Journal of Chromatography A
Volume 1153, Issues 1-2, 15 June 2007, Pages 74-89
Advances in Sample Preparation - Part II
doi:10.1016/j.chroma.2007.01.028 | How to Cite or Link Using DOI
Copyright © 2007 Elsevier B.V. All rights reserved.
Cited By in Scopus (6)
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Review
Recent developments in sample preparation
for chromatographic analysis of
carbohydrates
M.L. Sanza and I. Martínez-Castro
a
, a,
Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas (CSIC),
C/Juan de la Cierva, 3 E-28006 Madrid, Spain
Available online 12 January 2007.
Abstract
Carbohydrates are a very important group of compounds due to their roles as structural
materials, sources of energy, biological functions and environmental analytes; they are
characterized by their structural diversity and the high number of isomers they present. While
many advances have been made in carbohydrate analysis, the sample preparation remains
difficult. This review aims to summarize the most important treatments which have been recently
developed to be applied prior to the analysis of carbohydrates by chromatographic techniques.
Due to the multiplicity of structures and matrices, many different techniques are required for
clean-up, fractionation and derivatization. A number of new techniques which could be
potentially adequate for carbohydrate characterization have also been revised.
Keywords: Sample treatment; Carbohydrates ; Fractionation; Derivatization; Clean-up;
Chromatographic techniques
Article Outline
1. Introduction
2. Main processes in carbohydrate sample preparation
2.1. Clean-up
2.1.1. Filtration
2.1.2. Extraction
2.1.2.1. Liquid extraction
2.1.2.2. Solid phase extraction
2.1.2.3. Miscellaneous
2.2. Fractionation
2.2.1. Membrane fractionation
2.2.1.1. Ultrafiltration and nanofiltration
2.2.1.2. Dialysis and microdialysis
2.2.1.3. Liquid membranes
2.2.1.4. Polymeric membrane extraction
2.2.2. Carbon fractionation
2.2.3. Size exclusion chromatography
2.2.4. Field flow fractionation
2.2.5. Ion exchange chromatography
2.2.6. Simulated moving bed chromatography
2.2.7. Microbiological treatments
2.2.8. Supercritical fluid extraction
2.2.9. Pressurized liquid extraction
2.2.10. Molecularly imprinted polymers
2.2.11. Hydrophilic interaction chromatography
2.3. Chemical treatments
2.3.1. Hydrolysis
2.3.2. Derivatization
2.3.2.1. Derivatives for LC and CE
2.3.2.2. Derivatives for GC
2.3.3. Derivatives used for structural analysis
2.3.4. Thermochemolysis
3. Concluding remarks
Acknowledgements
References
1. Introduction
Carbohydrates are probably the most abundant organic molecules on the Earth, since they are
present in the cells of all living organisms and are the main constituents of plants and trees,
representing the major form of photosynthetically assimilated carbon in the biosphere. They
comprise up to 75 wt% of vascular plant tissues as structural polysaccharides [1] and [2]
Carbohydrates can be divided into classes of monosaccharides, oligosaccharides and
polysaccharides. All of them are polar molecules which encompass a high diversity of molecular
size, glycosidic linkage and functionality. They are always constituted by carbon, hydrogen and
oxygen but may contain other elements such as nitrogen, sulphur, phosphor, etc. They are
characterized by the high number of hydroxyl groups but other functional groups such as
carbonyl, carboxyl, amino, ether, etc. could be present. Carbohydrates also occur linked to
proteins or lipids which form glycoproteins or glycolipids, respectively. These two classes of
substances are especially interesting in the biochemistry field and will not be considered here.
In recent times big advances in analytical techniques for carbohydrate determination are taking
place: improvements in resolution and sensitivity [3], multiple couplings, oligosaccharide arrays
and glyco-chips [4], etc. which make necessary a corresponding advancement in those
techniques dealing with sample preparation.
Carbohydrates are subjects of chromatographic analysis in different fields: agriculture, food,
medicine, organic chemistry, biology, environmental science [2], [5] and [6] and also in
historic/archaeological studies, old paintings, etc. [7].
Regarding sample preparation it is necessary to take into account some physical and chemical
properties:
- Molecular weight, molecular size and shape: their molecular weights range within that of a
tetrose (C4H8O3) and those of polysaccharides with several thousands of monosaccharide units
which form polymers with straight- or branched-chain. The monosaccharide units are usually in
the form of hemiacetal rings. The number and types of isomers they are capable of displaying is
higher than that of any other group of compounds.
- Solubility in water and polar solvents is a function of molecular weight: they are generally
water-soluble and for many polymers complex equilibria can be expected, such as gelification,
helix formation, adsorption, etc., but there are a number of insoluble matrices such as cellulose
and other polysaccharides. Solubility in alcohols and polar solvents like pyridine, dimethyl
sulfoxide and dimethylformamide is restricted to small saccharides. The word “sugars” usually
refers to water-soluble carbohydrates.
- Although some saccharides are hygroscopic or thermally unstable, they can generally stand at
room temperature and are air-stable.
- Thermal stability: is very low; carbohydrates easily undergo dehydration and further
degradation such as the well-known caramelization reactions.
- Very high chemical reactivity: they react with acids, bases, amino groups and many reagents. It
is easy to make derivatives or to introduce protecting groups for specific functional groups; but
in order to keep the molecules unchanged it is necessary to hold pH near neutrality and to avoid
isomerization or decomposition reactions which easily take place in alkaline or acid media.
Isomerization of glucose to fructose has been confirmed even during some chromatographic
processes under alkaline conditions [8] and [9].
- They do not have properties of fluorescence emission and they only absorb in UV at very short
wavelengths.
Carbohydrates are becoming increasingly important due to their biological properties such as
prebiotic or antiadhesive effects [10] and [11] and the necessity of developing analytical methods
which can allow their characterization is growing. Putting aside the biochemical applications,
analyses of carbohydrates in most fields have different requirements according to their
objectives: it can be convenient to fractionate by molecular weight, or to discriminate between
two groups of molecules with different functional groups.
This review describes the main classes of processes of sample preparation that can be necessary
for chromatographic analysis of carbohydrates in different matrices, based on the classification
proposed by Smith [12]: to remove interferents (clean-up), to separate some carbohydrates
from other carbohydrates or to increase their concentration (fractionation) and to convert the
analyte into a more suitable form for detection or separation (chemical treatments). Most of the
techniques here described can be applied to HPLC, GC or CE analysis mainly depending on the
characteristics of the sample to be analysed (monosaccharides, oligosaccharides or
polysaccharides, simple matrices or complex mixtures, etc.). The literature cited covers the last
few years and is intended to be representative of the field rather than comprehensive.
2. Main processes in carbohydrate sample preparation
Fig. 1 shows a diagram of the main processes required for carbohydrate analysis. In some
foods, beverages or pharmaceutical products, sample preparation for carbohydrate analysis can
be limited to a simple dilution and filtration procedure [13] and [14]; however, all the above
mentioned steps (clean-up, fractionation, hydrolysis and/or derivatization) may be necessary for
complex samples.
Full-size image (32K)
Fig. 1. Diagram of sample preparation prior to chromatographic analysis of carbohydrates.
View Within Article
2.1. Clean-up
This procedure is usually carried out to discard insoluble material, lipids and proteins, desalt the
sample and remove impurities. These steps are essential for the success of many techniques used
for carbohydrate analysis such as gas, liquid chromatography and capillary electrophoresis.
Clean-up is also directed towards removing from the sample those substances with
chromatographic behaviour similar to that of sugars which may act as potential interferents
(carboxylic acids, polyphenols, etc.). Old methods were based on precipitation with ethanol and
subsequent filtration or centrifugation of the insoluble material [15]. Currently, some of them
continue to rely on standard methods and regulations [16] but modern procedures such as
membrane filtration are preferred for more complex mixtures.
Clean-up procedures can be grouped into filtration and extraction techniques.
2.1.1. Filtration
It is well known that filtration is an important part of sample preparation mainly for HPLC and
CE analysis to avoid insoluble material blocking the column. Different types of filters including
paper, glass fiber and membrane filters are used [12]. Moreover, other filtration techniques based
on the use of membranes such as ultrafiltration (UF), nanofiltration (NF) or reverse osmosis
(RO) are commonly utilized to obtain high-purity carbohydrates from complex matrices [17],
[18], [19] and [20] which are useful for both biological applications and food analysis. These
techniques allow the purification of carbohydrates and the concentration of the analyte over the
matrix in order to decrease the detection limit [21] and [22]. A combined membrane processing
system with UF and NF membranes was evaluated by Kamada et al. [23] showing a promising
effectiveness for purifying and concentrating oligosaccharides from chicory rootstock. Wang et
al. [24] studied the use of NF membranes in diafiltration mode to separate inorganic electrolytes
(NaCl, KCl, MgCl, Na2SO4 and MgSO4) from carbohydrates. Different models of transport
mechanism through NF membranes have also been designed to predict the results of purification
of oligosaccharides under different operation conditions [25].
In carbohydrate chemistry the use of these procedures for sample preparation has not only
been applied to the purification of samples: these techniques also constitute an important tool for
carbohydrate fractionation. Therefore, they will be discussed more extensively in the
fractionation section.
While several advances have been made in chromatographic techniques in an attempt to decrease
the time of analysis and increase the resolution and the sensitivity, sample preparation process is
still tedious and frequently lacks automatization. However, some advances have recently been
achieved in membrane extraction techniques hyphenated with HPLC, GC and CE [21]. High
performance anion exchange chromatography (HPAEC) on-line or off-line coupled with
electrospray ionization (ESI) or matrix assisted laser desorption ionization time of flight
(MALDI-TOF) appears to be a useful tool for oligosaccharide analysis. However, the high pH of
the mobile phase employed in HPAEC (pH ≈ 13) involving the instability of the oligomers for
long periods of time (see Section 1) and the presence of large amounts of sodium acetate, make
the desalting of the mobile phase necessary for detection by MS. The use of on-line membrane
suppressors has been found to be successful for this purpose [26] and [27]; however, in these
applications only sodium is removed and the presence of acetic acid can disturb MALDI-TOF
analysis. Other assays have been developed using a cation membrane suppressor connected in
series to an anion membrane suppressor [28]. This system has showed good results for on-line
desalting of HPAEC eluent [28] and [29].
A new method for carbohydrate pre-purification based on the use of a two-dimensional liquid
chromatographic system has been suggested by Rogatsky and Stein [30]. This automated system
was used to isolate and concentrate carbohydrates present in small amounts from biological
matrices prior to LC–ESI–MS–MS analysis.
2.1.2. Extraction
As indicated by Smith [12], extraction is the oldest and most basic sample preparation method
used to purify the analyte of interest from interfering substances or from a matrix using solvents.
Carbohydrates have been extracted using traditional methods such as solid–liquid and liquid–
liquid extractions. Enhanced solvent extraction methods like supercritical fluids or accelerated
solvent extraction systems are used at present as an interesting alternative for the analysis of
pesticides and non-polar substances. Very few of the latter techniques are applied to
carbohydrates since they are more orientated towards fractionation; therefore they will be
discussed later.
Precipitation of undesirable compounds with reagents is another way of extracting
carbohydrates from complex matrices. Different reagents such as Carrez are often used to
precipitate proteins or lipids prior to carbohydrate analysis [31], whereas precipitation with
acetate buffer/acetonitrile or perchloric acid has also been used [32]. To extract extracellular
bacterial polysaccharides from culture media containing proteins, trichloroacetic acid (TCA) and
enzyme treatments have commonly been employed to precipitate and hydrolyse the proteins,
respectively [33].
2.1.2.1. Liquid extraction
Numerous standard sample preparation methods are based on liquid–liquid extraction (LLE) as
described in a recent review [21]. However, this technique still has several drawbacks: difficult
automatization and large volumes of organic solvent are involved. Extraction of carbohydrates
from aqueous media is a difficult task due to their wide structural variety and their low solubility
in organic solvents.
Xylose and other sugars have been extracted from complex matrices using boronic acid
extractants [34], whereas Matsumuto et al. [35] formed ion-pairs with boronate anion and
quaternary ammonium cation for carbohydrate extraction. A macrotricyclic cage has been used
to extract monosaccharides from water to chloroform [36] and the use of carrier-mediated
extraction has been shown to be possible [37]. The same authors [38] have investigated 14
different solvents such as hydrocarbons, aromatics, alcohols and methyl tert-butyl ethers for the
selective recovery of neutral sugars (6 monosaccharides and 3 disaccharides) from aqueous
media, using primary, secondary and tertiary amines as carriers. The extraction rates varied
between 20 and 90%, but several points need to be clarified (for example, the interaction aminecarbonyl which could give rise to Maillard reaction).
The effect of hot water on the extraction of carbohydrates from different matrices has been
reviewed by King [39] utilizing several examples from the literature. On the other hand, alcohols
have been used for the extraction of total soluble carbohydrates from complex matrices.
Different alcohols (methanol, ethanol, isopropanol) at different concentrations (10–80%) allow
the extraction of complex mixtures of carbohydrates to be obtained for subsequent analysis as
has been shown by several authors [40], [41], [42], [43] and [44].
2.1.2.2. Solid phase extraction
Solid phase extraction (SPE) has commonly been used to purify samples prior to their analysis
[45], [46] and [47]. In SPE, samples are percolated through a solid sorbent, previously
conditioned, which is placed in a cartridge or column. The analytes of interest are retained by the
sorbent and further eluted in a small volume of an appropriate solvent [48]. Solid sorbents and
solvents should be chosen according to the analyte. This technique is easily automated, and is
flexible and environmentally friendly. Moreover, SPE also allows the hyphenation with
chromatographic techniques such as HPAEC-PAD (pulsed amperometric detection) [49] which
simplify sample analysis. The advantages and disadvantages of this technique have been
summarized [50], whereas new stationary phases, strategies and on-line systems have been
reviewed by Gilar et al. [51].
SPE seems to be the most popular alternative to removing interferences with the same chemical
behaviour as carbohydrates [52]. The main characteristics of this technique as applied to
carbohydrate analysis have been reviewed by Herbreteau [32]. Carbohydrates are commonly
eluted with water, diluted sulphuric acid or water modified with organic solvents, depending on
the sorbent.
SPE C18 cartridges are commonly employed to purify carbohydrates prior to their analysis
[32], [53], [54], [55] and [56] but other sorbents such as styrene-divinylbenzene [52]
aminopropyl silica [57], and ion-exchange phases are also used. Kitahara and Copeland [58]
compared the interaction between starch molecules and C8 and C18 cartridges. Differences of
adsorption were based on the hydrophobic character of starch. These authors demonstrated that
glucose and maltose were not adsorbed on either sorbent. Maltoheptaose was not retained in C8,
however, (1–4)-α-glucans were completely adsorbed on C18. In addition, fractionation of these
glucans based on their degree of polymerization (DP) could be carried out by this technique.
Schiller et al. [59] used three-step SPE to separate carbohydrates from a plant dry extract:
firstly, a hydrophobic polystyrene/divinylbenzene copolymer cartridge was used followed by
anion- and cation-exchange cartridges.
Cartridges based on hydrophilic interaction chromatography (HILIC) have been used for
desalting of glycans using a micro-scale SPE device packed with an HILIC sorbent; the glycans
were eluted using 25–50 μl of solvent and analyzed directly without derivatization using
MALDI-MS [60]. Graphitized cartridges have also been used for removing impurities such as
salts, detergents, proteins, peptidic material, etc. [61] and [62] from glycans for their subsequent
analysis and permit the separation of groups of oligosaccharides [63].
Nakano et al. [62] compared the use of three SPE cartridges (cation-exchange, C18 and
graphitized carbon) for removing peptides and glycopeptides from glycans prior to their analysis.
Authors found that N-glycans were not efficiently separated from interferences using C18 and
cation exchange cartridges. However, graphitized carbon cartridges were successfully used for
this purpose.
Removal of detergents and possible hydrophobic contaminants from small quantities of
oligosaccharides has been carried out by Huang et al. [64] on a small-batch addition of a
hydrophobic resin directly into the sample.
2.1.2.3. Miscellaneous
Solid phase microextraction (SPME) is based on the use of a fibre coated with a liquid
(polymer), a solid (sorbent), or a combination of both. The analytes are absorbed in the case of
liquid coatings or adsorbed in the case of solid coatings and further analysed by direct
introduction into the gas chromatograph. Recently, a new technique based on SPME with
helical-solid-sorbent has been developed [65] and applied to the analysis of carbohydrates
[66]. Per-O-methylated mono- and disaccharides were extracted using a helical support coated
with polydimethylsiloxane (PDMS). The helical solid sorbent reduces the extraction time and
improves the GC resolution.
Ultrasonic waves are also used for faster extraction of substances adsorbed onto solid materials.
This method has been used for the analysis of soluble carbohydrates from particulate organic
matter from the sea [67] and polysaccharides in marine sediments [68].
A microwave oven and a steam treatment reactor have been used to extract hemicellulosic
oligomers from spruce wood [69]. The oligomers obtained in the heat-treated liquid fraction
were also separated by gel filtration [70].
2.2. Fractionation
Most sugars are isomers which only differ in the configuration of hydroxylic groups and
molecular weight distribution. The characterization of these carbohydrates is not an easy task
and sometimes requires the use of different techniques for their previous fractionation to simplify
the sample before subsequent analysis. These techniques should provide the enrichment of the
samples without losses of the analytes; otherwise, the use of an internal standard becomes
necessary.
2.2.1. Membrane fractionation
Different membrane techniques have been used for sample preparation in analytical chemistry
[71]. In this section, we are going to remark on the recent applications of the main techniques to
carbohydrate fractionation based on the use of different technologies (ultrafiltration,
nanofiltration, dialysis, membrane-assisted solvent extraction) or different materials that
membranes are made of (supported liquid membranes, polymeric membranes).
2.2.1.1. Ultrafiltration and nanofiltration
Microfiltration (MF) and ultrafiltration (UF) processes have widely been used to purify,
concentrate and fractionate carbohydrates in biotechnology and the fermentation industry [72],
[73] and [74]. However, although acceptable results have been obtained in the separation of
oligosaccharides from polysaccharides, the separation of low molecular weight carbohydrates
can become a difficult task with these techniques. Swennen et al. [75] suggested the use of UF
membranes with different molecular mass cut-off to fractionate arabinoxylooligosaccharides in
their different degrees of polymerization (DP) and substitution (DS) whereas Confer and Logan
[76] used UF membranes to monitor the molecular weight distribution of polysaccharides in
solution during degradation.
Nanofiltration (NF) can be a useful tool for the fractionation of carbohydrate mixtures. The
separation properties of nanofiltration membranes were evaluated in 1997 by Urano et al. [77]
for model oligosaccharide fractions and applied to the purification of oligosaccharides obtained
from the artichoke. Aydogan et al. [78] stated that the NF separation of carbohydrates with
differing molecular sizes in several glucosyl units was not viable due to its poor selectivity.
Nishizawa et al. [79] studied the use of a membrane reactor system with a NF membrane to
separate glucose from a mixture of fructooligosaccharides (FOS), whereas Lopez Leiva and
Guzman [80] used cross-flow nanofiltration with a polyethylensulphone membrane for the
fractionation of lactose hydrolysates. More recently, Goulas et al. [81] evaluated the pressure,
feed concentration and filtration temperature effects of cross-flow nanofiltration working in full
recycled mode to fractionate oligosaccharide mixtures (model systems for mono-, di- and
trisaccharides). Further studies of these authors [82] demonstrated the efficiency of NF
membranes (cellulose acetate) in diafiltration mode to separate monosaccharides from di- and
trisaccharides with minor losses of these carbohydrates. UF membrane gave rise to a similar
separation of these carbohydrates but with higher losses of di- and oligosaccharides (UF
membrane pores are typically much larger than those of NF membranes). Nanofiltration
processes have been also used by other authors to obtain oligosaccharide fractions free from
monosaccharides using different kinds of membranes such as thin film composite membranes
[83], [84] and [85], cellulose acetate membranes [83], tubular ceramic membranes [86] and [87],
etc. Yuan et al. [88] also stated the energy-saving advantages of nanofiltration as compared to
other conventional methods for oligosaccharide purification.
2.2.1.2. Dialysis and microdialysis
Although the main applications of dialysis and microdialysis relative to carbohydrates have
been focused on their purification and concentration [89], [90] and [91], these techniques have
also been used to fractionate these compounds. Nilsson et al. [92] used microdialysis to elucidate
the molecular structures of starch, previously hydrolysed using different enzymes (β-amylase,
pullulanase and isoamylase). Simultaneous sampling fractionation and clean-up was achieved
with this technique which was used on-line with HPAEC-PAD. One of the most useful
applications of dialysis is the separation of carbohydrates of high molecular weight, such as
oligosaccharides and polysaccharides, from those of lower molecular weight [43], [93] and [94].
2.2.1.3. Liquid membranes
Liquid membranes of the “water–oil–water” type consist of an organic phase placed between two
aqueous phases [95]. The best liquid membrane configuration for industrial applications is the
supported liquid membranes (SLM). The use of these membranes for analytical chemistry was
first described by Audunsson [96]. The membrane is constituted by an organic solvent, which is
held by capillary forces in the pores of hydrophobic porous membranes [67]. SLM has been used
for the selective transport of some alditols (erythritol, threitol, ribitol and xylitol) [97], as well as
sugars and deoxy-sugars [95], [98] and [99]. The separation of sugars through SLM is based on
the use of specific carriers that form complexes with the isomeric carbohydrates. Boronic acid
derivatives [100] and [101] and neutral lipophilic macrocycles designed on different sugar
receptors [95], [99], [102] and [103] have been used as carriers for sugar transport.
Plasticized liquid membranes have also been proposed to separate fructose from glucose to
provide sufficient concentration of fructose for the formation of high fructose syrup [104].
Moreover, these membranes have been used to separate sucrose, glucose and fructose from crude
sources such as molasses, sugar cane juice and beet sugar juice.
The proposed industrial applications could easily be adapted to analytical conditions.
2.2.1.4. Polymeric membrane extraction
This technique uses a polymeric membrane, usually a silicone rubber membrane, which is more
stable than SLM and with a considerably higher lifetime; however, there are less possibilities for
chemical tuning of the separation process [71]. The transport of small saccharides through
plasticized polymeric membranes has been investigated by Riggs and Smith [105]. These authors
discovered that plasticized cellulose triacetate membranes containing large amounts of
trioctylmethylammonium chloride were stable and selectively permeable to neutral mono- and
disaccharides. They also studied the saccharide diffusion in relation to the size of the saccharide,
the carrier cation and carrier anion [105] and [106].
2.2.2. Carbon fractionation
Column chromatography with activated charcoal and Celite™ has been widely used to
fractionate carbohydrates since 1950 when it was first proposed by Whistle and Durso [107].
The use of different ethanol concentrations allows the selective extraction of the carbohydrates
previously adsorbed onto the charcoal column, depending on their degree of polymerization.
Considering AOAC method [108], selective extraction of monosaccharides can be achieved
using 1% (v/v) ethanol in water, disaccharides with 5% (v/v) ethanol and oligosaccharides with
50% (v/v) ethanol. Baker et al. [109] demonstrated that the use of borate ions in alkali
environments in the eluent accelerated the elution of certain carbohydrates from charcoal–
celite columns and also that, chromate, molibdate and tungstate ions reacted with polyalcohols
and caused changes in their separation.
Different authors have used this technique to remove monosaccharides from samples constituted
by complex mixtures of carbohydrates such as honey [110], [111], [112], [113] and [114]. The
presence of structurally similar carbohydrates, the existence of large amounts of
monosaccharides and the low contents of oligosaccharides in honey make it difficult to obtain
oligosaccharide patterns by chromatographic techniques. Therefore, separation of the most
abundant monosaccharides (glucose and fructose) previous to oligosaccharide analysis must be
carried out. In a recent study, Morales et al. [115] have substituted the use of activated charcoal
columns for an agitation and filtration process of charcoal in ethanol solutions to analyse honey
oligosaccharides removing mono and disaccharides. Fig. 2 shows the chromatographic profiles
obtained by HPAEC-PAD of a mixture of maltodextrins (A) and the fraction of mono and
disaccharides collected after charcoal treatment (B). Only small amounts of trisaccharides were
observed in this fraction. Using this method the fractionation of carbohydrates was similar to
that achieved with the celite:charcoal column, however, it was simpler and less time-consuming.
Full-size image (9K)
Fig. 2. HPAEC-PAD chromatographic profiles of a mixture of maltodextrins (a) and the fraction
of mono- and disaccharides collected after treatment with activated charcoal and 10% ethanol
(b). G1–G7: maltodextrins from DP1 to DP7.
View Within Article
The combination of activated charcoal:celite columns with other techniques of fractionation
constitutes a useful tool to isolate oligosaccharides, permitting their subsequent characterization.
In this sense, Wei et al. [116] developed a method for the fractionation of oligosaccharides from
honeydew. They used charcoal columns eluted with isopropyl alcohol followed by a
fractionation on Bio-gel P2. Other authors [117] have used charcoal:celite columns, preparative
liquid chromatography and gel permeation chromatography for the isolation of oligosaccharides
from a commercial beet medium invert syrup.
Montane et al. [118] have optimized the purification of xylooligosaccharides by activated
charcoal adsorption from lignin compounds which were more retained than the oligosaccharides
onto the activated charcoal.
Similar to the use of graphitized carbon columns for HPLC analysis of oligosaccharides [119],
[120], [121] and [122], graphitized carbon cartridges are becoming increasingly popular due to
the unique selectivity, high preparative capacity and chemical inertness of the carbon. Moreover,
graphitized cartridges have been used to separate monosaccharides from the higher molecular
weight carbohydrates of honey [123]. Elution of oligosaccharides was carried out using water
containing an organic modifier (alcohol, acetonitrile or trifluoroacetic acid) at different
temperatures.
2.2.3. Size exclusion chromatography
Size exclusion chromatography (SEC) is one of the most useful tools currently employed for the
fractionation of carbohydrates (oligo- and polysaccharides). In SEC, carbohydrates are
separated according to their molecular size, this separation also depending on the ratio of their
molecular dimensions to the average diameter of the pores of the stationary phase.
A considerable number of applications of this technique have been used in the separation of
carbohydrates for analytical purposes in the last few years [29], [70], [124], [125], [126],
[127], [128], [129] and [130]. Some authors have used SEC columns connected in series to
fractionate oligosaccharides from different molecular weights such as Lundqvist et al. [131] for
galactoglucomannans extracted from Picea abies and Sun et al. [132] for xylo-oligosaccharides.
The advances in SEC of cellulose and related polysaccharides have been recently reviewed by
Eremeeva [133]. The application of SEC to cellulose and other polysaccharides is not
straightforward due to their crystalline structure which does not allow solvents to penetrate easily
into the cellulose fibres and to break the intramolecular hydrogen bonds. Riccardi et al. [134]
have compared three methods (ethanol precipitation, dialysis and SEC on a desalting gel) to
separate simple carbohydrates from an exopolysaccharide, obtaining the best results with SEC.
Finke et al. [135] investigated the productivity (grams of carbohydrate separated per hour) and
resolution of the preparative continuous annular SEC. This system consists of two concentric
cylinders forming an annulus into which the stationary phase is packed [136]. Finke et al. [135]
compared the annular SEC with the fixed bed conventional gel chromatography. The
productivity of annular chromatography was 25-fold higher than that of the conventional
method.
SEC has also been established as a form of high performance liquid chromatography and several
advances, mainly in stationary phases and detection systems, have been developed.
Improvements and drawbacks of SEC have been summarised [137] and [138].
2.2.4. Field flow fractionation
Field flow fractionation (FFF) was first described by Giddings [139] and is based on the
separation of compounds in an open unpacked channel. The sample (in a small volume) is
injected at the inlet end of the channel and pushed through it by a longitudinal laminar transport
mobile phase flow. At the same time a perpendicular field is applied, driving the compounds to
one of the walls. The separation is a balance of the mass transport caused by the field and the
oppositely acting Brownian motion [140]. This method shares some similarities with SEC,
electophoresis and ultrafiltration. FFF applications to carbohydrates are based on the
fractionation of polysaccharides (dextran, pullulan, starch, cellulose derivatives, heteroglycans),
which cannot be fractionated by other techniques such as SEC [140] and [141].
2.2.5. Ion exchange chromatography
Ion exchange chromatography (IEC) has been widely used by the sugar industry to fractionate
mono- and oligosaccharides.
Sugars, as weak electrolytes, show little interaction with anion exchange resins in an aqueous
medium. These interactions are increased with the formation of sugar complexes with borates
and the mechanism of separation is mainly ion exchange [32].
For cation exchange resins the retention mechanism involves a mixture of different effects:
ligand exchange, exclusion mechanism, complex formation, ion–dipole interactions and
hydrogen bonds [32]. Vente et al. [142] have recently studied the influence of Ca2+, Na+ and K+
as cation exchange resins to separate monosaccharides and oligosaccharides. K+ loaded resin had
a stronger adsorption of sugars than the Na+ loaded resin. The retention was also related with the
number of equatorial-axial oriented sugar OH groups for complexation with the cation. K+ was
more suitable to separate glucose from oligosaccharides whereas Ca2+ was the best choice to
separate fructose from oligosaccharides.
Stefansson and Westerlund [143] have revised the ability of sugars to complex with metal
cations in chromatographic systems formed by ion-exchange resins. They pointed out the
selectivity of ligand-exchange chromatography at alkaline pHs, where strong complexation
between carbohydrates and suitable metal ions take place. The strength depends on the ionic
size of the cation and on the shape and flexibility of the complexing molecule [144]. Excellent
separations have been obtained with lanthanide cations [145]. Mechanisms and conditions to
improve selectivity have been extensively studied [146] and [147].
Anion- or cation-exchange resins have been commonly used, alone or in combination with SEC,
to fractionate oligosaccharides such as human milk oligosaccharides [148], cationised starch
[149], etc. Paull and Nesterenko [150] have summarised the most significant recent advances
made in stationary phases for ion exchange chromatography in complex matrices.
2.2.6. Simulated moving bed chromatography
Simulated moving bed (SMB) chromatography was invented in the 1960s and it has been used in
the sugar industry for many years for large scale separations (palatinose–trehalulose
fractionations) [151]. This technique is based on the adsorption of the samples in columns
connected in series. The adsorbents are usually a combination of size-exclusion and ion
exchange gels, although other stationary phases can be used. The system is divided into four
zones with at least one column each, connected by valves which can be individually opened and
closed. A recycling pump inside the system delivers the mobile phase through the columns
(flows out of zone 4 and is recycled to zone 1). The feed and the fresh eluent are constantly
introduced into the unit at two different injection points, whereas the extract stream enriched
with the more retained components and the raffinate stream enriched with the less retained
components leave the unit by the two withdrawal points. The flow of adsorbent is simulated by
shifting the injection and collection positions one column forward in the direction of the liquid
flow at a constant time interval [151], [152] and [153].
Sugar separation is usually designed by computer simulation by selecting different parameters
such as column phases and dimensions, total column number, eluent, switch time interval, etc.
[151].
This technique has been applied to the separation of binary mixtures of carbohydrates such as
glucose–fructose [154] and more recently to the separation of lactose from complex mixtures of
human milk oligosaccharides [153] using different stationary phases.
2.2.7. Microbiological treatments
The use of yeast to remove certain carbohydrates can also be considered as a fractionation
technique. Baumgartner et al. [155] used dried yeast (Saccharomyces bayanus) to remove
carbohydrates from carob powder for the purpose of analyzing cyclitols. After yeast treatment,
the remaining carbohydrates were removed by anion-exchange chromatography.
More recently, Saccharomyces cerevisiae has been used to selectively remove mono and
disaccharides [156]. S. cerevisiae showed a high specificity for the removal of some mono- and
disaccharides such as glucose, fructose, mannose, galactose and α-linked disaccharides.
However, rhamnose, sorbose, β-linked disaccharides and oligosaccharides with four or more
monosaccharide units were not removed. This technique has also been used to remove
monosaccharides from honey samples [85] and it is currently applied to the analysis of minor
compounds in honey in our laboratory.
Immobilized cells of the bacterium Z. mobilis have been used [157] to remove glucose, fructose
and sucrose present in food grade oligosaccharide mixtures. These carbohydrates were
removed within 12 h without any pH control or nutrient addition.
2.2.8. Supercritical fluid extraction
Supercritical fluid extraction (SFE) is based on the utilization of a fluid in supercritical
conditions. A pure supercritical fluid (SCF) is a gas or liquid at a temperature and pressure above
the critical point. Its properties are intermediate between those of a liquid and those of a gas and
its solubility behaviour is similar to that of a liquid while penetration into a solid matrix is
facilitated by the high diffusivity. As a consequence, the extraction becomes faster than for
conventional liquid extraction; also the extraction conditions can be easily controlled. This
technology is suitable for extraction and purification of a variety of compounds, especially those
with lipophile properties, such as lipids, pesticides and essential oils. As carbohydrates are
very polar compounds, SFE has been used to extract less polar compounds from sugar matrices.
Solubility of carbohydrates in supercritical carbon dioxide is low [158] but it shows a
remarkable increase when alcohols are added as polar co-solvents [159]. It is possible to separate
glucose and fructose using supercritical carbon dioxide and ethanol, starting with aqueous
solutions of the two sugars [160]. In spite of these findings, very few attempts to use SFE for
fractionation of carbohydrates have been made. The viability of selective fractionation of
mixtures of lactose–lactulose and galactose–tagatose using supercritical CO2 with a co-solvent
based on ethanol:water and isopropanol:water, respectively, has been recently evaluated [161]
and [162].
2.2.9. Pressurized liquid extraction
Pressurized liquid extraction (PLE) is a recently introduced technique which allows the isolation
of different compounds in a short time using small volumes of solvent. Accelerated solvent
extraction (ASE) is the registered name of the commercial equipment. It works with high
pressure and controlled temperature and can replace the classical liquid–solid extractions
(Soxhlet, sonication, etc.). It allows the use of solvents in subcritical conditions and it has been
largely applied to non-polar analytes but very scarcely used for very polar compounds.
Nevertheless, solubilities of low-molecular weight sugars in different alcohols may be modulated
in order to allow the fractionation of mixtures using these techniques.
Subcritical water extraction (SWE) has been scarcely applied to the fractionation of
carbohydrates, since the overheated water may act as an effective acid–base catalyst [163]
promoting sugar degradation and formation of HMF and other by-products. In spite of the
decrease in recovery, some separations have been assayed: sugars from hemicellulose
hydrolyzates [164] and even subcritical fluid chromatography of monosaccharides and polyols
[165].
ASE has been assayed for polysaccharides from several species of Linghzi (Chinese mushrooms)
[166]. It was performed with an ASE 200™ from Dionex, using water at 120 °C and 1500 psi for
5 min of extraction (two cycles). The total amount of extracted carbohydrates was found to be
higher than that obtained when using sonication. Some attempts have been done in our
laboratory to fractionate disaccharide mixtures with ASE 300™ [167] using different alcohol
concentrations and also to obtain oligosaccharide fractions from honey (unpublished results).
Although results are very promising, further work is being done in order to optimize PLE-based
procedures.
2.2.10. Molecularly imprinted polymers
Molecular imprinting is a well-developed tool in analytical chemistry which can be used as a
very efficient fractionating method. Molecularly imprinted polymers (MIPs) are systems capable
of recognizing specific molecules with a sorption capacity dependent on the properties and
template concentration of the surrounding medium.
Different molecularly imprinted polymers have been developed for carbohydrate recognition
with two main objectives: a number of works are directed towards obtaining specific traps for
glucose which do not bind fructose for biomedical purposes [168] while others are searching for
new separation systems for HPLC (and hence a high specificity is necessary).
The use of MIPs for racemic resolution of free sugars has been known for many years [169] and
[170]. However, recently, several works have been devoted to the development of specific MIPs
for glucose, focusing especially on determining their binding capacities with respect to fructose.
Poly(allylamine hydrochloride) was non-covalent imprinted with glucose phosphate monosodium salt which produced MIP hydrogels capable of binding glucose in quantitative and
isomerically specific ways [171]. Isomeric specificity in hydrogels imprinted for glucose was
demonstrated by higher binding capacities of glucose than those of fructose in the same
polymers. A novel, solution-to-surface imprinting method based on the different higher-order
conformations adopted by boronic-acid-appended poly(l-lysine), in the presence of sugars was
developed by Friggeri et al. [172]. The application of a d-glucose-imprinted polymer interface
for the selective detection of d-glucose over d-fructose was demonstrated successfully.
New networks based on star polymers were designed to be responsive and recognitive. Oral and
Peppas [173] obtained over 300% more uptake for d-glucose compared to d-fructose using
molecular imprinting with d-glucose and cross-linking with poly(ethylene glycol)
dimethacrylate. Polymers based on polyacrylates with improved ability to bind saccharides have
been recently designed [174]. Polymer selectivity for discrimination of α- and β-glycosidic bonds
as well as the effect of the number of glucose rings was assayed [175].
Porphyrin-based polymers have been prepared for carbohydrate recognition, and assayed with
several n-octyl-d-glucopyranosides [176]. Each porphyrin-based polymer demonstrates high
affinity and differential selectivity for carbohydrates similar in structure.
In spite of the high selectivity achieved with several systems, MIPs are mainly prepared for
chromatographic analysis and they have been very scarcely used for sample preparation. Perhaps
less selective (and less expensive) polymers could be designed in order to make easier the
fractionation of saccharide groups in different samples (food, cosmetics, environment, …).
2.2.11. Hydrophilic interaction chromatography
HILIC has been applied to carbohydrate analysis for more than 30 years although not under
this name [177]. Retention is caused by partitioning of the carbohydrate between a waterenriched layer of stagnant eluent on a hydrophilic stationary phase and a relative hydrophobic
bulk eluent. As previously mentioned HILIC SPE has been used as a sample preparation
technique, not only for clean-up, but also for carbohydrate fractionation. An interesting and
very long fractionation was carried out to isolate (S)-malic acid-1-O-d-glucopyranoside from the
morel mushroom (Morchella deliciosa) through multistep ultrafiltration on 10 and 0.5 kDa, GPC
on Shephadex G-15, and semipreparative HILIC on TSK-GelAmide-80. The glycoside was
identified by ESI and NMR and showed to be responsible for the umami taste of the morel
extract [178].
2.3. Chemical treatments
2.3.1. Hydrolysis
Hydrolysis is a necessary step before chromatographic analysis of different high molecular
weight polysaccharides. Acid hydrolysis has been used for many years for this purpose using
mainly trifluoroacetic acid (TFA) or HCl at different concentrations and thermal conditions [179]
and [180]. Polysaccharides can be also subjected to methanolysis with hydrochloric acid in
methanol for hydrolysis [22]. Enzymatic depolymerization of big molecules using different
enzymes is also very extended and has been summarized in several reviews [181], [182] and
[183]. The determination of the main neutral sugars in pectin [184] has been carried out using a
new method which involves a mild chemical attack (0.2 M TFA at 80 °C) followed by an
enzymatic hydrolysis.
Among recent advances of hydrolysis it is necessary to remark on the application of microwave
irradiation. Microwaves are gaining popularity for different heating treatments including
methanolysis and hydrolysis of saccharides [185]. An analytical procedure for the
characterisation of polysaccharides and identification of plant gums in very old polychrome
paintings was based on hydrolysis with 2 M TFA assisted by microwaves, clean-up of the
hydrolysate by ion-exchange resin, and analysis by high-performance anion-exchange
chromatography with pulsed amperometric detection [7]. Hydrolysis time was reduced to
20 min. A new procedure based on microwave irradiation with a catalytic amount of potassium
persulfate promoted the total hydrolysis on alumina support of eight different seed gums [186].
Different hydrolysis protocols used by marine biogeochemists to extract sugars from various
marine matrices including sinking particulate organic matter (POM), dissolved organic matter
(DOM), ultrafiltrated dissolved organic matter (UDOM), and sediments have been revised [5]. It
was demonstrated that mild and strong hydrolysis gives comparable results for open ocean
samples. It was also verified that most of the published sugar data obtained by chromatographic
techniques are related to suspended or sinking POM and sediments, and that there is a need for
data on sugar composition in DOM and UDOM, which involves further analytical difficulties for
chromatographic analysis.
2.3.2. Derivatization
Derivatives of carbohydrates are prepared for two different purposes: to increase volatility (for
GC) and to enhance sensitivity (for LC and CE); the type and requirements of both are different
and will be treated separately.
Besides the general conditions for analytical derivatization (reactions have to be fast and
complete, giving stable derivatives without by-products) [187], some additional requirements
arise when working with carbohydrates. It is necessary to take into account that the only
functional group common to all saccharides is the hydroxyl. Some useful derivatization reactions
are amenable to aldoses and not to ketoses; some very common sugars have no reducing group
(sucrose, trehaloses, raffinose, etc.) which makes a difference when the carbonyl group has to
react; sugar acids can exist as open forms or lactones (which will give different derivatives)
depending on the pH; and so on.
The state-of-the-art in derivatives preparation has been included in some reviews dedicated to
chromatographic analysis of sugars [188] and [189], therefore, in this section we only resume the
more recent publications on this subject.
2.3.2.1. Derivatives for LC and CE
Carbohydrates can be analyzed in liquid phase without derivatization, using UV detectors at
low wavelengths, refractive index or light scattering detectors; IEC is very often connected to
amperometric detectors which detect free sugars.
Nevertheless, in the search for enhancing sensitivity and selectivity, different methods to
introduce chromogenic and fluorogenic groups in the saccharide molecules have been proposed.
Derivatives can be prepared before the analysis (precolumn derivatives) or by making a chemical
reaction after separation but before detection. The last type of derivatives will not be considered
as “sample preparation” and will be not be discussed here.
Precolumn derivatives are applicable to both HPLC and CE. This subject was revised in 1996
[190], 1998 [191] and 1999 [192] and more recently in 2003 [189].
Most methods are based on the condensation of a carbonyl group in carbohydrates with
primary amines to give a Schiff base which is then reduced to a N-substituted glycosil amine.
The primary amine has to posses the desired chromophore or fluorophore substituent, usually an
aromatic ring. Reductive amination has been carried out with 2-aminopyridine (2-AP), different
trisulphonates, esters of p-amino benzoic acid, 2-aminoacridone, etc.
Carbohydrates possessing a free amino group can react with O-phtalaldehyde (OPA) to be
transformed into OPA derivatives or with phenyl isothiocyanate. p-Nitrobenzoates have been
used to substitute all the hydroxyl groups in mono-, di-, and trisaccharides [193] and
perbenzoylation has been used for the sensitive detection of monosaccharides [189].
Besides the derivatives cited in the above mentioned reviews, new reactions have been assayed.
2-Methyl-3-oxo-4-phenyl-2,3-dihydrofuran-2-yl acetate was developed for the analysis of
primary amines and aminated carbohydrates by means of HPLC, CE and MALDI/MS [194].
The obtained derivatives could be successfully analyzed by these techniques.
Oligosaccharides were derivatizated with various aminobenzoic esters (methyl, ethyl and butyl)
and aminobenzonitrile [195] for micelar electrokinetic chromatography (MEKC) with sodium
dodecyl sulphate (SDS). Butyl aminobenzoate derivatives gave the longest retention times and
the best resolution of the sugars, whereas derivatives of aminobenzonitrile gave the worst
separation which was attributed to their high polarity. The method was optimized and applied to
maltooligosaccharides and to human milk oligosaccharides.
p-Aminobenzoic acid (p-AMBA) was used for derivatization of neutral sugars, amino sugars and
uronic acids, enabling fluorescence or photometric detection [196]. p-AMBA and other reagents,
i.e., p-AMBA propyl ester, 1-aminopyrene, 2-(2-aminophenyl)indole, and 4-aminoazobenzene,
were compared to derivatize neutral sugars, amine sugars and uronic acids for RP-HPLC analysis
[197]. Separation was complete and detection limits suitable; p-AMBA allowed the
determination of target compounds in landfill leachates and lysimeter percolates.
A novel reagent, 4-(3-methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid (PMPA) was assayed [198]
in order to improve methods based on 1-phenyl-3-methyl-5-pyrazolone (PMP) developed by
Honda et al. [199]. PMPA derivatives from 17 neutral sugars, 2 aminosugars, 3 N-acetylaminosugars and 2 uronic acids were chromatographed. Eight of them were selected for
validation: linearity was good and detection limits ca. 10 pmol. The PMP method was simplified
by Zhang et al. [200] and applied to determination of monosaccharides and oligosaccharides in
Aloe powder and food by MEKC and HPLC (Fig. 3).
Full-size image (9K)
Fig. 3. Separation of PMP-labeled carbohydrates in Aloe powder by (a) MEKC and (b) HPLC.
1: Analysis of aloe powder and 2: analysis of the hydrolysate of aloe powder. (Reprinted from
[200], with the permission from Elsevier © 2002).
View Within Article
A method of ultramicroanalysis for mono- and disaccharides using CE with laser-induced
fluorescence (LIF) detection was based on conversion to 7-nitro-2,1,3-benzoxadiazole (NBD) Nmethyl glycosylamines [201]. The reaction consisted of two steps (reductive N-methylamination
followed by condensation with NBD). Conditions were mild and did not cause desialylation.
Attomols could be detected (amount injected).
Sialic acids require mild procedures which have been revised [202].
Carbohydrates in milk powder were derivatized with p-nitroaniline by irradiation with
microwaves prior to HPLC analysis with UV detection. Reaction was complete in 5 min and the
sensitivity was high (detection limits within 3.3 and 5.1 μg/mL, 10 and 15 nmol) [203].
In spite of the wide possibilities available to choose derivatives for LC or CE, new procedures
continue to appear either to improve those already existing or to develop new applications.
Derivatives obtained through reductive amination are commonly used, since there are many
possibilities to select an amino compound with the required properties; but the analysis of nonreducing sugars and/or polyalcohols requires derivatization of hydroxyl groups. Maximum
sensitivity has been reached with LIF (laser-induced fluorescence) but many procedures may be
carried out with less sensitive procedures.
2.3.2.2. Derivatives for GC
Carbohydrates always need to be converted into volatile compounds before GC analysis. The
classical methods consist of replacing all the active-hydrogen atoms by non-polar substituents.
Methyl, trifluoroacetyl, trimethylsilyl and tert-butyldimethylsilyl ethers have been the most
popular derivatives, allowing GC analysis of saccharides with up to four to five monosaccharide
units, as well as sugar alcohols, aminosugars and acids. These reactions give a different
compound for every anomeric form of the sugar (up to six), which can be easily separated in
capillary columns [187]; this fact can be considered as an advantage for equilibrium studies of
single carbohydrates, but an inconvenience when a complex mixture has to be separated.
In recent times, some well established derivatization methods have been improved. A classical
method for GC analysis of carbohydrates in fruit products was modified introducing a preequilibration time of the sample in dry pyridine at 50 °C for 20 min before the addition of the
trimethylsilylimidazole (TMSI) [204]. This method was shown to give significantly lower
variation coefficients for sugars in apple juice concentrates, pure and adulterated with syrup,
when compared to the original capillary GC method.
Several attempts have been made to reduce the number of chromatographic peaks of each
derivatized carbohydrate. Very stable alditol acetates may be obtained by reduction and
acetylation, but their volatility is low and these derivatives are only adequate for
monosaccharides. An automated derivatization instrument has been developed for preparation of
alditol acetates for GC–MS profiling of bacterial carbohydrates [205] which was also applied
for the determination of muramic acid in organic dust [206].
Another commonly used approach is to convert the free carbonyl groups into oximes or N-alkyl
oximes before silylation, which affords only two peaks for each reducing sugar (corresponding to
E and Z isomers of the oxime). These compounds are also quite stable and hence have become
very popular. The oximation–silylation method has been modified and optimized [207]: aniline
was found to be the best reaction medium for oxime formation with hydroxylamine
hydrochloride, and N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) was the most efficient of
the assayed silylation reagents. Short analysis time was achieved with few by-products. The
method was evaluated for arabinose, xylose, fructose, glucose, sucrose and salicin (internal
standard) and found to be suitable for processed food analysis.
Aldononitrile acetates are derivatives from aldoses which give a unique peak for every sugar; but
they are not reliable for ketoses [187].
Methods for quantitative analysis of di-O-isopropylidene acetate (IPAc) derivatives of hexoses
or pentafluorobenzoyl (PFBz) esters have been reported [208] in order to study multiple
isotopomers by chemical ionization GC–MS. The PFBz derivative was about 100-fold more
sensitive than that of acetate: the useful range for isotopic tracer studies was 25–2500 pmol for
electron impact (EI) analysis of the acetate derivative and 0.1–55 pmol for nitrogen chemical
ionization (NCI) analysis of PFBz derivative (sample amount injected). EI–GC/MS analysis of
the IPAc derivative was preferred for most studies where sample size was not limited.
A type of carbohydrate derivative that retains the charge on the anomeric carbon of the original
monosaccharide (dialkyl-dithioacetal acetates) was optimized for the quantitative evaluation of
13
C distribution into isotopomers of 13C-labeled aldoses and ketoses [209]; these derivatives were
well suited for measuring isotopic enrichment into the characteristic anomeric carbon of aldose
sugars and could probably facilitate the global analysis of metabolic flux in carbohydrate
pathways.
Microwave-assisted preparation of sugars and organic acids for simultaneous determination in
citric fruits by GC was accomplished [210] using a domestic microwave oven. Sugars were
converted to trimethylsilylated oximes in 5 min, avoiding filtration and centrifugation.
Recoveries were within 95–100% of the assayed sugars.
TMS oximes were prepared in the presence of the fruit matrix for quantitation of sugar alcohols,
mono-, di- and trisaccharides in sour cherries; the GC–MS procedure was very reproducible,
RSD% averaging 2.3 for components present in concentrations ≥1% and 3.6 for those present in
concentrations <1% [211]. The procedure was further successfully extended to other fruits [212].
Also Brazilian propolis was directly derivatized [213].
Although some of the existing procedures for preparing GC derivatives are quite satisfactory,
and some of them have even been improved [207] and [211], one of the goals of these methods –
to achieve only one chromatographic peak for each individual sugar – seem to need further work.
2.3.3. Derivatives used for structural analysis
The so-called methylation analysis is an analytical method largely used for structural studies of
polysaccharides. It involves methylation of the free hydroxyls in the intact polysaccharide,
cleavage of the glycosidic links and acetylation of the new formed hydroxyls. Several similar
methods have been developed, based on achieving monosaccharides with two different types of
substituents, one of them marking the position of the glycosidic linkage in the original
polysaccharide, and the other helping to make the molecule volatile. All these methods allow the
monosaccharide components and positions of the glycosydic linkages to be studied in the
original polysaccharide [214].
Classical techniques have been revised and compared [215] in order to obtain a rapid method for
simultaneous identification of furanosides and pyranosides in heteropolysaccharides.
A novel method for the methylation of polysaccharides using microwave (MW) irradiation has
been described [216]. Seed gum from Cyamopsis tetragonolobus (Guar) was fully methylated
with dimethyl sulphate and sodium hydroxide using 100% microwave power for 4 min in 68%
yield. The completely methylated seed gum thus obtained was hydrolyzed by 70% formic acid
followed by 0.5N H2SO4 under full microwave power for 1.16 and 1.66 min, respectively. The
partially methylated monosaccharides were separated and identified.
Glycerol was used to improve solubilization of high molecular weight polysaccharides prior to
methylation for linkage analysis [217]. Four model polysaccharides: neutral, acidic (both within
1000–2000 kDa), dextran 40 kDa and 2000 kDa showed significant increases in derivatization
yield as measured by recovery of partially methylated alditol acetates.
More work should be conducted on this subject in order to continue to improve the above
mentioned results.
2.3.4. Thermochemolysis
Thermochemolysis of carbohydrates was described by Fabbri and Helleur in 1999 [218] as an
interesting characterization method of insoluble polysaccharides. Permethylated deoxy aldonic
acids were identified as key products allowing identification of the original molecules. Challinor
reviewed in 2001 [219] the developments of the thermally assisted hydrolysis and methylation
(THM) techniques in analytical pyrolysis. THM is applied in pyrolysis coupled to GC and GC–
MS to polysaccharides in environmental, food and agricultural studies. Thermally assisted
hydrolysis and alkylation were applied to aerosol samples [6]. Levoglucosan, and other
compounds of polysaccharide origin were identified as their methyl ester and ether derivatives
among the products of thermally assisted methylation. The mechanisms involved in these
reactions have been discussed by Schwarzinger [220] and [221].
This a very specific field where further developments in instrumentation are the key to
advancement.
3. Concluding remarks
Sample preparation methods for chromatographic analysis are in continuous progress;
nevertheless, evolution seems to be faster in certain fields such as volatiles or pesticides, whereas
others like carbohydrates need special attention. The dissimilarity in saccharide properties and
structures could be partly responsible for this. New techniques involving vapour-phase
transference or low polarity media are clearly unsuitable for carbohydrates; but a considerable
number of new techniques could be potentially adequate for carbohydrate characterization to
which more research should be dedicated to reach this objective.
Although alternative techniques such as helical-solid-sorbent extraction or the use of ultrasonic
waves have been used for carbohydrate clean-up, the traditional filtration and extraction
procedures are still the most widely used techniques. Treatments with alcohols and the use of
solid phase extraction are especially convenient for carbohydrate purification. Nevertheless,
the trend of clean-up processes is orientated towards greater automatization, the on-line systems
being a promising way of carbohydrate purification.
Before discussing the utility of the fractionation techniques, it is necessary to distinguish
between the separation of carbohydrates depending on their molecular weight and the
separation of isomers. Most of the previously mentioned techniques can be successfully used for
the first aim; however, the fractionation of carbohydrates with the same molecular weight
which only differs in their monosaccharide composition or glycosidic linkages becomes a
difficult task.
It is well known that the most common technique used for DP fractionation is SEC followed by
IEC. Activated charcoal columns have also been widely used; nevertheless the selectivity of this
technique towards the different structures of the carbohydrate limits its application. Moreover,
the use of charcoal columns can be tedious, although in recent times graphitized cartridges are
gaining popularity. The membrane techniques can also be a good alternative to these
methodologies, although selection of membrane should be carefully considered depending on the
carbohydrate. Moreover, membrane techniques offer good possibilities for automation.
Ricciardi et al. [134] have compared three methodologies (ethanol precipitation, dialysis and
SEC) to separate simple carbohydrates from an exopolysaccharide (EPS). Ethanol
precipitation was faster and less expensive, however losses of EPS could be observed. Dialysis
and SEC recovered 100% of the EPS, however, SEC was a simpler and less expensive technique.
We have recently compared three fractionation techniques (nanofiltration, activated charcoal and
yeast treatments) to separate monosaccharides from oligosaccharides in honey [85]. Gas
chromatographic profiles of untreated honey and the samples obtained by the three methods are
shown in Fig. 4. The nanofiltration process did not show the complete removal of
monosaccharides, although a great diminution of these sugars was observed. On the contrary,
almost 100% of monosaccharides were removed using activated charcoal and yeast treatments.
However, some di- and trisaccharide concentrations decreased due to the selectivity of the
fractionation, while nanofiltration preserved the original profile.
Full-size image (47K)
Fig. 4. GC profile of TMS-oximes of carbohydrates in honeydew honey (a) untreated, (b) after
nanofiltration process, (c) after fermentation with yeast, (d) after activated charcoal treatment. 1:
Monosaccharide, 2: disaccharides and 3: trisaccharides; i.s.: internal standard. (Redrawn from
[85], with the permission from ACS © 2005).
View Within Article
The separation of isomers is not a straightforward procedure. Traditional techniques such as IEC
or activated charcoal are not orientated towards this purpose. Therefore, other techniques such as
MIPs, PLE, SFE or SLM have been developed. Nevertheless, all these techniques have been
assayed with simple mixtures and their applications are scarce. Some assays comparing SFE and
PLE [166] and [167] are being carried out in our laboratory with simple disaccharide mixtures.
Both techniques are showing promising results in the separation of carbohydrates whereas PLE
seems to present higher recovery than SFE.
More experiments should be done to compare: effectiveness, recovery and feasibility of all the
above mentioned techniques which could encourage their applicability.
According to the derivatization methodologies, in spite of the number of proposed reactions, an
optimal derivative does not exist. The desired sensitivity, the nature of the analytes, and the
instrument to be employed in every specific problem determine the type of derivative to be
chosen.
From the very extensive literature which has been examined, it can be deduced that during recent
years the advances in some aspects of carbohydrate pre-treatments have been scarce; some
promising techniques which have been successfully used in other fields have hardly been applied
to saccharides. It is worth noting that some attempts are being made in order to extend the
application area of several techniques. Some fractionation methods used at present in the sugar
industry could be easily devised at analytical scale level (as happened with SFE). A special case
is that of derivatization, which seems to progress faster in HPLC/CE area than in GC, where the
existing derivatives are still not totally satisfactory.
Acknowledgements
This work was supported by projects API2003-007 (financed by the EU and Instituto Nacional
de Investigación y Tecnología Agraria y Alimentaria) and ANALISYC S-505/AGR-0312
(financed by Comunidad de Madrid).
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Corresponding author. Tel.: +34 91 562 29 00; fax: +34 91 564 48 53.
Journal of Chromatography A
Volume 1153, Issues 1-2, 15 June 2007, Pages 74-89
Advances in Sample Preparation - Part II
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