lecture 10 carbohydrate analysis

1
Carbohydrate analysis
Pham Van Hung, PhD
Carbohydrate
• Monosaccharides
– Glucose
– Fructose
– Galactose
• Disaccharides
–Maltose
–Sucrose
– Lactose
• Olygosaccharides
• Polysaccharides
– Starch
–Pectin
– Cellulose
1. Monosaccharides and Oligosaccharides
A. Chemical Method
B. Enzymatic Method
1. Glucose oxidase
2. Hexokinase
C. Chromatography Method
1. Thin layer chromatography
2. Gas chromatography
3. Liquid column chromatography
2. Polysaccharides
Determination
Sample preparation
2
Phenol-Sulfuric acid

(total carbohydrate)
• Dehydration reaction
• Produce various furan derivatives
• Condense with themselves and other
product to produce brown and black
substances
• Condense with phenol to produce colored
compounds
Procedure
Somogyi-Nelson Method
(Total Reducing Sugar)
• Reducing sugar have an aldehyde group
• React with an oxidizing agent to produce a carboxylic
group.
• Cu(I) reduces an arsenomolybdate complex (ammonium
molybdate [(NH4)6Mo7O24]+sodium arsenate
(Na2HAsO7)+Sulfuric acid) to produce an intense, stable
blue color.
Procedure
3
Enzyme methods
• Glucose oxidase
• Hexokinase
Glucose Oxidase System
D-Glucose + O
2
Gluconic Acid + H
2
O
2

H
2
O
2
+ 0 - Dianisidine 2 H
2
O + Oxidized 0-Dianisidine
(Colorless) (Brown)

OCH
3
H
3
CO
H
2
NNH
2
H
3
CO OCH
3
HN
NH
Glucose Oxidasse
Peroxidase
Gluconic Acid, Glucouronic Acid and Gluconolactone
H
C
COH

CHO
COH
C
CH
2
O
H
O
O
C
OH
COH
CHO
COH
C
CH
2
O
O
HO
H
H
H
H
H
Gluconic Acid Gluconolactone
C
H
O
COH

CHO
COH
C
C
OH
O
OH
H
Glucouronic Acid
H
H
H
H
H
Hexokinase System
Glucose + ATP G - 6 - P + ADP
G - 6 - P + NADP
+
Gluconate - 6 - phosphate + NADPH + H
+
NADP
+
: Nicotinamide Adenine Dinucleotide Phosphate
NADPH : Reduced Nicotinamide Adenine Dinucleotide Phosphate
The NADPH formed in this reaction is stoichiometric with the amount of glucose.
NADPH is measured at 334 nm.
Hexokinase
G-6-P DH
G-6-P DH: Glucose-6-phosphate dehydrogenase
ATP: Adenosine triphosphate, coenzyme used as an energy carrier in the

cells of all known organisms
4
G-6-Phosphate + NADP
+
Gluconate-6-phosphate + NADPH + H
H
C
COH
CHO
COH
C
CH
2
OPOH
O
OH
O
O
+
NAPD
+
NADPH + H
+
C
H
OH
COH
CHO
COH
C

CH
2
OPOH
O
OH
O
H
NADP
+
P
O
O
O
O
P
O
O
O
CH
2
CH
2
O
OH
OPO3H2
N
N
N
N
NH

2
O
OH
OH
N
CONH
2
-
-
+
NADPH
P
O
O
O
O
P
O
O
O
CH
2
CH
2
O
OH
OPO3H2
N
N
N

N
NH
2
O
OH
OH
-
-
N
HH
+ H
+
CONH
2
Qualitative Analysis
Thin Layer chromatography
Silica gel as stationary phase
Quantitation by densitomer
A
B
C
A
B
S
Solvent
Rf =
A
S
For Spot A
5

Requirement: Compounds must be volatile.
Sugars form Trimethylsilyl Ether.
Si SiCl
CH
3
CH
3
CH
3
O
CH
3
CH
3
CH
3
Ether linkage Trimethyl chlorosilane
Gas Chromatography
OH
O
OH
OH
HO
CH
2
OH
1
2
3
4

5
6
+
Si
CH
3
CH
3
CH
3
5Cl
O-Si(CH
3
)
3
O
O-Si(CH
3
)
3
O-Si(CH
3
)
3
(CH
3
)
3
-Si-O
CH

2
O-Si(CH
3
)
3
1
2
3
4
5
6
5HCl+
Derivation of Glucose with Trimethylchlorosilane
Glucose Trimethylchlorosilane
Liquid Chromatography
Fructose
Glucose
Maltotriose
Maltose
Stationary Phase: Waters Associates. 840380
Solvent: H
2O/CH3CN
Polysaccharides
Quantification depends on chemical or enzymatic
hydrolyses of them into monomers and analysis of the
monomers.
Qualitative Analyses
1. Amylose + I
-
Blue color

Use spectrometer for determining the amylose - iodine blue
color
2. Amylopectin + I
-
Reddish color
6
Total starch
The end!