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

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

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

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

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 Sugar Copper Oxide

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

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

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???

Explanation of results Onion: +ve Benedict’s, +ve Biuret 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 reducing sugars -presence of proteins -presence of fats -presence of proteins

Explanation of results 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 reducing sugars -presence of starch -not enough proteins or fats -presence of starch -not enough proteins or fats