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Chemical Analysis: Detecting Sugars, Proteins and Lipids

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1 Chemical Analysis: Detecting Sugars, Proteins and Lipids
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 % 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

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
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

10 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

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