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! . 1 Carbohydrate analysis Pham Van Hung, PhD Carbohydrate • Monosaccharides – Glucose – Fructose – Galactose •. Polysaccharides Determination Sample preparation 2 Phenol-Sulfuric acid (total carbohydrate) • Dehydration reaction • Produce various furan derivatives • Condense