Download Lab 4 - Chemical Analysis of Organic

LAB 4
Chemical Analysis: Detecting Sugars,
Proteins and Lipids
Detection of Organic molecules using
stains
• materials:
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–
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–
Onion
Potato
Apple
Peanut
• stains:
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–
–
–
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
Open, reducing form of glucose
• 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
Benedict’s
Sugar
aldehyde
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
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–
–
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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
-presence of reducing sugars
-presence of proteins
– 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
Explanation of results
• Apple: +ve Benedict’s
-presence of reducing sugars
85% water
-presence of starch
0.3% protein – 11 types of amino acids
-not enough proteins or fats
0.4% fats
12% carbohydrates – all three monosaccharides +
starch
– 2.3% fiber
– 0.6% organic acids – e.g. citric acid
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–
–
–
• Potato: +ve Potassium Iodide
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–
–
–
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78% water
2.2% protein
0.1% fat
18% starch
0.4% fiber
-presence of starch
-not enough proteins or fats