Lecture 1.WET METHODS OF CARBOHYDRATE
ANALYSES
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. These OH’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
In Glucuronic acid C2, C4, C5 OH’s are on same side
C
CO2H
H
O
OHH
C
CO2H
H
O
OHH
OH
H
OHH
HO
H OH
H
H
H
HO
HO
glucuronic acid galacturonic acid
Alditols• In Mannitol C2, C4,
C5 OH’s are not at same side in Fisher Projection
CH2OH
CH2OH
H
H
OH
OH
H
H
HO
HO
Mannitol
CH2OH
OH
OH
H
CH2OH
H
HO
H
Xylitol
Conformations
O
OH
OH
OHOH
CH2OH
-D glucopyranose
O
OH
OH
OH
OHCH2OH
-D glucopyranose
[a]25D
+19o +112o
Anomers
For aged solutions [a] 25D
= +52.7o
Rotations of Fresh Solutions
Reason: Mutarotation is the best evidence for the cyclic hemiacetal structure of D-(+)-glucose
Monosaccharides,Hemiacetal Formation II
C
C
C
O H
CH2OH
C
C O
H
..HH
H
H
OH
OH
HO
OHC
C
C
O
CH2OH
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 H
OH
H
C5 OH attacks aldehyde giving a pyranose ring (6 member structure)
C4 OH attacks aldehyde giving a furanose ring (5 member structure)
O
OH
OH
OHOH
CH2OHO
O
OH
OH
OH
OHCH2OH
CHO
OHOH
OH
OH
CH2OH
OH
OHOH
HOCH2OH
O
OH
OH
OHHOCH2OH
CHO
H OH
HO H
H OH
H OH
CH2OH
CHOOH
OH
OH
D glucose
OH
-D glucopyranose
CH2OH
-D glucofuranose
Mutarotation
-D glucofuranose
-D glucopyranose
Ring closure between C1 and C4 -OH
Ring closure between C1 and C5 -OH
• 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 (depending on analytical techniques i.e GC vs LC/MS)
• Polysaccharides – refer to polymers composed of a large
number of monosaccharides linked by glycosidic linkages
ex. Cellulose
oxygen bridge (ether-type orglycosidic bond)
anhydro-glucopyranose unit
Cellobiose
n = 1 -5000
OHOH
HO
CH2OHOO
CH2OH
HOOH
OOH
HOHO
CH2OHO
OOH
OHO
CH2OHO
Cellulose
b-D-anhydroglucopyranose units linked by (1,4)-glycosidic bonds
O O
O OO
OO
OH
CH2OH
HOHO
HOOH
CH2OHHO
OH
CH2OH
CH2OH
OHOH
HO
3'
4'n
1
2
3
4
5
6
2'5'
6'
1'
(potential aldehyde)
Non-ReducingEnd-Group
ReducingEnd-Group
PolysaccharidesPolysaccharides can be divided into two classes
– Homopolysaccharides• consist of only one kind of monosaccharide
ex cellulose
– Heteropolysaccharides• consist of two or more kinds of
monosaccharides
ex galactoglucomannans
Polysaccharides
Polysaccharides can not only have different sequences of monosaccharide units, but also different sequences of glycosidic linkages and different kinds of branching
– a very high degree of diversity for polysaccharides and their structure-function relationships
Plant PolysaccharidesThe conformation of individual monosaccharide residues in a polysaccharide is relatively fixed, however, joined by glycosidic linkages, they can rotate to give different chain conformations.
OOHO
HOO
OH
OHO
HOO
OH
HO OHO
HOO
OHOHO
HO O
OH
1,4 glycosidiclinkage 1,6 glycosidic
linkage
The different kinds of primary structures that result in secondary and tertiary structures give different kinds of properties
– water solubility, aggregation and crystallization, viscosity, gelation, etc.
Polysaccharides have a variety of functions
– Storage of chemical energy in photosynthesis
– Inducing Structural Integrity in plant cell walls
Plant Polysaccharides
StarchStarch is composed completely of D-
glucose – found in the leaves, stems, roots,
seeds etc in higher plants–stores the chemical energy produced
by photosynthesisMost starches are composed of two types
of polysaccharides - amylose and amylopectin–amylose - a mixture of linear
polysaccharides of D-glucose units linked a-(1-4) to each other• between 250-5,000 glucose residues
Starch Polymer Components
Amylose
Amylopectin (1 residue in every 20 is 16 linked to branch off)
The Components of Starch
O
HOO
OH
OH
O
HO
HO
OH
OHO
O
OH
OH
O
O
OHO
HO OH
O
(1-4)
Amylose Amylopectin
(1-4)
(1-6)O
HOO
OH
OH
O
HOO OH
OH
O
HOO
OH
OH
OHO
OH
OO
HOO OH
OH
O
O
HOO
HO
OH
O
Starch tertiary structure (Helix)
QUALITATIVE ANALYSIS
There various tests that can be used to detect the presence or absence of carbohydrates or sugars. Some of these are:
• Molisch Reaction• Anthrone Reaction• Iodine Test • Benedict Test
MOLISCH REACTION
In this reaction the furfural that is formed from the carbohydrate by the sulfuric acid condenses with the phenol to give the characteristic color.
PROCEDURETwo ml of a sample solution is placed in a test tube. Two drops of the Molisch reagent (a solution of α-napthol in 95% ethanol) is added. The solution is then poured slowly into a tube containing two ml of concentrated sulfuric acid so that two layers form.
MOLISCH REACTION
A positive test is indicated by the formation of a purple product at the interface of the two layers.
a negative test (left) and a positive test (right)
ANTHRONE REACTION
Anthrone, 9,10-dihydro-9-ketoanthracene reacts with many carbohydrates to give a green color.
PROCEDURE
1 ml of a sample solution is placed in a test tube. 2ml of a 0.2% of Anthrone in conconcentrated sulfuric acid is added. In the presence of carbohydrates a clear green color will appear and will rapidly increase in intensity until a dark blue-green solution results.
QUANTITATIVE ANALYSIS
The quantitative methods for the estimation of sugars and carbohydrates depend on the properties of reduction and optical rotation that the sugars have. Some of the quantitative methods used are;
• Munson and Walker Method• Iodide-Thiosulfate Method• Lane-Eynon Titrimetric Method
LANE-EYNON METHOD
This is a short and rapid method and often the most accurate method for the estimation of reducing sugars. It is based on a determination of the volume of a test solution required required to reduce completely a known volume of alkaline copper reagent. The end point is indicated by the use of an internal indicator, methylene blue.
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 up
to 250ml in a volumetric flask.• The solution is shaken thoroughly and
filtered.• 10ml 10% solution of potassium oxalate is to
100ml of the filtrate and made up to 500ml, shaken and filtered
LANE-EYNON METHOD
PROCEDURE• 10ml of the mixed Fehling reagent is placed in a 250ml
Erlenmeyer flask.• The sugar solution is transferred into a burette and
suspended over the Erlenmeyer flask.• 15ml of the sugar solution is added to the flask and heated to
boiling.• The solution is boiled for about 15 seconds and portions of
the sugar solution is added rapidly until only the faintest perceptible blue color remains.
• 2-5 drops of a 1% aqueous solution of methylene blue is added and heating is continued.
• The sugar solution is added dropwise until the titrtion is complete which is shown by the reduction of the dye.
LANE-EYNON METHOD
The amount of sugar may be calculated by the formula;
The factor is obtained in Literature, in which the factor for each titration from 15 to 50ml is given.
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