General methods for analysis of food carbohydrates
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Feb 24, 2020
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About This Presentation
Carbohydrate Analysis
Slide Content
GENERAL METHODS
FOR ANALYSIS OF
FOOD CARBOHYDRATES
Dr.Raj Kumar Kudari M.Pharm, PhD
Professor
Hindu College of Pharmacy, Guntur AP India.
FOR ANALYSIS OF
FOOD CARBOHYDRATES
Dr.Raj Kumar Kudari M.Pharm, PhD
Professor
Hindu College of Pharmacy, Guntur AP India.
Introduction
•Cereals,vegetables,fruits,rice,potatoes,legumes,andflour
productsarethemajorsourcesoffoodcarbohydrates.Thus,
naturallyoccurringsugarsareconsumedaspartofahealthydiet.
•Monosaccharides,sucrose,andpolysaccharidesarepresentinall
vegetables.Carbohydratesaresynthesizedbyallgreenplantsand
inthebodyareeitherabsorbedimmediatelyorstoredintheform
ofglycogen.Theycanalsobemanufacturedinthebodyfrom
someaminoacidsandtheglycerolcomponentoffats.Moreover,
sugarscanbeaddedtofoodsduringprocessingorpreparation,
mainlytoenhancefoodsensorialquality.Nutritionistsdividefood
carbohydratesintotwoclasses:
•A)Readilyavailablemono-,di,-orpolysaccharideslikeglucose,
fructose,lactose,dextrinsandstarch,
•B)Structuralpolysaccharidesofplantcellwalls(DietaryFiber),
complexpolysaccharideslikecellulose,pectinsandβ-glucans
•Cereals,vegetables,fruits,rice,potatoes,legumes,andflour
productsarethemajorsourcesoffoodcarbohydrates.Thus,
naturallyoccurringsugarsareconsumedaspartofahealthydiet.
•Monosaccharides,sucrose,andpolysaccharidesarepresentinall
vegetables.Carbohydratesaresynthesizedbyallgreenplantsand
inthebodyareeitherabsorbedimmediatelyorstoredintheform
ofglycogen.Theycanalsobemanufacturedinthebodyfrom
someaminoacidsandtheglycerolcomponentoffats.Moreover,
sugarscanbeaddedtofoodsduringprocessingorpreparation,
mainlytoenhancefoodsensorialquality.Nutritionistsdividefood
carbohydratesintotwoclasses:
•A)Readilyavailablemono-,di,-orpolysaccharideslikeglucose,
fructose,lactose,dextrinsandstarch,
•B)Structuralpolysaccharidesofplantcellwalls(DietaryFiber),
complexpolysaccharideslikecellulose,pectinsandβ-glucans
Introduction
•Inotherwords,availablecarbohydratesarethosethatare
hydrolyzedbyenzymesofthehumangastrointestinalsystem
whiletheunavailableonesarenothydrolyzedbyendogenous
humanenzymes(sugaralcohols,manyoligosaccharides,andnon
starchpolysaccharides)buttheycanbefermentedby
microorganismsinthelargeintestinetovaryingextentsandthen
absorbed.Sugars(glucose,sucrose,fructose,lactoseand
maltose),sugarpolyols(sorbitolandmannitol),oligosaccharides
(GOS,galactooligosaccharidesandFOS,fructooligosaccharides)
andpolysaccharides(starchandnonstarchpolysaccharides)have
beendescribedasthemajorclassesofcarbohydratesrelevantfor
humannutrition.
•Thefermentable-short-chaincarbohydrates-likeoligo-,di-and
monosaccharidesandPolyols(FODMAPS)thatcanbepoorly
absorbedbythesmallintestineprovidedifferenteffectson
gastrointestinalhealth.FODMAPscanbefoundinawidevariety
offoods.
•Inotherwords,availablecarbohydratesarethosethatare
hydrolyzedbyenzymesofthehumangastrointestinalsystem
whiletheunavailableonesarenothydrolyzedbyendogenous
humanenzymes(sugaralcohols,manyoligosaccharides,andnon
starchpolysaccharides)buttheycanbefermentedby
microorganismsinthelargeintestinetovaryingextentsandthen
absorbed.Sugars(glucose,sucrose,fructose,lactoseand
maltose),sugarpolyols(sorbitolandmannitol),oligosaccharides
(GOS,galactooligosaccharidesandFOS,fructooligosaccharides)
andpolysaccharides(starchandnonstarchpolysaccharides)have
beendescribedasthemajorclassesofcarbohydratesrelevantfor
humannutrition.
•Thefermentable-short-chaincarbohydrates-likeoligo-,di-and
monosaccharidesandPolyols(FODMAPS)thatcanbepoorly
absorbedbythesmallintestineprovidedifferenteffectson
gastrointestinalhealth.FODMAPscanbefoundinawidevariety
offoods.
Choice of Method of Analysis
•The analytical method that can be adopted depends on
the following:
a)characteristics of the carbohydrate.
b)Interfering substances
c)Solubility
d)Stability of the carbohydrate
e)Suitability of the method
f)Sample preparation
Most of the times coupled methods are adopted for
specific sugars.
Carbohydrate content can be measured after all other
components are measured.
• But! This can lead to erroneous results.!
Instead they can be Directly measured.!
% Carbohydrates = 100 -%Moisture -% Protein -% Lipid -% Mineral
•The analytical method that can be adopted depends on
the following:
a)characteristics of the carbohydrate.
b)Interfering substances
c)Solubility
d)Stability of the carbohydrate
e)Suitability of the method
f)Sample preparation
Most of the times coupled methods are adopted for
specific sugars.
Carbohydrate content can be measured after all other
components are measured.
• But! This can lead to erroneous results.!
Instead they can be Directly measured.!
% Carbohydrates = 100 -%Moisture -% Protein -% Lipid -% Mineral
.
•Types of sugars depending on the special
functional group are :
•neutral sugars
•acidic sugars
•amino sugars
•sugar alcohols & their isomers .
•Types of sugars depending on the special
functional group are :
•neutral sugars
•acidic sugars
•amino sugars
•sugar alcohols & their isomers .
.
1. CHROMATOGRAPHIC AND ELECTROPHORETIC METHODS
2. CHEMICAL METHODS
3. ENZYMATIC METHODS
4. PHYSICALMETHODS
5. IMMUNOASSAYS
General Applicable Methods of
Carbohydrate Analysis
1. CHROMATOGRAPHIC AND ELECTROPHORETIC METHODS
2. CHEMICAL METHODS
3. ENZYMATIC METHODS
4. PHYSICALMETHODS
5. IMMUNOASSAYS
General Applicable Methods of
Carbohydrate Analysis
1.A ) CHROMATOGRAPHICMETHODS
•Most powerful analyticaltechniques
Analysis of Type and Concentration of Mono
andOligo
•Commonly used to separate and identify
carbhydrates are
1.THIN LAYER CHROMATOGRAPHY ( TLC)
2.GAS CHROMATOGRAPHY, (GC)
3.HPLC
•Carbohydrates are separated basedon
their differential absorption
characteristics
•Most powerful analyticaltechniques
Analysis of Type and Concentration of Mono
andOligo
•Commonly used to separate and identify
carbhydrates are
1.THIN LAYER CHROMATOGRAPHY ( TLC)
2.GAS CHROMATOGRAPHY, (GC)
3.HPLC
•Carbohydrates are separated basedon
their differential absorption
characteristics
1.B) ELECTROPHORETICMETHODS
•Carbohydrates are separated by
electrophoresisafterbeingderivatized–
(makethemelectricallycharged)
•Solutionofderivatizedcarbohydratesis
appliedtoagelandavoltageisapplied
acrossthemedium.
•Thecarbohydrates arethenseparated
basedontheirsize
–THE SMALLER THE SIZE –FASTER IT MOVES
IN AN ELECTRICAL FIELD
•Carbohydrates are separated by
electrophoresisafterbeingderivatized–
(makethemelectricallycharged)
•Solutionofderivatizedcarbohydratesis
appliedtoagelandavoltageisapplied
acrossthemedium.
•Thecarbohydrates arethenseparated
basedontheirsize
–THE SMALLER THE SIZE –FASTER IT MOVES
IN AN ELECTRICAL FIELD
2.) CHEMICALMETHODS
•These methods are used to determine
Mono & Oligosaccharidesbecause most of
themare reducingsugars.
•Concentration of carbohydrates can be
determinedby:
1.GRAVIMETRIC METHODS
2.SPECTROPHOTOMETRY & COLORIMETRY
3.TITRATION METHODS
4.ACID HYDROLYSIS
Note:
•Non-reducing carb can be determined but they
have to behydrolyzed.
•These methods are used to determine
Mono & Oligosaccharidesbecause most of
themare reducingsugars.
•Concentration of carbohydrates can be
determinedby:
1.GRAVIMETRIC METHODS
2.SPECTROPHOTOMETRY & COLORIMETRY
3.TITRATION METHODS
4.ACID HYDROLYSIS
Note:
•Non-reducing carb can be determined but they
have to behydrolyzed.
3.)ENZYMATICMETHODS
•They base on the ability of the enzymes to catalyze
specificreactions. These methods are rapid, highly
specific, sensitive to even lowconcentrations.
•Little sample preparation is required
–Liquid Foods –can be testeddirectly
–Solid Foods –dissolved inwater
•Two commonly usedmethods:
1.To Allow the reaction to complete and measure the
concentration of theproduct
(Concentration of the product –Concentration of the Initial
Substrate.)
2.To Measure the initial rate of enzymereaction.
( ReactionRate -Substrate Concentration)
•They base on the ability of the enzymes to catalyze
specificreactions. These methods are rapid, highly
specific, sensitive to even lowconcentrations.
•Little sample preparation is required
–Liquid Foods –can be testeddirectly
–Solid Foods –dissolved inwater
•Two commonly usedmethods:
1.To Allow the reaction to complete and measure the
concentration of theproduct
(Concentration of the product –Concentration of the Initial
Substrate.)
2.To Measure the initial rate of enzymereaction.
( ReactionRate -Substrate Concentration)
4.) PHYSICALMETHODS
•These methods rely on change in physicochemical
characteristics asitscarbohydrate concentration
varies.
•Commonly UsedTechniques/Parameters are:
1.POLARIMETRY
2.REFRACTOMETRY (Refractive Index)
3.INFRARED RADIATION SPECTROSCOPY
4.DENSITY MEASUREMENT
•These methods rely on change in physicochemical
characteristics asitscarbohydrate concentration
varies.
•Commonly UsedTechniques/Parameters are:
1.POLARIMETRY
2.REFRACTOMETRY (Refractive Index)
3.INFRARED RADIATION SPECTROSCOPY
4.DENSITY MEASUREMENT
5.) IMMUNO-ASSAYS
Animmunoassay isabiochemical testthat
measures thepresence orconcentration of
amacromolecule orasmallmoleculeina
solutionthroughtheuseofanantibodyor
anantigen.
Themoleculedetectedbytheimmunoassay is
oftenreferredtoasan"ANALYTE" andisin
manycasesaproteinoracarbohydrate of
differentsizeandtypes,aslongastheproper
antibodiesthathavetheadequateproperties
fortheassayaredeveloped.
Analytes in biological liquids such
asserumorurinearefrequentlymeasured
usingimmunoassays formedicalandresearch
purposes.
Animmunoassay isabiochemical testthat
measures thepresence orconcentration of
amacromolecule orasmallmoleculeina
solutionthroughtheuseofanantibodyor
anantigen.
Themoleculedetectedbytheimmunoassay is
oftenreferredtoasan"ANALYTE" andisin
manycasesaproteinoracarbohydrate of
differentsizeandtypes,aslongastheproper
antibodiesthathavetheadequateproperties
fortheassayaredeveloped.
Analytes in biological liquids such
asserumorurinearefrequentlymeasured
usingimmunoassays formedicalandresearch
purposes.
5.) IMMUNO-ASSAYS cont’d
•Specific for low molecular weightcarbohydrates.
•Developedby:
1.Attaching the carbohydrate to aprotein
2.Injecting it into ananimal
3.Animal develops antibodies specific for the
carbohydratemolecule
4.Antibodies are extracted and used for
determining the concentration of the
carbohydrate.
•Immunoassays are extremely sensitive,
specific, easy to use, andrapid.
•Specific for low molecular weightcarbohydrates.
•Developedby:
1.Attaching the carbohydrate to aprotein
2.Injecting it into ananimal
3.Animal develops antibodies specific for the
carbohydratemolecule
4.Antibodies are extracted and used for
determining the concentration of the
carbohydrate.
•Immunoassays are extremely sensitive,
specific, easy to use, andrapid.
Independent of what ever analytical method
that is followed, The food carbohydrates are
analyzed under 4 categories.
The 4 categories are:
I) Total sugar Analysis
II) Mono and Disaccharide Analysis
III) Oligo and Polysaccharide Analysis
IV) Dietary fiber Analysis
that is followed, The food carbohydrates are
analyzed under 4 categories.
The 4 categories are:
I) Total sugar Analysis
II) Mono and Disaccharide Analysis
III) Oligo and Polysaccharide Analysis
IV) Dietary fiber Analysis
a.V
CHROMATOGRAPHIC
AND
ELECTROPHORETIC
METHODS
CHEMICAL
METHODS
ENZYMATIC
METHODS
PHYSICAL
METHODS
IMMUNO
ASSAYS
Oligosaccharide
analysis
(Size Exclusion
Chromatography)
Total Sugar
Analysis (Phenol
Sulphuricassay)
Oligosaccharid
e Analysis
Oligosacchar
ide Analysis
Glucose in
Blood
(Immuno
Assay)
Oligosaccharide
analysis
(High performance
Anion Exchange
Chromatography)
Total Sugars
(Anthrone
SulphuricAssay)
Glucose,
Fructose,
Galactose
Dietary Fibre
(Gravimetry)
Monosaccharides
(HPLC)
Total Sugars
(UronicAcid
Assay)
Sucrose,
Lactose,
Galacto
mannans,
Glucans
Disaccharides (Gas
Chomatography )
Mono &
Disaccharides
( Acid
Hydrolysis)
Dietary fibre
analysis
Acidic Sugars & Neutral
Sugars
(Gas Liqd
chromagraphy )
ANALYSIS OF CARBOHYDRATES BASED ON DIFFERENT METHODS
CHROMATOGRAPHIC
AND
ELECTROPHORETIC
METHODS
CHEMICAL
METHODS
ENZYMATIC
METHODS
PHYSICAL
METHODS
IMMUNO
ASSAYS
Oligosaccharide
analysis
(Size Exclusion
Chromatography)
Total Sugar
Analysis (Phenol
Sulphuricassay)
Oligosaccharid
e Analysis
Oligosacchar
ide Analysis
Glucose in
Blood
(Immuno
Assay)
Oligosaccharide
analysis
(High performance
Anion Exchange
Chromatography)
Total Sugars
(Anthrone
SulphuricAssay)
Glucose,
Fructose,
Galactose
Dietary Fibre
(Gravimetry)
Monosaccharides
(HPLC)
Total Sugars
(UronicAcid
Assay)
Sucrose,
Lactose,
Galacto
mannans,
Glucans
Disaccharides (Gas
Chomatography )
Mono &
Disaccharides
( Acid
Hydrolysis)
Dietary fibre
analysis
Acidic Sugars & Neutral
Sugars
(Gas Liqd
chromagraphy )
ANALYSIS OF CARBOHYDRATES BASED ON DIFFERENT METHODS
ANALYTICAL METHODS BASED ON TYPE OF CARBOHYDRATES
Total
Sugar
Analysis
Mono &
Disaccharide
Analysis
Oligosaccharid
e Analysis
Dietary
Fiber
Analysis
Phenol
Sulphuric
Acid Assay
Acid Hydrolysis Size Exclusion
Chromatography
(SEC)
Uppsala
Method
Anthrone
Sulphuric
Acid Assay
Gas Chromatography High
Performance
Anion Exchange
Chromatography
(HPAEC)
Enzymatic/
Gravimetric
Methods
Uronic Acid
Assay
HPLC Enzymatic
Analysis
_____
EnzymaticAnalysis:
1)Glucose
2)Galactose and
Lactose
3)Fructose, Glucose,
and Sucrose
4)Glucans
5)Galactomannans
_____ _____
Total
Sugar
Analysis
Mono &
Disaccharide
Analysis
Oligosaccharid
e Analysis
Dietary
Fiber
Analysis
Phenol
Sulphuric
Acid Assay
Acid Hydrolysis Size Exclusion
Chromatography
(SEC)
Uppsala
Method
Anthrone
Sulphuric
Acid Assay
Gas Chromatography High
Performance
Anion Exchange
Chromatography
(HPAEC)
Enzymatic/
Gravimetric
Methods
Uronic Acid
Assay
HPLC Enzymatic
Analysis
_____
EnzymaticAnalysis:
1)Glucose
2)Galactose and
Lactose
3)Fructose, Glucose,
and Sucrose
4)Glucans
5)Galactomannans
_____ _____
Monosaccharide andOligosaccharides
•Amount of preparation depends on the nature of
thefood
•Aqueous solutions require littlepreparation.
But
PHYSICALLY ASSOCIATED orCHEMICALLY BOUNDneed to
beisolated.
Method of Isolation depends on :
1.Carbohydratetype
2.Food MatrixType
3.Purpose of theAnalysis
Prior to Analysis SamplePreparationis Needed
•Amount of preparation depends on the nature of
thefood
•Aqueous solutions require littlepreparation.
But
PHYSICALLY ASSOCIATED orCHEMICALLY BOUNDneed to
beisolated.
Method of Isolation depends on :
1.Carbohydratetype
2.Food MatrixType
3.Purpose of theAnalysis
Prior to Analysis SamplePreparationis Needed
General methods for preparation of
Carbohydrate Sample for analysis
EXTRACTION
a)Liquid-Liquid Extraction
b) Super Critical fluid extraction
c) Pressurized Extraction
d) Field Flow Extraction
FILTERATION
a) Membrane Filtration
CHEMICAL TREATMENT
a) Hydrolysis
b) derivatization
Carbohydrate Sample for analysis
EXTRACTION
a)Liquid-Liquid Extraction
b) Super Critical fluid extraction
c) Pressurized Extraction
d) Field Flow Extraction
FILTERATION
a) Membrane Filtration
CHEMICAL TREATMENT
a) Hydrolysis
b) derivatization
Process ofSamplePreparation
Dried
(undervacuum)
Ground to a
finepowder
Defatted
(by solventhexane or
chloroformextraction)
TO AVOID THERMAL
DEGRADATION
TO ENHANCE SOLVENT
EXTRACTION
Dried
(undervacuum)
Ground to a
finepowder
Defatted
(by solventhexane or
chloroformextraction)
TO AVOID THERMAL
DEGRADATION
TO ENHANCE SOLVENT
EXTRACTION
Sample Preparation
•Beforeanalyzingforanyclassofcarbohydrate,
whetheritismonosaccharideorinsolublecellulosic
material(Polysaccharide)thesamplemustbe
preparedsoastoremoveunwantedsubstancesthat
caninterferewithanalysis.
•Forsamplesthatareessentiallysugarsolutions(juice,
honey)verylittlesamplepreparationisrequired.
•Forothersamples,suchasoilseeds,cerealsor
wholefoods:
»fats
»proteins
»pigments
»vitamins
»minerals
»variousothercompounds
shouldberemovedpriortoanalysis.
•Beforeanalyzingforanyclassofcarbohydrate,
whetheritismonosaccharideorinsolublecellulosic
material(Polysaccharide)thesamplemustbe
preparedsoastoremoveunwantedsubstancesthat
caninterferewithanalysis.
•Forsamplesthatareessentiallysugarsolutions(juice,
honey)verylittlesamplepreparationisrequired.
•Forothersamples,suchasoilseeds,cerealsor
wholefoods:
»fats
»proteins
»pigments
»vitamins
»minerals
»variousothercompounds
shouldberemovedpriortoanalysis.
Sample Preparation (cont’d)
•Thereareseveraldetailedproceduresforthe
removalofthesesubstancespriortothe
analysisofsimplesugarsorpolysaccharides.
•Itshouldbenotedthattheextentofsample
preparationrequiredisalsodependentonthe
analyticaltechniqueand/orequipmentbeing
used.
•Generally,samplesaredriedandgroundfirst,
followedbyadefattingstep.
•Dryingcanbedoneundervacuum,at
atmosphericpressure,orforsamplesthatare
sensitivetoheat,inafreezedryer.
•Thereareseveraldetailedproceduresforthe
removalofthesesubstancespriortothe
analysisofsimplesugarsorpolysaccharides.
•Itshouldbenotedthattheextentofsample
preparationrequiredisalsodependentonthe
analyticaltechniqueand/orequipmentbeing
used.
•Generally,samplesaredriedandgroundfirst,
followedbyadefattingstep.
•Dryingcanbedoneundervacuum,at
atmosphericpressure,orforsamplesthatare
sensitivetoheat,inafreezedryer.
Sample Preparation cont’d
Defatting of Carbohydrates
Samples, once ground to a specified mesh size, are
defatted using a nonpolar solvent such as hexane or
chloroform. Low molecular weight carbohydrates can
then be extracted using hot 80% ethanol.
The ethanol extract will contain :
mineral salts, pigments, and organic acids as well as low
molecular weight sugars and proteins.
The residue will contain:
proteins and high molecular weight CARBOHYDRATES
including cellulose, pectin, starch, and any food gums
(hydrocolloids) that may be present.
HENCE RESIDUE SHOULD BE COLLECTED
FOR FURTHER REFINEMENT.
Defatting of Carbohydrates
Samples, once ground to a specified mesh size, are
defatted using a nonpolar solvent such as hexane or
chloroform. Low molecular weight carbohydrates can
then be extracted using hot 80% ethanol.
The ethanol extract will contain :
mineral salts, pigments, and organic acids as well as low
molecular weight sugars and proteins.
The residue will contain:
proteins and high molecular weight CARBOHYDRATES
including cellulose, pectin, starch, and any food gums
(hydrocolloids) that may be present.
HENCE RESIDUE SHOULD BE COLLECTED
FOR FURTHER REFINEMENT.
Sample Preparation cont’d
•Removal of Proteins
•Proteins are removed from samples using a protease enzyme
papain.
•Removal of Water soluble and insoluble Polysaccharides
•Water soluble polysaccharides can be extracted using water and
separated from insoluble material by centrifugation or filtration.
•Depending on the compound of interest in the sample, an
enzymatic treatment with α-amylase and/or amyloglucosidase
can be used to get rid of starch.
•In this case starch is hydrolyzed to glucose, which can be
separated from high molecular weight polysaccharides by
dialysis or by collecting the high molecular weight material as a
precipitate after making the solution to 80% ethanol.
•Glucose is soluble in 80% ethanol while polysaccharide material
is not.
•Removal of Proteins
•Proteins are removed from samples using a protease enzyme
papain.
•Removal of Water soluble and insoluble Polysaccharides
•Water soluble polysaccharides can be extracted using water and
separated from insoluble material by centrifugation or filtration.
•Depending on the compound of interest in the sample, an
enzymatic treatment with α-amylase and/or amyloglucosidase
can be used to get rid of starch.
•In this case starch is hydrolyzed to glucose, which can be
separated from high molecular weight polysaccharides by
dialysis or by collecting the high molecular weight material as a
precipitate after making the solution to 80% ethanol.
•Glucose is soluble in 80% ethanol while polysaccharide material
is not.
Independent of what ever analytical method
that is followed, The food carbohydrates are
analyzed under 4 categories.
The 4 categories are:
I) Total sugar Analysis
II) Mono and Disaccharide Analysis
III) Oligo and Polysaccharide Analysis
IV) Dietary fiber Analysis
that is followed, The food carbohydrates are
analyzed under 4 categories.
The 4 categories are:
I) Total sugar Analysis
II) Mono and Disaccharide Analysis
III) Oligo and Polysaccharide Analysis
IV) Dietary fiber Analysis
I) Total Sugar Analysis
Total sugar analysis is carried out utilizing Chemical
and Spectrophotometric Methods
1)Phenol–Sulfuric Acid Assay
2)Anthrone Sulphuric Acid Assay
3)Uronic Acid Assay
Total sugar analysis is carried out utilizing Chemical
and Spectrophotometric Methods
1)Phenol–Sulfuric Acid Assay
2)Anthrone Sulphuric Acid Assay
3)Uronic Acid Assay
1) Phenol–Sulfuric Acid Assay
Theory
Inthepresence ofstrongacidsandheat,
carbohydrates undergoaseriesofreactionsthatleads
totheformation offuranderivatives suchas
furanaldehydeandhydroxymethyl furaldehyde.
Adehydrationreactionisfollowedbytheformationof
furanderivatives,whichthencondensewiththemselves
orwithphenoliccompounds toproducedarkcolored
complexes,displayingsomefuranderivativesandthe
carbohydratesfromwhichtheyoriginate.
ThedevelopedcomplexabsorbsUV-Visiblelight,andthe
absorbanceisproportionaltothesugarconcentrationin
alinearfashion.
Anabsorbance maximum isobserved at490nmfor
hexosesand480nmforpentosesanduronicacidsas
measured by a UV-Visible spectrophotometer .
Theory
Inthepresence ofstrongacidsandheat,
carbohydrates undergoaseriesofreactionsthatleads
totheformation offuranderivatives suchas
furanaldehydeandhydroxymethyl furaldehyde.
Adehydrationreactionisfollowedbytheformationof
furanderivatives,whichthencondensewiththemselves
orwithphenoliccompounds toproducedarkcolored
complexes,displayingsomefuranderivativesandthe
carbohydratesfromwhichtheyoriginate.
ThedevelopedcomplexabsorbsUV-Visiblelight,andthe
absorbanceisproportionaltothesugarconcentrationin
alinearfashion.
Anabsorbance maximum isobserved at490nmfor
hexosesand480nmforpentosesanduronicacidsas
measured by a UV-Visible spectrophotometer .
.
1) Phenol–Sulfuric Acid Assay cont’d
Procedure
•80% Phenol solution is added to a glass test tube
containing a clear sample solution.
•Concentrated sulfuric acid is added in a rapid stream
directly to the surface of the liquid in the test tube.
•The mixture is thoroughly combined using a VORTEX
MIXERand then permitted to stand a sufficient time
to allow for color development.
•The solution absorbance is read at 480 -490nm
using a spectrophotometer, depending on the type of
sugar present.
•Mixing and standing time should be kept the same for
all samples to assure reproducible results.
1) Phenol–Sulfuric Acid Assay cont’d
Procedure
•80% Phenol solution is added to a glass test tube
containing a clear sample solution.
•Concentrated sulfuric acid is added in a rapid stream
directly to the surface of the liquid in the test tube.
•The mixture is thoroughly combined using a VORTEX
MIXERand then permitted to stand a sufficient time
to allow for color development.
•The solution absorbance is read at 480 -490nm
using a spectrophotometer, depending on the type of
sugar present.
•Mixing and standing time should be kept the same for
all samples to assure reproducible results.
.
1) Phenol–Sulfuric Acid Assay cont’d
Quantification
•Acalibrationcurveisconstructedusingthesugarbeing
assayed.
•Astock1mg/mlaqueoussugarstandardsolutionis
usedtoprepare5or6standardsrangingfrom10to100
µg/ml.
•Eachstandardissubjectedtothereactionprocedure
outlinedabove,transferredtoacuvette,andits
absorbancereadat480-490nm.
•Theabsorbanceoftheblankshouldbesubtractedfrom
theabsorbanceofthestandardsmanually,ortheblank
shouldbeusedtozerothespectrophotometer.
•Agraphofabsorbancevs.concentrationisconstructed
andtheamountofanalyteisderivedfromthe
calibrationcurve.
1) Phenol–Sulfuric Acid Assay cont’d
Quantification
•Acalibrationcurveisconstructedusingthesugarbeing
assayed.
•Astock1mg/mlaqueoussugarstandardsolutionis
usedtoprepare5or6standardsrangingfrom10to100
µg/ml.
•Eachstandardissubjectedtothereactionprocedure
outlinedabove,transferredtoacuvette,andits
absorbancereadat480-490nm.
•Theabsorbanceoftheblankshouldbesubtractedfrom
theabsorbanceofthestandardsmanually,ortheblank
shouldbeusedtozerothespectrophotometer.
•Agraphofabsorbancevs.concentrationisconstructed
andtheamountofanalyteisderivedfromthe
calibrationcurve.
1) Phenol–Sulfuric Acid Assay cont’d
Applications
•Thephenol-sulfuricmethodiswidelyusedtodetermine
thetotalconcentrationofcarbohydratesinasample.
•Itcanbeusedonlipid-freeextractsfromcereals,seeds,
andplants,providedthesampleisinsolution,andis
appropriateforbothreducingandnon-reducingsugars.
•Itisadvantageousinthatthereagentsarelowcostand
readilyavailable,therequiredequipmentisminimal,
andtheassayissimple.
•Additionally,itcanbeusedtoquantify
monosaccharides, oligosaccharides, and
polysaccharides.
Applications
•Thephenol-sulfuricmethodiswidelyusedtodetermine
thetotalconcentrationofcarbohydratesinasample.
•Itcanbeusedonlipid-freeextractsfromcereals,seeds,
andplants,providedthesampleisinsolution,andis
appropriateforbothreducingandnon-reducingsugars.
•Itisadvantageousinthatthereagentsarelowcostand
readilyavailable,therequiredequipmentisminimal,
andtheassayissimple.
•Additionally,itcanbeusedtoquantify
monosaccharides, oligosaccharides, and
polysaccharides.
1) Phenol–Sulfuric Acid Assay cont’d
Applications
•Absorptioncurvesarecharacteristicfordifferentsugars;
therefore,thismethodprovidesthemostaccurate
resultswhenappliedtosamplescontainingonlyone
typeofcarbohydrate.
•Thisassayhasbeenusedtoquantifytotalsugarsina
samplecontainingmorethanonetypeofcarbohydrate.
•Inthiscase,glucoseisoftenusedtoconstructthe
calibrationcurveandtheresultsarethenapproximate
onlyandshouldbestatedasglucoseequivalents.
Applications
•Absorptioncurvesarecharacteristicfordifferentsugars;
therefore,thismethodprovidesthemostaccurate
resultswhenappliedtosamplescontainingonlyone
typeofcarbohydrate.
•Thisassayhasbeenusedtoquantifytotalsugarsina
samplecontainingmorethanonetypeofcarbohydrate.
•Inthiscase,glucoseisoftenusedtoconstructthe
calibrationcurveandtheresultsarethenapproximate
onlyandshouldbestatedasglucoseequivalents.
Vortex Mixer
2) AnthroneSuphuricAcidMethod
Theanthronereactionisthebasisofarapidand
convenientmethodforthedeterminationof
carbohydrates,eitherfreeorpresentin
polysaccharides.
Theory
Similartothephenol–sulfuricacidassay,theanthrone
methodisbasedonthecondensationoffuranldehyde
derivatives,generatedbycarbohydratesinthe
presenceofastrongacid,withareagent,inthiscase
anthrone(9,10dihydro-9-ozoanthracene)toproduce
coloredcompounds.
Thereactionofcarbohydratesinastronglyacidic
environmentwithanthroneresultsinablue-green
colorandtheabsorbanceisreadat620nm.
Theanthronereactionisthebasisofarapidand
convenientmethodforthedeterminationof
carbohydrates,eitherfreeorpresentin
polysaccharides.
Theory
Similartothephenol–sulfuricacidassay,theanthrone
methodisbasedonthecondensationoffuranldehyde
derivatives,generatedbycarbohydratesinthe
presenceofastrongacid,withareagent,inthiscase
anthrone(9,10dihydro-9-ozoanthracene)toproduce
coloredcompounds.
Thereactionofcarbohydratesinastronglyacidic
environmentwithanthroneresultsinablue-green
colorandtheabsorbanceisreadat620nm.
.
2.Anthronone SuphuricAcid Method cont’d
•Operating Procedure
•A cooled mixture of 2% anthrone in concentrated sulfuric
acid is mixed with an aliquot of a clear sample solution
containing the sugar being assayed.
•After incubation in a temperature-controlled environment
for sufficient time to allow color development, the solution
is poured into an appropriate spectro photometric cuvette
and the absorbance measured at 620nm.
•
•Quantification
•Similar to the phenol–sulfuric acid assay, the anthrone
reaction is non-stoichiometricand therefore requires the
construction of a standard curve for quantitative purposes.
•
2.Anthronone SuphuricAcid Method cont’d
•Operating Procedure
•A cooled mixture of 2% anthrone in concentrated sulfuric
acid is mixed with an aliquot of a clear sample solution
containing the sugar being assayed.
•After incubation in a temperature-controlled environment
for sufficient time to allow color development, the solution
is poured into an appropriate spectro photometric cuvette
and the absorbance measured at 620nm.
•
•Quantification
•Similar to the phenol–sulfuric acid assay, the anthrone
reaction is non-stoichiometricand therefore requires the
construction of a standard curve for quantitative purposes.
•
.
2.Anthronone SuphuricAcid Method cont’d
•Applications
•The anthrone–sulfuric method is most applicable to solutions
containing one type of hexose because even sugars with similar
structures result in different rates and quantities of color
development.
•Other sugars, such as pentoses and hexuronic acids, will also
react to produce colored compounds that absorb at the same
wavelength, but this only becomes a problem if they are
present in a solution above a certain level.
•This assay can also be used for quantitative analysis of oligoand
polysaccharides provided only one type is present in solution.
•
•The anthrone method has been modified for use with a micro-
plate, thus permitting the analysis of many samples within a
short period of time and reducing the quantity of reagent
needed.
2.Anthronone SuphuricAcid Method cont’d
•Applications
•The anthrone–sulfuric method is most applicable to solutions
containing one type of hexose because even sugars with similar
structures result in different rates and quantities of color
development.
•Other sugars, such as pentoses and hexuronic acids, will also
react to produce colored compounds that absorb at the same
wavelength, but this only becomes a problem if they are
present in a solution above a certain level.
•This assay can also be used for quantitative analysis of oligoand
polysaccharides provided only one type is present in solution.
•
•The anthrone method has been modified for use with a micro-
plate, thus permitting the analysis of many samples within a
short period of time and reducing the quantity of reagent
needed.
*Principle
H2SO4
+
Furfura
l
Anthrone
Blue
Green
Complex
H2SO4
+
Furfura
l
Anthrone
Blue
Green
Complex
Materials
1.Anthronereagent (0.2% in conc. H2SO4).
2.Glucose(10mg/100ml).
3.Colorimeter orspectrophotometer.
1.Anthronereagent (0.2% in conc. H2SO4).
2.Glucose(10mg/100ml).
3.Colorimeter orspectrophotometer.
3) Analysis of Uronic Acids (m-HydroxydiphenylMethod)
•Uronicacidsareaclassofacidsugarswithboth
carbonylandcarboxylicacidfunctionalgroups.
•Theyaresugarsinwhichtheterminalcarbon's
hydroxyl group hasbeen oxidized toa
carboxylicacid.
•Colorimetricmethods fordetermining uronic
acidsaresimilartothephenol–sulfuricacidand
theanthroneassays.
•Carbazole isanaromaticheterocyclicorganic
compound.Ithasatricyclicstructure,consisting
oftwosix-membered benzene ringsfusedon
eithersideofafive-membered nitrogen-
containingring.
•Uronicacidsareaclassofacidsugarswithboth
carbonylandcarboxylicacidfunctionalgroups.
•Theyaresugarsinwhichtheterminalcarbon's
hydroxyl group hasbeen oxidized toa
carboxylicacid.
•Colorimetricmethods fordetermining uronic
acidsaresimilartothephenol–sulfuricacidand
theanthroneassays.
•Carbazole isanaromaticheterocyclicorganic
compound.Ithasatricyclicstructure,consisting
oftwosix-membered benzene ringsfusedon
eithersideofafive-membered nitrogen-
containingring.
3) Analysis of Uronic Acids (m-HydroxydiphenylMethod) cont’d
•Two types of Tests are present:
1) Carbazole Assay
2)Hydroxy diphenylAssay
•The carbazole assay (reported by Dishe) involves :
mixing a sample containing uronic acid
with concentrated sulfuric acid,
heating it at 100°C,
cooling it and then reacting it with 0.1% carbazole in ethanol.
•Aftersufficienttimeforcolordevelopment,absorbanceisreadat
520-535nm.Modificationstothisassayincludealterationstothe
timingofstepsandreagentconcentrations.
•Thecarbazoleassay,whilesimple,rapid,andsensitivesuffers
interferencesfromhexosesandpentoses.Thereplacementofcarbazole
withm-hydroxydiphenylincreasesthespecificityandsensitivityofthe
assay.
•Two types of Tests are present:
1) Carbazole Assay
2)Hydroxy diphenylAssay
•The carbazole assay (reported by Dishe) involves :
mixing a sample containing uronic acid
with concentrated sulfuric acid,
heating it at 100°C,
cooling it and then reacting it with 0.1% carbazole in ethanol.
•Aftersufficienttimeforcolordevelopment,absorbanceisreadat
520-535nm.Modificationstothisassayincludealterationstothe
timingofstepsandreagentconcentrations.
•Thecarbazoleassay,whilesimple,rapid,andsensitivesuffers
interferencesfromhexosesandpentoses.Thereplacementofcarbazole
withm-hydroxydiphenylincreasesthespecificityandsensitivityofthe
assay.
.
3.Analysis of UronicAcids Cont’d
Theory
•Whileallcarbohydrates reactinconcentrated acidtoform
coloredcompounds, uronicacidsreactwithcarbazoleor
•m-hydroxydiphenyl inastronglyacidicenvironment toform
PINKCOLORED complexes.Absorbancemeasurements are
readat520nm-535nmincreaselinearlywithuronicacid
concentrationfrom0to100µg/ml.
Procedure
•Asamplesolutionisthoroughly mixedwithsulfuricacid
containingtetraborateinatesttubeandplacedinaboiling
waterbathfor5minutes.Afterrapidcoolinginanicewater
bath,m-hydroxydiphenyl isaddedtoeachsampletesttubeand
vortexedtoensureadequatemixing.Theabsorbance foreach
sampleisreadafterallowingthecolortodevelopfor20
minutes.Asampleblank(containingasamplesolvent)should
bepreparedatthesametimeasthesamples.
3.Analysis of UronicAcids Cont’d
Theory
•Whileallcarbohydrates reactinconcentrated acidtoform
coloredcompounds, uronicacidsreactwithcarbazoleor
•m-hydroxydiphenyl inastronglyacidicenvironment toform
PINKCOLORED complexes.Absorbancemeasurements are
readat520nm-535nmincreaselinearlywithuronicacid
concentrationfrom0to100µg/ml.
Procedure
•Asamplesolutionisthoroughly mixedwithsulfuricacid
containingtetraborateinatesttubeandplacedinaboiling
waterbathfor5minutes.Afterrapidcoolinginanicewater
bath,m-hydroxydiphenyl isaddedtoeachsampletesttubeand
vortexedtoensureadequatemixing.Theabsorbance foreach
sampleisreadafterallowingthecolortodevelopfor20
minutes.Asampleblank(containingasamplesolvent)should
bepreparedatthesametimeasthesamples.
.
3.Analysis of Uronic Acids Cont’d
Quantification
•Asolutionofanappropriateuronicacidstandard(i.e.,
galacturonicacid)isusedtoprepareseveraldilutions
andtheseareusedtoprepareastandardcurve.
•Agraphofconcentrationvs.absorbanceisconstructed
andthesampleabsorbancereadingplottedalongthe
curvetoobtainconcentrationvalues.
•Standardcurvestypicallyrangefrom10to100µg/ml.
Applicability
•Them-hydroxydiphenylassaycantoleratethe
presenceofnon-uronicacidsugars,upto~200µg/ml
hasbeenreportedbuthigherconcentrationsofneutral
sugarsmayartificiallyincreaseabsorbancereadings.
•Additionally,thepresenceofproteininasamplemay
interferewiththeabsorbances.Thismethod is
appropriateforthequantificationofpecticmaterialin
fruitsandvegetablesandhasbeenadaptedforuse
withamicro-plate.
3.Analysis of Uronic Acids Cont’d
Quantification
•Asolutionofanappropriateuronicacidstandard(i.e.,
galacturonicacid)isusedtoprepareseveraldilutions
andtheseareusedtoprepareastandardcurve.
•Agraphofconcentrationvs.absorbanceisconstructed
andthesampleabsorbancereadingplottedalongthe
curvetoobtainconcentrationvalues.
•Standardcurvestypicallyrangefrom10to100µg/ml.
Applicability
•Them-hydroxydiphenylassaycantoleratethe
presenceofnon-uronicacidsugars,upto~200µg/ml
hasbeenreportedbuthigherconcentrationsofneutral
sugarsmayartificiallyincreaseabsorbancereadings.
•Additionally,thepresenceofproteininasamplemay
interferewiththeabsorbances.Thismethod is
appropriateforthequantificationofpecticmaterialin
fruitsandvegetablesandhasbeenadaptedforuse
withamicro-plate.
II) Mono & Disaccharide Analysis
1) ACID HYDROLYSIS
2) GAS CHROMATOGRAPHY
3) H P L C
4) HIGH PERFORMANCE ANION EXCHANGE
CHROMATOGRAPHY (HPAEC)
5) ENZYMATIC ANALYSIS
.
1) ACID HYDROLYSIS
2) GAS CHROMATOGRAPHY
3) H P L C
4) HIGH PERFORMANCE ANION EXCHANGE
CHROMATOGRAPHY (HPAEC)
5) ENZYMATIC ANALYSIS
.
1 Acid Hydrolysis
•Inthepresenceofastrongacidandheat,the
glycosidic bond between monosaccharide
residuesinapolysaccharideiscleaved.
•Duringthisreaction,onemoleculeofwateris
consumed foreveryglycosidiclinkagecleaved.
•During acid hydrolysis, released
monosaccharides aresusceptibletodegradation
inthepresenceofhotconcentratedacid.
•However,notallglycosidiclinkagesarecleaved
atthesamerateandthehydrolysistimemust
besufficienttohydrolyzealllinkagesinthe
sample.
•Thesetwoneedsmustbebalanced,theneed
forhydrolysisofsufficientstrengthandlength
topermitcompletehydrolysis,butnotsolong
soastoleadtosampledegradation.
•Inthepresenceofastrongacidandheat,the
glycosidic bond between monosaccharide
residuesinapolysaccharideiscleaved.
•Duringthisreaction,onemoleculeofwateris
consumed foreveryglycosidiclinkagecleaved.
•During acid hydrolysis, released
monosaccharides aresusceptibletodegradation
inthepresenceofhotconcentratedacid.
•However,notallglycosidiclinkagesarecleaved
atthesamerateandthehydrolysistimemust
besufficienttohydrolyzealllinkagesinthe
sample.
•Thesetwoneedsmustbebalanced,theneed
forhydrolysisofsufficientstrengthandlength
topermitcompletehydrolysis,butnotsolong
soastoleadtosampledegradation.
1 Acid Hydrolysis cont’d
•
•Sulfuricacidandtrifluoraceticacid(TFA)
arecommonly usedforhydrolysis.
•Ithasbeenreportedthatsulfuricacidis
superiortoTFAforthehydrolysisof
fibroussubstratessuchaswheatbran,
straw, apples, and microcrystalline
cellulose.
•However,sulfuricacidcanbedifficultto
removepost-hydrolysisanditspresence
caninterferewithsomeanalyses.ASTFA
ISVOLATILE ANDCANBEEASILY REMOVED
PRIORTOANHPLCANALYSIS.
•
•Sulfuricacidandtrifluoraceticacid(TFA)
arecommonly usedforhydrolysis.
•Ithasbeenreportedthatsulfuricacidis
superiortoTFAforthehydrolysisof
fibroussubstratessuchaswheatbran,
straw, apples, and microcrystalline
cellulose.
•However,sulfuricacidcanbedifficultto
removepost-hydrolysisanditspresence
caninterferewithsomeanalyses.ASTFA
ISVOLATILE ANDCANBEEASILY REMOVED
PRIORTOANHPLCANALYSIS.
1 Acid Hydrolysis (cont’d)
•HYDROLYSIS FORDIETARYFIBER
•Ahydrolysisprocedureusingsulfuricacidappropriatefor
watersolubledietaryfibermaterialinfoodshasbeen
outlined.Itrequiresmixingthesamplematerialwith1M
sulfuricacidandheatingat100°Cfor2.5hours.
•HYDROLYSIS FORNEUTRAL POLYSACCHARIDES
•Another recommended procedure for neutral
polysaccharides involvesmixing2to5mgofaccurately
weigheddrysamplewith0.1to0.25mlof2MHCLand
heatingat100°Cfor2to5hours.
•Degradation of released sugars will be evident as a
decrease in monosaccharide concentration as hydrolysis
time increases.
•HYDROLYSIS FORACIDICSUGARS
•Samplescontainingacidicsugarresiduessuchaspectins
andcertainfungalpolysaccharides canbedifficultto
hydrolyzequantitativelyusingtraditionalmethods that
employTFAorsulfuricacid.
•Incaseswhere quantitative hydrolysis cannot be
achieved,qualitativeinformationcanbeobtainedusing
anappropriate chromatographic technique post–acid
hydrolysis,andtheuronicacidcontentisdetermined
usingSPECTROPHOTOMETRIC METHOD
•HYDROLYSIS FORDIETARYFIBER
•Ahydrolysisprocedureusingsulfuricacidappropriatefor
watersolubledietaryfibermaterialinfoodshasbeen
outlined.Itrequiresmixingthesamplematerialwith1M
sulfuricacidandheatingat100°Cfor2.5hours.
•HYDROLYSIS FORNEUTRAL POLYSACCHARIDES
•Another recommended procedure for neutral
polysaccharides involvesmixing2to5mgofaccurately
weigheddrysamplewith0.1to0.25mlof2MHCLand
heatingat100°Cfor2to5hours.
•Degradation of released sugars will be evident as a
decrease in monosaccharide concentration as hydrolysis
time increases.
•HYDROLYSIS FORACIDICSUGARS
•Samplescontainingacidicsugarresiduessuchaspectins
andcertainfungalpolysaccharides canbedifficultto
hydrolyzequantitativelyusingtraditionalmethods that
employTFAorsulfuricacid.
•Incaseswhere quantitative hydrolysis cannot be
achieved,qualitativeinformationcanbeobtainedusing
anappropriate chromatographic technique post–acid
hydrolysis,andtheuronicacidcontentisdetermined
usingSPECTROPHOTOMETRIC METHOD
2Gas-Liquid Chromatography (GC)
•Gas-liquidchromatography isatechniquewherebycomponents
inamixtureareseparatedbasedontheirdegreeofaffinityfor
orinteractionwithaLIQUIDSTATIONARY PHASE.Inthecaseof
GC,thesamplecomponents aredissolvedinagasphaseand
movedthroughaverysmallborecolumn,theinteriorofwhich
iscoatedwiththestationaryphase.
•These separations occur athighpressure and high
temperatures.Components inthesamplemixturewithahigh
affinityforthestationaryphasewillstayinthecolumnlonger
andelutelaterthanthosewithlessaffinityforthestationary
phase.
•Thedegreeofaffinityfororinteractionwiththestationary
phasethatamolecule hasisgoverned byitsstructure,
properties,andthechemistryofthestationaryphasebeing
used.
•Gas-liquidchromatography isatechniquewherebycomponents
inamixtureareseparatedbasedontheirdegreeofaffinityfor
orinteractionwithaLIQUIDSTATIONARY PHASE.Inthecaseof
GC,thesamplecomponents aredissolvedinagasphaseand
movedthroughaverysmallborecolumn,theinteriorofwhich
iscoatedwiththestationaryphase.
•These separations occur athighpressure and high
temperatures.Components inthesamplemixturewithahigh
affinityforthestationaryphasewillstayinthecolumnlonger
andelutelaterthanthosewithlessaffinityforthestationary
phase.
•Thedegreeofaffinityfororinteractionwiththestationary
phasethatamolecule hasisgoverned byitsstructure,
properties,andthechemistryofthestationaryphasebeing
used.
2 Gas-Liquid Chromatography (GC) cont’d
Derivatization
•TheprerequisiteofaGCseparationinthesample
mustbevolatile.Giventhatmonosaccharides are
notvolatile,theymustbederivatizedpriorto
analysis.
•NEUTRAL MONOSACCHARIDES AND ACIDIC
MONSACCHARIDES arederivatized.
•Theessentialelements ofthisderivitization
procedurearethereductionofneutralsugarsto
alditols(acyclicSugarAlcoholsformallyderivedfrom
analdosebyreductionofthecarbonylfunctionalgroup)
andtheirsubsequentacetylation.
•Theresultingalditolacetatesarethendissolvedina
suitablesolventandinjectedontoaGCcolumn.
Derivatization
•TheprerequisiteofaGCseparationinthesample
mustbevolatile.Giventhatmonosaccharides are
notvolatile,theymustbederivatizedpriorto
analysis.
•NEUTRAL MONOSACCHARIDES AND ACIDIC
MONSACCHARIDES arederivatized.
•Theessentialelements ofthisderivitization
procedurearethereductionofneutralsugarsto
alditols(acyclicSugarAlcoholsformallyderivedfrom
analdosebyreductionofthecarbonylfunctionalgroup)
andtheirsubsequentacetylation.
•Theresultingalditolacetatesarethendissolvedina
suitablesolventandinjectedontoaGCcolumn.
2. Gas-Liquid Chromatography (Cont’d)
Neutral Sugars
•Thestartingmaterialmustbeadrysampleofoneormore
monosaccharides .Polysaccharide material must firstbe
HYDROLYZED and theacidremoved priortoanalysis.
Trifluoraceticacidworkswellforthispurposebecauseitis
volatileandcanbeeasilyremovedbyrotaryevaporation.
•Thedrysamplecontainingasmallamount(~10mg)ofaccurately
weighed monosacharide materialandinositolhexa-acetate
(internalstandard)ismixedwithasolutionofsodiumborohydride
inammonium hydroxidetoconvertmonosaccharides intoalditols.
•Aceticacidisaddedtoacidifythesampleanddestroyexcess
sodiumborohydrideafterthereactionhasreachedcompletion.
Themixtureisdried(rotaryevaporationorunderastreamof
nitrogen)andmethanolisaddedandremovedbydryingwitha
nitrogenstreamseveraltimes.Treatmentwithmethanolremoves
borateionsasvolatilemethylborate.
•Whenthefinalportionofmethanolhasbeenremovedandthesample
isdry,themixtureofalditolsisacetylatedbyaddingaceticanhydride
andheatingitat121°Cforafewhours.Afewdropsofwaterare
addedtothereactionvialtodestroyanyresidualaceticanhydrideand
theentiremixtureisbroughttodryness.Theresultingalditolacetates
aresolubilizedinmethylenechlorideinpreparationforGCanalysis.
Neutral Sugars
•Thestartingmaterialmustbeadrysampleofoneormore
monosaccharides .Polysaccharide material must firstbe
HYDROLYZED and theacidremoved priortoanalysis.
Trifluoraceticacidworkswellforthispurposebecauseitis
volatileandcanbeeasilyremovedbyrotaryevaporation.
•Thedrysamplecontainingasmallamount(~10mg)ofaccurately
weighed monosacharide materialandinositolhexa-acetate
(internalstandard)ismixedwithasolutionofsodiumborohydride
inammonium hydroxidetoconvertmonosaccharides intoalditols.
•Aceticacidisaddedtoacidifythesampleanddestroyexcess
sodiumborohydrideafterthereactionhasreachedcompletion.
Themixtureisdried(rotaryevaporationorunderastreamof
nitrogen)andmethanolisaddedandremovedbydryingwitha
nitrogenstreamseveraltimes.Treatmentwithmethanolremoves
borateionsasvolatilemethylborate.
•Whenthefinalportionofmethanolhasbeenremovedandthesample
isdry,themixtureofalditolsisacetylatedbyaddingaceticanhydride
andheatingitat121°Cforafewhours.Afewdropsofwaterare
addedtothereactionvialtodestroyanyresidualaceticanhydrideand
theentiremixtureisbroughttodryness.Theresultingalditolacetates
aresolubilizedinmethylenechlorideinpreparationforGCanalysis.
2 .Gas-Liquid Chromatography (Cont’d)
Acidic Sugars
• Aswithneutralpolysaccharides, polysaccharides
containinguronicacidsmustbehydrolyzedanddried
priortoanalysis.Itisverydifficulttoobtaina
quantitativeyieldofacidicmonosaccharides usingacid
hydrolysis,andwhenthisisrequired,itisadvisableto
useaspectrophotometric method.Asamplecontaininga
small amount (~10mg) ofaccurately weighed
carbohydrate materialisdissolvedinsodiumcarbonate
andthentreatedwithsodiumborohydride.
•Aceticacidisaddedtodestroyexcessborohydrideand
borateions,removed withmethanol asdescribedfor
neutralmonosaccharides .Theresultantmixtureofaldonic
acidsandaldoses(fromneutralsugarsifpresent)ismade
intoTMSderivatives(TRIMETHYLSILYL (TMS)ETHERSAREA
WAYTODERIVATIZEFUNCTIONAL GROUPS PRIORTOGC
ANALYSIS)bytreatingthedryresiduewithamixture
containing pyridine, hexamethyl-disilazane, and
trifluoraceticacid.Aninternalstandard, suchas
docosane,shouldbeusedforquantification.
•
Acidic Sugars
• Aswithneutralpolysaccharides, polysaccharides
containinguronicacidsmustbehydrolyzedanddried
priortoanalysis.Itisverydifficulttoobtaina
quantitativeyieldofacidicmonosaccharides usingacid
hydrolysis,andwhenthisisrequired,itisadvisableto
useaspectrophotometric method.Asamplecontaininga
small amount (~10mg) ofaccurately weighed
carbohydrate materialisdissolvedinsodiumcarbonate
andthentreatedwithsodiumborohydride.
•Aceticacidisaddedtodestroyexcessborohydrideand
borateions,removed withmethanol asdescribedfor
neutralmonosaccharides .Theresultantmixtureofaldonic
acidsandaldoses(fromneutralsugarsifpresent)ismade
intoTMSderivatives(TRIMETHYLSILYL (TMS)ETHERSAREA
WAYTODERIVATIZEFUNCTIONAL GROUPS PRIORTOGC
ANALYSIS)bytreatingthedryresiduewithamixture
containing pyridine, hexamethyl-disilazane, and
trifluoraceticacid.Aninternalstandard, suchas
docosane,shouldbeusedforquantification.
•
2 Gas-Liquid Chromatography (Cont’d)
Quantification
•Aflameionizationdetector(FID)isthemostcommonly used
detector.WithanFID,quantificationrequiresusinganinternal
standardandtheformulationofresponsefactors(RF).
•Response factorsareused tocorrectGCresponse to
monosaccharides andlossesarisingfrom hydrolysis and
derivatization.Theyareobtainedforeachmonosacharide by
subjectingamixtureofstandardmonosaccharides corresponding
tothemonosaccharides presentinthesampletothesame
hydrolyticandderivatizationconditions.Onceresponsefactors
havebeendeterminedforeachmonosacharide present,theyare
usedtodeterminethepercentcontentofeachmonosaccharide
residue,%M,inthesampleaccordingtothefollowingequation:
•%M = (RF ×A
M×W
S×F ×100)/(A
S×S)
•where RF = the response factor for each monosaccharide,
A
M= peak area for monosaccharide in sample,
W
S= weight in mg of internal standard in sample
solution,
F is a factor for converting monosaccharides to polysaccharide residues
(0.88 for pentoses and 0.90 for hexoses),
• A
S= peak area of internal standard in sample solution, and
• S is the dry weight in mg of starting sample material.
Quantification
•Aflameionizationdetector(FID)isthemostcommonly used
detector.WithanFID,quantificationrequiresusinganinternal
standardandtheformulationofresponsefactors(RF).
•Response factorsareused tocorrectGCresponse to
monosaccharides andlossesarisingfrom hydrolysis and
derivatization.Theyareobtainedforeachmonosacharide by
subjectingamixtureofstandardmonosaccharides corresponding
tothemonosaccharides presentinthesampletothesame
hydrolyticandderivatizationconditions.Onceresponsefactors
havebeendeterminedforeachmonosacharide present,theyare
usedtodeterminethepercentcontentofeachmonosaccharide
residue,%M,inthesampleaccordingtothefollowingequation:
•%M = (RF ×A
M×W
S×F ×100)/(A
S×S)
•where RF = the response factor for each monosaccharide,
A
M= peak area for monosaccharide in sample,
W
S= weight in mg of internal standard in sample
solution,
F is a factor for converting monosaccharides to polysaccharide residues
(0.88 for pentoses and 0.90 for hexoses),
• A
S= peak area of internal standard in sample solution, and
• S is the dry weight in mg of starting sample material.
2 Gas-Liquid Chromatography (Cont’d)
Advantages
•GCanalysisofcarbohydratesisadvantageousbecauseitis
anestablishedtechniqueandmuchofthemethod
optimizationhasbeendone.
•Italsorequiressmallsamplesizesandisverysensitive,a
vitaladvantagewhenonlyasmallamountofsampleis
available.
•Disadvantages
•Thedisadvantagesofthistechniqueoriginatechieflyfrom
thepreparatorysteps.Ifeitherthereductionorthe
acetylationstepsdonotproceedtocompletion,thequantity
ofderivatizedsugarswillbeunderestimated.
•Inshort,thereisampleopportunityforsampleloss.
Additionally,preparationmayappearprohibitivelylaborious,
especiallywhencomparedtoHPLCtechniques.
Advantages
•GCanalysisofcarbohydratesisadvantageousbecauseitis
anestablishedtechniqueandmuchofthemethod
optimizationhasbeendone.
•Italsorequiressmallsamplesizesandisverysensitive,a
vitaladvantagewhenonlyasmallamountofsampleis
available.
•Disadvantages
•Thedisadvantagesofthistechniqueoriginatechieflyfrom
thepreparatorysteps.Ifeitherthereductionorthe
acetylationstepsdonotproceedtocompletion,thequantity
ofderivatizedsugarswillbeunderestimated.
•Inshort,thereisampleopportunityforsampleloss.
Additionally,preparationmayappearprohibitivelylaborious,
especiallywhencomparedtoHPLCtechniques.
3) High Performance Liquid Chromatography (HPLC)
SimilartoGC,HPLCisaseparationtechniquewhereby
compounds inamixtureareseparatedonastationary
phase.Inthiscase,themobilephase(eluant)
containingthesampleandthestationaryphaseare
bothliquids.
UnlikeGC,HPLCseparationisafunctionofthe
compatibility (differingcompatibility) ofsample
components fortheeluantandstationaryphase.
AHPLCseparationcanbemanipulated bychanging
theconcentrationand/ormakeupoftheeluant.
Forexample,acompound thatisverycompatiblewith
thestationaryphase(andthereforeonethatstayson
thecolumnlongerandeluteslater)canbeforcedoff
thecolumnfasterbychangingthesolventmakeup
suchthatsamplecomponents favoritversusthe
stationaryphase.
SimilartoGC,HPLCisaseparationtechniquewhereby
compounds inamixtureareseparatedonastationary
phase.Inthiscase,themobilephase(eluant)
containingthesampleandthestationaryphaseare
bothliquids.
UnlikeGC,HPLCseparationisafunctionofthe
compatibility (differingcompatibility) ofsample
components fortheeluantandstationaryphase.
AHPLCseparationcanbemanipulated bychanging
theconcentrationand/ormakeupoftheeluant.
Forexample,acompound thatisverycompatiblewith
thestationaryphase(andthereforeonethatstayson
thecolumnlongerandeluteslater)canbeforcedoff
thecolumnfasterbychangingthesolventmakeup
suchthatsamplecomponents favoritversusthe
stationaryphase.
3) High Performance Liquid Chromatography (HPLC) cont’d
UnderthebroadcategoryofHPLCthereareseveral
sub-typescharacterizedbythetypeofstationary
andmobilephasesemployed andthereforethe
chemistryoftheseparation.
Innormalphasechromatography thestationaryphase
ishydrophilicandthemobilephasevariesinits
hydrophilic/hydrophobic naturedepending onthe
samplecomponents beingseparated.
Inreversephasechromatography, thestationary
phaseishydrophobic.
UnderthebroadcategoryofHPLCthereareseveral
sub-typescharacterizedbythetypeofstationary
andmobilephasesemployed andthereforethe
chemistryoftheseparation.
Innormalphasechromatography thestationaryphase
ishydrophilicandthemobilephasevariesinits
hydrophilic/hydrophobic naturedepending onthe
samplecomponents beingseparated.
Inreversephasechromatography, thestationary
phaseishydrophobic.
4) High Performance Anion Exchange Chromatography
(HPAEC)
High performance anion exchange chromatography
(HPAEC), anincreasingly popular choiceforthe
separationofcarbohydrates, ischaracterized byan
anionicstationaryphaseandamobilephasewithahigh
pH.
AthighpH(10to14),carbohydratehydroxylgroupsionize
andtheirseparationisbasedontheirdifferingaffinity
fortheoppositelychargedstationaryphaseandthe
mobilephase.Asamplecontainingmonosaccharides,
eitherasitsnaturalstateorafterahydrolysisstep,must
beseparated chromatographically sothateach
monosaccharide canbeidentifiedandquantified.
Thischromatography isbased onthefactthat
carbohydrates inastronglyalkalineenvironment will
ionize,therebyrenderingthemamenable toseparation
onanionexchange column.HPAECcolumnsusedfor
carbohydratesarecoatedwithananionexchangeresin.
(HPAEC)
High performance anion exchange chromatography
(HPAEC), anincreasingly popular choiceforthe
separationofcarbohydrates, ischaracterized byan
anionicstationaryphaseandamobilephasewithahigh
pH.
AthighpH(10to14),carbohydratehydroxylgroupsionize
andtheirseparationisbasedontheirdifferingaffinity
fortheoppositelychargedstationaryphaseandthe
mobilephase.Asamplecontainingmonosaccharides,
eitherasitsnaturalstateorafterahydrolysisstep,must
beseparated chromatographically sothateach
monosaccharide canbeidentifiedandquantified.
Thischromatography isbased onthefactthat
carbohydrates inastronglyalkalineenvironment will
ionize,therebyrenderingthemamenable toseparation
onanionexchange column.HPAECcolumnsusedfor
carbohydratesarecoatedwithananionexchangeresin.
4) High Performance Anion Exchange Chromatography (HPAEC)
cont’d
Forexample,theDionex(Sunnyvale, CA)PA1column,
optimized fortheseparation ofmonosacharides,
disaccharides, oligosaccharides andlowmolecular
weight polysaccharides, iscomposed of10µm
nonporous beadscoveredinaquaternaryamineanion
exchangematerial.
HPAEC systemstypicallyusesodiumhydroxideasthe
eluanttoseparate mono anddisaccharides, while
eluantsforlargermolecules oftenincludesodium
acetatetoincreaseionicstrength.
Thedetector ofchoiceforHPAEC isthepulsed
amperometric detector(PAD).Ingeneral,amperometry
measures thechangeincurrentresultingfromthe
oxidationorreductionofacompound atanelectrode.In
PAD,itisthechange incurrentresultingfrom
carbohydrate oxidationatagoldorplatinumelectrode
thatismeasured.
HPAEC-PADpermitstheseparationandquantificationof
bothneutralandacidicmonosaccharides inone
analyticalrun.
cont’d
Forexample,theDionex(Sunnyvale, CA)PA1column,
optimized fortheseparation ofmonosacharides,
disaccharides, oligosaccharides andlowmolecular
weight polysaccharides, iscomposed of10µm
nonporous beadscoveredinaquaternaryamineanion
exchangematerial.
HPAEC systemstypicallyusesodiumhydroxideasthe
eluanttoseparate mono anddisaccharides, while
eluantsforlargermolecules oftenincludesodium
acetatetoincreaseionicstrength.
Thedetector ofchoiceforHPAEC isthepulsed
amperometric detector(PAD).Ingeneral,amperometry
measures thechangeincurrentresultingfromthe
oxidationorreductionofacompound atanelectrode.In
PAD,itisthechange incurrentresultingfrom
carbohydrate oxidationatagoldorplatinumelectrode
thatismeasured.
HPAEC-PADpermitstheseparationandquantificationof
bothneutralandacidicmonosaccharides inone
analyticalrun.
4) High Performance Anion Exchange Chromatography (Cont’d)
Quantification
Quantifyingtheamountofeachindividualmono-,di-,oligo-,or
polysaccharide fromanHPAECchromatogram isquitesimple
provided4to5dilutionsofappropriatestandardsarerunwitha
setofsamples.
ThesoftwareassociatedwiththeHPLCsystemwillintegrate
peaks,providepeakareas/heights,andgiveconcentration
valuesprovidedstandardswithknown concentrations have
beenrun.Itisimportanttorunstandardscorresponding to
sampleconstituentsbecausePADresponsevarieswiththe
analyte.
Detectorresponse decreases withtheincreasingdegreeof
polymerization therefore,therelativepercentage ofeach
component inachromatogram (andthereforethesample)
cannotbedeterminedbypeakareasalone,especiallywhenthe
samplecontainsmono-,di-,oligo-,andpolysaccharides.
Sugarconcentrationvaluesobtainedforacidhydrolysatesneedto
befurtheradjustedtoaccountforthemoleculeofwaterthat
wasaddedtoeachresidueuponhydrolysis.Forhexosesthis
requiresaconversionfactorof0.90andforpentosesafactorof
0.88isused.
Quantification
Quantifyingtheamountofeachindividualmono-,di-,oligo-,or
polysaccharide fromanHPAECchromatogram isquitesimple
provided4to5dilutionsofappropriatestandardsarerunwitha
setofsamples.
ThesoftwareassociatedwiththeHPLCsystemwillintegrate
peaks,providepeakareas/heights,andgiveconcentration
valuesprovidedstandardswithknown concentrations have
beenrun.Itisimportanttorunstandardscorresponding to
sampleconstituentsbecausePADresponsevarieswiththe
analyte.
Detectorresponse decreases withtheincreasingdegreeof
polymerization therefore,therelativepercentage ofeach
component inachromatogram (andthereforethesample)
cannotbedeterminedbypeakareasalone,especiallywhenthe
samplecontainsmono-,di-,oligo-,andpolysaccharides.
Sugarconcentrationvaluesobtainedforacidhydrolysatesneedto
befurtheradjustedtoaccountforthemoleculeofwaterthat
wasaddedtoeachresidueuponhydrolysis.Forhexosesthis
requiresaconversionfactorof0.90andforpentosesafactorof
0.88isused.
4) High Performance Anion Exchange Chromatography (Cont’d)
Advantages/Disadvantages
TheadvantageofHPAECfortheanalysisofcarbohydrates isthat
samplesdonotrequirederivatizationandtheanalysisitselfis
usuallyquitefast.
Inthepast,disadvantages ofHPLCoriginatedwithdetection
systems,whichforthemostpartwerenotverysensitive.
Refractiveindexdetectorshavetraditionallybeenthedetectorof
choice,becausemoresensitivedetectors,forexample UVor
fluorescence detectors,arenotappropriate foranalyzing
carbohydrates sincecarbohydrates donotcontainmoietiesthat
respondtothesedetectionsystems.
HPAEC-PADhasovercomethisdisadvantage,enablingtheseparation
andquantificationofmonosaccharides withlowdetectionlimits.
Inaddition,usingsodiumhydroxideaseluantisinexpensiveand
relativelysafe.Becausecarbohydrates aretypicallysolubleinthe
mobile phase, derivatization isunnecessary andsample
preparation isusuallylimitedtotheremoval ofinterfering
substances suchaslipidsandproteins.ThePADisalso
appropriateforusewithgradientelution.
•
Advantages/Disadvantages
TheadvantageofHPAECfortheanalysisofcarbohydrates isthat
samplesdonotrequirederivatizationandtheanalysisitselfis
usuallyquitefast.
Inthepast,disadvantages ofHPLCoriginatedwithdetection
systems,whichforthemostpartwerenotverysensitive.
Refractiveindexdetectorshavetraditionallybeenthedetectorof
choice,becausemoresensitivedetectors,forexample UVor
fluorescence detectors,arenotappropriate foranalyzing
carbohydrates sincecarbohydrates donotcontainmoietiesthat
respondtothesedetectionsystems.
HPAEC-PADhasovercomethisdisadvantage,enablingtheseparation
andquantificationofmonosaccharides withlowdetectionlimits.
Inaddition,usingsodiumhydroxideaseluantisinexpensiveand
relativelysafe.Becausecarbohydrates aretypicallysolubleinthe
mobile phase, derivatization isunnecessary andsample
preparation isusuallylimitedtotheremoval ofinterfering
substances suchaslipidsandproteins.ThePADisalso
appropriateforusewithgradientelution.
•
5) Enzymatic Analysis
Enzymaticmethodsfordeterminingsugarcontentrelyonthe
abilityofanenzymetocatalyzeaspecificreactionandemploy
asuitablemethodformonitoringtheprogressionofthe
reactionortheconcentrationofreactionproduct.
Enzymaticmethodsarehighlyspecific,usuallyrapidandsensitive
tolowsugarconcentrations.
Thereareenzymaticmethodsformostcommonsugars,andfor
somesuchasglucoseseveraldifferentenzymescanbeused,
andtheseassaysareavailableinkitform.
Enzymaticmethodsforthequantitativeanalysisofglucose,
fructose,sucrose,lactose,andgalactosearepresented.
Enzymaticmethodshavebeendevelopedforthequantification
ofvariouspolysaccharides,includingβ-glucan,starch,
galactomannans,locustbeangumandguargum.
Enzymaticmethodsfordeterminingsugarcontentrelyonthe
abilityofanenzymetocatalyzeaspecificreactionandemploy
asuitablemethodformonitoringtheprogressionofthe
reactionortheconcentrationofreactionproduct.
Enzymaticmethodsarehighlyspecific,usuallyrapidandsensitive
tolowsugarconcentrations.
Thereareenzymaticmethodsformostcommonsugars,andfor
somesuchasglucoseseveraldifferentenzymescanbeused,
andtheseassaysareavailableinkitform.
Enzymaticmethodsforthequantitativeanalysisofglucose,
fructose,sucrose,lactose,andgalactosearepresented.
Enzymaticmethodshavebeendevelopedforthequantification
ofvariouspolysaccharides,includingβ-glucan,starch,
galactomannans,locustbeangumandguargum.
5) Enzymatic Analysis
Thesegenerallyrequireenzymatichydrolysisofthe
polymerfollowedbyenzymaticdeterminationofthe
releasedmonosaccharidesusingoneofthemethods.
i) Glucose OxidaseMethod
a) Estimation of Glucose
ii) HexokinaseMethod
a) Estimation of Fructose, Glucose and Sucrose
iii) Galactose Dehydrogenase& Galactose OxidaseMethod
a) Estimation of Galactose and Lactose
Thesegenerallyrequireenzymatichydrolysisofthe
polymerfollowedbyenzymaticdeterminationofthe
releasedmonosaccharidesusingoneofthemethods.
i) Glucose OxidaseMethod
a) Estimation of Glucose
ii) HexokinaseMethod
a) Estimation of Fructose, Glucose and Sucrose
iii) Galactose Dehydrogenase& Galactose OxidaseMethod
a) Estimation of Galactose and Lactose
i) GLUCOSE OXIDASE METHOD : A) ESTIMATION OF GLUCOSE
Theory
Oneoftheearliestandmostwidelyusedenzymesforthequantitative
determinationofglucoseisGLUCOSEOXIDASE.Thishighlyspecific
enzyme,whichcanbeobtainedfromPenicilliumnotatumand
Aspergillisniger,catalyzestheoxidation(2HYDROGENATOMS
REMOVED)ofβ-D-gluco-pyranosetoformD-glucono-1,5-lactone,
whichisashortlivedspeciesthathydrolyzestoyieldD-gluconic
acid.
ThereactionofglucosewithglucoseoxidasealsoyieldsH
2O
2ina
ratioof1:1.
•Earlymethodsfordetectingthequantityofglucoseinasample
aftertreatmentwithglucoseoxidasereliedonthevolumetric
titrationofgluconicacidformed.
•Inclinicallabreactionsperoxidaseisusedincombinationwitha
chromogentoyieldacoloredcomplexinthepresenceofH
2O
2.
•Glucoseisoxidizedtoyieldgluconicacidandperoxideinthe
presenceofglucoseoxidaseandperoxidasecatalyzestheoxidation
ofachromogen(o-dianisidine)inthepresenceofH
2O
2,thereby
enablingquantitativemeasurementspectrophotometricallywhen
anappropriatestandardcurvehasbeenestablished.
Theory
Oneoftheearliestandmostwidelyusedenzymesforthequantitative
determinationofglucoseisGLUCOSEOXIDASE.Thishighlyspecific
enzyme,whichcanbeobtainedfromPenicilliumnotatumand
Aspergillisniger,catalyzestheoxidation(2HYDROGENATOMS
REMOVED)ofβ-D-gluco-pyranosetoformD-glucono-1,5-lactone,
whichisashortlivedspeciesthathydrolyzestoyieldD-gluconic
acid.
ThereactionofglucosewithglucoseoxidasealsoyieldsH
2O
2ina
ratioof1:1.
•Earlymethodsfordetectingthequantityofglucoseinasample
aftertreatmentwithglucoseoxidasereliedonthevolumetric
titrationofgluconicacidformed.
•Inclinicallabreactionsperoxidaseisusedincombinationwitha
chromogentoyieldacoloredcomplexinthepresenceofH
2O
2.
•Glucoseisoxidizedtoyieldgluconicacidandperoxideinthe
presenceofglucoseoxidaseandperoxidasecatalyzestheoxidation
ofachromogen(o-dianisidine)inthepresenceofH
2O
2,thereby
enablingquantitativemeasurementspectrophotometricallywhen
anappropriatestandardcurvehasbeenestablished.
a)Estimation of Glucose cont’d
Aliquots of sample solution are mixed with a buffered solution
containing glucose oxidase, peroxidase, and the chosen
chromogen and incubated under temperature controlled
conditions for a specified period of time (time and
temperature dependant on a chosen analytical method).
After sufficient time for color development, the absorbance is
read at an appropriate wavelength. For example, when
using method which uses o-dianisodinehydrochloride as
chromogen in 0.1 M acetate buffer at pH 5.5, sample
solutions are incubated at 30°C for 5 min and absorbance
read at 525 nm against a reagent blank.
Quantification
A calibration curve is constructed by plotting absorbance vs.
concentration for 5 separate glucose dilutions.
Quantification is achieved using the calibration curve.
Applications
•Glucose oxidase is a highly specific enzyme. Therefore this
method is applicable for use with samples that contain
other sugars.
Aliquots of sample solution are mixed with a buffered solution
containing glucose oxidase, peroxidase, and the chosen
chromogen and incubated under temperature controlled
conditions for a specified period of time (time and
temperature dependant on a chosen analytical method).
After sufficient time for color development, the absorbance is
read at an appropriate wavelength. For example, when
using method which uses o-dianisodinehydrochloride as
chromogen in 0.1 M acetate buffer at pH 5.5, sample
solutions are incubated at 30°C for 5 min and absorbance
read at 525 nm against a reagent blank.
Quantification
A calibration curve is constructed by plotting absorbance vs.
concentration for 5 separate glucose dilutions.
Quantification is achieved using the calibration curve.
Applications
•Glucose oxidase is a highly specific enzyme. Therefore this
method is applicable for use with samples that contain
other sugars.
ii) HEXOKINASE METHOD :
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE
HEXOKINASE is another enzyme frequently used in the
quantitative determination of glucose. Glucose reacts with
hexokinase in the presence of ADENOSINE TRIPHOSPHATE
(ATP) TO FORM GLUCOSE -6-PHOSPHATE + ADENOSINE
DIPHOSPHATE (ADP).
Glucose-6-phosphate reacts with glucose -6-phosphate
dehydrogenase in the presence of nicotinamide-adenine
dinucleotide (NAD) to produce 6 -phosphogluconate and
NADH.
NADH concentration is measured spectrophotometrically at
340 nm, or the reaction can be modified so that it can be
amendable to colorimetric determination
.
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE
HEXOKINASE is another enzyme frequently used in the
quantitative determination of glucose. Glucose reacts with
hexokinase in the presence of ADENOSINE TRIPHOSPHATE
(ATP) TO FORM GLUCOSE -6-PHOSPHATE + ADENOSINE
DIPHOSPHATE (ADP).
Glucose-6-phosphate reacts with glucose -6-phosphate
dehydrogenase in the presence of nicotinamide-adenine
dinucleotide (NAD) to produce 6 -phosphogluconate and
NADH.
NADH concentration is measured spectrophotometrically at
340 nm, or the reaction can be modified so that it can be
amendable to colorimetric determination
.
.
GLUCOSE ESTIMATION
GLUCOSE ESTIMATION
ii) HEXOKINASE METHOD :
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE
cont’d
FRUCTOSE ESTIMATION
Theory
Fructose can also be quantitatively assayed using hexokinase.
Glucose and fructose can be assayed together using
HEXOKINASE,
GLUCOSE-6-PHOSPHATE DEHYDROGENASE,
PHOSPHOGLUCOSE ISOMERASE (PGI)
to catalyze specific reactions.
In the presence of ATP and hexokinase, glucose and fructose
are phosphorylated to glucose -6-phosphate and fructose -6-
phosphate (F-6-P) respectively.
Adding NAD and glucose -6-phosphate dehydrogenase oxidizes
G-6-P to gluconate-6-phosphate and results in the
formation of NADH which can be measured at 340nm.
(NADH is measured at 340nm)
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE
cont’d
FRUCTOSE ESTIMATION
Theory
Fructose can also be quantitatively assayed using hexokinase.
Glucose and fructose can be assayed together using
HEXOKINASE,
GLUCOSE-6-PHOSPHATE DEHYDROGENASE,
PHOSPHOGLUCOSE ISOMERASE (PGI)
to catalyze specific reactions.
In the presence of ATP and hexokinase, glucose and fructose
are phosphorylated to glucose -6-phosphate and fructose -6-
phosphate (F-6-P) respectively.
Adding NAD and glucose -6-phosphate dehydrogenase oxidizes
G-6-P to gluconate-6-phosphate and results in the
formation of NADH which can be measured at 340nm.
(NADH is measured at 340nm)
ii) HEXOKINASE METHOD :
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE
cont’d
SUCROSEESTIMATION
Addingphospho-glucoseisomerasechangesF-6-PintoG-6-P,
whichisthenoxidizedtogluconate-6-phosphateinthepresence
ofNADandleadstotheformationofNADH.
Sucrosemayalsobeassayedusinghexokinaseorglucoseoxidase
byfirsttreatingitwithINVERTASEtoreleaseglucoseand
fructose.
(INVERTASEIS AN ENZYME THAT SPLITS SUCROSE
INTO ITS COMPONENT PARTS : GLUCOSE AND FRUCTOSE.)
NAD=Nicotinamideadenine dinucleotide is a coenzyme (PARTLY
MADE FROM NICOTINAMIDE -VITAMIN B3)found in all living cells. It
is a dinucleotide, consisting of two nucleotides joined
through their phosphate groups.
(NADH=Reduced form of NAD.)
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE
cont’d
SUCROSEESTIMATION
Addingphospho-glucoseisomerasechangesF-6-PintoG-6-P,
whichisthenoxidizedtogluconate-6-phosphateinthepresence
ofNADandleadstotheformationofNADH.
Sucrosemayalsobeassayedusinghexokinaseorglucoseoxidase
byfirsttreatingitwithINVERTASEtoreleaseglucoseand
fructose.
(INVERTASEIS AN ENZYME THAT SPLITS SUCROSE
INTO ITS COMPONENT PARTS : GLUCOSE AND FRUCTOSE.)
NAD=Nicotinamideadenine dinucleotide is a coenzyme (PARTLY
MADE FROM NICOTINAMIDE -VITAMIN B3)found in all living cells. It
is a dinucleotide, consisting of two nucleotides joined
through their phosphate groups.
(NADH=Reduced form of NAD.)
Reactions
Sucrose is broken into Glucose and Fructose when
reacted with Enzyme Invertase
1. D-Fructose + ATP
Hexokinase
→ Fructose-6-phosphate + ADP
2. Fructose-6-phosphate
Phospho glucose Isomerase
→Glucose-6-Phosphate
3. Glucose-6-phosphate + NAD
Dehydrogenase
→ Gluconate-6-phosphate
+ NADH
(NADH is measured at 340nm)
Sucrose is broken into Glucose and Fructose when
reacted with Enzyme Invertase
1. D-Fructose + ATP
Hexokinase
→ Fructose-6-phosphate + ADP
2. Fructose-6-phosphate
Phospho glucose Isomerase
→Glucose-6-Phosphate
3. Glucose-6-phosphate + NAD
Dehydrogenase
→ Gluconate-6-phosphate
+ NADH
(NADH is measured at 340nm)
ii) HEXOKINASE METHOD :
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE cont’d
Procedure
Buffer,NAD,ATP,andsamplesolutioncontaining
glucoseandfructosearecombined inatest
tube.
Amixture ofhexokinase and glucose-6-
phosphate dehydrogenase isaddedandafter
sufficienttimeforthereactiontoproceed,the
solutionabsorbanceisreadat340nm.
Theabsorbance isreadagainafteradding
phospho-glucoseisomerase.
Ablankshouldbesubjected tothesame
procedureasthesampleandusedtosetzero
inthespectrophotometer .
Adetailedproceduretodeterminebothglucose
andfructoseisavailable.
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE cont’d
Procedure
Buffer,NAD,ATP,andsamplesolutioncontaining
glucoseandfructosearecombined inatest
tube.
Amixture ofhexokinase and glucose-6-
phosphate dehydrogenase isaddedandafter
sufficienttimeforthereactiontoproceed,the
solutionabsorbanceisreadat340nm.
Theabsorbance isreadagainafteradding
phospho-glucoseisomerase.
Ablankshouldbesubjected tothesame
procedureasthesampleandusedtosetzero
inthespectrophotometer .
Adetailedproceduretodeterminebothglucose
andfructoseisavailable.
ii) HEXOKINASE METHOD :
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE cont’d
Quantification
Glucoseisquantifiedfromabsorbance valuesobtained
afteradding hexokinase andglucose-6-phosphate
dehydrogenase .
Toquantifyfructoseinthesamesample,absorbance
valuesreadafteraddingphospho-glucoseisomeraseare
correctedforinitialglucoseabsorbancevalues.
ThequantityofNADHproducedisstoichiometricwiththe
quantityofglucoseandfructose.
Applications
Thismethodisappropriateforthedeterminationofglucose
andfructoseinmanydifferentvarietiesoffoodstuffs
includingjam,honey,andicecream,aslongasthey
havebeentreatedtoremoveinterferingsubstancessuch
aslipidsandproteins.
Samplesshouldbeclear,relativelycolorless,andfreefrom
precipitatedmaterial.
Solutionsthatareturbidorcontaininterferingmattercan
befilteredortreatedwithCarrezreagents for
clarification.
A) ESTIMATION OF FRUCTOSE, GLUCOSE AND SUCROSE cont’d
Quantification
Glucoseisquantifiedfromabsorbance valuesobtained
afteradding hexokinase andglucose-6-phosphate
dehydrogenase .
Toquantifyfructoseinthesamesample,absorbance
valuesreadafteraddingphospho-glucoseisomeraseare
correctedforinitialglucoseabsorbancevalues.
ThequantityofNADHproducedisstoichiometricwiththe
quantityofglucoseandfructose.
Applications
Thismethodisappropriateforthedeterminationofglucose
andfructoseinmanydifferentvarietiesoffoodstuffs
includingjam,honey,andicecream,aslongasthey
havebeentreatedtoremoveinterferingsubstancessuch
aslipidsandproteins.
Samplesshouldbeclear,relativelycolorless,andfreefrom
precipitatedmaterial.
Solutionsthatareturbidorcontaininterferingmattercan
befilteredortreatedwithCarrezreagents for
clarification.
iii) GALACTOSE DEHYDROGENASE & GALACTOSE OXIDASE METHOD
A) ESTIMATION OF GALACTOSE AND LACTOSE
•GALACTOSE ESTIMATION:
•D-GalactosehasbeenassayedenzymaticallyusingGALACTOSEDEHYDROGENASE
andGALACTOSEOXIDASE.Inthepresenceofoxygen,galactoseisoxidizedtoD-
galacto-hexodialdo-1,5-pyranosebygalactoseoxidase.Thisreactiongenerates
hydrogenperoxide,whichcanbemeasuredCOLORIMETRICALLYinthepresence
ofhydrogendonors.
•Alternately,D-galactoseisoxidizedtogalactonicacidbyNADwhenβ-galactose
dehydrogenaseispresent,andtheresultantformationofNADHcanbe
monitored SPECTRO-PHOTOMETRICALLY .
•LACTOSEESTIMATION:
•Lactosecanalsobedetectedasgalactoseaftertreatmentwithβ-
galactosidase,anenzymethatcatalyzesthehydrolysisoflactoseto
D-glucoseandD-galactose.Bothgalactoseandlactosemaybe
determinedinasamplebycorrectingthelactosecontentofa
sampleobtainedpost–β-galactosidasedigestionbysubtractingthe
freegalactosedeterminedintheabsenceoftheenzyme.
•Enzymaticmethodsfordetermininglactoseandgalactoseare
availableintheliteratureandinkitform.
A) ESTIMATION OF GALACTOSE AND LACTOSE
•GALACTOSE ESTIMATION:
•D-GalactosehasbeenassayedenzymaticallyusingGALACTOSEDEHYDROGENASE
andGALACTOSEOXIDASE.Inthepresenceofoxygen,galactoseisoxidizedtoD-
galacto-hexodialdo-1,5-pyranosebygalactoseoxidase.Thisreactiongenerates
hydrogenperoxide,whichcanbemeasuredCOLORIMETRICALLYinthepresence
ofhydrogendonors.
•Alternately,D-galactoseisoxidizedtogalactonicacidbyNADwhenβ-galactose
dehydrogenaseispresent,andtheresultantformationofNADHcanbe
monitored SPECTRO-PHOTOMETRICALLY .
•LACTOSEESTIMATION:
•Lactosecanalsobedetectedasgalactoseaftertreatmentwithβ-
galactosidase,anenzymethatcatalyzesthehydrolysisoflactoseto
D-glucoseandD-galactose.Bothgalactoseandlactosemaybe
determinedinasamplebycorrectingthelactosecontentofa
sampleobtainedpost–β-galactosidasedigestionbysubtractingthe
freegalactosedeterminedintheabsenceoftheenzyme.
•Enzymaticmethodsfordetermininglactoseandgalactoseare
availableintheliteratureandinkitform.
iii) GALACTOSE DEHYDROGENASE & GALACTOSE OXIDASE METHOD
A) ESTIMATION OF GALACTOSE AND LACTOSE cont’d
ReactionTheory
•GalactoseisoxidizedtogalactonicacidbyNADinthe
presenceofgalactosedehydrogenase.NADH
generatedbythisreactionispresentinsolutionata
concentrationproportionaltothegalactosecontent.
Lactose
1. D-Lactose + H
2O
β -Glactosidase
→ D-Glucose + D-Galactose
Galactose
2. D-Galactose + NAD
+Dehydrogenase
→ Galactonic acid
+ NADH + H
+
A) ESTIMATION OF GALACTOSE AND LACTOSE cont’d
ReactionTheory
•GalactoseisoxidizedtogalactonicacidbyNADinthe
presenceofgalactosedehydrogenase.NADH
generatedbythisreactionispresentinsolutionata
concentrationproportionaltothegalactosecontent.
Lactose
1. D-Lactose + H
2O
β -Glactosidase
→ D-Glucose + D-Galactose
Galactose
2. D-Galactose + NAD
+Dehydrogenase
→ Galactonic acid
+ NADH + H
+
iii) GALACTOSE DEHYDROGENASE & GALACTOSE OXIDASE METHOD
A) ESTIMATION OF GALACTOSE AND LACTOSE cont’d
Procedure
Samplescontaininggalactoseexclusivelyaremixedwithabuffer
containingNAD,theinitialabsorbance isreadat340nmand
then measured again afterincubation withgalactose
dehydrogenase .
Lactosecontaining samples mustfirstbetreatedwithβ-
galactosidase TOCONVERT LACTOSE TOGLUCOSE AND
GALACTOSE andthenincubatedwithgalactosedehydrogenase .
Bothgalactoseandlactosecanbedeterminedinoneassayusing
thesetwoenzymes.Blanks(eachcontainingallrequired
enzymesandbufferbutnosample)needtobepreparedatthe
sametimeasthesamples.
Quantification
ThequantityofNADHformedisstoichiometricwiththeamountof
galactosepresentinthesample.
Theabsorptiondifferencesforthesamplesandblanksmustbe
determinedfirstbysubtractinginitialabsorbancevalues(pre–
galactosedehydrogenase addition)fromthefinalabsorbance
values(post–galactosedehydrogenase addition).
A) ESTIMATION OF GALACTOSE AND LACTOSE cont’d
Procedure
Samplescontaininggalactoseexclusivelyaremixedwithabuffer
containingNAD,theinitialabsorbance isreadat340nmand
then measured again afterincubation withgalactose
dehydrogenase .
Lactosecontaining samples mustfirstbetreatedwithβ-
galactosidase TOCONVERT LACTOSE TOGLUCOSE AND
GALACTOSE andthenincubatedwithgalactosedehydrogenase .
Bothgalactoseandlactosecanbedeterminedinoneassayusing
thesetwoenzymes.Blanks(eachcontainingallrequired
enzymesandbufferbutnosample)needtobepreparedatthe
sametimeasthesamples.
Quantification
ThequantityofNADHformedisstoichiometricwiththeamountof
galactosepresentinthesample.
Theabsorptiondifferencesforthesamplesandblanksmustbe
determinedfirstbysubtractinginitialabsorbancevalues(pre–
galactosedehydrogenase addition)fromthefinalabsorbance
values(post–galactosedehydrogenase addition).
iii) GALACTOSE DEHYDROGENASE & GALACTOSE OXIDASE METHOD
A) ESTIMATION OF GALACTOSE AND LACTOSE cont’d
Applications
Thisassayisapplicablefordetermininggalactoseconcentration
insolutionsthatarefreeoffatandprotein.Samplestobe
analyzedshouldbeclearandlessthan0.5g/Ltotalgalactose
andlactose.
Samplescontainingproteincanbetreatedwithperchloricacid
orCarrezreagent,whichprecipitatesproteinsandabsorbs
somecoloredcompounds.Theenzymaticassayforlactose
andgalactosecanbeusedformanydifferentfoodproducts,
INCLUDINGMEAT,DAIRY,ANDBAKERYPRODUCTS,provided
sampletreatmentincludesstepstoextractsugarsand
removeinterferingsubstances.
GalactosedehydrogenasealsooxidizesL-arabinose,therefore,
thepresenceofarabinosewillinterferewiththeanalysis.
A) ESTIMATION OF GALACTOSE AND LACTOSE cont’d
Applications
Thisassayisapplicablefordetermininggalactoseconcentration
insolutionsthatarefreeoffatandprotein.Samplestobe
analyzedshouldbeclearandlessthan0.5g/Ltotalgalactose
andlactose.
Samplescontainingproteincanbetreatedwithperchloricacid
orCarrezreagent,whichprecipitatesproteinsandabsorbs
somecoloredcompounds.Theenzymaticassayforlactose
andgalactosecanbeusedformanydifferentfoodproducts,
INCLUDINGMEAT,DAIRY,ANDBAKERYPRODUCTS,provided
sampletreatmentincludesstepstoextractsugarsand
removeinterferingsubstances.
GalactosedehydrogenasealsooxidizesL-arabinose,therefore,
thepresenceofarabinosewillinterferewiththeanalysis.
Galactosereactions
•β-D-Galactose-1-Phosphate β-D-Galactose
(catalyzed by Alkaline Phosphatase)
•β-D-Galactose+ NAD
+
+ H2OD-Galactono[1,4] lactone+NADH
+
+H
+
(catalyzed by Galactosedehydrogenase)
MEASURED UV INTENSITY AT 340 NM
•NADH
+
+Color reagent (bright yellow)Color Reagent (blue/violet)+ NAD
(catalyzed by Diaphorase)
MEASURED COLOR INTENSITY AT 550 NM
•β-D-Galactose-1-Phosphate β-D-Galactose
(catalyzed by Alkaline Phosphatase)
•β-D-Galactose+ NAD
+
+ H2OD-Galactono[1,4] lactone+NADH
+
+H
+
(catalyzed by Galactosedehydrogenase)
MEASURED UV INTENSITY AT 340 NM
•NADH
+
+Color reagent (bright yellow)Color Reagent (blue/violet)+ NAD
(catalyzed by Diaphorase)
MEASURED COLOR INTENSITY AT 550 NM
.III) Oligosaccharide & Polysaccharide Analysis
1)Size Exclusion Chromatography
2) High Performance Anion Exchange Chromatography
(HPAEC)
3) Enzymatic Analysis
1)Size Exclusion Chromatography
2) High Performance Anion Exchange Chromatography
(HPAEC)
3) Enzymatic Analysis
III) Oligosacharide& Polysaccharide Analysis
•Oligosaccharides released by partial acid
hydrolysis/enzymatic attack on polysaccharide
material.
•Forexample,the(1,4)linkageattachedtoa(1,3)
linkedglucoseunitinmixedlinked(1,3)(1,4)-β-D-
glucanispreferentiallycleavedby(1,3)(1,4)-β-D-
glucan-4-glu-canohydrolase (lichenase).
•Theoligosaccharides released from alichenase
digestionofβ-glucansareprimarily3-O-β-cellobiosyl
and 3-O-β-cellotriosyl-D-glucose; the relative
proportionofwhichisindicativeofthedominant
polysaccharidestructure.
•Methodsforanalyzingoligosaccharides aresimilarto
themethodsofanalyzingmonosaccharides .
•Forexample,theextractionofoligosaccharides from
foodproductsisaccomplished asformonosaccharides,
withhot80%ethanol.
•Oligosaccharides released by partial acid
hydrolysis/enzymatic attack on polysaccharide
material.
•Forexample,the(1,4)linkageattachedtoa(1,3)
linkedglucoseunitinmixedlinked(1,3)(1,4)-β-D-
glucanispreferentiallycleavedby(1,3)(1,4)-β-D-
glucan-4-glu-canohydrolase (lichenase).
•Theoligosaccharides released from alichenase
digestionofβ-glucansareprimarily3-O-β-cellobiosyl
and 3-O-β-cellotriosyl-D-glucose; the relative
proportionofwhichisindicativeofthedominant
polysaccharidestructure.
•Methodsforanalyzingoligosaccharides aresimilarto
themethodsofanalyzingmonosaccharides .
•Forexample,theextractionofoligosaccharides from
foodproductsisaccomplished asformonosaccharides,
withhot80%ethanol.
III) Oligosacharide& Polysaccharide Analysis
•Theycanbehydrolyzedwithacidorenzymes totheir
constituent monosaccharides and thehydrolysate
subjectedtoanalysisbychromatographic, chemicalor
enzymaticmethods.
•Inaddition,sizeexclusiontechniques suchashigh
performance sizeexclusion chromatography orgel
permeation chromatography canbeusedtoseparate
oligosaccharidemixturesbysize.
Methods adopted are:
•1)Size Exclusion Chromatography [GEL
FILTRATION]
[MOLECULAR SIEVE CHROMATOGRAPHY]
•2) High Performance Anion Exchange
Chromatography (HPAEC)
•3) Enzymatic Analysis
•Theycanbehydrolyzedwithacidorenzymes totheir
constituent monosaccharides and thehydrolysate
subjectedtoanalysisbychromatographic, chemicalor
enzymaticmethods.
•Inaddition,sizeexclusiontechniques suchashigh
performance sizeexclusion chromatography orgel
permeation chromatography canbeusedtoseparate
oligosaccharidemixturesbysize.
Methods adopted are:
•1)Size Exclusion Chromatography [GEL
FILTRATION]
[MOLECULAR SIEVE CHROMATOGRAPHY]
•2) High Performance Anion Exchange
Chromatography (HPAEC)
•3) Enzymatic Analysis
1)Size Exclusion Chromatography
•Size-exclusion chromatography, is
achromatographic method in which molecules in solution
are separated by their SIZE, and in some casesMOLECULAR
WEIGHT.
•Sizeexclusion chromatography isachromatographic
techniquewhereby moleculesinasampleareseparated
basedontheirsize.Moleculesinaneluantstream(usually
abuffer)aredirectedintoacolumnfilledwithagelpacking
ofclearlydefinedporesize.Thesmallermoleculesina
samplegetheldupinthepores,thereforespendingmore
timeinthecolumnandelutinglaterthanlargermolecules,
whichessentiallypassthroughthespacesbetween the
poresandelutefirst.
•Separationisinfluencedbythesizeoftheporesinthe
columnpacking.Effluentfromthecolumnismonitored
usingoneormoredetectors.Refractiveindexdetectorsare
oftenusedaloneorincombination withlightscattering
and/orviscosmetricdetectors.
•[SECSEPARATES PROTEINS BASED ON THEIR SIZES
(HYDRODYNAMIC RADII)ANDNOTBYABSOLUTE MOLECULAR
WEIGHT]
•Size-exclusion chromatography, is
achromatographic method in which molecules in solution
are separated by their SIZE, and in some casesMOLECULAR
WEIGHT.
•Sizeexclusion chromatography isachromatographic
techniquewhereby moleculesinasampleareseparated
basedontheirsize.Moleculesinaneluantstream(usually
abuffer)aredirectedintoacolumnfilledwithagelpacking
ofclearlydefinedporesize.Thesmallermoleculesina
samplegetheldupinthepores,thereforespendingmore
timeinthecolumnandelutinglaterthanlargermolecules,
whichessentiallypassthroughthespacesbetween the
poresandelutefirst.
•Separationisinfluencedbythesizeoftheporesinthe
columnpacking.Effluentfromthecolumnismonitored
usingoneormoredetectors.Refractiveindexdetectorsare
oftenusedaloneorincombination withlightscattering
and/orviscosmetricdetectors.
•[SECSEPARATES PROTEINS BASED ON THEIR SIZES
(HYDRODYNAMIC RADII)ANDNOTBYABSOLUTE MOLECULAR
WEIGHT]
1)Size Exclusion Chromatography cont’d
•Sizeexclusionchromatography hasbeenusedasa
method forseparatingandcharacterizingmixturesof
oligosaccharides basedonsize.Dextrins,orstarch
hydrolysates, canberathercomplex mixtures of
oligosaccharides withthesizeandproportionoflinear
andbranchedoligomersvarydependingonthestarting
material(ricestarch,cornstarch,etc.)andhydrolysis
method(enzymes,acid)evenwhentheDEisthesame.
•[DEXTROSEEQUIVALENTVALUE(DE);DEXTRINSWITHDE<20
ARENAMEDASMALTODEXTRINS]
•Dextrins[DEXTRINSAREWATER-SOLUBLE&HYDROLYZED STARCHES]
ofDErangingfrom4to25wereanalyzedonaSEC
system equipped with multi-angle light-scattering
detector,enablingtheacquisitionofinformationwith
respecttomolecularweightdistribution.
•Incombination withhighperformance anionexchange
chromatography (HPAEC),whichprovidedinformationaboutthe
relativeoccurrenceofindividualoligosaccharides,SECcoupled
withamolecularweightsensitivedetectorallowedamore
completedextrinsampleprofilethantheDEvaluealone(which
onlyreflectsthenumberofreducingends).
•Sizeexclusionchromatography hasbeenusedasa
method forseparatingandcharacterizingmixturesof
oligosaccharides basedonsize.Dextrins,orstarch
hydrolysates, canberathercomplex mixtures of
oligosaccharides withthesizeandproportionoflinear
andbranchedoligomersvarydependingonthestarting
material(ricestarch,cornstarch,etc.)andhydrolysis
method(enzymes,acid)evenwhentheDEisthesame.
•[DEXTROSEEQUIVALENTVALUE(DE);DEXTRINSWITHDE<20
ARENAMEDASMALTODEXTRINS]
•Dextrins[DEXTRINSAREWATER-SOLUBLE&HYDROLYZED STARCHES]
ofDErangingfrom4to25wereanalyzedonaSEC
system equipped with multi-angle light-scattering
detector,enablingtheacquisitionofinformationwith
respecttomolecularweightdistribution.
•Incombination withhighperformance anionexchange
chromatography (HPAEC),whichprovidedinformationaboutthe
relativeoccurrenceofindividualoligosaccharides,SECcoupled
withamolecularweightsensitivedetectorallowedamore
completedextrinsampleprofilethantheDEvaluealone(which
onlyreflectsthenumberofreducingends).
1)Size Exclusion Chromatography Cont’d
•Conventional or low pressure size exclusion
chromatography predatesSEC.Lowpressuresystems
employlargecapacitycolumns(i.e.,1.6×70cmand
larger)singlyorseveralinseriesthatoperateata
relativelylowpressurecompared toSECandseparate
largersamplevolumes.
•Therefore,theyareoftenusedforpreparative orsemi-
preparativepurposes,tocleansamples(removesaltsorother
smallmolecularweightmaterialfromasample)orisolatea
specificfractionofinterestinasamplepriortofurtheranalysis.
•Eluentconcentrationisoftenmonitoredwithadetector(i.e.,RI
detector)soachromatogram ofsampledistributionisobtained.
•Alternately,aeluentcontainingseparatedmaterialiscollected
intubesandthetubecontentsanalyzedusinganotheranalytical
technique,suchasthephenol–sulfuricacidassay.Byplotting
thetubenumbervs.thecarbohydrate concentration,acurve
representingsampledistributioncanbeobtained.
•Forexample,largeandsmallmolecularweightstarchfractions
innativeandacid-modifiedstarchesfromcereals,pulses,and
tuberswereseparatedonaSepharose CL4Bgel(Pharmacia
FineChemicals,Sweden)at30ml/hwithwateraseluent.
•Conventional or low pressure size exclusion
chromatography predatesSEC.Lowpressuresystems
employlargecapacitycolumns(i.e.,1.6×70cmand
larger)singlyorseveralinseriesthatoperateata
relativelylowpressurecompared toSECandseparate
largersamplevolumes.
•Therefore,theyareoftenusedforpreparative orsemi-
preparativepurposes,tocleansamples(removesaltsorother
smallmolecularweightmaterialfromasample)orisolatea
specificfractionofinterestinasamplepriortofurtheranalysis.
•Eluentconcentrationisoftenmonitoredwithadetector(i.e.,RI
detector)soachromatogram ofsampledistributionisobtained.
•Alternately,aeluentcontainingseparatedmaterialiscollected
intubesandthetubecontentsanalyzedusinganotheranalytical
technique,suchasthephenol–sulfuricacidassay.Byplotting
thetubenumbervs.thecarbohydrate concentration,acurve
representingsampledistributioncanbeobtained.
•Forexample,largeandsmallmolecularweightstarchfractions
innativeandacid-modifiedstarchesfromcereals,pulses,and
tuberswereseparatedonaSepharose CL4Bgel(Pharmacia
FineChemicals,Sweden)at30ml/hwithwateraseluent.
1)Size Exclusion Chromatography Cont’d
•Oligosaccharidesarisingfromenzymaticdegradation
ofcarrageenanhavealsobeenseparated
successfullyonalowpressureSECsystem.Using
Super-dex30
•Usinggel-permeationchromatographyisrootedinthe
factthatrelativelylargesamplesizesmaybeapplied
tothecolumnandthereforeseparatedfractionscan
becollectedinrelativelylargeamounts.
•Additionally,ithasbeenreportedthatconventional
SECseparatessomeclassesofoligosaccharides,
suchasahomologousseriesofmalto-dextrins,with
betterresolutionthanHPSEC.
•OneofthedisadvantagesoflowpressureSEC
systemsisthetimerequiredforasinglerun,often
requiringmanyhoursorevendays.
•Oligosaccharidesarisingfromenzymaticdegradation
ofcarrageenanhavealsobeenseparated
successfullyonalowpressureSECsystem.Using
Super-dex30
•Usinggel-permeationchromatographyisrootedinthe
factthatrelativelylargesamplesizesmaybeapplied
tothecolumnandthereforeseparatedfractionscan
becollectedinrelativelylargeamounts.
•Additionally,ithasbeenreportedthatconventional
SECseparatessomeclassesofoligosaccharides,
suchasahomologousseriesofmalto-dextrins,with
betterresolutionthanHPSEC.
•OneofthedisadvantagesoflowpressureSEC
systemsisthetimerequiredforasinglerun,often
requiringmanyhoursorevendays.
2) High Performance Anion Exchange Chromatography (HPAEC)
•UsingHPAEC forcarbohydrate analysisishighlightedagain
whentheseparationofoligosaccharides isconsidered.The
combination ofcolumn packing,eluentcomposition, and
sensitiveelectrochemical detection enables thebaseline
resolutionofahomologous seriesofoligosaccharidessuchas
dextrins(starchhydrolysates)uptoDP40.
•Unlikemonosaccharide analysis,wherethemobilephaseis
typicallysodium hydroxide alone, mobile phases for
oligosaccharidescontainsodiumacetateaswelltoincreaseionic
strength andtoensure adequate pushing offofthe
oligosaccharides fromthecolumn.TheDionexCarbopacPA1
columnhasbeenusedtoseparatedextrinsuptoDP30usinga
mobilephasecombinationof80%Aand20%Battimezerowith
alineargradientto90%Band10%A(whereA=100mMNaOH
andB=100mMNaOHcontaining600mMNaOAC).
•Inthecaseofdextrins,samplepreparationconsistsonlyof
dryingthesample,dilutinganappro-priateamountwithwater,
andfiltering(0.45umfilter)priortoinjection.HPAEChasalso
beenusedasapreparative technique toseparate the
oligosaccharidesinbeer.Again,usingaNaOH/NaOACbuffer,an
effluantwascollectedfromthecolumnin20secondfractions
andsubjectedtofurtheranalysis.
•UsingHPAEC forcarbohydrate analysisishighlightedagain
whentheseparationofoligosaccharides isconsidered.The
combination ofcolumn packing,eluentcomposition, and
sensitiveelectrochemical detection enables thebaseline
resolutionofahomologous seriesofoligosaccharidessuchas
dextrins(starchhydrolysates)uptoDP40.
•Unlikemonosaccharide analysis,wherethemobilephaseis
typicallysodium hydroxide alone, mobile phases for
oligosaccharidescontainsodiumacetateaswelltoincreaseionic
strength andtoensure adequate pushing offofthe
oligosaccharides fromthecolumn.TheDionexCarbopacPA1
columnhasbeenusedtoseparatedextrinsuptoDP30usinga
mobilephasecombinationof80%Aand20%Battimezerowith
alineargradientto90%Band10%A(whereA=100mMNaOH
andB=100mMNaOHcontaining600mMNaOAC).
•Inthecaseofdextrins,samplepreparationconsistsonlyof
dryingthesample,dilutinganappro-priateamountwithwater,
andfiltering(0.45umfilter)priortoinjection.HPAEChasalso
beenusedasapreparative technique toseparate the
oligosaccharidesinbeer.Again,usingaNaOH/NaOACbuffer,an
effluantwascollectedfromthecolumnin20secondfractions
andsubjectedtofurtheranalysis.
2) High Performance Anion Exchange Chromatography (HPAEC) cont’d
•Oligosaccharides (composed ofglucose,galactose,and
xylose)releasedbytheactionofcellulaseonseed
xyloglucanwereseparatedonaCarboPakPA100column
(Dionex,Sunnyvale,CA)usinggradientelutionofsodium
hydroxide andsodium acetatebuffers.Oligosaccharides
suchasisomaltose,kojibiose,gentiobiose,nigerose,and
maltosefromdifferentvarietiesofhoneyhavealsobeen
separatedusingHPAEC-PAD.
•Notlimitedtotheseparationofneutraloligosaccharides,
oligogalacturonicacidsfromstrawberryjuicehavebeen
separatedonaHPAECsystemusingasodiumhydroxide
gradientandaCarboPacPA-100column.Oligogalacturonic
acidsresultingfrompectinenzymatic depolymerisation
wereseparated onaMono-Qanionexchange column
(Pharmacia, Upsala,Sweden) usinggradient elution
(Na
2SO
4inphosphate buffer)anddetectedonaphoto-
diodearraydetector.Theanionexchangeresinpermitted
goodresolutionofoligomers.
•Oligosaccharides (composed ofglucose,galactose,and
xylose)releasedbytheactionofcellulaseonseed
xyloglucanwereseparatedonaCarboPakPA100column
(Dionex,Sunnyvale,CA)usinggradientelutionofsodium
hydroxide andsodium acetatebuffers.Oligosaccharides
suchasisomaltose,kojibiose,gentiobiose,nigerose,and
maltosefromdifferentvarietiesofhoneyhavealsobeen
separatedusingHPAEC-PAD.
•Notlimitedtotheseparationofneutraloligosaccharides,
oligogalacturonicacidsfromstrawberryjuicehavebeen
separatedonaHPAECsystemusingasodiumhydroxide
gradientandaCarboPacPA-100column.Oligogalacturonic
acidsresultingfrompectinenzymatic depolymerisation
wereseparated onaMono-Qanionexchange column
(Pharmacia, Upsala,Sweden) usinggradient elution
(Na
2SO
4inphosphate buffer)anddetectedonaphoto-
diodearraydetector.Theanionexchangeresinpermitted
goodresolutionofoligomers.
2) High Performance Anion Exchange Chromatography (HPAEC) cont’d
•Themaindisadvantage ofusingHPAEC-PADfor
analysingoligosaccharides istheabsence of
adequate standards formany oligosaccharide
types(i.e.,oligosaccharides fromβ-glucan,
malto-oligosaccharides over DP 7),which
preventstheirquantification.
•Inaddition,pulsed amperometric detector
responseisnotequivalentforallsampletypes
and,infact,detectorresponsedecreaseswiththe
increaseofDP;thereforepurifiedstandardsare
required inorder tofacilitateaccurate
quantification.
•Themaindisadvantage ofusingHPAEC-PADfor
analysingoligosaccharides istheabsence of
adequate standards formany oligosaccharide
types(i.e.,oligosaccharides fromβ-glucan,
malto-oligosaccharides over DP 7),which
preventstheirquantification.
•Inaddition,pulsed amperometric detector
responseisnotequivalentforallsampletypes
and,infact,detectorresponsedecreaseswiththe
increaseofDP;thereforepurifiedstandardsare
required inorder tofacilitateaccurate
quantification.
3) Enzymatic Analysis
•Enzymatic hydrolysis ofoliogsaccharides coupled with
chromatographic separation andidentificationofreleased
oligosaccharidesiscommonly usedtoquantifyoligosaccharides
inbothsimpleandcomplexfoodsystems.
•Thismethodhassuccessfullybeenappliedtotheanalysisof
fructo-oligosaccharides andinulin(AOAC997.08).Inulinand
oligofructosearefructans,(2→1)linkedβ-D-fructofuranosyl
unitswithorwithoutaterminalglucose,foundnaturallyin
chicory,Jeruselem artichoke,andonions.Bothinulinand
oligofructosearepolydispersemixtureswithDPvaluesranging
from2to60and2to10,respectively.
•Thesefructanshavereceivedmuchattentioninrecentyears
basedontheirpostulatedpositivenutritionalbenefits,including
improvedlaxationandthestimulationofbeneficialgutmicro
flora,andtheirpotentialusesasfunctionalfoodadditives.
•Becauseofinulins’largerDPitislesssolublethanoligofructose
andabletoformmicro-crystalsinsolutionthatimpartafat-like
mouth feel,enabling ittofunctionasafatmimetic.
Oligofructoseismoresolublethaninulinandsweettasting,
makingitappropriateforuseasahumectants inbakedgoods
andabinderingranolabarswithimproved nutritional
functionalityvs.cornsyrup.
•Enzymatic hydrolysis ofoliogsaccharides coupled with
chromatographic separation andidentificationofreleased
oligosaccharidesiscommonly usedtoquantifyoligosaccharides
inbothsimpleandcomplexfoodsystems.
•Thismethodhassuccessfullybeenappliedtotheanalysisof
fructo-oligosaccharides andinulin(AOAC997.08).Inulinand
oligofructosearefructans,(2→1)linkedβ-D-fructofuranosyl
unitswithorwithoutaterminalglucose,foundnaturallyin
chicory,Jeruselem artichoke,andonions.Bothinulinand
oligofructosearepolydispersemixtureswithDPvaluesranging
from2to60and2to10,respectively.
•Thesefructanshavereceivedmuchattentioninrecentyears
basedontheirpostulatedpositivenutritionalbenefits,including
improvedlaxationandthestimulationofbeneficialgutmicro
flora,andtheirpotentialusesasfunctionalfoodadditives.
•Becauseofinulins’largerDPitislesssolublethanoligofructose
andabletoformmicro-crystalsinsolutionthatimpartafat-like
mouth feel,enabling ittofunctionasafatmimetic.
Oligofructoseismoresolublethaninulinandsweettasting,
makingitappropriateforuseasahumectants inbakedgoods
andabinderingranolabarswithimproved nutritional
functionalityvs.cornsyrup.
3) Enzymatic Analysis
•Bothinulinandoligofructoseinafoodproductmaybe
quantifiedbysubjectingahotwaterextracttoan
enzymatictreatmentthatpermitsfructandetermination
bydifference (sugar content before andafter
treatment)
•Freefructoseandsucrosearedeterminedintheoriginal
sample,freeglucoseandglucosefromstarchare
determined afteramylo-glucosidase treatment, and
totalglucoseandtotalfructosearedetermined after
fructozymhydrolysis.
•Glucoseandfructosefromfructansarethendetermined
bydifference.HPAEC-PADisideallysuitedforthe
analysisoftheseenzyme hydrolysates because it
enablesbaselineresolutionofallsugarsofinterestand
facilitatesstraightforwardquantification.
•A)Analysisofβ-D-Glucans(CerealPolysaccharides)
•B)Analysis of Galactomannans
•Bothinulinandoligofructoseinafoodproductmaybe
quantifiedbysubjectingahotwaterextracttoan
enzymatictreatmentthatpermitsfructandetermination
bydifference (sugar content before andafter
treatment)
•Freefructoseandsucrosearedeterminedintheoriginal
sample,freeglucoseandglucosefromstarchare
determined afteramylo-glucosidase treatment, and
totalglucoseandtotalfructosearedetermined after
fructozymhydrolysis.
•Glucoseandfructosefromfructansarethendetermined
bydifference.HPAEC-PADisideallysuitedforthe
analysisoftheseenzyme hydrolysates because it
enablesbaselineresolutionofallsugarsofinterestand
facilitatesstraightforwardquantification.
•A)Analysisofβ-D-Glucans(CerealPolysaccharides)
•B)Analysis of Galactomannans
A) Analysis of β-D-Glucans(Cereal Polysaccharides)
Theory
•glucansarecellwallpolysaccharidesfoundincereal
grainssuchasoatsandbarley,wheatandryegrains.
•Analysisofβ-glucanrequiresenzymatichydrolysis
usingβ-glucan-4-glucanohydrolase(LICHENASE)to
yieldoligosacchridesandonsubsequenthydrolysis
withβ-glucosidase,releasedGLUCOSEisassayed
usingglucoseoxidaseasoutlinedingluosanalysis.
(1 → 3) (1 → 4)-β-D-Glucan
Lichenase
→ 1 → 3 linked cellodextrins
(1→3)linkedcellodextrins
β-glucosidase
→Glucose
Theory
•glucansarecellwallpolysaccharidesfoundincereal
grainssuchasoatsandbarley,wheatandryegrains.
•Analysisofβ-glucanrequiresenzymatichydrolysis
usingβ-glucan-4-glucanohydrolase(LICHENASE)to
yieldoligosacchridesandonsubsequenthydrolysis
withβ-glucosidase,releasedGLUCOSEisassayed
usingglucoseoxidaseasoutlinedingluosanalysis.
(1 → 3) (1 → 4)-β-D-Glucan
Lichenase
→ 1 → 3 linked cellodextrins
(1→3)linkedcellodextrins
β-glucosidase
→Glucose
A) Analysis of β-D-Glucans(Cereal Polysaccharides)
Procedure
•Anaccuratelyweighedamountofdrysample(usuallyflouror
millingfraction)ismixedwithphosphatebuffer(pH6.5),
boiledbriefly,andmixedthoroughly.
•Themixtureisincubatedwithhighpuritylichenaseat50°C,
combinedwithsodiumacetatebuffer(pH4.0)andcentrifuged.
•Aportionofthesupernatantistreatedwithglucosidaseand
anotherportionwithacetatebuffertoserveasablank.
•Afterincubationat40°Cwithglucosidasethesamplesare
dilutedandglucosedeterminedenzymaticallyusingthe
glucoseoxidase-peroxidasemethod(Section2.3.4.1).
•Standardscontaining50and100µg/mlglucose,reagent
blanks(containingacetatebufferandglucoseoxidase-
peroxidasereagent)andflourcontrolswithknownβ-glucan
valuesarepreparedandrunwitheachsetofsamples.
Procedure
•Anaccuratelyweighedamountofdrysample(usuallyflouror
millingfraction)ismixedwithphosphatebuffer(pH6.5),
boiledbriefly,andmixedthoroughly.
•Themixtureisincubatedwithhighpuritylichenaseat50°C,
combinedwithsodiumacetatebuffer(pH4.0)andcentrifuged.
•Aportionofthesupernatantistreatedwithglucosidaseand
anotherportionwithacetatebuffertoserveasablank.
•Afterincubationat40°Cwithglucosidasethesamplesare
dilutedandglucosedeterminedenzymaticallyusingthe
glucoseoxidase-peroxidasemethod(Section2.3.4.1).
•Standardscontaining50and100µg/mlglucose,reagent
blanks(containingacetatebufferandglucoseoxidase-
peroxidasereagent)andflourcontrolswithknownβ-glucan
valuesarepreparedandrunwitheachsetofsamples.
A) Analysis of β-D-Glucans(Cereal Polysaccharides) Cont’d
Quantification
•Absorbance valuesfromglucosestandardsareusedto
evaluatepercentglucoseinthesample.
•Whencalculatingthepercentglucoseitisimportantto
includeaconversionfactorof0.9toaccountforthe
differenceinmolecularweightoffreeglucosevs.glucose
inpolysaccharides.
Applicability
•Thisassayisappropriateforusewithdryfloursand
millingfractionsofcerealgrainssuchasrye,oats,barley,
wheat,andunsweetened cerealsprovidedalloftheabove
havebeenmilledtopassa0.5mmmesh.
•Thisassaymayalsobeusedforsamplescontaining
simplesugarsbyextractingthesamplewith50%ethanol
toremovethesesugars(whichcanartificiallyincrease
measuredglucosevalues)priortolichenasetreatment.
•Assaystepshavebeenmodified andoptimized for
differentapplicationsincludingfloursandmillingfractions
fromcerealgrainsandβ-glucaninmalt,wort,andbeer.
•
Quantification
•Absorbance valuesfromglucosestandardsareusedto
evaluatepercentglucoseinthesample.
•Whencalculatingthepercentglucoseitisimportantto
includeaconversionfactorof0.9toaccountforthe
differenceinmolecularweightoffreeglucosevs.glucose
inpolysaccharides.
Applicability
•Thisassayisappropriateforusewithdryfloursand
millingfractionsofcerealgrainssuchasrye,oats,barley,
wheat,andunsweetened cerealsprovidedalloftheabove
havebeenmilledtopassa0.5mmmesh.
•Thisassaymayalsobeusedforsamplescontaining
simplesugarsbyextractingthesamplewith50%ethanol
toremovethesesugars(whichcanartificiallyincrease
measuredglucosevalues)priortolichenasetreatment.
•Assaystepshavebeenmodified andoptimized for
differentapplicationsincludingfloursandmillingfractions
fromcerealgrainsandβ-glucaninmalt,wort,andbeer.
•
B)Analysis of Galactomannans
Galactomannans
•Galactomannansarepolysaccharidesconsistingofaβ-1--
---4linkedmannosylbackbonesubstitutedtovarying
degreesattheC-6positionbyanα-galactoseunit.The
levelofsubstitutionvariesdependingongalactomannan
source.
•Examples:
galactomannanfromguarhasagalactose:mannose
(G:M)is1:2
Locustbeangumhasgalactose:mannose(G:M)is1:4.
Anenzymaticmethodforquantitativelydetermining
galactomannanshasbeendevelopedbasinguponthe
knownG:Mratiosofcommongalactomannans.
Galactomannans
•Galactomannansarepolysaccharidesconsistingofaβ-1--
---4linkedmannosylbackbonesubstitutedtovarying
degreesattheC-6positionbyanα-galactoseunit.The
levelofsubstitutionvariesdependingongalactomannan
source.
•Examples:
galactomannanfromguarhasagalactose:mannose
(G:M)is1:2
Locustbeangumhasgalactose:mannose(G:M)is1:4.
Anenzymaticmethodforquantitativelydetermining
galactomannanshasbeendevelopedbasinguponthe
knownG:Mratiosofcommongalactomannans.
B)Analysis of Galactomannanscont’d
Galactomannanscont’d
•Theory
•Theconcentrationofgalactomannaninasampleis
determinedfromthequantityofgalactosereleasedbyα-
galactosidaseafterβ-mannanasedigestion.Galactoseis
quantifiedspectrophotometricallyaftertreatmentwith
NADandgalactosedehydrogenase.
Galactomannan
β-mannanase
→Oligosaccharidemixture
Oligosaccharidemixture
α-galactosidase
→Galactose+
manno-oligosaccharides
Galactomannanscont’d
•Theory
•Theconcentrationofgalactomannaninasampleis
determinedfromthequantityofgalactosereleasedbyα-
galactosidaseafterβ-mannanasedigestion.Galactoseis
quantifiedspectrophotometricallyaftertreatmentwith
NADandgalactosedehydrogenase.
Galactomannan
β-mannanase
→Oligosaccharidemixture
Oligosaccharidemixture
α-galactosidase
→Galactose+
manno-oligosaccharides
B)Analysis of Galactomannanscont’d
Galactomannanscont’d
Procedure
•Dryfloursamplesfromguarorlocustbeangumare
extractedwith80%ethanoltoremovesimplesugarsand
oligosaccharideswhichmaycontaingalactose.
•Thegalactomannanpresentissolubilizedbyboilingthe
flourinabufferfollowedbyincubationat40°Cwithβ-
mannanase.
•Samplemixturesarecentrifugedtoseparateinsoluble
materialandportionsofthesupernatantareincubated
withα-galactosidasetoreleasegalactoseandanother
portionistreatedwithanacetatebuffer(blank).
Galactoseisquantitativelydeter-minedinthesample
aftertreatmentwithNADandgalactosedehydrogenase.
Galactomannanscont’d
Procedure
•Dryfloursamplesfromguarorlocustbeangumare
extractedwith80%ethanoltoremovesimplesugarsand
oligosaccharideswhichmaycontaingalactose.
•Thegalactomannanpresentissolubilizedbyboilingthe
flourinabufferfollowedbyincubationat40°Cwithβ-
mannanase.
•Samplemixturesarecentrifugedtoseparateinsoluble
materialandportionsofthesupernatantareincubated
withα-galactosidasetoreleasegalactoseandanother
portionistreatedwithanacetatebuffer(blank).
Galactoseisquantitativelydeter-minedinthesample
aftertreatmentwithNADandgalactosedehydrogenase.
B)Analysis of GalactomannansCont’d
Quantification
•Blankabsorbancevaluesaresubtractedfromsampleabsorbancesand
theresultingvaluesusedtoquantitativelydeterminegalactoseby
accountingforsampledilutions,theconversionoffreegalactoseto
anhydro-galactose,andafactortorelatefreegalactoseto
galactomannancontent(×0.90).Theaveragepercentageofgalactosein
guarandlocustbeangumis38and22%,respectively.
Applicability
•Thismethodcanbeusedforanalysingseedfloursprovidedthe
galactose:mannoseratioofthegalactomannanisknown.
•Itcanalsobeusedwhenlowmolecularweightsugarsarepresent
inthesampleprovidedtheyareremovedviaethanolextraction
priortoanalysis.Thepresenceof1→6linkedα-D-galactoseunits
insomeoligosaccharides,suchasraffinoseandstachy-ose,are
alsosusceptibletoα-galactosidase;theseoligosaccharideswill
arti-ficiallyincreasethegumcontentiftheyarenotremoved
beforeenzymetreatment.
Quantification
•Blankabsorbancevaluesaresubtractedfromsampleabsorbancesand
theresultingvaluesusedtoquantitativelydeterminegalactoseby
accountingforsampledilutions,theconversionoffreegalactoseto
anhydro-galactose,andafactortorelatefreegalactoseto
galactomannancontent(×0.90).Theaveragepercentageofgalactosein
guarandlocustbeangumis38and22%,respectively.
Applicability
•Thismethodcanbeusedforanalysingseedfloursprovidedthe
galactose:mannoseratioofthegalactomannanisknown.
•Itcanalsobeusedwhenlowmolecularweightsugarsarepresent
inthesampleprovidedtheyareremovedviaethanolextraction
priortoanalysis.Thepresenceof1→6linkedα-D-galactoseunits
insomeoligosaccharides,suchasraffinoseandstachy-ose,are
alsosusceptibletoα-galactosidase;theseoligosaccharideswill
arti-ficiallyincreasethegumcontentiftheyarenotremoved
beforeenzymetreatment.
.
IV)Dietary Fiber Analysis
1)Uppsala Method
2)Enzymatic -Gravimetric Methods
IV)Dietary Fiber Analysis
1)Uppsala Method
2)Enzymatic -Gravimetric Methods
Dietary fiber
Dietary fiberis the indigestible portion of food derived
from plants. It has two main components:
1)SOLUBLE FIBER
2)INSOLUBLE FIBER
1)Solublefiber,whichdissolvesinwater,isreadilyfermentedin
thecolonintogasesandphysiologicallyactivebyproducts,and
canbeprebioticandviscous.Itdelaysgastricemptyingwhichin
turncancauseanextendedfeelingoffullness.
2)Insoluble fiber,which doesnotdissolveinwater,is
metabolicallyinertandprovidesbulking,oritcanbeprebioticand
metabolicallyfermentinthelargeintestine.Bulkingfibersabsorb
water asthey move through the digestive system,
easingdefecation.
Dietaryfiberscanactbychangingthenatureofthecontentsof
the gastrointestinal tract and by changing how
othernutrientsandchemicalsareabsorbed.Sometypesofsoluble
fiberabsorbwatertobecome agelatinous,viscoussubstance
whichisfermentedbybacteriainthedigestivetract.Sometypes
ofinsolublefiberhavebulkingactionandarenotfermented.
Dietary fiberis the indigestible portion of food derived
from plants. It has two main components:
1)SOLUBLE FIBER
2)INSOLUBLE FIBER
1)Solublefiber,whichdissolvesinwater,isreadilyfermentedin
thecolonintogasesandphysiologicallyactivebyproducts,and
canbeprebioticandviscous.Itdelaysgastricemptyingwhichin
turncancauseanextendedfeelingoffullness.
2)Insoluble fiber,which doesnotdissolveinwater,is
metabolicallyinertandprovidesbulking,oritcanbeprebioticand
metabolicallyfermentinthelargeintestine.Bulkingfibersabsorb
water asthey move through the digestive system,
easingdefecation.
Dietaryfiberscanactbychangingthenatureofthecontentsof
the gastrointestinal tract and by changing how
othernutrientsandchemicalsareabsorbed.Sometypesofsoluble
fiberabsorbwatertobecome agelatinous,viscoussubstance
whichisfermentedbybacteriainthedigestivetract.Sometypes
ofinsolublefiberhavebulkingactionandarenotfermented.
Dietary Fiber Cont’d
Lignin,amajordietaryinsolublefibersource,mayalter
therateandmetabolismofsolublefibers.Othertypesof
insolublefiber,notablyresistantstarch,arefully
fermented.
Somebutnotallsolubleplantfibersblockintestinal
mucosaladherenceandtranslocationofpotentially
pathogenicbacteriaandmaythereforemodulate
intestinalinflammation,aneffectthathasbeen
termedcontrabiotic.
Chemically,dietaryfiberconsistsofnon-
starchpolysaccharidessuchasarabinoxylans,cellulose,
andmanyotherplantcomponentssuchasresistant
starch,resistantdextrins,inulin,lignin,chitins,pectins,
beta-glucans,andoligosaccharides.
Lignin,amajordietaryinsolublefibersource,mayalter
therateandmetabolismofsolublefibers.Othertypesof
insolublefiber,notablyresistantstarch,arefully
fermented.
Somebutnotallsolubleplantfibersblockintestinal
mucosaladherenceandtranslocationofpotentially
pathogenicbacteriaandmaythereforemodulate
intestinalinflammation,aneffectthathasbeen
termedcontrabiotic.
Chemically,dietaryfiberconsistsofnon-
starchpolysaccharidessuchasarabinoxylans,cellulose,
andmanyotherplantcomponentssuchasresistant
starch,resistantdextrins,inulin,lignin,chitins,pectins,
beta-glucans,andoligosaccharides.
Dietary Fiber Cont’d
Theterm"fiber"issomethingofamisnomer,sincemany
typesofsocalleddietaryfiberarenotactuallyfibrous.
Foodsourcesofdietaryfiberareoftendividedaccording
towhethertheyprovidesolubleorinsolublefiber.Plant
foodscontainbothtypesoffiberinvaryingdegrees,
accordingtotheplant'scharacteristics.
Advantagesofconsumingfiberaretheproductionof
healthfulcompoundsduringthefermentationofsoluble
fiber,andinsolublefiber'sabilitytoincreasebulk,soften
stool,andshortentransittimethroughtheintestinal
tract.
Adisadvantageofadiethighinfiberisthepotentialfor
significantintestinalgasproductionandbloating.
Theterm"fiber"issomethingofamisnomer,sincemany
typesofsocalleddietaryfiberarenotactuallyfibrous.
Foodsourcesofdietaryfiberareoftendividedaccording
towhethertheyprovidesolubleorinsolublefiber.Plant
foodscontainbothtypesoffiberinvaryingdegrees,
accordingtotheplant'scharacteristics.
Advantagesofconsumingfiberaretheproductionof
healthfulcompoundsduringthefermentationofsoluble
fiber,andinsolublefiber'sabilitytoincreasebulk,soften
stool,andshortentransittimethroughtheintestinal
tract.
Adisadvantageofadiethighinfiberisthepotentialfor
significantintestinalgasproductionandbloating.
Dietary Fiber Cont’d
•Dietaryfiberistheediblepartsofplantsoranalogous
carbohydratesthatareresistanttodigestionandabsorptioninto
thehumansmallintestinewithcompleteorpartialfermentation
inthelargeintestine.Dietaryfiberincludespolysaccharides,
oligosaccharides,lignin,andassociatedplantsubstances.
•Dietaryfiberspromotebeneficialphysiologicaleffectsincluding
laxation,andbloodcholesterolattenuation,andbloodglucose
attenuation.In1970s,itwasunderstoodthatitisaresponseto
theobservedphysiologicalbehaviorofplantcellwallmaterialin
thehumandigestivesystemandthepostulatedbenefitsofthis
behavior.
•DigestiveEnzymesareimpermeableintoDietaryfiber.This
waslaterexpandedtoincludeallindigestiblepolysaccharides
such as gums, mucilages, modified celluloses,
oligosaccharides,andpectins.Theseadditionalsubstances
behavedphysiologicallyinasimilarmannertocompounds
includedintheoriginaldefinitionofDietaryfiber.Theywere
edibleandnotdigestedandnotabsorbedinthesmallintestine.
•Dietaryfiberistheediblepartsofplantsoranalogous
carbohydratesthatareresistanttodigestionandabsorptioninto
thehumansmallintestinewithcompleteorpartialfermentation
inthelargeintestine.Dietaryfiberincludespolysaccharides,
oligosaccharides,lignin,andassociatedplantsubstances.
•Dietaryfiberspromotebeneficialphysiologicaleffectsincluding
laxation,andbloodcholesterolattenuation,andbloodglucose
attenuation.In1970s,itwasunderstoodthatitisaresponseto
theobservedphysiologicalbehaviorofplantcellwallmaterialin
thehumandigestivesystemandthepostulatedbenefitsofthis
behavior.
•DigestiveEnzymesareimpermeableintoDietaryfiber.This
waslaterexpandedtoincludeallindigestiblepolysaccharides
such as gums, mucilages, modified celluloses,
oligosaccharides,andpectins.Theseadditionalsubstances
behavedphysiologicallyinasimilarmannertocompounds
includedintheoriginaldefinitionofDietaryfiber.Theywere
edibleandnotdigestedandnotabsorbedinthesmallintestine.
Dietary Fiber Cont’d
•Insolublefibersuchacelluloseisassociatedwithincreasing
fecalbulkandimprovinglaxation.
•Solublefiber,whichincludesβ-glucan,psylliumgum,and
pectin,isassociatedwithloweringcholesterolandmoderating
postprandialbloodglucoselevels.Solublefiberisbelievedto
bindcholesterolandbileacids,increasingtheirexcretion.
•Alternately,ithasbeensuggestedthatshortchainfattyacids
producedbybacterialfermentationoffibermaybeneficially
affectlipidmetabolism.Withrespecttoplasmaglucoselevels,
itisthoughtthatviscositydevelopmentinthegutcausedby
thepresenceofsolublefiber(β-glucan,psylliumgum,andguar
gum)themovementofglucoseintothebloodstream,
flatteningthebloodglucosepeakthatusuallyoccursaftera
meal.Additionally,manyfibersourcescontainbothsoluble
andinsolublefiberandthereforeprovidethebenefits
associatedwithbothtypesoffiber.
•Insolublefibersuchacelluloseisassociatedwithincreasing
fecalbulkandimprovinglaxation.
•Solublefiber,whichincludesβ-glucan,psylliumgum,and
pectin,isassociatedwithloweringcholesterolandmoderating
postprandialbloodglucoselevels.Solublefiberisbelievedto
bindcholesterolandbileacids,increasingtheirexcretion.
•Alternately,ithasbeensuggestedthatshortchainfattyacids
producedbybacterialfermentationoffibermaybeneficially
affectlipidmetabolism.Withrespecttoplasmaglucoselevels,
itisthoughtthatviscositydevelopmentinthegutcausedby
thepresenceofsolublefiber(β-glucan,psylliumgum,andguar
gum)themovementofglucoseintothebloodstream,
flatteningthebloodglucosepeakthatusuallyoccursaftera
meal.Additionally,manyfibersourcescontainbothsoluble
andinsolublefiberandthereforeprovidethebenefits
associatedwithbothtypesoffiber.
THERE ARE TWO FUNDAMENTALLY DIFFERENT
APPROACHES TO ANALYZE DIETARY FIBER:
1)Uppsala Method
2)Enzymatic -Gravimetric Methods
Uppsala Method
TheUppsalamethodisbasedonthe
priorremovaloffreesugarsandstarch,
includingtreatmentwiththermostable
amylases,followedby the
determinationofneutralnon-starch
polysaccharideresiduesasalditol
acetatesbyGLC.
APPROACHES TO ANALYZE DIETARY FIBER:
1)Uppsala Method
2)Enzymatic -Gravimetric Methods
Uppsala Method
TheUppsalamethodisbasedonthe
priorremovaloffreesugarsandstarch,
includingtreatmentwiththermostable
amylases,followedby the
determinationofneutralnon-starch
polysaccharideresiduesasalditol
acetatesbyGLC.
Uppsala Method
The Uppsala method requires measuring the
NEUTRAL SUGARS,
URONIC ACIDS (PECTIN MATERIAL),
KLASON LIGNIN (NON-CARBOHYDRATE DIETARY FIBER)
and summing these components to obtain a dietary fiber value.
Procedure:
Thesampleisfirstsubjectedtoanamylaseandamyloglucosidase
enzyme-digestiontoremovestarch.Starchhydrolysatesand
lowmolecularweightsugarsareseparatedfromsolublefiber
usingan80%ethanolprecipitation,leavingaresiduecontaining
bothsolubleandinsolublefiber.Neutralsugarsare
determinedafterderivitisationastheiralditolacetatesbyGC,
uronicacidsareassayedcolorimetrically,andKlasonligninis
determinedgravimetrically.
The Uppsala method requires measuring the
NEUTRAL SUGARS,
URONIC ACIDS (PECTIN MATERIAL),
KLASON LIGNIN (NON-CARBOHYDRATE DIETARY FIBER)
and summing these components to obtain a dietary fiber value.
Procedure:
Thesampleisfirstsubjectedtoanamylaseandamyloglucosidase
enzyme-digestiontoremovestarch.Starchhydrolysatesand
lowmolecularweightsugarsareseparatedfromsolublefiber
usingan80%ethanolprecipitation,leavingaresiduecontaining
bothsolubleandinsolublefiber.Neutralsugarsare
determinedafterderivitisationastheiralditolacetatesbyGC,
uronicacidsareassayedcolorimetrically,andKlasonligninis
determinedgravimetrically.
2) Enzymatic -Gravimetric Methods
•Thesecondmethodisameasurebydifferencewhere
dietaryfiberresidueisisolated,dried,weighed,and
thenthisweightisadjustedfornon-dietaryfiber
material.Enzymatic-gravimetricofficialmethodinvolves
subjectingasampletoasuccessionofenzymes(α-
amylase,amyloglucosidase,protease)toremove
digestiblematerial,anethanolprecipitationstepto
isolatenon-starchpolysaccharide,andfinallyanashand
proteindetermination.
•Thequantityofproteinandashissubtractedfromthe
dryresidueweighttoobtainatotaldietaryfibervalue.
Thismethodhasbeenmodifiedtopermitthe
determinationoftotalsolubleandinsolubledietary
fiber.
•Thesecondmethodisameasurebydifferencewhere
dietaryfiberresidueisisolated,dried,weighed,and
thenthisweightisadjustedfornon-dietaryfiber
material.Enzymatic-gravimetricofficialmethodinvolves
subjectingasampletoasuccessionofenzymes(α-
amylase,amyloglucosidase,protease)toremove
digestiblematerial,anethanolprecipitationstepto
isolatenon-starchpolysaccharide,andfinallyanashand
proteindetermination.
•Thequantityofproteinandashissubtractedfromthe
dryresidueweighttoobtainatotaldietaryfibervalue.
Thismethodhasbeenmodifiedtopermitthe
determinationoftotalsolubleandinsolubledietary
fiber.
2) Enzymatic -Gravimetric Methods cont’d
•With appropriate methods forthe
measurement ofthemajorityofdietary
fiberconstituents,recenteffortshave
focusedonestablishingofficialmethods
forclassesofcompounds thatbehave
physiologicallylikedietaryfiber,butare
solublein80%ethanolandhence
excludedfromcommonly usedofficial
methods formeasuring dietaryfiber.
Thesecompounds includefructans,which
arefoundinonions,leeks&chicory.
•With appropriate methods forthe
measurement ofthemajorityofdietary
fiberconstituents,recenteffortshave
focusedonestablishingofficialmethods
forclassesofcompounds thatbehave
physiologicallylikedietaryfiber,butare
solublein80%ethanolandhence
excludedfromcommonly usedofficial
methods formeasuring dietaryfiber.
Thesecompounds includefructans,which
arefoundinonions,leeks&chicory.
2) Enzymatic -Gravimetric Methods cont’d
•Polydextrose,alsosolublein78%ethanol
owingtoitsextensivelybranchedstructure,can
beanalyzedusingthismethod.Fructansinclude
bothinulinandoligofructose,whichratherthan
beingsinglemolecular species,arepoly-
dispersemixturesoffructosepolymers.while
inulintypicallyrangesfrom~2to60,butthere
isvariationinthisrangedependingontheinulin
source.BecausetheDPofthesecompounds
varies,theirsolubilityin80%ethanolvaries,
andthereforetheirinclusionintotaldietary
fiber(TDF)measurements isnotcomplete.
•Polydextrose,alsosolublein78%ethanol
owingtoitsextensivelybranchedstructure,can
beanalyzedusingthismethod.Fructansinclude
bothinulinandoligofructose,whichratherthan
beingsinglemolecular species,arepoly-
dispersemixturesoffructosepolymers.while
inulintypicallyrangesfrom~2to60,butthere
isvariationinthisrangedependingontheinulin
source.BecausetheDPofthesecompounds
varies,theirsolubilityin80%ethanolvaries,
andthereforetheirinclusionintotaldietary
fiber(TDF)measurements isnotcomplete.
2) Enzymatic -Gravimetric Methods Cont’d
Giventhenutritionalimplicationsofdietaryfiberintake,
itsquantitativedeterminationinfoodproductsisvery
important,butthemeasurementofdietaryfiberhas
beenhamperedbycontroversiessurroundingitsvery
definitionandthefactthatdietaryfiberencompassesa
classofcompoundswithdisparateproperties.
Therearenowseveralmethodsinplacethatpermit
themeasurementofTDFandadditionalmethods
tospecificallymea-surethosecompounds(i.e.,
polydextroseandfructans)excludedfrom
conventionaldietaryfibermethods
Giventhenutritionalimplicationsofdietaryfiberintake,
itsquantitativedeterminationinfoodproductsisvery
important,butthemeasurementofdietaryfiberhas
beenhamperedbycontroversiessurroundingitsvery
definitionandthefactthatdietaryfiberencompassesa
classofcompoundswithdisparateproperties.
Therearenowseveralmethodsinplacethatpermit
themeasurementofTDFandadditionalmethods
tospecificallymea-surethosecompounds(i.e.,
polydextroseandfructans)excludedfrom
conventionaldietaryfibermethods
.
THANK YOU
THANK YOU
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