Biomolecule Identification © Copyright 2012 by Dr. Raj Bawa. All rights reserved. Contact Information: Bawa Biotech LLC, 21005 Starflower Way, Ashburn, Virginia 20147, USA (Tel: 703‐582‐1745; 703‐723‐0034; Fax: 571‐223‐1844; Email: [email protected]).
All experiments will be performed by groups of 4-5 students at lab tables under the supervision of Dr. Bawa.
Each group should obtain the following materials and stock solutions before beginning the experiments.
Equipment and Materials (per group)
Salad oil (lipid), glucose, starch, egg albumen
Benedict's reagent
Biuret reagent
Iodine reagent (iodine potassium iodide or IKI)
Sudan IV
Disposable pipettes
24 clean test tubes
3 beakers – to mix stock solutions
1-liter beaker – as the water bath
Weighing paper – for balance
Unknown Samples
Distilled water
Hot plate
Stirring rods
Wax pencil
Toothpicks
Stock Solutions
(1)
1% glucose stock solution - Weigh 1.0 g glucose on the balance and add it to 100 ml warm
distilled water in a beaker. Stir until dissolved.
(2)
0.5% starch stock solution - Add 0.5 g cornstarch to 100 ml warm water in a beaker. Stir until
dissolved.
(3)
10% protein stock solution - Dissolve 1 g dry egg albumen in 10 ml warm water.
I. Benedict’s Test ‐ Detection of Reducing Sugars (Monosaccharides and Disaccharides) The Benedict’s test is a semi-quantitative test for measuring the amount of reducing sugars like glucose.
Benedict's reagent is a blue solution which, when heated in the presence of simple sugars
(monosaccharides) or some disaccharides, turns a range of different colors depending on the amount of the
sugar present. The color change is dependent upon the reduction (gain of electrons) of the aldehyde and
ketone groups present in the reducing sugar molecules. In other words, the sugar molecule acts as a
reducing agent (itself gets oxidized) and brings about reduction of the Benedict’s reagent. In an alkaline
solution, sugars can reduce (i.e., donate or give electrons) various metallic compounds to free metals and
other forms.
Benedict's reagent is an alkaline solution of copper sulfate (giving it its blue color), sodium citrate, and
sodium carbonate. If the mixture of glucose and Benedict's reagent is heated, the soluble, blue, divalent
copper ion (cupric copper or Cu++) of the copper sulfate is reduced to the insoluble, red-orange, monovalent copper ion (cuprous copper or Cu+) of cuprous oxide (Cu2O), giving the solution its reddish-orange
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color. However, note that depending on the amount of reducing sugar present in your sample, the
precipitate that is formed at the bottom of the test tube can range in color from green to yellow to orangered to brown:
green orange red brown
Benedict’s test identifies reducing sugars. All monosaccharides and almost all disaccharides (except
sucrose) are able to reduce the Benedict’s reagent. In other words, when the Benedict’s reagent is added
to a sample containing reducing sugars and then the mixture is heated, the blue solution will change to
reddish-orange. The disaccharide sucrose does not reduce the Benedict’s reagent because its functional
groups are involved in bonding two consecutive monosaccharides together and thus not available for a
chemical reaction with the Benedict’s reagent. Starches are also non-reducing sugars.
Q. Identify the test tube below that contains glucose:
Determine the accuracy and sensitivity of Benedict's reagent by testing it against solutions of protein, lipids,
starch. Number test tubes #1-5. Always place numbers near the top of the test tube so that they will not melt
off in the water bath. Pipette 1 ml of the indicated solution into each test tube:
#1 - 1% glucose
#2 - 0.5% starch
#3 - lipid
#4 - protein
#5 - distilled water (control)
#6 - unknown Sample
Add 1 ml Benedict's reagent to all tubes (#1 - #6), swirl to mix, and then place all tubes simultaneously in
a large beaker 1/3 full of tap water boiling on a hot plate. Heat tubes for 2-3 minutes, then remove the
tubes with a test tube holder, remix, and allow cooling. Observe and record all color changes, and volume
and color of precipitate formed. Be sure to discuss your results that you record in the following table with
Dr. Bawa:
Test Tube
#1
#2
#3
#4
#5
#6
Solution Tested
Color/Results
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Q. Did you get a positive Benedict test in the starch tube? Why?
Q. Why was this test negative for starch, a substance composed of many glucose molecules?
Q. Why did we include a test tube containing distilled water only?
Q. What is a monomer and what is a polymer? Give examples of each.
Q. How do you know that the formation of the reddish-orange precipitate was not the result of
impurities in the water used to formulate the sample solutions #1 to #4?
Q. Is there a relationship between the intensity of the color formed and the glucose concentration?
Q. What is a positive control? What is a negative control? Give examples of each.
II. Iodine Test ‐ Detection of Starch/Polysaccharide/Carbohydrate A blue-black dark precipitate results when the yellow iodine solution is added to starches (amylose). Note
that the color may range from brown to blue-black and depends upon the amount of starch present. The
dye is thought to be trapped within the structure of the large starch molecule. Number test tubes #1-6 and
pipette 1 ml of indicated solutions into each:
#1 - 1% glucose
#2 - 0.5% starch
#3 - lipid
#4 - protein
#5 - distilled water (control)
#6 - unknown Sample
Add 2-3 drops of iodine solution into each tube. Swirl tubes to mix. Observe and record results below:
Test Tube
#1
#2
#3
#4
#5
#6
Solution Tested
Color/Results
Q. Did any substance tested above (other than starch) show a positive test? If so, explain.
Q. If the starch sample is heated for 5-10 minutes on the water bath, could you get a positive Benedict’s
test for reducing sugars in a sample that contain starch? Why? [Hint: hydrolysis]
Q. Define monomers and polymers. Give examples of each.
III. Sudan IV Test ‐ Detection of Lipids/Fats/Triglycerides Dyes are often used to indicate the presence of certain types of molecules. They are especially effective
when they attach or form complexes with a specific class of molecules in a mixture. Such is the case with
Sudan IV, which binds with non-polar lipid molecules but does not bind to polar molecules such as water
(“like dissolves like”).
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A positive Sudan IV test is shown below:
The generalized structure of a lipid is shown below:
Number test tubes #1-6 and pipette 1 ml of the indicated solution into each:
#1 - 1% glucose
#2 - 0.5% starch
#3 - lipid
#4 - protein
#5 - distilled water (control)
#6 - unknown Sample
Add a very small amount of Sudan IV dye (powder) into each test tube using a toothpick. This may be
accomplished by picking up a few grains of the dye with a toothpick and then dropping the toothpick
containing the Sudan IV dye powder into the solution. Swirl tubes to mix. Note and record all color
changes in the table below:
Test Tube
Solution Tested
Color/Results
#1
#2
#3
#4
#5
#6
Q. Did the Sudan IV dye dissolve in any of the tubes that contained water?
Q. In what substance did it dissolve?
Q. What can you conclude about the solubility of the Sudan IV dye?
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IV. Biuret Test ‐ Identification of Proteins Biuret reagent is a blue solution containing sodium hydroxide (NaOH) and copper sulfate (CuSO4) (It
does not contain any Biuret compound). The copper atoms of the Biuret reagent react with peptide bonds
of a protein or polypeptide (small fragments of a protein) to produce a violet-purple color change. A deep
violet color indicates the presence of proteins and a light pink color indicates the presence of peptides.
The reaction of the Biuret solution with peptide bonds of results in the formation of the Biuret compound
as shown below:
Number test tubes #1-6 and pipette 1 ml of the indicated solution into each:
#1 - 1% glucose
#2 - 0.5% starch
#3 - lipid
#4 - protein
#5 - distilled water (control)
#6 - unknown Sample
To each test tube, add 5 to 6 drops of the Biuret reagent. If a purple-violet color is not obtained, add more
of the reagent drop-by-drop, but avoid getting a blue color. Record the final color observed in each tube
below:
Test Tube
#1
#2
#3
#4
#5
#6
Solution Tested
Color/Results
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