine from the formulation was greater than 99%,
demonstrating that the decomposition of glucosamine during chitosan hydrolysis at the
specified conditions has minimal impact on assay accuracy [Yan and Evenocheck, 2012].

Method validation

To verify the detector response linearity, five concentrations of glucosamine standards

were analyzed and the results plotted as peak area counts against glucosamine concentration.
The correlation coefficient R2 is over 0.999 between the two parameters. The assay
repeatability and intermediate precision were determined for the relative standard deviation of
the test results from two analysts. The relative standard deviations (RSD) of five replicate
analyses (repeatability) for the chitosan and formulations are 0.93 % and 1.67 % respectively,
for analyst one, and 5.25 % and 5.49 % respectively, for analyst two. The overall RSD
(reproducibility) were 4.52 % for the analysis of chitosan and 3.84 % for the analysis of the
formulation. The measured glucosamine content was within 5% of the nominal content for
both chitosan and the formulations. The recovery of chitosan from the matrix samples were
calculated as percentage of the glucosamine spike. Chitosan recovery for the three
formulations (50, 100, and 150% of target chitosan) was between 99 and 103%,
demonstrating the accuracy of the method.

CONCLUSIONS
Carbohydrates are widely distributed in biological systems providing various functions.
Research of their medicinal uses and applications in consumer products has significantly
increased over the years. Because of their structural diversity, analytical methods for their
determination are quite varied. HPLC has been extensively used in carbohydrate analysis due
to its versatility and separation capabilities. Its different separation modes, coupled with
different detection methods, can meet the high analytical demands and provide useful tools
for the clinical and routine chemical analysis of carbohydrates.

REFERENCES

Anumula, K. R., (1994). Quantitative determination of monosaccharides in glycoproteins by

high-performance liquid chromatography with highly sensitive fluorescence detection.
Anal. Biochem., 220, 275-283.
 High Performance Liquid Chromatography for Carbohydrate Analysis 17

Anumula, K. R., (2006). Advances in fluorescence derivatization methods for high-

performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal.
Biochem., 350, 1-23.
Aoki, K., Tiemeyer, M., (2010). The glycomics of glycan glycuronylation in Drosophila
Melanogaster. Methods in Enzymol., 480, 297-321.
Barraud, P., Stott, S., (2008). Glycolipids: Biomarkers for Neural Stem and Progenitor Cells.
In: Sasaki, D. (Ed.), Glycolipids: New Research. New York: Nova Science Publishers
Bruggink, C., Maurer, R., Herrmann, H., Cavalli, S., Hoefler, F., (2005). Analysis of
carbohydrates by anion exchange chromatography and mass spectrometry. J.
Chromatogr. A, 1085, 104-109.
Bruno, A. E., Krattiger, B., (1995). On-Column Refractive Index Detection of Carbohydrates
Separated by HPLC and CE. J. Chromatogr. Library, 58, 431-446.
Churms, S. C., (2002a). High performance hydrophilic interaction chromatography of
carbohydrates with polar sorbents. J. Chromatogr. Library, 66, 121-163.
Churms, S. C., (2002b). Modern size-exclusion chromatography of carbohydrates and
glycoconjugates. J. Chromatogr. Library, 66, 267-303.
Clarke, A. J., (1993). Compositional analysis of peptidoglycan by HPAEC. Anal. Biochem.,
212, 344-350.
D’Amboise, M., Noel, D., Hanai, T., (1980). Characterization of bonded-amine packing for
liquid chromatography and high-sensitivity determination of carbohydrates. Carbohydr.
Res., 79, 1-10.
De Castro, C., Parrilli, M., Holst, O., Molinaro, A., (2010). Microbe-associated molecular
patterns in innated immunity: extraction and chemical analysis of gram negative bacterial
lipopolysaccharides. Methods in Enzymol., 480, 89-115.
Didraga, M., Barroso, B., Bischoff, R., (2006). Recent Developments in Proteoglycan
Purification and Analysis. Current Pharmaceutical Analysis, 2, 323-337.
Einbu, A., Grasdalen, H., Varum, K. M., (2007). Kinetics of hydrolysis of chitin/chitosan
oligomers in concentrated hydrochloric acid. Carbohydr. Res., 342, 1055-1062.
El Rassi, Z., (2002). Reversed-phase and hydrophobic interaction chromatography of
carbohydrates and glycoconjugates. J. Chromatogr. Library (Vol. 66, pp. 41-102).
Esko, J. D., Doering, T. L., Raetz, C. R. H., (2009). Eubacteria and Archaea. In A. Varki, R.
D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart M. E.
Etzler (Eds.), Essentials of Glycobiology. Cold Spring Harbor (NY): Cold Spring Harbor
Laboratory Press.
Franck, A., (2006). Inulin. In Stephen, A. M., Phillips, G. O., Williams, P. A. (Eds.), Food
Polysaccharides and Their Applications (2nd ed.). Boca Raton: Taylor & Francis.
Freeze, H. H., Kranz, C., (2006). Endoglycosidase and Glycoamidase Release of N-Linked
Oligosaccharides. Curr. Protoc. Protein Sci., 45.
Fu, D., O'Neill, R. A., (1995). Monosaccharide composition analysis of oligosaccharides and
glycoproteins by high-performance liquid chromatography. Anal. Biochem., 227, 377-
384.
Geyer, H., Geyer, R., (2006). Strategies for analysis of glycoprotein glycosylation. Biochim.
Biophys. Acta 1764, 1853-1869.
Harazono, A., Kobayashi, T., Kawasaki, N., Itoh, S., Tada, M., Hashii, N., Yamaguchi, T.,
(2011). A comparative study of monosaccharide composition analysis as a carbohydrate
test for biopharmaceuticals. Biologicals, 39, 171-180.
18 Xun Yan

Hardy, M. R., Townsend, R. R., Lee, Y. C., (1988). Monosaccharide analysis of

glycoconjugates by anion exchange chromatography with pulsed amperometric detection.
Anal. Biochem., 170, 54-62.
Harvey, D. J., (2001). Identification of protein-bound carbohydrates by mass spectrometry.
Proteomics, 1, 311-328.
Harvey, D. J., (2011). Analysis of carbohydrates and glycoconjugates by matrix-assisted laser
desorption/ionization mass spectrometry: An update for the period 2005-2006. Mass
Spectrometry Reviews, 30, 1-100.
Hase, S., (2002). Pre- and post-column detection-oriented derivatization techniques in HPLC
of carbohydrates. J. Chromatogr. Library, 66, 1043-1069.
Honda, S., (1996). Postcolumn derivatization for chromatographic analysis of carbohydrates.
J. Chromatogr. A, 720, 183-199.
Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K., Nakamura, J., (1989). High-
performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-
absorbing and electrochemically sensitive 1-phenyl-3-methyl-5-pyrazolone derivatives.
Anal. Biochem., 180, 351-357.
Ji, D., Roman, M., Zhou, J., Hildreth, J., (2007). Determination of chondroitin sulfate content
in raw materials and dietary supplements by high-performance liquid chromatography
with ultraviolet detection after enzymatic hydrolysis: single-laboratory validation. J
AOAC Int., 90, 659-669.
Jumel, K., (2002). Molar mass determination of complex bioglycopolymers by size exclusion
chromatography and light scattering detection. J. Chromatogr. Library, 66, 305-345.
Kakehi, K., Honda, S., (1986). Profiling of carbohydrates, glycoproteins and glycolipids. J.
Chromatogr., 379, 27-55.
Kakehi, K., Uedo, M., Suzuki, S., Honda, S., (1993). Determination of hyaluronic acid by
HPLC of oligosacharides derived therefrom as 1-(4-methoxy) phenyl-3-methyl-
pyrazolone derivatives. J. Chromatogr., 630, 141-146.
Kesselmeier, J., Heinz, E., (1987). Separation of molecular species of plant glycolipids and
phospholipids by HPLC. Methods in Enzymol., 148, 650-661.
Lacourse, W. R., (2002). Pulsed electrochemical detection of carbohydrates at noble metal
electrodes following liquid chromatographic and electrophoretic separation. J.
Chromatogr. Library, 66, 905-946.
Lafosse, M., Herbreteau, B., (2002). Carbohydrate analysis by LC and SFC using
evaporative light scattering detection. J. Chromatogr. Library, 66, 1101-1134.
Lamari, F. N., Kuhn, R., Karamanos, N. K., (2003). Derivatization of carbohydrates for
chromatographic, electrophoretic and mass spectrometric structure analysis. J.
Chromatogr. B, 793, 15-36.
Lee, K. B., Loganathan, D., Merchant, Z. M., Linhardt, R. J., (1989). Carbohydrate analysis
of glycoproteins. Appl. Biochem. Biotechnol., 23, 53-80.
Lennarz, W. J., (1980). The Biochemistry of Glycopoteins and Proteoglycans. New York:
Plenum Press.
Li, Q., Dunn, E. T., Grandmaison, E. W., Goosen, M. F. A., (1996). Applications and
properties of chitosan. In: Goosen, M. F. A. (Ed.), Applications of Chitin and Chitosan
(pp. 3-29). Boca Raton: CRC Press.
Malcolm, E. W., Green, J. W., Swenson, H. A., (1964). A study of the borate–carbohydrate
complex. J. Chem. Soc., 1964, 4669-4676.
 High Performance Liquid Chromatography for Carbohydrate Analysis 19

Marino, K., Bones, J., Katta, J. J., Rudd, P. M., (2010). A systematic approach to protein
glycosylation analysis: a path through the maze. Nat. Chem. Biol., 6, 713-723.
Merry, A. H., Neville, D. C. A., Royle, L., Matthews, B., Harvey, D. J., Dwek, R. A., Rudd,
P. M., (2002). Recovery of Intact 2-Aminobenzamide-Labeled O-Glycans Released from
Glycoproteins by Hydrazinolysis. Anal. Biochem., 304, 91-99.
Moreau, R. A., Doehlert, D. C., Welti, R., Isaac, G., Roth, M., Tamura, P., Nuñez, A., (2008).
The identification of mono-, di-, tri-, and tetragalactosyl-diacylglycerols and their natural
estolides in oat kernels. Lipids, 43, 533-548.
Mort, A. J., Pierce, M. L., (2002). Preparation of carbohydrates for analysis by modern
chromatography and electrophoresis. J. Chromatogr. Library, 66, 3-38.
Nilsson, B., Svensson, S., (1979). A new method for degradation of the protein part of
glycoproteins: isolation of the carbohydrate chains of asialofetuin. Carbohydr. Res., 72,
183-190.
Paulsen, B. S., Olafsdottir, E. S., Ingolfsdottir, K., (2002). Chromatography and
electrophoresis in speparation and characterization of polysaccharides from lichens. J.
Chromatogr. A, 967, 163-171.
Paulus, A., Klockow, A., (1996). Detection of carbohydrates in capillary electrophoresis. J.
Chromatogr. A, 720, 353-376.
Pecina, P., Bonn, G., (1984). High performance liquid chromatographic elution behaviour of
alchohols, aldehydes, ketones, organic acids and carbohydrates on a strong cation
exchange stationary phase. J. Chromatogr., 287, 245-258.
Raetz, C. R. H., (1990). Biochemistry of endotoxins. Annu. Rev. Biochem., 59, 129-170.
Raetz, C. R. H., Whitfield, C., (2002). Lipopolysaccharide endotoxins. Annu. Rev. Biochem.,
71, 635-700.
Kumar, M. N., Muzzarelli, R. A., Muzzarelli, C., Sashiwa, H., Domb, A. J. , (2004). Chitosan
chemistry and pharmaceutical perspectives. Chem. Rev., 104, 6017-6084.
Robyt, J. F., (1998). Essentials of Carbohydrate Chemistry. New York: Springer-Verlag.
Rohrer, J. S., Cooper, G. A., Townsend, R. R., (1993). Identification, quantification, and
characterization of glycopeptides in reversed-phase HPLC separations of glycoprotein
proteolytic digests. Anal. Biochem., 212, 7-16.
Rosenfeld, J. M. (2003). Derivatization in the current practice of analytical chemistry. Trends
in Analytical Chemistry, 22, 785-798.
Shabrukova, N., Shestekova, L. M., Zainetdinova, D. R., Gamayurova, V. S., (2002).
Research of acid hydrolyses of chitin-glucan and chitosan-glucan complexes. Chemistry
and Computational Simulation. Butlerov Communications, 2, 57-59.
Slimestad, R., Vagen, I. M., (2006). Thermal stability of glucose and other sugar aldoses in
normal phase HPLC. J. Chromatogr. A, 1118, 281-284.
Snyder, L. R., Kirkland, J. J., Dolan, J. W., (2010). Introduction to Modern Liquid
Chromatography (3rd ed.). NJ: John Wiley & Sons.
Stahl, M., von Broke, A., Bayer, E., (2002). Mass spectrometry of oligosaccharides. J.
Chromatogr. Library, 66, 961-1042.
Strydom, D. J., (1994). Chromatographic separation of 1-phenyl-3-methyl-5-pyrazolone-
derivatized neutral, acidic and basic aldoses. J. Chromatogr. A, 678, 17-23.
Svejcar, J., Robertson, W. V., (1967). Micro separation and determination of mammalian
acidic glycosaminoglycans (mucopolysaccharides). Anal. Biochem., 18, 333-350.
 20 Xun Yan

Thiemermann, C., (2002). Interactions between lipoteichoic acid and peptidoglycan from
Staphylococcus aureus: a structural and functional analysis. Microbes and Infection, 4,
927-935.
Toan, N. V., Ng, C. H., Aye, K. N., S., T. T., Stevens, W. F., (2006). Production of high-
quality chitin and chitosan from preconditioned shrimp shells. J. Chem. Technol.
Biotechnol., 81, 1113-1118.
Van Die, I., Van Stijin, C. M., Geyer, H., Geyer, R., (2010). Structural and functional analysis
of glycosphingolipids of Schistosoma Mansoni. Methods in Enzymol., 480, 117-140.
Varki, A., Lowe, J. B. (2009). Biological Roles of Glycans. In: Varki, A., Cummings, R. D.,
Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E. (Eds.),
Essentials of Glycobiology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Vyios, D. H., Karamanos, N. K., Tsigano, C. P., (2002). Advances in analysis of
glycosaminoglycans: its application for the assessment of physiological and pathological
state of connective tissues. J. Chromatogr. B, 781, 21-38.
Yamada, K., Kakehi, K., (2011). Recent advances in the analysis of carbohydrates for
biomedical use. J. Pharm.Biomed. Anal., 55, 702-727.
Yan, X., Evenocheck, H. (2012). Chitosan analysis using acid hydrolysis and HPLC/UV.
Carbohydr. Polym., 87, 1774-1778.
Zaia, J., (2004). Mass spectrometry of oligosaccharides. Mass Spectrometry Reviews, 23, 161-
227.
Zhang, R., Zhang, Z., Liu, G., Hidaka, Y., Shimonishi, Y., (1993). High-performance liquid
chromatographic determination of neutral and amino monosaccharides by ultraviolet and
fluorescence detection of sugar 9-fluorenylmethoxycarbonyl hydrazones and 9-
fluorenylmethoxycarbonyl amino sugars at picomole and sub-picomole levels. J.
Chromatogr., 646, 45-52.
Zhang, Y., Lee, Y. C., (2002). High-performance anion-exchange chromatography of
carbohydrates on pellicular resin columns. J. Chromatogr. Library, 66, 207-250.
Zhou, J., Waszkuc, T., Mohammed, F., (2005). Determination of Glucosamine in Raw
Materials and Dietary Supplements Containing Glucosamine Sulfate and/or Glucosamine
Hydrochloride by High-Performance Liquid Chromatography with FMOC-Su
Derivatization: Collaborative Study. J. AOAC Int., 88, 1048-1058.

View publication stats