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