lecture 10 carbohydrate analysis

Phenol-Sulfuric acidtotal carbohydrate • Dehydration reaction • Produce various furan derivatives • Condense with themselves and other product to produce brown and black substances • C

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

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

Enzyme methods

• Glucose oxidase

• Hexokinase

Glucose Oxidase System

H2O2+ 0 - Dianisidine 2 H2O + Oxidized 0-Dianisidine

OCH3

H3CO

H3CO OCH3

Glucose Oxidasse

Peroxidase

Gluconic Acid, Glucouronic Acid and Gluconolactone

H

C

C OH C HO

C OH C

CH 2 O H O

O C

OH

C OH

C

HO

C OH

C

CH 2 O

O

H

H

H

H

H

H

C

H O

C OH C HO

C OH 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

G-6-Phosphate + NADP + Gluconate-6-phosphate + NADPH + H

H

C

C OH C HO

C OH C

CH 2 O P OH O

OH

O O

C

H OH

C OH

C

HO

C OH

C

CH 2 O P OH

O

OH

O

H

NADP

P

O

O O O

P O O

O CH2 CH2

O

OH OPO3H2

N

N N N NH2

O

OH OH

N CONH2

-+

NADPH

P

O

O O O

P O O

O CH 2

CH 2

O

N

N N N

NH 2

O

OH OH

-N

H H

+ 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

Requirement: Compounds must be volatile.

Sugars form Trimethylsilyl Ether

CH3

CH3

CH3 O

CH3

CH3

CH3

Ether linkage Trimethyl chlorosilane

Gas Chromatography

OH O

OH

OH HO

CH2OH

1

2 3 4 5 6

CH3

CH3 CH3

5Cl

O-Si(CH3)3 O

O-Si(CH3)3

O-Si(CH3)3 (CH3)3-Si-O

CH2O-Si(CH3)3

1

2 3 4 5 6

5HCl

+

Derivation of Glucose with Trimethylchlorosilane

Liquid Chromatography

Fructose

Glucose

Maltotriose Maltose

Stationary Phase: Waters Associates 840380

Solvent: H2O/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

-Total starch

The end!