disaccharides determination: a review of analytical...
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Critical Reviews in Analytical Chemistry
ISSN: 1040-8347 (Print) 1547-6510 (Online) Journal homepage: http://www.tandfonline.com/loi/batc20
Disaccharides Determination: A Review ofAnalytical Methods
Marta Pokrzywnicka & Robert Koncki
To cite this article: Marta Pokrzywnicka & Robert Koncki (2018) Disaccharides Determination:A Review of Analytical Methods, Critical Reviews in Analytical Chemistry, 48:3, 186-213, DOI:10.1080/10408347.2017.1391683
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Disaccharides Determination: A Review of Analytical Methods
Marta Pokrzywnicka and Robert Koncki
Department of Chemistry, University of Warsaw, Warsaw, Poland
ABSTRACTDisaccharides are determined mainly for dietetic purposes, hence the most analyses are carried out forfood and drink samples. Its content can also be used to profile groceries in order to identify the origin andquality of the products. They also can be an indicator of the rate of metabolism as well as for the control ofsome technological and biotechnological processes. Unfortunately most of technological analysis areperformed with nonselective polarimetry methods. Sugars due to specific physicochemical properties ofcompounds are difficult to determine with classical analytical techniques. The most commondisaccharides are composed of several types of monomers connected by a different configuration of theglycosidic bond, therefore, there are subject of the same characteristic reactions. This often enforces theneed for pre-separation of sample components. Therefore, nowadays the most popular analyticalmethodologies for disaccharides determination are based on chromatographic and electrophoretictechniques. An alternative is enzymes application that allow both selective recognition of target analyteand its conversion to easy detected product, allowing detection by relatively simple conventionalanalytical methods. Another approach is the use of advanced chemometric methodologies for computingof data obtained from some spectroscopic techniques. This article is a review of the recent analyticalliterature devoted to non-selective and selective methods for disaccharide determination in real samples.
KEYWORDSChromatography;Disaccharides;Electrophoresis; Enzymaticmethods; Spectroscopy
1. Introduction
From a huge group of sugars, disaccharides are the most popu-lar analytes especially in food industry and agriculture. Amongthem the most popular are sucrose, maltose and lactose(Fig. 1), which were the main analytes reported in 85% of publi-cations cited in this review. It is no surprise that the most oftenanalysed disaccharide is sucrose (68%). In the 19th century thetable sugar became strategic resource with special dotation forproduction and quality control improvement. For many yearsit was recognized as the best source of energy, nowadays it is adietician’s nightmare. WHO considers it as one of the majoritysource of obesity and dental caries and strongly recommendreduction of intake.[1] Therefore sucrose is most often deter-mined in food products, especially soft drinks[2–14] andsweets.[13–17] Because of its plant origin, sucrose is also deter-mined in fruits and vegetables[17–26] (with particular emphasison sugar cane[27] and sugar beet[28–31]) and other plant mate-rial,[32–38] where it is an indicator of regular plant growth.[36]
There are also some examples of sucrose assays in urine[39–41]
and blood plasma[40–43] with special application to blood-brainbarrier permeability investigations.[43]
Similar concern as target analyte share lactose (37%) andmaltose (32%). Lactose, characteristic for mammals milk, isdetermined exactly in milk[44–50] as well as in cheeses,[51]
yogurts[52] and different dairy products.[53] Considering thatabout 50–75% of human population (data from different
sources[51,54]) suffer from lactose intolerance, not surprisingnumber of publications reported new methods for lactose assay.Finally, maltose occurs mostly in cereals grains. Except cerealproduct[55,56] and starch hydrolates[57,58] it is often determinedin alcoholic drinks[59] especially in beer[60–63] where togetherwith glucose it is a marker of progress of fermentation processas well as a marker of quality of final product. Of course notonly these three disaccharides are targets of analytical interest.There are also reported several methods for determination oftrehalose,[34,64–68] lactulose,[69–73] isomaltose,[56,74] cellobi-ose,[68,75–78] xylobiose,[75,79] mellibiose[78,80] and more.
Selective methods of disaccharides determination involvemany analytical challenges. Most of known disaccharides are iso-mers, with the same molecular formula, molecular weight andalmost the same functional group. Only small structural changessuch as the inversion of groups at a single chiral carbon atom or achange in the position of the carbonyl group, decide about differ-ent sweetness, solubility, and chemical reactivity. This diversity isclear even for monosaccharides, for example when simple mono-saccharides like glucose, galactose and fructose are compared. Incase of disaccharides it becomesmore complicated. Disaccharidesare compounds with acetal bond between anomeric carbon ofone monosaccharide molecule and any hydroxyl group of secondmonosaccharide, so more isomer variations appear. In Figure 2the structures of ten different disaccharides are presented. Each ofthem is built of two glucose units only and differ by anomeric
CONTACT Marta Pokrzywnicka [email protected] Department of Chemistry, University of Warsaw, Pasteura 1, Warsaw, 02-093, Poland.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/batc.© 2018 Taylor & Francis Group, LLC
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY2018, VOL. 48, NO. 3, 186–213https://doi.org/10.1080/10408347.2017.1391683
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form and order of bounded carbon atom in second molecule.Eight of presented compounds are reducing sugars and theyundergo the same characteristic assays based on Fehling’s,[81]
Smogy-Nelson[82] or Summer[83] methods. Other differences aretaste (for example different sweetness rate or bitter in case of gen-tiobiose[84]) and susceptibility to digestives enzymes. What isobvious, because of different configuration of stereogenic carbonseach of them have different specific rotation angle. These proper-ties could be a base for selective determination method, unfortu-nately effect of the light beam refraction is additive, and thereforesuch approach is useless in case of quantitative analysis of sugarmixtures. Nerveless, polarimetry together with other non-selec-tive techniques such as refractometry and hydrometry are cur-rently used in industrial analysis.[85]
2. Non-selective analytical methods
Similarity in disaccharides structures and properties causesmost of analytical methods non-selective. Therefore in manycases information about disaccharide content, especially in
food and beverages, is limited to total sugar content – theparameter which presents the total concentration disaccharidesand related monosaccharides in sample. This parameter pro-vides satisfactory information from nutritional point of view.In case of these assays there is no necessary to apply any selec-tive techniques. For such purposes, several methods based onnon-selective chemical reactions or physical properties of sugarsolutions have been developed.
2.1. Chemical methods
For estimation of total sugar content often phenol-sulphuricacid assay[86] is applied. This method is useful for determina-tion of reducing and non-reducing sugars in complex samples,also in the presence of salts and proteins residues. The assay isbased on measure of colour of aromatic complex absorbance at490 nm. The method was developed in the middle of XX cen-tury but is still applied because of its simplicity and availabilityof reagents. Also 3,4-dimethylphenol forming colour adductsexhibiting absorption maximum at 510 nm wavelength can be
Figure 1. Structures of three common disaccharides: sucrose, maltose and lactose.
Figure 2. Examples of disaccharides composed of two glucose units.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 187
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applied for total sugar determination.[87] However because ofdifferent absorptivity of individual sugars, this method can besuccessfully applied only under specific conditions.
Inmany cases almost all sugars presented in sample have reduc-ing properties caused mainly by monosaccharides and reducingdisaccharides. There is a lot of methods for reducing sugars deter-mination and some of them are presented in Table 1. Predomi-nantly these are modifications of classic Fehling’s methoddeveloped in the middle of XIX century[81] based on the reductionof cupric ion to cuprous oxide. The main differences are in thetitrant and titrand. That could be dissimilarities in reaction condi-tions like in Loof–Schorl[88] and Ofner’s[89] methods or exchangethe role of sample and titrant as in case of Lane and Eynonsmethod,[88] where Fehlings solution with known cupric concentra-tion are titrated with sugar solution. Copper based reaction can bealso applied for spectrophotometric detection. Cuprous ions canreact with neocuproine to produce a yellow complex that stronglyabsorb the light of 457 nm wavelength. It is obvious that the deter-mination methods based on reducing properties of sugars arestrongly influenced by other reductans (almost the same methodsare applied for estimation of total antioxidant capacity assays)
Methods based on ferricyanide reduction also have somevariation. Except iodometric Mohr method[92] or simple photo-metric measurements of decay of ferricyanide absorption at480 nm wavelength[92,93] also formation of Prussian Blue, after
addition of ferric nitrate can be applied allowing observation ofabsorption increase at 690 nm wavelength.[35] Under flow anal-ysis condition, thanks to following measurements of hexacya-noferrate obtained after reduction by sugars from non-hydrolysed and hydrolysed sample, quite selective determina-tion of sucrose was possible in beet juices and syrups.[31]
Also benzoic acid derivatives containing nitrogen as 3,5-dinitro-salycilic acid (DNS)[83,91] and p-hydroxybenzoic acid hydrazide(PAHBAH)[94–96] can be reduced by sugars and form colouredproducts that absorb in visible range at 540 and 410 nm wave-length, respectively. An interesting case ismethylamine. Its reactionwith reducing sugars leading to formation product, which colourdepends on sugar structure.With monosaccharides it forms yellowproduct that absorb at 400 nm, whereas with oligosaccharides (dis-accharides and trisaccharides) violet- carmine adduct is formedthat absorbs light of 540 nm wavelength.[97] This way the discrimi-nation and determination of chosen saccharide groups is possibleas well as estimation of total reducing disaccharide content only ifoligosaccharides composed of higher number of monosaccharidesunits are not present.
Some reactions of reducing sugars allow to distinguish aldo-ses and ketoses. For example Seliwanoff’s test is characteristicreaction of ketohexoses, which in acidic solutions are convertedinto hydroxymethylfurfural forms that react with resorcinol togive red complexes with strong, characteristic absorption peaksat 398 and 480 nm wavelength. This qualitative test has beensuccessfully applied for quantitative photometric determinationof lactulose in pharmaceutical samples.[98] Hydroxymethylfur-fural can react also with cysteine hydrochloride–tryptophanreagent producing pink chromophore with maximum absorp-tion at 518 nm. This reaction also has been applied for determi-nation of lactulose in pharmaceutical products.[99]
So-called Raybin test based on reaction of sugar with 5-diaz-ouracil gives positive results (bluish precipitate) for compoundscontaining 1,2-glycosidic bond such as sucrose, raffinose or sta-chyose, while palantinose and melezitose also containing thisbond type yielded a reddish-brown precipitate. This test hasbeen adapted for photometric determination of sucrosetogether with rafinose in honeys samples.[100]
Reducing sugars in alkaline solution tautomerize to form eno-diols. These compounds can react with zirconyl chloride to formfluorescence derivatives. Tautomerisation to enodiols is muchfaster for ketoses, so under adequate conditions (lower reactiontemperature and shorter time) selective fructose determination canbe achieved. Similar fluorescent complexes with zirconyle chlorideare formed by fructose-based disaccharides, so this method is alsouseful for fluorometric determination of sucrose.[7]
Finally, in case of reducing sugars determination it is worthto mention about qualitative osazone test.[101] At high tempera-tures reducing sugars react with phenyl hydrazyne to form yel-low crystals called osazones. For each reducing sugar crystalswith different structure, precipitation time and melting pointare formed. Comparison of those parameters is useful for sac-charides differentiation.
2.2. Physical methods
Physical techniques developed for investigations of liquidslike polarimetry, hydrometry and refractometry also belong to
Table 1. Methods for reducing sugars determination. Table 1. Examples of non-selective chemical methods for determination of reducing sugars.
Method Reaction Detection Source
Lane and Eynon’smethod*
sugars reduce cupric ionsto cuprous oxide
end point titrationindicated bymethylene blue
[88]
Ofner’s method* sugars reduce cupric ionsto cuprous oxideexcess of cupric ionsare reduced bypotassium iodide
iodine titrated by sodiumthiosulphate withstarch as indicator
[89]
Loof -Schorlmethod*
sugars reduce cupric ionsto cuprous oxideexcess of cupric ionsare reduced bypotassium iodide
iodine titrated by sodiumthiosulphate withstarch as indicator
[88]
Knight and Allenmethod*
sugars reduce cupric ionsto cuprous oxide
residual cupric ions aretitrated with EDTAwith murexide asindicator
[88]
Munson andWalkermethod
sugars reduce cupric ionsto cuprous oxide
cuprous oxide are driedand weight
[89]
Smogyi- Nelsonmethod
sugars reduce cupric ionsto cuprous oxidecuprous oxide reducedmolybdic acid tomolybdenum blue
photometricdetermination ofmolybdenum blue at500 nm.
[82,86]
Sumner’s method sugars reduce 3,5-dinitrosalycilic acid to3-amino-5-nitrosalicylic acid
photometricdetermination of 3-amino-5-nitrosalicylicacid at 540 nm.
[83,91]
Hagedorn-Jensenmethod
sugars reduceferricyanide toferrocyanide
iodometricallydetermination offerricyanide by theMohr method
[92]
photometricdetermination offerricyanide at480 nm.
�official methods recommended by ICUMSA.
188 M. POKRZYWNICKA AND R. KONCKI
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non-selective methods of disaccharides determination. How-ever, there are not applied to total sugar determination becausecontributions of each solute (sugar) in total liquid density,refraction index and light beam polarization are individual andnot simply additive as in case of chemical stoichiometric reac-tions reported above. Those techniques are widely applied insamples where influence for refractive index is expected onlyfrom single disaccharide.[9,15] However, due to measurementsimplicity and non-expensive equipment, from over 100 years,these methods are still widely used for routine analysis in sugarindustry. There are also so called Saccharimeters the speciallydesigned polarimeters and refractometers with scale recalcu-lated from angle of rotation or refractive index to sucrose con-centration. Analysis is very simple because proper values aretabulated.
Obviously, density, rotation angle and refractive index dependon temperature. Additionally, polarization and refractive indexdepend also on wavelength and therefore the measurements haveto be performed under precisely specified conditions: temperatureof 20�C (293K), and wavelength of the D line of sodium(589.3 nm) as standard and symbolized by nD. It should be empha-sized that accuracy of such measurements is based on assumptionof substance purity. Although the result of analysis is true only ifsingle sugar is present in sample, for fast routine assays suchmethod is sufficient. These techniques could be applied to analysisof complex samples after sugars separation. However, in the analyt-ical practice only refractometry has found wider application asdetection technique in liquid chromatography. Recently[15], thetechniques based on refractive index difference were applied fordetermination of sucrose content. In case of candy floss analysisretro-reflected beam interference based on refractive index detec-tion has been applied For cola drinks analysis instead of conven-tional refractometer measurements were carried in photometricflow cell. They were based on detection of light deflected by Schlie-ren effect formed due to refractive index gradient.[9] The result ofmeasurements were presented using Brix degrees.
Brix (�Bx) and related scales (Balling �Bg, Plato �P) arewidely applied for sugar liquids characterization. One Brix,Balling and Plato degree is percentage by weight of sucrose inpure water solution. Difference between these three indices isin reference temperature and closeness of measurements values(3,5 and 6 decimal places for �Bx, �Bg, and �P, respectively).Nowadays the most popular and the most used is the Brixindex. Plato index is sometimes applied in brewing industry.Generally these units are closely connected with concentrationof sucrose in pure water but sometimes the industry uses theseunits somewhat loosely to refer to any sweet solids in a prod-uct.[102] In case of sugars other than sucrose Brix is called the“apparent Brix” and is always a relative value. The Brix indexcan be determinated both by hydrometry and refractometry.Specially designed devices have scale both in [kg/m3]/ [g/cm3]or nD and Brix. However, in some cases values obtained byhydrometer and refractometers can differ each other, especiallyin case of “apparent Brix” measurements and samples withcomplex matrix. For example analysis of orange juice samplesrequired special correction because of sample acidity.[103]
An interesting issue is industrial analysis of sucrose. Nowadaysall commonly accepted methods for table sugar assay are codifiedby ICUMSA (International Commission for Uniform Methods of
Sugar Analysis). ICUMSA Methods Book.[104] contains descriptionof official methods recommended for quality control in sugarindustry, both for sucrose and impurities (lead, arsenic, copper,iron, etc.) concentrations determination as well as tabulated valuesof specific rotation, refractive index and density of sucrose solu-tions. This book contains also regulations concerning some selec-tive determinations based on chromatographic and enzymaticmethods, reported in the next paragraphs of this review.
3. Selective analytical methods
Taking into account rather high content of sugars in real sam-ples and practically unlimited accessibility of these samples(mainly food and agriculture products), very low detection lim-its and wide determination ranges are not extremely importantparameters of modern methods developed for analysis ofsugar-containing products. A crucial analytical factor of thesemethods is a selectivity allowing detection of particular saccha-ride in the presence of complex matrix of sample additionallycontaining various other very similar sugars. Currently thereare three main trends for the development of methods for selec-tive determination of disaccharides. The most popular andeffective are various separation techniques like chromatographyand electrophoresis. The second direction is the development ofbioselective methods based on almost specific recognition ofanalyte by enzymes and its conversion into easily detectedproduct. A third relatively new direction, also dedicated forsample analysis without analyte separation, is the application ofadvanced spectroscopic methods (IR, Raman) combined withuse of sophisticated chemometric tools.
3.1. Separation-based methods
Nowadays the most dynamically developed and widely reported inthe analytical literature separative technique for disaccharidesdetermination is liquid chromatography. Some recent papersdescribing application of various chromatographic methods fordisaccharide determination are cited in Table 2. High PerformanceLiquid Chromatography (HPLC) is the official technique for rou-tine sugars analysis recommended by AOAC International (Associ-ation of Official Analytical Chemists).[86] HPLC gives bothqualitative (identification of the carbohydrate) and, with peak inte-gration, quantitative information. The analysis is rapid, applicableto samples with a wide range of sugar concentrations, precise andaccurate and do not required derivatization of carbohydrates.
Liquid chromatography for disaccharide separations exploit dif-ferences in polarity (HPLC normal and reversed- phase, HILIC,HTLC) or electrical charge (IEC: cation, anion exchange and ionexclusion) of target molecules. The most popular chromatographicmode is HLPC with normal-phase configuration. In this modecommonly applied stationary phase is silica gel with amino groups,whereas acetonitrile–water (40–95% acetonitrile) is used as amobile phase. The elution order is monosaccharides, disaccharidesand finally higher oligosaccharides. The gradient elution allows toavoid interferences caused by sugar alcohols (mannitol can coelutewith maltose and lactose while inositol with sucrose). In reversedphase HPLC the hydrophobic stationary phase is silica gel withadded alkyl chains, for example C18 column.[42] Thismode of sepa-ration has been used for separation of mono- di- and trisaccharides
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 189
-
Table2.
ApplicationofLiqu
idchromatograph
yford
isaccharides
determ
ination.
Chromatograph
icseparatio
n
Detectio
ncolumn
elution
Determined
disaccharid
esOtherdeterm
ined
substance
Sample
Reference
Normal-Phase
HPLC
ELSD
Zorbax
RX-SIL(m
odified
byethylene
diam
ine)
isocratic
acetonitrile-water
(72%
:28%
)with
0.03%
ethylene
diam
ineand0.05%
ammoniumhydroxide
sucrose,maltose,lactose
glucose,fructose,raffinose
Drin
ks(app
lejuicepineapple
juiceorange
juicegrapewine
liquor)
[10]
ELSD
carbohydratecolumn
Isocratic
acetonitrile–w
ater
(70%
:30%
)sucrose,maltose,lactose
glucose,fructose,galactose
raffinose
fruits,vegetables,grains,seeds
andleaves
[32]
ELSD
Phenom
enex
Luna
5uNH2100A
isocratic
acetonitrile-water
(82.5%
:17.5%
)sucrose
glucose,fructose,sorbitol
fruits(peach,app
lewatermelon,
cherry)
[23]
ELSD
SpherisorbNH2
gradient
from
81%acetonitrile
/19%
waterto
75%
acetonitrile
/25%
waterover
40min
maltose
glucose,fructose
beer
[62]
ELSD
PrevailCarbohydrateES
isocratic
acetonitrile-water
(80%
:20%
)lactose,lactulose
glucose,fructose,galactose
synthetic
samples
[105]
YMCPack
Polyam
ine
Zorbax
Carbohydrate
Analysis
UnisonUK-Am
inoHT
ELSD
andC-CA
DNH2-Krom
asil
isocratic
acetonitrile–w
ater
(70%
:30%
)sucrose,maltose,lactose
glucose,fructose
maltotriose
sauces,syrup
s,jellies,glazes,
hone
anddairy
products
[13]
CAD
ShodexAsahipak
NH2P-50E4
gradient
from
90%acetonitrile
/10%
waterto
77%
acetonitrile
/23%
waterover
22minandkept
constant
until40
min
sucrose,maltose
glucose,fructose,erythritol,
xylitol,sorbitol,mannitol,
maltitol
drinks
(juices,nectarsand
syrups)
[106]
RID
Tracer
carbohydratescolumn
isocratic
acetonitrile–w
ater
(75%
:25%
)sucrose,lactose,lactulose
glucose,fructose,galactose
milk-based
form
ulae
[69]
RID
SupelcosilLC-NH2
isocratic
acetonitrile–w
ater
(80%
:20%
)trehalose
—Selaginella
lepidophyllaplant
[67]
RID
Zorbax
Carbohydate
aminopropyl
isocratic
acetonitrile–w
ater
(82%
:18%
)sucrose,maltose,lactose
glucose,fructose,m
annose,
sorbito
l,xylitol
wine,juices,honey,dairy
products,biscuits
[14]
RID
UltraAminoColumn
isocratic
acetonitrile–w
ater
(75%
:25%
)lactose,lactulose
—HeatTreated
Milk
[44]
PinacleIIAm
ino
isocratic
acetonitrile–w
ater
(75%
:25%
)2PinacleIIAm
ino(in
series)
isocratic
acetonitrile–w
ater
(75%
:25%
)RID
Zorbax
Carbohydate
aminopropyl
isocratic
acetonitrile–w
ater
(82%
:18%
)sucrose,maltose,lactose
glucose,fructose,m
annose,
sorbito
l,xylitol
wine,juices,honey,dairy
products,biscuits
[14]
RID
UltraAminoColumn
isocratic
acetonitrile–w
ater
(75%
:25%
)lactose,lactulose
—heattreatedmilk
[44]
PinacleIIAm
ino
isocratic
acetonitrile–w
ater
(75%
:25%
)2PinacleIIAm
ino(in
series)
isocratic
acetonitrile–w
ater
(75%
:25%
)RID
PrevailCarbohydrateES
isocratic
acetonitrile–w
ater
(75%
:25%
)lactose,lactulose
—conservedmilk
[45]
RID
Agilent
Zorbax
Carbohydrate
isocratic
acetonitrile–w
ater
(80%
:20%
)sucrose
glucose,fructose,sorbitol,
organicacids
grapefruitpu
lps
[24]
RID
SpherisorbAm
ino
isocratic
acetonitrile–w
ater
(70%
:30%
)lactulose
–lactose-free
milk
[107]
190 M. POKRZYWNICKA AND R. KONCKI
-
ESIM
S/MS
PrevailCarbohydrateES
isocratic
acetonitrile–
ammoniumform
ate
(70%
:30%
)
lactose,lactulose
ESI/M
SAcqu
ityBEHam
ide
gradient
from
95%ofacetonitrile/5
%0.1%
ammoniainwaterisocratic
for
2min70%ofacetonitrile/3
0%0.1%
ammoniainwaterfor2
minand60%of
acetonitrile/4
0%0.1%
ammoniain
waterfor2
min
sucrose
glucose,fructose,kestose,
nystose
datesfruits
[22] MS/MS
Acqu
ityBEHam
ide
isocratic
acetonitrile–0.1M
ammoniumhydroxidein
water(80%
:20%
)
sucrose
—ratp
lasm
a,blood,andbrain
homogenate
[43]
MS/MS
Acqu
ityBEHam
ide
gradient
from
75%of
0.1%
ammoniumhydroxidein
acetonitrile/2
5%0.1%
ammoniumhydroxidein
waterto
67.5%of0.1%
ammoniumhydroxidein
acetonitrile/3
2.5%
0.1%
ammoniumhydroxidein
waterfor1
0min,to62.5%of
0.1%
ammoniumhydroxide
inacetonitrile/37.5%0.1%
ammoniumhydroxidein
waterfor2
minand75%of
0.1%
ammoniumhydroxide
inacetonitrile/25%0.1%
ammoniumhydroxidein
waterfor0
.1minthen
isocratic
for6
min
isom
altose
pannose,isom
altotriose
milk
powder
[74]
MS/MS
ZIC-
HILIC
gradient
from
75%acetonitrile
/25%
5mMofam
monium
acetateinwater
to40%
acetonitrile
/60%
5mMof
ammoniumacetateinwater
in10
min
sucrose,lactulose
raffinose,m
annitol
urine
[39]
AscentisSi
gradient
from
80%acetonitrile
/20%
5mMofam
monium
acetateinwater
to65%
acetonitrile
/35%
5mMof
ammoniumacetatein
waterin6min
SupelcocilLC-NH2
gradient
from
75%acetonitrile
0.05%form
icacid/25%
H2O
0.05%form
icacidto
40%
acetonitrile
0.05%form
icacid/60%
H2O
0.05%
form
icacidin6min
(Continuedon
nextpage)
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 191
-
Table2.
(Continued)
Chromatograph
icseparatio
n
Detectio
ncolumn
elution
Determined
disaccharid
esOtherdeterm
ined
substance
Sample
Reference
chem
iluminescence
Krom
asilNH2
isocratic
acetonitrile–w
ater
(70%
:30%
)lactose
glucose,fructose,xylose,
arabinose
grapefruitsextracts
[108]
Reversed
-Phase
HPLC
DAD
andRID
Aminex
HPX-87H
isocratic
3mMsulfuric
acid
lactose
glucose,fructose,organicacids
goat’smilk
yogu
rts
[52]
MS
Alltech
C18
gradient
from
90%0.1%
acetic
acidinwater/1
0%acetonitrile
with
0.1%
acetic
acidto
100%
0.1%
aceticacid
inwater
over8min,isocratic
for3
min,to0.1%
aceticacid
inwater/10%acetonitrile
with
0.1%
aceticover3min
andisocraticallyat90%0.1%
aceticacidinwater/10%
acetonitrile
with
0.1%
acetic
for3
min,for
atotalrun
time
of17
min
sucrose
—equine
serum
[42]
Hydroph
ilicInteractionCh
romatog
raph
y[HILIC]
QTO
FMS
XamideColumn
gradient
5%,10%
or20%of
100mMam
moniumform
ate
(pH3.2)in:0%acetonitrile/
100%
water
to80%
acetonitrile/2
0%waterfor
60min
lowmolecularweigh
theparindisaccharid
eheparin
—[109]
ESI/TOFM
S/MS
Zorbax
NH2am
inopropylsilica
gradient
from
88%8mM
ammoniumform
atein
acetonitrile/1
2%8mM
ammoniumform
ateinwater
isocratic
for1
0minto
80%
8mMam
moniumform
atein
acetonitrile/2
0%8mM
ammoniumform
ateinwater
for8
min,to75%8mM
ammoniumform
atein
acetonitrile/2
5%8mM
ammoniumform
ateinwater
for4
min,to70%8mM
ammoniumform
atein
acetonitrile/3
0%8mM
ammoniumform
ateinwater
for5
min,and
isocratic
for
2min
sucrose,mellibiose
glucose,fructose,galactose,
raffinose,m
anninotriose,
stachyose,verbascose
crud
eandprocessedRadix
Rehm
anniae
[80]
ELSD
Acclaim
Trinity
P2isocratic
acetonitrile–100
mM
ammoniumform
iatebu
ffer
pH3.65
(80%
:20)
lactulose
—milk
[72]
192 M. POKRZYWNICKA AND R. KONCKI
-
HighTemperature
Liqu
idCh
romatog
raph
y[HTLC]
ELSD
Hypercarb
isocratic
water
sucrose,maltose,lactose
glucose,fructose,galactose
milk,orang
eandmandarin
efruits
[110]
Ion-Exchange
Chromatog
raph
y[HPA
EC](anionexchange)
PAD
CarboPac
PA1
gradient
from
16mMsodium
hydroxideto
250mMsodium
hydroxidefor6
0min
maltose,lactose,trehalose,cellobiose
glucose,galactose,rafinose,
ribose,rham
nose,arabinose,
ethanolsorbitol,glycerol,
arabito
l,erythrito
l
yeastculturesandferm
entatio
nbroths
[76]
CarboPac
MA1
isocratic
480mMsodium
hydroxide
PAD
CarboPac
PA10
isocratic
5mMsodium
hydroxide
sucrose
glucose,fructose
wastewaterfrom
thebeverage
indu
stry
[66]
PAD
CarboPac
PA10
isocratic
50mMpotassium
hydroxide
sucrose,lactose
glucose,fructose
chocolate
[16]
PAD
CarboPac
PA10
gradient
87.5mMsodium
hydroxideisocratic
for
10min,to500mMsodium
hydroxidefor0
.1min,
isocratic
for7
min
maltose,isomaltose
glucose,rib
ose
bloodserum
[111]
PAD
CarboPac
PA20
isocratic
8mMsodium
hydroxide
lactose
glucose,galactose
naturally
“lactosefree”hard
cheese
[51]
PAD
CarboPac
PA20
isocratic
water
sucrose
glucose,fructose,galactose,
arabinose,mannose,
rham
nose,m
annitol
greencoffeebean
[33]
PAD
AminoPac
PA10
gradient
from
17.5mMsodium
hydroxideto
25mMsodium
hydroxidefor2
min,to
37.5mMsodium
hydroxide
for3
min,to90
mMsodium
hydroxidefor6
min,2
min
isocratic,to100mMsodium
hydroxidefor2
min,to
175mMsodium
hydroxide
for5
min,to175mMsodium
hydroxideand3mMsodium
acetatefor0
.1min,to
175mMsodium
hydroxide
and7mMsodium
acetatefor
8min,to175mMsodium
hydroxideand10
mM
sodium
acetatefor5
min,to
175mMsodium
hydroxide
and20
mMsodium
acetate
for7
min,to75
mMsodium
hydroxideand60
mM
sodium
acetatefor5
min,
10minisocratic
maltose,isomaltose
glucose,fructose,arabinose,
maltotriose,isomaltotriose,
panose,m
altotertose,
aminoacids
ricewines
[59]
PAD
CarboPac
PA20
isocratic
1mMsodium
hydroxide
sucrose
glucose,fructose,arabinose,
mannose,xylose
aqueousextractsand
hydrolysates
ofbiom
ass
[112]
(Continuedon
nextpage)
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 193
-
Table2.
(Continued)
Chromatograph
icseparatio
n
Detectio
ncolumn
elution
Determined
disaccharid
esOtherdeterm
ined
substance
Sample
Reference
PAD
CarboPac
PA200
gradient
0.1M
sodium
hydroxide
isocratic
for9
min,to0.1M
sodium
hydroxideand0.04M
sodium
acetatefor1
7min,to
0.1M
sodium
hydroxideand
0.25Msodium
acetatefor
0.1min,isocraticfor1
4min
cellobiose,xylobiose
linearxylo-oligosaccharides
and
cello-o
ligosaccharides
lignocellulosicprocessing
products
[75]
PAD
Ham
ilton
RCX-30
gradient
from
50mMsodium
hydroxide25
mMsodium
acetateto
50mMsodium
hydroxide100mMsodium
acetatefor1
5min,isocratic
for1
0min
maltose,isomaltose
glucose,malto-oligosaccharides
wheatflour
[55]
PAD
CarboPac
PA10
gradient
2mMsodium
hydroxideisocratic
for5
min,
to5mMsodium
hydroxide
for3
min,isocraticfor2
min,
to20
mMsodium
hydroxide
for2
min,isocraticfor3
min,
to30
mMsodium
hydroxide
for1
min,isocraticfor2
min,
to40
mMsodium
hydroxide
for3
min,isocraticfor1
min,
to45
mMsodium
hydroxide
for3
min,isocraticfor2
min
sucrose,lactose,trehalose
glucose,fructose,arabinose,
ribose,xylose
rawsugar
[65]
ESI/M
SCarboPac
PA20
gradient
3mMpotassium
hydroxideisocratic
for
30min,to80
mMpotassium
hydroxidefor5
min
sucrose,lactose,trehalose
glucose,fructose,m
annitol,
glucosylglycerol
intracellularextractsof
cyanobacteria
[113]
ELSD
CarboPac
PA1
isocratic
32mMpotassium
hydroxide
sucrose
glucose,fructose,arabinose,
xylose
drinks
(cola,orange
juice,
watermelon
juice)
[12]
Ion-Exchange
Chromatog
raph
y[HPC
EC](catio
nexchange)
ESI/M
SIOA-1000
9mm
isocratic
20mMform
icacidand
10mMtrichloroacetic
acid
sucrose,lactulose,sucralose
rham
nose,erythritol
urineandbloodplasma
[40]
RID
Carbosep
Corgel87H3
isocratic
5mMsulfuric
acid
sucrose
glucose,fructose,sorbitol
apple’sleafandfruitp
eel
[25]
RID
RNMCarbohydrateNaC
isocratic
water
xylobiose
xylo-oligosaccharides
enzymaticallyhydrolysed
pulp
[79]
RID
SugarP
akI(Ca
2C)
isocratic
water
lactose,lactulose
—heattreatedmilk
[44]
RID
RezexRSO-OligosaccharideIE
(AgC
mode)
isocratic
water
maltose
glucose,fructose
beer
[61]
IEXC
a2C
isocratic
water
Ion-ExclusionCh
romatog
raph
y
RID
Bio-RadAm
inex
HPX
87H
Isocratic
0.005n
phosph
oricacid
sucrose
glucose,fructose,organicacids
fruitjuices
[114]
Abbreviatio
ns:CAD
-Charged
AerosolD
etectio
n;C-CA
D-C
orona-Ch
argedAe
rosolD
etector;DAD
-Diode
ArrayDetector;ESI-Electrospray
Ionizatio
n;ELSD
-EvaporativeLigh
tScatteringDetectio
n;MS-MassSpectrom
etry;M
S/MS-Tan-
demMassSpectrom
etry;PAD
-PulsedAm
perometric
Detectio
n;QTO
FMS-Quadrup
olTime-Of-Flight
MassSpectrom
etry;RID-R
efractiveIndexDetector;TO
FMS-Time-Of-Flight
MassSpectrom
etry.
194 M. POKRZYWNICKA AND R. KONCKI
-
by groups. A main disadvantage of this technique concerns mono-saccharides separation (short retention time results in elution as asingle unresolved peak). There could be also problem in the pres-ence of anomers that can results in peak doubling and/orbroadening.
For HPLC systems a variety of detectors can be coupled:Refractive Index Detector (RID),[14,24,44,45,67,69] EvaporativeLight Scattering Detectors (ELSD),[10,23,32,62] various MassSpectrometry techniques (tandem MS,[39,43,74] ESI MS[22,115])Charged Aerosol Detectors (CAD)[13,106] or even chemilumi-nescence.[108] A simple and economic RID seems to be the bestsolution for determination of separated sugars although it isless sensitive then other types of detectors. Unfortunately dueto its strongly dependence on solvent, type RID cannot beapplied in case of gradient elution. On the other hand, ELSDand MS required solvent evaporation.
An alternative HPLC mode for separating polar compounds ishydrophilic interaction liquid chromatography (HILIC). In thiscase a polar-hydrophilic stationary phase, characteristic for normalphase mode, is coupled with also polar water-containing, mobilephase characteristic for reversed phase.[116] But unlike in reversed-phase chromatography, gradient elution HILIC begins with a low-polarity organic solvent and elutes polar analytes by increasing thepolar aqueous content.[42,80] Such inversion of elution order allowsshortening of analysis when the target analyte is a single particularsaccharide. Another variation of HPLC is high temperature liquidchromatography (HTLC).[110] In HTLC thanks to raising separa-tion temperature to about 100�C there is a possibility to apply purewater as eluent without elongate time of analysis and degradationin resolution.
Carbohydrate separation can be achieved also by High Per-formance Thin Layer Chromatography (HPTLC).[117] Silica gel60 F254 plates with dropped of 1 mL of sample were developedat room temperature with a mobile phase of acetonitrile: water(8.5:1.5, v/v). Before determination sample was derivatives byaniline diphenylamine o-phosphoric acid. This method wasapplied for determination of maltose and total sugars in insweet potato (Ipomoea batatas L.).
Disaccharides separation based on their electrical charge is alsopossible, because carbohydrates are very weak acids (pKa values>12).[118] In strongly alkaline solutions some carbohydrate hydroxylgroups are ionized allowing sugars separation on anion-exchangecolumns. The employed mobile phases are simple and inexpensivesodium hydroxide[33,51,65,66,76,111,112] or potassium hydrox-ide[16,113,114] solutions, with or without addition of acetatesalt.[55,59,75] Also water may be used,[33] but then postcolumn addi-tion of a electrolyte solution is required for obtain adequate detec-tion conditions. This separation mode is very often connected withPulsed Amperometric Detection (PAD). Amperometric detectionhave a lot of advantages. Under specific pH and voltage conditionsonly carbohydrates will undergo the redox reaction. Coupling thismethod with chromatographic separation additionally increasesselectivity of detection. The development of so-called triple-pulsedamperometric detector,[118] solve the problem of electrode poison-ing by accumulation of oxidized products on its surface andallowed electrochemical detection for carbohydrates. The entirecleaning process takes milliseconds and is ongoing throughout therun. Because electrode reaction relies on oxidation of carbohydratehydroxyl and aldehyde groups, this detector is suitable for both
reducing and nonreducing carbohydrates. Also example of applica-tion of ELSD detector coupled with cation exchange separation isdescribed.[114] This type of detector requires evaporation of eluentbefore detection step and therefore the non-volatile potassium saltsin the basic eluent has to be removed by a suppressor.
In cation exchange liquid chromatography stationary phasesare often resin loaded with one of a variety of metal counterions Ca2C,[44,61] NaC[79] or AgC[61], which react selectively withthe weakly negatively charged hydroxyl groups of sugar mole-cules. The selectivity of this process is controlled with theappropriate choice of resin type and of the metallic speciesbonded to it, as well as by the temperature of column (columnsnormally are operated at elevated temperatures to increase itsefficiency). The mobile phase is typically water. The mechanismof separation is based on the strength of the bonding betweencis-glycols of sugar molecules with the cation loaded on the col-umn. The elution order is related to the number and strengthof cis-glycol complexes formed and takes place in the order ofdecreasing molecular weight.[86]
Ion exclusion chromatography (IEC) also found applicationin disaccharide determination.[114] In this techniques at ionexchange resin the ionic substances are rejected while non-ionic or partially ionized substances are retained and separatedby partition between the liquid inside the resin particles andthe liquid outside the particles. In effect the ionic substancespass quickly through the column. Non-ionic or partially ion-ized substances are held up and eluted more slowly.
Modern liquid chromatography techniques require specializedlaboratory equipment and are often connectedwith high consump-tion of eluent – mostly expensive chemically ultra-pure solvents.Moreover, the most often applicable normal-phase HPLC meth-ods, where organic solvents are applied, stay in contradiction togreen chemistry assumptions. From this point of view Gas Chro-matography (GC) seems to be better solution for disaccharidesdetermination. Without consumption of expensive ultra-purereagents it allows to determine much more analytes in the courseof single run.[119] In the Table 3 some papers from last 20 yearsdevoted to GC determination of disaccharides are collected. Inalmost all cases disaccharides have been determined together withmonosaccharides and sugar alcohols.
Sugars are non-volatile and thermally unstable compounds.Their determination with GC have to be preceded by the deriv-atization process, often complicated and time consuming. Thisderivatization required before separation step is the main prac-tical drawback strongly limiting the development of GC fordisaccharide analytics. For GC sugars determination methyl,acetate, trifluoroacetate and trimethylsilyl derivatives can beapplied.[132] Nowadays the most popular are trimethylsilyls(TMS) and trimethylsilyl oximes (TMSO). Trimethylsilylationof carbohydrates is a simple reaction and because non-volatilereagents or by-products are involved, the complete reactionmixture can be injected directly into the gas chromatograph,avoiding clean-up stages. Trimethylsilyl derivatives of carbohy-drates acquire different chemical properties, molecule increasevolatility and thermal stability and decrease polarity.[133] It isworth to mention that TMSO derivatised disaccharides are iso-meric molecules with monosaccharide ring structure and anopen chain with the oxime group. The only difference is in theposition of the substituents so they can have similar mass
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 195
-
Table3.
ApplicationofGas
Chromatograph
yford
isaccharides
determ
ination.
Chromatograph
icseparatio
n
Detectio
ncolumn
temperature
Determined
disaccharid
esOtherdeterm
ined
substance
Sample
Reference
FID
TR-1capillarycolumn(60m
£0.32
mmi.d.;0.25
mmfilm
of100%
methylpolysiloxane)
80� C
for2
minto
240�Cat5
� C/m
inleftfor5
minto
280�Cat20
� C/m
inleftfor1
5min
sucrose,maltose,lactose,m
elibiose
glucose,fructose,xylose,
mannose,galactose,sorbitol,
myo-inositol
tobacco
[36]
FID
ZB-5(30m
£0.25
mmi.d.;0.25mm
film
of5%
phenylmethylpolysiloxane)
100�Cto
180�Cat4
� C/m
inleftfor
2minto
215�Cat2�C/minto
325�C
at3�C/minleftfor1
0min
sucrose,maltose
trehalose,turanose,
cellobiose,palantinose,isom
altose
glucose,fructose,raffinose,
pann
ose
honeys
[120]
FID
SPB-1(30m£
0.25
mmi.d.;0.25mm
film
ofcrosslinkedmethylsilicone)
170�Cfor1
0minto
215�Cat15
� C/m
into
240�Cat1
� C/m
into
320�Cat
5�C/minleftfor2
0min
sucrose,maltose,a
,a-trehalose,
a,b-trehalose,turanose,cellobiose,
kojibiose,lam
inaribiose,nigerose,
maltulose,trehalulose,palantin
ose,
melibiose,gentio
biose,isom
altose
—honeys
[121]
Rtx-65
TG(25m£
0.25
mmi.d.;
0.1m
mfilm
ofcrossbond35%
dimethyl-65%
diph
enyl
polysiloxane)
200�Cfor2
0minto
270�Cat15
� C/
minto
290�Cat1�C/minto
300�C
at15
� C/m
inleftfor4
0min
FID
OV-101(25m£
0.25
mmi.d.)
180�Cto
280�Cat2�C/minto
290�Cat
10� C
/minleftfor1
5min
sucrose,maltose,lactose,m
altulose
glucose,fructose,galactose
enteralformulations
[122]
FID
CP-SIL5CB(25m£
0.25
mmi.d.;
0.25mmfilm
ofmethylsilicone)
180�Cfor3
6minto
300�Cat10
� C/m
inleftfor5
0min
sucrose
glucose,fructose,m
annose,
galactose,mannitol,
bornesito
l,myo-in
osito
l
coffeeandcoffeesubstitutes
[123]
MS
FID
SPB-1(25m£
0.25
mmi.d.;0.25mm
film
ofcrosslinkedmethylsilicone)
200�Cfor2
0minto
270�Cat15
� C/m
inleftfor4
0min
sucrose
glucose,fructose,inositols
fruitjuices
[124]
MS
FID
fusedsilicacapillarycolumn(7.5m
£0.25
mm)
180�Cfor2
0minto
270�Cat20
� C/m
inleftfor3
0min
sucrose
glucose,fructose,m
yo-in
osito
lorange
juice
[125]
MS
fusedsilicacapillarycolumn(22m£
0.25
mm)
MS
HP-5msUltraInert(15
m£
0.25
mmi.
d.;0.25m
mfilm
of5%
-phenyl)-
methylpolysiloxane)
180�Cfor2
minto
320�Cat7�C/min
leftfor2
min
lactose
—milk,cheese,yogh
urt
[126]
MS
SPB-1(30m£
0.25
mmi.d.;0.25mm
film
ofcrosslinkedmethylsilicone)
270�C
sucrose,maltose,lactose,
a,a-trehalose,turanose,cellobiose,
kojibiose,lam
inaribiose,nigerose,
maltulose,palantin
ose,melibiose,
isom
altose,sophorose,epilactose,
lactulose,leucrose
3-O-b-D-
galactopyranosyl-D-arabinose,
galactobioses,mannobioses
——
[119]
MS
TBR-1(30m£
0.25
mmi.d.;0.25mm
film)
200�Cfor1
5minto
270�Cat15
� C/m
into
290�Cat1�C/minLeftfor3
0min
lactose,a,a-trehalose,cellobiose,
laminaribiose,gentio
biose,
soph
orose
catechin,epicatechin,ethyl-
glucoside,glyceryl
–glucosides
wines
[127]
MS
DP-5MS(30m£
0.25
mm;0.25m)
50� C
to130�Cat30
� C/m
into
300�Cat
10� C/m
insucrose,maltose,m
elibiose
hexoses,hexoltioles,pentose
andpentosealcohols
cerealandpseudo
cerealflour
[128]
MS
DB-5or
HP-5M
S(30m
£0.25
mm;
0.25mmfilm)
50� C
to250�Cover58
min
sucrose,maltose,trehalose,
mannitol,sorbito
lstabilizersform
icrobial
preparations
[129]
MS
Rtx-65TG
(25m
£0.25
mmi.d.;
0.1mm
film
ofCrossbond35%
dimethyl–65%diph
enyl
polysiloxane)
170�Cfor1
0minto
215�Cat15
� C/m
into
240�Cat1
� C/m
into
320�Cat
5�C/minleftfor2
0min
sucrose,maltose,a
,a-trehalose,
a,b-trehalose,turanose,cellobiose,
kojibiose,lam
inaribiose,nigerose,
maltulose,trehalulose,palantin
ose,
melibiose,gentio
biose,isom
altose
raffinose,1-kestose,6-kestose,
pann
ose,erlose,neokestose,
malezito
se,m
altotriose,
isom
altotriose
honeys
[130,131]
Abbreviatio
ns:M
S-MassSpectrom
etry;FID-FlameIonizatio
nDetector.
196 M. POKRZYWNICKA AND R. KONCKI
-
spectra. Moreover, their retention time could also be similar.That is why coupling of gas chromatography with mass spec-trometry could give foul analytical information about disac-charides contents.[119] For GC disaccharide detection FlameIonization Detectors (FID) and Mass Spectrometers (MS) aremainly applied. MS offer advantages of both qualitative andquantitative information. Depending on the used detector,nitrogen (for FID) or helium (for MS) is applied as carrier gas.
A relatively novel and attractive alternative for LC and GCtechniques is Capillary electrophoresis (CE). CE is an electri-cally driven separation technique with many advantages suchas significant cost-effectiveness, high separation speed and largenumber of theoretical plates. CE requires minimal amounts(only microliters) of buffer, organic solvents and additives. Acommon problem with CE is its low detection sensitivitycaused by extremely low sample injection volume (nanoliters)and small inner diameter of the capillary. On the other hand,CE can be easily coupled with a variety of optical and electro-chemical detectors. CE systems have successfully applied fordisaccharides separation and determination. Some examples ofsuch analytical applications are collected in Table 4. However,none of them is officially recommended analytical method.
Separation principles of disaccharides in EC systems is simi-lar to this applied in anion exchange chromatography. Thehigh pH value is required, that is why the most common back-ground electrolyte is 50–75 mM sodium hydroxide. Capillarylength, critical for adequate separation resolution, range from10[11] to 120 cm.[63] Further improvement of separation effec-tivity can be obtained by chemical modifications of active sur-face of used capillaries.[77] There are also described applicationof microchip in place of conventional fused silica capillary.[134]
Disaccharide detection in capillary electrophoresis may seemproblematic. In case of electrochemical detection, because ofhigh voltage applied during separation, detector electrodesshould be somehow separated from capillary. Detector electro-des are placed about 50mm opposite to the capillary outlet.[135]
In case of photometric detection because of lack of chromo-phores for carbohydrates, precolumn derivatization[136] orother transformation[137] is required. There is also possibility toindirect absorbance detection. In this case an ionic chromo-phore is added to background electrolyte for example: 2,6-pyri-dinedicarboxylic acid, maleic acid and phthalic acid,[77] sorbicacid,[138] 1-naphthylacetic acid[63] or 2,6-pyridinedicarboxylicacid.[139] The detector receives a constant signal due to the pres-ence of these substances. The analyte displaces some of theseions, and detector signal decreases during the passage of ananalyte through the detector. Similarly chemiluminescencedetection can be applied with presence of luminol in back-ground electrolyte.[140]
3.2. Bioselective methods
Sugars as natural compounds are participate in many biotransfor-mation processes catalysed by various enzymes. Several biocatalyticpathways of such transformations are useful in the analytical chem-istry of sugars. The enzymes and enzymatic pathways involved inbiorecognition and biodetection for three main disaccharides areshown in Figures 3–5. In almost all cases enzymes involved in sac-charide metabolism are highly selective. Often they exhibit both
substrate and reaction specifity. This specific biorecognition of dis-accharides by respective enzymes can be applied in analyticalchemistry for the development of highly selective methods fordetermination of particular analyte without the need of separationof sugars and other components of samplematrix.
As can be seen from Figures 3–5, in the course of severalenzymatic disaccharides biotransformations various specificco-products, mediators and intermediates are consumed or cre-ated. This way enzymatic biorecognition could be easily cou-pled with various kinds of simple chemical detectors andsensors. A type of applied detector is closely related with classof last enzyme in biotransformation path of target sugar. Areview of detection methods coupled with enzymatic recogni-tion of disaccharides reported in the literature is presented inTable 5. Except amperometry, spectrophotometry, conductom-etry, fluorimetry and chemiluminometry, several detectiontechniques based on potentiometry[50,144] and ion selectivedevices[145–148] are also possible. Light addressable potentiome-try[144] and differential pHmetry[50] are based on acidificationby phosphorylation reaction catalyzed by glucokinase (GK E.C.2.6.1.2) or hexokinase (HK E.C. 2.7.1.1). Ion-Sensitive FieldEffect Transistors (ISFET) are sensitive for hydrogen ions gen-erated during reaction with dehydrogenases (describedexamples concern glucose[147–149] and galactose[148] dehydro-genases), or generated after electrolysis of hydrogen perox-ide.[146] A bit more complicated is situation with ElectrolyteIsolator Semiconductor (EIS). This device sensitive for fluorideions required application of 4-fluoroaniline as HPR mediator.For disaccharides determination also entalpimetric measure-ments of temperature changes during single[27] or multistepenzymatic reactions can be utilized.[150] Examples of applica-tion of Fourier Transform Near Infrared Spectroscopy(FTNIR)[151] or coulometry[152] have been also reported.
Predominantly, the biosensing schemes for disaccharides arebased on sequence of enzymatic conversions of target analyteinto detectable final species (Figs. 3–5) These so-called cascadeenzyme reactions, consisting of at least two even to foursteps,[29,58,145,161] realized in the analytical practise are listed inTable 6. Most of them are based on specific enzymatic hydroly-sis of target disaccharide to respective monosaccharides (thefirst step of enzymatic cascade) and then on their enzymaticallycatalysed oxidation (the second step) allowing detection usingconventional instrumental methods (Tab. 5). The enzymeapplied in the first step defines which disaccharide will be bio-recognized and determined, whereas further enzymes convertintermediate products into final detectable species. For exam-ple, amperometric detection of lactose could be performedusing only two enzymes: b-galactosidase (b-Gal, E.C. 3.2.1.23),glucose oxidase (GOx, E.C. 1.1.3.4).[48] However, in somecases[47,53] horseradish peroxidase (HPR E.C. 1.11.1.7) improv-ing detection of enzymatically generated hydrogen peroxide isalso implemented into biosensing system. Anotherextraordinary applied enzyme is mutarotase (Mut, E.C.5.1.3.3),[3,26,146,153,158,160,165] because most of the hydrolasesdecompose disaccharides into a- glucose, whereas the nextenzymes (glucose oxidase (GOx, E.C. 1.1.3.4) or glucose dehy-drogenases (GDh, E.C. 1.1.1.47)) are specific for b-D-glucose.However, this enzyme is not crucial in these biosensing path-ways, because mutarotation can occurs spontaneously
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 197
-
Table4.
ApplicationofCapillaryElectrophoresisford
isaccharides
determ
ination.
Detectio
ncapillarydimensions
background
electrolyte
voltage
Determined
disaccharid
esOther
determ
ined
substance
Sample
Reference
DAD
indirectdetectionof
maleicacidat210nm
61.5cm
effectiveleng
th138.2mMsodium
hydroxide,
40mMmaleicacid,5
mM1-
tetradecyl--3-
methylim
idazoliumchlorid
e
¡21.7kV
sucrose,lactose,
cellobiose,
xylose,fructose,glucose,
galactose,raffinose,
cellotriose,cellotetraose,
cellopentaose,cellohexaose
biom
ass
[77]
70cm
totallength
22.5mmID
DAD
directdetectionat278
and240nm
71.5cm
effectiveleng
th130mMsodium
hydroxide,36
mM
disodium
phosph
ate
C17kV
xylobiose
xylo-oligosaccharides
enzymaticallyhydrolysed
pulp
[79]
80cm
totallength
50mm
IDDAD
indirectdetectionof
sorbicacidat254nm
8.5cm
effectiveleng
th20
mMsorbicacid,40mMsodium
hydroxide,0.2mMcetyl
trimethylammonium
brom
ide
C25kV
sucrose
glucose,fructose
honey
[138]
60cm
totallength
50mm
IDDAD
270nm
absorbing
interm
ediateform
atted
byph
otooxidatio
n
60cm
totallength
98mMsodium
hydroxide,120mM
sodium
chlorid
e¡1
4kV
sucrose,lactose
glucose,fructose
post-explosion
residu
es,
smokedevice,cough
syrup,applejuice,red
wine
[137]
50mm
ID
DAD
directdetectionat
270nm
60cm
effectiveleng
th130mMsodium
hydroxide,36
mM
disodium
phosph
ate
C10kV
cellobiose,trehalose
fructose,fucose,galactose,
glucose,myo-in
osito
l,mannitol,mannose,
rham
nose,ribose,sorbito
l,xylose
PinotN
oirred
wines
[68]
50cm
totallength
50mm
ID
DAD
indirectdetectionof
1-naph
thylaceticacidat
222nm
120cm
totallength
1mMsolutio
nof1-naph
thylacetic
acidadjusted
topH
12.5with
sodium
hydroxide
C25kV
sucrose,maltose
glucose,fructose,m
altotriose
wort
[63]
75mm
ID
DAD
directdetectionat
280nm
afterp
recolumn
derivatizationwith
p-am
inobenzoicacid
57cm
totallength
20mMsodium
tetraborate
C20kV
maltose
glucose,malto-oligosaccharides
beers,orange
andplum
juices
[136]
75mm
ID
DAD
indirectdetectionof
2,6-pyrid
inedicarboxylic
acidat275nm
104cm
effectiveleng
th112.5
20mM2,6-pyrid
inedicarboxylic
acid,0,5mM
cetyltrimethylammonium
hydroxideadjusted
topH
12.1
with
1Msodium
hydroxide
¡25kV
sucrose,lactose
glucose,fructose
yogu
rt,orang
ejuice,sake
mash,pickledapricot
[139]
cmtotallength
50mm
ID
Chem
iluminescence
65cm
totallength
0.018gluminolin10
mL0.02M
sodium
hydroxidesolutio
nof
10%dimethylsulfoxide
C15kV
sucrose
fructose,rhamnose,cyclodextrin
–[140]
25mm
ID
Cond
uctometric
4cm
effectiveleng
th75
mMsodium
hydroxide
C5kV
sucrose
glucose,fructose,ribose
energy
drinks
[11]
10cm
totallength
10mm
IDAm
perometric
(special
design
edgraphene–
cobaltmicrosphere
hybridpasteelectrodes)
40cm
totallength
75mMsodium
hydroxide
C12kV
sucrose,lactose
glucose,fructose,m
annitol
Honey,m
ilk[141]
25mm
ID
Amperometric
(special
design
edgraphene–
copp
ercomposite
electrodes)
40cm
totallength
75mMsodium
hydroxide
C12kV
sucrose,lactose
glucose,fructose,m
annitol
honey,milk,peach,
banana
[135]
25mm
ID
Amperometric
(nano-NiO
modified
carbon
paste
electrode)
27cm
totallength
50mMsodium
hydroxide
C10kV
sucrose
glucose,fructose,m
annitol
honey
[142]
25mm
ID
Amperometric
40cm
totallength
75mMsodium
hydroxide
C12kV
sucrose
paeoniflorin,paeonoside,
glucose,andfructose
MoutanCortex
[143]
25mm
IDAm
perometric
8cm
totallength
100mMsodium
hydroxide
100V
sucrose,lactose,trehalose
glucose,fructose,galactose,
mannose,xylose
honey
[134]
1mmID
onmicrochip
DAD
-Diode
ArrayDetector.
198 M. POKRZYWNICKA AND R. KONCKI
-
(effectively in the presence of phosphate ions[166]). Sometimesadditional enzymes are used in the developed bioanalytical sys-tems for eliminations of interferences. For example speciallydesigned bioreactors with immobilized glucose oxidase (GOx,E.C. 1.1.3.4) and catalase (Cat, E.C. 1.11.1.6) have been appliedfor elimination influences from glucose in the course of sucrosedetermination.[26,165]
There are also some enzymatic paths that allow selectivedetermination only selected disaccharide without theirhydrolysis to respective monosaccharides.[5,27,46,54,58,151,156]
Sucrose phosphorylase (SP E.C. 2.4.1.1) decomposes sucroseto glucose 1-phosphate, a substance which further enzy-matic conversion (Fig. 3) is not interfered by glucose pres-ent in the sample.[5,156] Similar biosensing scheme (Fig. 4)has been developed for maltose using maltose phosphory-lase (MP E.C. 2.4.1.8) in the first biorecognition step.[58]
Lactose can be oxidized by cellobiose dehydrogenase (CDHE.C. 1.1.99.18) and concentration of acceptor are mea-sured.[46,54] However such approach is useful only in case ofsamples that do not contain cellobiose and maltose.[170] Sin-gle enzymatic reaction can be applied also for sucrose deter-mination after hydrolysis with invertase, but in these casesuncommon detection method have to be applied: thermom-etry for measuring of heat produced in the course of bioca-talyzed[27] reaction or subtle changes of Infra-Red spectrabetween substrate and product.[151]
Commercially available photometric assay kits for disaccha-ride determination are based on two steps cascade enzymaticreactions.[107,171–174] These kits contain soluble enzymes, how-ever as can be seen from table 6 a large number of bioanalyticalsystems dedicated for disaccharide determination is based onimmobilized enzymes. They are immobilized in the form of
Figure 3. Analytically useful enzymatic pathways for detection of sucrose.
Figure 4. Analytically useful enzymatic pathways for detection of lactose.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 199
-
mono- or polyenzymatic bioreactors or as biosensing layersintegrated with respective detectors (biosensors). These biosen-sors[27,46,53,54,57,70,146,148,150,156,157,167,175] and bioreac-tors[3,5,26,64,70,71,155,160,161,165] are often applied in systemsdesigned for flow analysis. Also measurements with freeenzyme are performed under flow analysis conditions,[17,153]
although it is connected with high enzyme consumption. Suchapproach (flow analysis) allows mechanization of multistepanalytical procedure and offers highly reproducible conditionsof biochemical processes (precise control of reaction time, tem-perature, reagent mixing etc.) and detection, what is especiallyimportant in case of reported here biocatalytic analytical meth-ods due to their kinetic, non- stationary character.
Not only enzymes could be applied as biocatalytic mate-rials for selective determination disaccharides. Severalmicroorganisms, natural sources of commercially availableenzyme preparation, were used for development of biosens-ing devices. Yeast cell were successfully used instead ofinvertase[166] and coupled with GOx on surface of biosen-sor. Saccharomyces crevisiae caused fermentation of glucoseobtained from catalysed by lactase lactose hydrolysis andallow determination with carbon dioxide electrode.[176] Con-sumption of oxygen by specially grown mutants of Escheri-chia coli K12 enable to monitoring of sucrose, maltose andlactose.[175] A maltase-displayed bacteria and glucose dehy-drogenase-displayed bacteria were co-immobilized on multi-
Figure 5. Analytically useful enzymatic pathways for detection of maltose.
Table 5. Possible detection methods defined by the last enzyme in biocatalytic path, Table 5 Detection type according to last enzyme type in cascade enzymatic path.
Final enzyme type
Oxidases: GOx,GaOx, PyOx
Dehydrogenases: GDh, GaDh,FDh, G6PDh, CDh Peroxidase: HrP
Detection technique Amperometry oxygen consumption,[17,48,57,153,154]H2O2 detection[3,26,155]
reduction of acceptor[46,54,71,156,157]
oxidation of mediator [47,53]
Conductometry increased in conductivity afterlactone dissociation [158,159]
— —
Spectrophotometry H2O2 chromogenic reactions[160] with G6PDH absorbance of
NADPH (340 nm) [29,58,64,161];with FDH reduction of MTTto MTT formazan (570 nm)[41,162]
product of reaction of 4aminoantrypine and Phenol4 sulphonic acid salt(500 nm) [163]
Fluorimetry — Fluorescence of NADPH(ex- 340 nm; em 460 nm) [5]
reduction of Amplex Red tohigh fluorescent resorufin(ex- 550 nm; em-585 nm)[28]
Chemiluminometry oxidation of luminol by H2O2[164,165]
— —
Abbreviations: CDh- cellobiose dehydrogenase; FDh- fructose dehydrogenase; G6PDh- glucose-6 phosphate dehydrogenase; GDh- glucose dehydrogenase; GaDh- galac-tose dehydrogenase; GOx- glucose oxidase; GaOx- galactose oxdase; HrP- horseradish peroxidase; MTT- 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide;NADPH- Nicotinamide adenine dinucleotide phosphate; PyOx- pyranose oxidase.
200 M. POKRZYWNICKA AND R. KONCKI
-
Table6.
Enzymaticdeterm
inationofdisaccharid
es.
Cascadeenzymaticreactio
nsequ
ence
Target
Analyte
1st
enzyme
2nd
enzyme
3rd
enzyme
4th
enzyme
detection
method
immobilizatio
nmethod
Other
determ
ined
sugars
Sample
Reference
immob
ilizedenzyme(biosensor)
sucrose
invertase
mutarotase
glucoseoxidase
horseradish
peroxidase
EIS
cross-linking
with
GA
glucose
—[145]
sucrose
invertase
mutarotase
glucoseoxidase
—Conductometry
cross-linking
with
BSA
andGA
glucose
orange
andapplejuices
[158]
sucrose
invertase
mutarotase
glucoseoxidase
—ISFET
photo-crosslinkable
polymer(PVA
SbQ)
glucose
—[146]
sucrose
invertase
glucoseoxidase
catalase
—Calorim
etry
covalent
attachmentto
activated
with
GA
glucose
—[150]
sucrose
sucroseph
osph
orylase
phosph
ogluco-m
utase
glucose-6ph
osph
ate
dehydrogenase
—Am
perometry
entrappedincarbon
pasteelectrodematrix
—pineapple,peachapple
juice
[156]
sucrose
invertase
fructose
dehydrogenase
——
Amperometry
entrapmentw
itha
dialysismem
brane
fructose,glucose
cond
ensedmilk,
referencematerial
[157]
sucrose
invertase
glucosedehydrogenase
——
ISFET
cross-linking
with
BSA
andGA
glucose
—[147]
sucrose
invertase
glucokinase
——
Ligh
tadd
ressable
potentiometry
cross-linking
with
BSA
andGA
glucose
—[144]
sucrose
invertase
glucosedehydrogenase
——
ISFET
cross-linking
with
GA
glucose
—[148]
sucrose
invertase
——
—Thermom
etry
cross-linking
with
BSA
andGA
—sugarcanejuice
[27]
lactose
b-galactosidase
glucoseoxidase
horseradishperoxidase
—Am
perometry
entrapmentw
ithin
dialysismem
brane
glucose
chocolate,dairy
samples
[53]
lactose
b-galactosidase
glucoseoxidase
horseradishperoxidase
—Am
perometry
cross-linking
with
GA
glucose
milk
[47]
lactose
b-galactosidase
glucoseoxidase
horseradishperoxidase
—Am
perometry
—glucose
milk,cheese,yogh
urt
[126]
lactose
b-galactosidase
mutarotase
glucoseoxidase
—Am
perometry
cross-linking
with
GAand
b-cyclodextrin
and
coveredby
nafion
glucose
—[49]
lactose
b-galactosidase
glucoseoxidase
——
Conductometry
cross-linking
with
BSA
andGA
glucose
milk
[159]
lactose
b-galactosidase
glucoseoxidase
——
Amperometry
cross-linking
with
gelatin
eandGA
glucose
milk
[48]
lactose
b-galactosidase
glucoseoxidase
——
Voltammetry
cross-linking
with
GA,
covalent
bond
edwith
polyazetidine
glucose
—[167]
lactose
b-galactosidase
glucosedehydrogenase
——
ISFET
cross-linking
with
GA
glucose
—[149]
lactose
b-galactosidase
galactosedehydrogenase
——
ISFET
cross-linking
with
GA
galactose
—[148]
lactose
lactase
galactoseoxidase
——
Amperometry
Lang
muir-Blodgetfi
lmof
poly(3-hexyl
thioph
ene)/stearic
acid
galactose
—[168]
lactose
cellobiosedehydrogenase
——
—Am
perometry
physicaladsorptio
n,entrapmentw
itha
dialysismem
brane
—milk
[46,54]
maltose
amylo-
glucosidase
mutarotase
glucoseoxidase
horseradish
peroxidase
EIS
cross-linking
with
GA
glucose
—[54]
maltose
a–g
lucosidase
glucokinase
—Ligh
tadd
ressable
potentiometry
cross-linking
with
BSA
andGA
glucose
—[145]
maltose
a–g
lucosidase
glucosedehydrogenase
——
ISFET
cross-linking
with
GA
glucose
—[144]
(Continuedon
nextpage)
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 201
-
Table6.
(Continued)
Cascadeenzymaticreactio
nsequ
ence
Target
Analyte
1st
enzyme
2nd
enzyme
3rd
enzyme
4th
enzyme
detection
method
immobilizatio
nmethod
Other
determ
ined
sugars
Sample
Reference
maltose
amylo-
glucosidase
glucoseoxidase
——
Cyclicvoltammetry
physicaladsorptio
nglucose
beer
[148]
maltose
amylo-
glucosidase
glucoseoxidase
——
Amperometry
cross-linking
with
BSA
andGA
glucose
starch
hydrolysate
[60]
maltose
a–g
lucosidase
pyranose
oxidase
——
Amperometry
cross-linking
with
chito
san,carbon
nanotube
andGA
glucose,galactose,xylose
beer
[57]
lactulose
b-galactosidase
(inbioreactor)
fructose
dehydrogenase
——
Amperometry
cross-linking
with
BSA
andGA
fructose
milk
[154]
immob
ilizedenzyme(bioreactor)
sucrose
invertase
phosph
oglucose
isom
erase
hexokinase
glucose-6ph
osph
ate
dehydrogenase
Spectrophotometry
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
glucose,fructose
synthetic
samples
[161]
sucrose
invertase
mutarotase
glucoseoxidase
catalase
Amperometry
covalent
attachmentto
Amino-Cellulofine
activated
byGA
glucose,fructose
(byFD
H)C
ocacola,kiwi,apple,
banana,m
andarin
[26]
sucrose
invertase
mutarotase
glucoseoxidase(in
solutio
n)catalase
Chem
iluminescence
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
glucose
Pepsi,coke,cereal,cake
mix
[165]
sucrose
invertase
mutarotase
glucoseoxidase
—Am
perometry
cross-linking
with
BSA
andGAon
pig’ssm
all
intestine
glucose
fruitjuices
[3]
sucrose
invertase
mutarotase
glucoseoxidase
—Spectrophotometry
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
glucose
—[160]
sucrose
invertase
mutarotase
glucoseoxidase
—Am
perometry
cross-linking
tocellulose
mem
branewith
BSA
andGA
glucose
—[155]
sucrose
sucroseph
osph
orylase
phosph
ogluco-m
utase
glucose-6ph
osph
ate
dehydrogenase
—Fluorometry
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
—ionicsoftdrink,cola,
orange
juice
[5]
sucrose
invertase
glucoseoxidase
——
Chem
iluminescence
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
glucose
—[164]
sucrose
invertase
glucoseoxidase
(biosensor)
——
Amperometry
Glucose
oxidasesand
wich
mem
braneand
invertasebearingsilk
reactor
glucose
—[169]
sucrose
invertase
——
—FT-NIRspectroscopy
covalent
attachmentto
silicon
chipsilanized
with
ATPS
and
form
ylated
with
GA
—softdrinks
[151]
lactose
lactase
glucoseoxidase
——
Chem
iluminescence
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
glucose
—[164]
202 M. POKRZYWNICKA AND R. KONCKI
-
maltose
amylo-
glucosidase
glucoseoxidase
——
Chem
iluminescence
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
glucose
—[164]
lactulose
b-galactosidase
(insolutio
n)fructose
dehydrogenase
——
Amperometry
covalent
attachmentto
CPGallkylam
inated
with
APTS
and
form
ylated
with
GA
fructose
milk
[71]
trehalose
trehalase
hexokinase
(insolutio
n)glucose-6ph
osph
ate
dehydrogenase(in
solutio
n)
—Spectrophotometry
covalent
attachmentto
Epoxyresin
glucose
ferm
entatio
nbroth
[64]
non-
immob
ilizedenzyme
sucrose
invertase
hexokinase
phosph
oglucose
isom
erase
glucose-6ph
osph
ate
dehydrogenase
Spectrophotometry
—glucose,fructose
beet
root
[29]
sucrose
invertase
mutarotase
glucoseoxidase
—Am
perometry
—glucose
sugarb
eetm
olase
[153]
sucrose
invertase
glucoseoxidase
horseradishperoxidase
—Fluorim
etry
—glucose
sugarb
eet
[28]
sucrose
invertase
glucoseoxidase
horseradishperoxidase
—Spectrophotometry
—glucose
coffeebeans
[163]
sucrose
invertase
fructose
dehydrogenase
——
Spectrophotometry
—fructose
bloodserum,urin
e[41]
sucrose
invertase
glucoseoxidase
——
Amperometry
—glucose
orange
andapplejuice,
icecream,condensed
milk,jellies,greenpea,
corn,w
heat,peanuts
[17]
lactose
b-galactosidase
glucokinase
hexokinase
—DifferentialpH
techniqu
e—
glucose
milk
[50]
lactose
lactase
glucoseoxidase
——
Amperometry
—infant
form
ula
[17]
maltose
maltose
epimerase
maltose
phosph
orylase
phosph
ogluco-m
utase
glucose-6ph
osph
ate
dehydrogenase
Spectrophotometry
——
potato
solublestarch
[58]
maltose
a–g
lucosidase
glucoseoxidase
——
Amperometry
—corn
andmaltsyrup
[17]
maltose
a–g
lucosidase
glucoseoxidase
——
Coulom
etry
—glucose
—[152]
lactulose
b-galactosidase
hexokinase
——
DifferentialpH
techniqu
e—
glucose,fructose
milk
[50]
lactulose
b-galactosidase
fructose
dehydrogenase
——
Spectrophotometry
—fructose
milk
[162]
Abbreviatio
ns:APTS-3-am
inopropyltriethoxysilane;BSA-b
ovineserumalbu
min;CPG
-controlledporous
glass;EIS-electrolyteisolator
semicondu
ctor;FT-NIR-FourierTransform
NearInfraredSpectroscopy;G
A-glutaraldehyde;ISFET-
Ion-Sensitive
FieldEffectTransistor.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 203
-
walled carbon nanotubes modified glassy carbon electrodeto obtain electrochemical biosensor for maltose and glu-cose.[177] In the course of another investigations[178] therepressor of Escherichia coli lac operon has been engineeredas altered effector for selective recognition of lactulose.
Whereas biorecognition of various sugars using enzymesand biocatalytic materials is widely reported in the analyticalliterature, till now the immunoassays for disaccharide determi-nation are not developed. It is practically impossible to grownan antibody for sucrose due to lack of its immunogenicity.Although maltose and lactose antibodies are available, theiranalytical application is not reported in the literature.
3.3. Spectroscopic methods
Disaccharides, like all organic compounds, have reach characteris-tic spectra in Infra-Red (IR) range of wavelength with specificabsorption bands (table 7). The application of IR spectroscopy fordisaccharides determination required absorbance measurements atmany wavelengths simultaneously and then the use of special mul-tivariate statistical techniques to relate spectral data with the con-centration of the chosen component. Such techniques as multiplelinear regression (MLR) or partial least squares (PLS) regressionequation can predict the concentration of each constituent fromthe absorbance values at selected wavelengths. Because of the struc-tural similarities of sugars the spectra can overlap for each other.The baseline can variate in various apparatus and because of ambi-ent conditions (especially temperature). In case of aqueous solutionthere is high background spectrum of water. Sample surface imper-fections can effect in nonlinear, inhomogeneous and anisotropiclight scattering. In some cases high frequency detector noise canoccur. Obviously, many other compounds present in the samplecan contain similar functional group and thus can interfere.[179]
For IR data processing and interpretation rather sophisticatedchemometric methods are required. In experiments from two[181]
to more than fifty[182] spectra are recorded and averaged. In manycases also further data handling are processed. For smoothing andresolving overlapping peaks first and second derivatives computa-tion[20,182,183] or Savitzky-Golay (SG) filter[8,19,30,73,180,184–186] couldbe applied. Because spectra included very extensive data StandardNormal Variates (SNV),[8,18,30,184,185,187] Principal ComponentAnalysis (PCA)[18,56] or Genetic Algorithm (GA)[187] could be
applied to reduce the dimensionality of the data in order to extractmain and remove insignificant information. GA together withArti-ficial Neural Network (ANN) could also be coupled to define ana-lyte origin when group of samples is compared.[20] To obtaincalibration curves the Partial Least Square Regression (PLS) aremostly applied, however the use of Multiple Linear Regression(MLR)[187] and Principal Component Regression (PCR)[4] is alsoreported in the literature. The PLS and PCR use data reductiontechniques to extract from all extensive data much smaller amountof new variables representative for most of the variability in sam-ples. These newly defined variables can be used to create a calibra-tion curve or develop a regression equation to predict theconcentration of disaccharide in sample. In those methods, it is notnecessary to reduce data dimension, as it is in MLR where only alimited number of wavelengths are used. Examples of applicationof IR for disaccharide determination using various chemometrictools for data processing are presented in table 8. As can be seenfrom this table, chemometrically supported IR spectroscopy is use-ful for analysis of real samples having quite complexmatrix.
It is worth to notice that not only IR spectroscopy can be sup-ported by advanced chemometric methods to estimate disacchar-ides content in sample. There is also example of measurements invisible range with the use of second derivative spectra in combina-tion with PLS regression modelling for determination of sucroseand trehalose in olive leaves.[34] Some electrochemical techniquesmay require statistical data treatment to could be applied for selec-tive disaccharide determination. An Electrochemical ImpedanceSpectroscopy (EIS) was applied for determination of sucrose, glu-cose, fructose and total sugar content in pineapple fruit.[21,189] Afterstandard addition, measurement was performed and ANN techni-ques was applied to predict specific mathematical models for eachone of determined sugars. PLS method was also used to model therelationship between the EISmeasurements and the s