LAB 4

1. LAB 4 Chemical Analysis: Detecting Sugars, Proteins and Lipids

2. Detection of Organic molecules using stains • materials: • Onion • Potato • Apple • Peanut • stains: • Benedict’s - presence of reducing sugars • Biuret’s – presence of proteins • Sudan IV – presence of fats • Potassium Iodide presence of starch/polysaccharides

3. Potassium Iodide • starch divided up into two fractions • amylose – helical and unbranched • amylopectin - branched • amylose forms a blue-black complex when it combines with the iodide ions found in a potassium iodide solution made for detecting starches • this potassium iodide solution is also called Lugol’s reagent • actually a mixture of potassium iodide (soluble)and elemental iodine (insoluble) • results in a soluble solution containing triiodide • the Lugol’s reagent interacts with the coiled structure of the amylose fraction of the starch – blue black color • specifically the triiodine molecule interacts with the polysaccharide • no color produced by amylopectin or monosaccharides or dissacharides

4. Benedict’s Reagent • used to detect the presence of reducing sugars • reducing sugar – sugar that in its open form bears aldehyde group • all monosaccharides (reagent converts fructose to glucose) • maltose, lactose • in nature – many foods possess both closed ring and open linear forms of sugars • under heat - copper of the reagent reacts with the aldehyde of the sugar to turn a specific color aldehyde Open, reducing form of glucose

5. Benedict’s Reagent • copper is reduced by the sugar to form copper oxide (red, insoluble) • color denotes amount of reaction (i.e. approximate amount of reducing sugar) • green - 0.5% sugar • many dissacharides produce this color • yellow – 1.0% • red - 2% or more • many monosaccharides produce this color • sucrose is not detected by this reagent – not a reducing sugar • many polysaccharides such as starch begin with a reducing sugar • may not be sufficient to turn the reagent’s color aldehyde Open, reducing form of glucose Benedict’s Copper Oxide Sugar

6. Biuret’s Reagent • chemical reagent that detects the presence of peptide bonds • presence of peptide bonds can produce a pink to violet color • pink – short chain polypeptides • violet – longer proteins • spectrophotometers can be used to measure the absorbance of these colored solutions = protein concentration • biuret = chemical compound with the formula H2NC(O)NHC(O)NH2 • yet Biuret’s reagent doesn’t contain biuret • it gives a positive reaction same as the peptide-like bonds in the biuret molecule • BR = potassium hydroxide and copper • potassium hydroxide increases the pH of the solution (alkaline) • copper forms violet-colored complexes in the alkaline environment of the test • the copper sulfate of the reagent water BR albumin BR + albumin

7. Sudan Staining • several different kinds of Sudan stains • all are used to stain lipids • different colors • Sudan III = red • Sudan IV = black • Sudan IV stains lipid droplets black in slide preparations • also can stain in liquid preparations Sudan III staining = oil and water

8. Explanation of results • Benedict’s • Onion – reddish color (+ve) • Peanut – blue (-ve) • Apple - +ve • Potato – yellowish color (+ve) • Biuret • Onion – +ve (pink) • Peanut - +ve (purple) • Apple – -ve (blue) • Potato – -ve (blue) • Sudan IV • Potassium Iodide • potato, apple – black staining, +ve result • peanut, onion – little to no staining • peanut – kind of gray???

9. Explanation of results -presence of reducing sugars -presence of proteins • Onion: +ve Benedict’s, +veBiuret • 1% protein (sulfurous odor of the onion) • 4% sugar – glucose, fructose and sucrose • fructose the most • sugar content increases with maturation • 2% soluble fiber in flesh – insoluble fiber in the skin • 0.1% fat • 98% water • Peanut: +ve Biuret, +ve Sudan IV • 25% protein – source of the essential amino acids histidine and arginine • 48% fat (7% saturated, 24% mono, 16% poly) – 41% considered oil • numerous fatty acids – palmitic, stearic, oleic, linolenic, linoleic • 0% sugar; 1% cellulose, 9% fiber • 21% carbohydrates – NO SIMPLE SUGARS -presence of fats -presence of proteins

10. Explanation of results -presence of reducing sugars -presence of starch -not enough proteins or fats • Apple: +ve Benedict’s • 85% water • 0.3% protein – 11 types of amino acids • 0.4% fats • 12% carbohydrates – all three monosaccharides + starch • 2.3% fiber • 0.6% organic acids – e.g. citric acid • Potato: +ve Potassium Iodide • 78% water • 2.2% protein • 0.1% fat • 18% starch • 0.4% fiber -presence of starch -not enough proteins or fats