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Lecture 1.WET METHODS OF
CARBOHYDRATE ANALYSES
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Nomenclature of Carbohydrates• D, L Defines the configuration at C5
D has the OH at Right in Fischer projectionL has the OH at Left in Fischer projection
• Gluco defines the configuration of the OH at C2, C4, C5. TheseOH’s are on same side while the C3 -OH is opposite to others
• α ,β defines the configuration of the OH at C1, the anomeric carbon• Pyran indicates 6 member ring size• Furan indicates 5 member ring size
Examples follow
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In Glucuronic acid C2, C4, C5 OH’s areon same side
C
CO 2H
H
O
OHH
C
CO 2H
H
O
OHH
OH
H
OHH
HO
H OH
H
H
H
HO
HO
glucuronic acid galacturonic acid
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Alditols• In Mannitol C2, C4,
C5 OH’s are not atsame side in Fisher
Projection
CH2OH
CH2OH
H
H
OH
OH
H
H
HO
HO
Mannitol
CH2OH
OH
OH
H
CH2OH
H
HO
H
Xylitol
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Conformations
O
OH
OH
OHOH
CH 2 OH
-D glucopyranose
O
OH
OH
OH
OH
CH 2 OH
-D glucopyranose
[a ] 25D +19
o +112 o
Anomers
For aged solutions
[a ] 25D = +52.7 o
Rotations ofFreshSolutions
Reason: Mutarotation is the best evidence for the cyclichemiacetal structure of D-(+)-glucose
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Monosaccharides,Hemiacetal Formation II
C
C
C
O H
CH 2OH
C
C O
H
..HH
H
H
OH
OH
HO
OHC
C
C
O
CH 2OH
C
C
HH
H
H
OH
OH
HO H
O
C
C C
C
HC HHO
CH2OH
O
H
OHH
H OH H
.. O
C
C C
C
C HHO
CH2OH
OHH
H OH HOH
H
C5 OH attacks aldehyde giving a pyranose ring (6 member structure)
C4 OH attacks aldehyde giving a furanose ring (5 member structure)
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O
OH
OH
OH
OH
CH 2 OH
O
O
OH
OH
OH
OH
CH 2 OH
CHO
OH
OHOH
OH
CH 2 OH
OH
OH
OH
HO
CH 2 OH
O
OH
OH
OHHO
CH 2 OH
CHO
H OH
HO H
H OH
H OH
CH 2 OH
CHOOH
OH
OH
D glucose
OH
-D glucopyranose
CH 2 OH
-D glucofuranose
Mutarotation
-D glucofuranose
-D glucopyranose
Ring closure betweenC1 and C4 -OH
Ring closure betweenC1 and C5 -OH
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• Oligosaccharides – consist of several monosaccharide
residues joined together with glycosidic
linkages
– di, tri, tetrasaccharides
(depending on the number of monosaccharides)
– up to 10 - 20 monosaccharides (dependingon analytical techniques i.e GC vs LC/MS)
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• Polysaccharides – refer to polymers composed of a large
number of monosaccharides linked by
glycosidic linkagesex. Cellulose
oxygen bridge (ether-type or glycosidic bond)
anhydro-glucopyranose unit
Cellobiose
n = 1 -5000
OH
OH
HO
CH2OHOO
CH2OH
HO
OH
OOH
HOHO
CH2OHO
OOH
OHO
CH2OHO
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Cellulose
-D-anhydroglucopyranose unitslinked by (1,4)-glycosidic bonds
O O
O OO
OO
OH
CH2OHHO
HOHO
OH
CH2OHHO
OH
CH 2OH
CH 2OH
OH
OHHO
3'
4'n
1
2
3
4
5
6
2'5'
6'
1'
(potential aldehyde)
Non-ReducingEnd-Group
ReducingEnd-Group
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Polysaccharides
Polysaccharides can be divided into two classes – Homopolysaccharides
• consist of only one kind of monosaccharideex cellulose
– Heteropolysaccharides• consist of two or more kinds of
monosaccharidesex galactoglucomannans
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Polysaccharides
Polysaccharides can not only havedifferent sequences of monosaccharideunits, but also different sequences of
glycosidic linkages and different kinds ofbranching – a very high degree of diversity for
polysaccharides and their structure-function relationships
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Plant Polysaccharides
The conformation of individualmonosaccharide residues in a polysaccharideis relatively fixed, however, joined byglycosidic linkages, they can rotate to give
different chain conformations.
OOHO
HOO
OH
O
HOHO
O
OH
HO O
HOHO
O
OHOHO
HOO
OH1,4 glycosidiclinkage 1,6 glycosidic
linkage
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The different kinds of primary structuresthat result in secondary and tertiarystructures give different kinds ofproperties
– water solubility, aggregation andcrystallization, viscosity, gelation, etc.
Polysaccharides have a variety offunctions
– Storage of chemical energy in
photosynthesis
Plant Polysaccharides
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StarchStarch is composed completely of D-
glucose – found in the leaves, stems, roots,
seeds etc in higher plants
– stores the chemical energy producedby photosynthesisMost starches are composed of two types
of polysaccharides - amylose andamylopectin – amylose - a mixture of linear
polysaccharides of D-glucose units
linked -(1-4) to each other
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Starch Polymer Components
Amylose
Amylopectin (1 residue in every 20 is 1 6 linked to branch off)
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The Components of Starch
O
HOO
OH
OH
O
HO
HO
OH
OHO
O
OH
OH
O
O
OHO
HO OH
O
(1-4)
AmyloseAmylopectin
(1-4)
(1-6)O
HOO
OH
OH
O
HOO OH
OH
O
HOO
OH
OH
O
HOOH
OO
HOO OH
OH
O
O
HOO
HO
OH
O
Starch tertiary structure (Helix)
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QUALITATIVE ANALYSIS
There various tests that can be used todetect the presence or absence ofcarbohydrates or sugars. Some of theseare:
• Molisch Reaction• Anthrone Reaction• Iodine Test• Benedict Test
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MOLISCH REACTION
In this reaction the furfural that is formed fromthe carbohydrate by the sulfuric acidcondenses with the phenol to give the
characteristic color.PROCEDURETwo ml of a sample solution is placed in atest tube. Two drops of the Molisch reagent(a solution of α-napthol in 95% ethanol) isadded. The solution is then poured slowlyinto a tube containing two ml of concentratedsulfuric acid so that two layers form.
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MOLISCH REACTION
A positive test is indicated by the formationof a purple product at the interface of thetwo layers.
a negative test (left) and a positive test
(right)
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ANTHRONE REACTION
Anthrone, 9,10-dihydro-9-ketoanthracenereacts with many carbohydrates to give agreen color.
PROCEDURE1 ml of a sample solution is placed in a testtube. 2ml of a 0.2% of Anthrone in
conconcentrated sulfuric acid is added. In thepresence of carbohydrates a clear greencolor will appear and will rapidly increase inintensity until a dark blue-green solutionresults.
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QUANTITATIVE ANALYSIS
The quantitative methods for theestimation of sugars and carbohydratesdepend on the properties of reduction andoptical rotation that the sugars have.Some of the quantitative methods usedare;
• Munson and Walker Method• Iodide-Thiosulfate Method• Lane-Eynon Titrimetric Method
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LANE-EYNON METHOD
This is a short and rapid method and oftenthe most accurate method for theestimation of reducing sugars. It is basedon a determination of the volume of a testsolution required required to reducecompletely a known volume of alkaline
copper reagent. The end point is indicatedby the use of an internal indicator,methylene blue.
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LANE-EYNON METHOD
SAMPLE PREPARATION• 12.5g of the sample is dissolved in water.• 25ml of 10% neutral lead acetate solution is
added.• Some alumina cream is added and made upto 250ml in a volumetric flask.
• The solution is shaken thoroughly and
filtered.• 10ml 10% solution of potassium oxalate is to100ml of the filtrate and made up to 500ml,shaken and filtered
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LANE-EYNON METHODPROCEDURE
• 10ml of the mixed Fehling reagent is placed in a 250mlErlenmeyer flask.
• The sugar solution is transferred into a burette andsuspended over the Erlenmeyer flask.
• 15ml of the sugar solution is added to the flask and heated toboiling.
• The solution is boiled for about 15 seconds and portions ofthe sugar solution is added rapidly until only the faintestperceptible blue color remains.
• 2-5 drops of a 1% aqueous solution of methylene blue isadded and heating is continued.• The sugar solution is added dropwise until the titrtion is
complete which is shown by the reduction of the dye.
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LANE-EYNON METHOD
The amount of sugar may be calculated bythe formula;
The factor is obtained in Literature, inwhich the factor for each titration from 15to 50ml is given.
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