FOOD ANALYSIS: Carbohydrate Analysis B. Pam Ismail [email protected] FScN 146 612 625 0147 FOOD ANALYSIS: Carbohydrate Analysis The following is/are a carbohydrate(s): A. Pectin B. Cellulose C. Lignin D. A & B E. B & C F. A & C G. All of the above H. None of the above 1 FOOD ANALYSIS: Carbohydrate Analysis The following is/are a carbohydrate(s): A. Pectin B. Cellulose C. Lignin D. A & B E. B & C F. A & C G. All of the above H. None of the above FOOD ANALYSIS: Carbohydrate Analysis The method used to determine starch gelatinization could be used to determine starch retrogradation A. True B. False 2 FOOD ANALYSIS: Carbohydrate Analysis Importance of Carbohydrates Importance of Analyzing Carbohydrates FOOD ANALYSIS: Carbohydrate Analysis Carbohydrate Classification CH2OH CH2OH HO O O OH OH OH Monosaccharides O Di and oligosaccharides (2-10 units) OH OH Polysaccharides CH2OH HOH C O O 2 OH o Starch HO O HO CH2OH o Dietary fiber OH OH CH2OH CH2OH O O OH OH O O O OH OH CH2OH CH2OH CH2OH CH2 CH2OH CH2OH O O O O OH OH O O OH OH OH OH O O O O O O O OH OH OH OH OH OH 3 FOOD ANALYSIS: Carbohydrate Analysis General Sample Preparation Drying o Vacuum oven Fat extraction o Soxhlet FOOD ANALYSIS: Carbohydrate Analysis Total Carbohydrate Analysis H (research purposes) HO O Phenol-sulfuric acid method O What happens to glycosidic linkages under acidic conditions? strongly acidic conditions furans monosaccharides (heat) CH2OH CH2OH O O enolizations, OH OH OH dehydrating reactions H O HO O OH OH O 4 FOOD ANALYSIS: Carbohydrate Analysis Total Carbohydrate Analysis (research purposes) Phenol-sulfuric acid method Furfural derivatives (furans) react with phenols to produce colored compounds (triarylmethane dyes) Absorption maxima between 480 and 490 nm OH OH absorption @ 490 nm concentration + H2SO4 2 O R OHC O R OH Absorption @ 490 nm 490 Absorption @ Not a Stoichiometric reaction concentration FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Somogyi-Nelson Colorimetric Munson-Walker Based on reducing Methods Cu+2 to Cu+1 Lane-Eynon Paper and TLC Chromatographic Methods HPLC GC Enzymatic Methods 5 FOOD ANALYSIS: Carbohydrate Analysis Extraction Solvent extraction o mono- and disaccharides are soluble in water and also in 80% ethanol o most polysaccharides and proteins are insoluble in 80% ethanol o mono/disaccharide extraction using hot 80% ethanol o neutral pH conditions required to prevent hydrolysis Carrez treatment o clarification with Carrez-reagents o Potassium hexacyanoferrate K4[Fe(CN)6] solution, ZnSO4 o Breaks emulsions o precipitates proteins and partially polysaccharides o absorbs colors FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Colorimetric For reducing sugars Methods Principle: Based on reduction of cupric ions in alkaline solution to cuprous ions by reducing sugars; reaction with a complex compound to generate color; amount of cuprous ions generated is measured to quantitate reducing sugars (gravimetric or titration) 6 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Colorimetric For reducing sugars Methods Principle: Based on reduction of cupric ions in alkaline solution to cuprous ions by reducing sugars; reaction with a complex compound to generate color; amount of cuprous ions generated is measured to quantitate reducing sugars (gravimetric or titration) CHO COOH H OH Cu2+ H OH HO H Cu2O + HO H - H OH OH H OH H OH H OH CH OH 2 CH2OH What is wrong? FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Colorimetric For reducing sugars Methods Lane-Eynon Somogyi-Nelson Munson-Walker Cu(II) complexes and Quantification: reagents uses: -Determination of Cu2O by -Citrate gravimetric procedures or titrimetric procedures (sodium - ethylenediaminetetraacetic Differences in Methods acid (EDTA) thiosulfate or potassium permanganate - tartrate - spectrophotometric assays - pH and reagent to create (reduction of arsenomolybdate, alkaline conditions (NaOH, blue color – 520 nm) KOH, Na2CO3) 7 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Colorimetric For reducing sugars Methods What carbohydrates do you analyze using the Somogyi-Nelson method and other reduction methods? How can you analyze sucrose? CH2OH HOH C O O 2 OH HO O HO CH2OH OH OH FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Very popular approach in the field of carbohydrate Enzymatic analysis Methods Enzymes are proteins with powerful catalytic activity (lower the activation energy) High specificity for both the compound to be converted and for the type of reaction enzyme reactant enzyme product 8 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Enzymatic food analysis involves determination of food Enzymatic constituents which are usually substrates for enzymes Since the final determination is a spectrophotometric Methods assay solutions have to be clarified, sometimes enzyme- inhibitors have to be removed Very often two types of reaction are necessary since it is not possible to measure the reactant or the product Measuring the reaction food Auxiliary reaction constituent A + B P + Q Indicator reaction P + C R + S C, R or S FOOD ANALYSIS: Carbohydrate Analysis glucose Auxiliary reaction A + B P + Q D-Glucose + ATP Hexokinase Glucose-6-phosphate + ADP Indicator reaction NADPH P + C R + S Glucose-6-phosphate + NADP+ G6P-DH 6-Phosphogluconate + NADPH + H+ G6P-DH - Glucose-6-phosphate-Dehydrogenase 9 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Enzymatic Methods Example Applications: o Monosaccharides Fructose, Glucose, Galactose o Oligosaccharides Lactose, Maltose, Sucrose o Polysaccharides Amylose, Amylopectin FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Enzymatic Methods Advantages: Usually specific for substance being measured Kits commercially available Generally have low detection limits Disadvantages: Interfering compounds maybe present Not always 100% specific 10 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Chromatographic Paper and TLC Methods mostly qualitative information, however, TLC also used for quantitation Visualized by autoradiography FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Chromatographic Paper and TLC Methods TLC: stationary phases: cellulose, silica or impregnated silica mobile phase, e.g.: similar to paper Paper chromatography: Chromatography; 1-butanol-acetone-H2O stationary phase: cellulose filter; 4:5:1 (v/v); 1-butanol-ethanol-H2O mobile phase: e.g. n-propanol/ethyl 2:1:2 (v/v) (for acetate impregnated silica) acetate/H2O (65/10/25 v/v/v); Silica impregnation: acetates (0.02 – 0.2 M) or phosphates (0.07 – 0.5M) improved separation/resolution, spots are sharpened 11 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Chromatographic HPLC Methods Anion-Exchange HPLC Normal-Phase HPLC Cation-Exchange HPLC Reversed-Phase HPLC FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Normal-Phase HPLC Stationary phase: unmodified silica strong interactions between free silanol groups of the silica gel and the hydroxyl groups of the carbohydrates good separation of carbohydrates almost impossible Use chemically modified silica stationary phases! Also called HILIC (Hydrophilic Interaction Liquid Chromatography) 12 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Normal-Phase HPLC Chemically modified silica stationary phases (HILIC): amine-bonded stationary phases (e.g. -C3H6NH2) mobile phase usually acetonitrile/water (50-85% acetonitrile) separation may be influenced by the column temperature formation of hydrogen bonds between amino groups and hydroxyl groups FOOD ANALYSIS: Carbohydrate Analysis Recap Questions How do we measure total carbohydrates? What are the preparatory steps for carbohydrate analysis? How do we extract mono, di and oligosaccharides? Name methods used to determine mono, di, and oligosaccharides What is the basis of carbohydrate analysis using enzymes? 13 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Normal-Phase HPLC ? Amine-bonded stationary phases Detection: usually RI-detection Drawbacks: gradient elution is not possible, insensitive and highly unspecific FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Normal-Phase HPLC Drawbacks of amine-bonded stationary phases: H+ O H OH H limited column life due to the formation C H+ of Schiff bases between the amino H C OH H C NR HNRC R-NH2 group and the sugar molecules HO C H H C OH H C OH -H O H C OH HO C H 2 HO C H H C OH H C OH H C OH Modifiers CH2OH H C OH H C OH CH2OH CH OH Glucoseglucose 2 ImineImin (Schiff Other modifications available for uses in carbohydrate analysis: base) … diol-bonded (e.g., -C3H6OC2H3OHCH2OH) stationary phases cyano-bonded stationary phases (e.g., -C2H4CN) 14 FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Reversed-Phase HPLC Stationary phase: e.g. RP-18-columns Used for the analysis of gluco-oligomers with water as mobile phase Separation of different monosaccharides is difficult….Why? What can be done? Reversed-phase chromatography is usually affected by anomers, resulting in peak doubling or broadening (use amine groups in mobile phase to acceleraate anomerization) FOOD ANALYSIS: Carbohydrate Analysis Mono- and Oligosaccharide Analysis Anion-Exchange HPLC In solution of high pH carbohydrate hydroxyl groups