Practical Assignment 1: Biomacromolecules [50 Marks]

You are provided with 8 unknown liquid samples A, B, C, D, E, F, G and H and test tubes. These
samples were equally treated in the following experiments.

Experiment 1:
Procedure:
1) Take 5 ml of each sample and put in 8 separate test tube s
2) Using a grease pencil, number the tubes A1, B1, C1 etc.
3) Add one drop of Lugols reagent solution to all test tubes.
*Samples B, C and D had blue-black color; others have the brown color of Lugols reagent.
4) Repeat steps 1 to 2 and add 4 5 drops of saliva collected from the volunteer student
5) Set the tubes in a water bath @ 37C for hour or more.
6) Remove the tubes from the water bath and test contents by adding one drop of Lugols reagent.
*You note all have the same brown color.

Experiment 2:
Procedure:
1) Place 5 ml of Reagent X into 8 test tubes and label as in experiment 1 above.
2) Add four to five drops of each of the 8 samples.
3) Place tubes in a beaker water-bath over a Bunsen burner; boil for 2 minutes.
4) Remove the tubes and let cool slowly in a test tube rack.
*Samples A, B and F develop an orange color with an orange-red precipitate
5) Repeat steps 1 to 2 (replacing Reagent X with Barfoeds reagent) and heat the test tubes for 15-20
min, followed by cooling.

Experiment 3:
Procedure:
1) Into each of the 8 test-tubes containing the samples, add 5 ml of Seliwanoffs reagent.
2) Place the test tubes into a beaker water-bath over a Bunsen burner; boil for 1 minute.
3) Note any color change.
*A deep cherry red color was observed in Samples G and F, while sample A gave a slight or faint
pink color. Sample B gave a colour which was somewhat closer to cherry red.

Experiment 4:
Procedure:
1) Add 5 ml of 5% sample G to each of two test tubes; label these Tube 1 and Tube 2.
2) To Tube 1, add 5 mL of Reagent X and bring to a boil in the beaker water bath.
3) Remove Tube 1 from heat immediately.
4) To Tube 2, add two drops of 1M HCl and boil in the beaker water bath for 1 minute.
5) Remove Tube 2 from heat and neutralize with two drops of 1M NaOH.
6) Test the contents of Tube 2 with 5 mL of Reagent X.
*Tube 1 remained blue while Tube 2 turned orange with an orange-red precipitate.

Experiment 5:
Procedure:
1) Add 3 ml of 10% NaOH and one drop of 1% CuSO
4
to 5mL of the 8 samples in test tubes and mix
well.
2) Add additional drops of CuSO
4
until a color change is seen.
*A mauve (deep-purple or violet) color is observed in Samples B and D.

Experiment 6:
Procedure:
1) Mix 2 mL of 1% alcohol 3 mL of each of the 8 samples
2) Add 0.5 ml of 0.1% freshly prepared Ninhydrin to the 5 mL alcoholic mixture prepared above
3) Heat in a water bath and note color formation.
*A deep blue/purple color was observed in samples B and E.
4) Repeat steps 1 to 2 using 5 mL of 1% albumin solution.
5) Heat to boiling the albumin to which Ninhydrin was added.
*No deep blue/purple was observed

Experiment 7:
Procedure:
To 2 ml of each of the 8 samples, 3mL of 70% ethanol was added, followed by vigorous mixing. After
10 min observations were noted. A milky emulsion was observed only in Samples B and E.

Experiment 8:
You are provided with additional samples I and J. To 5 mL of each of these samples, 3 mL Bial's
reagent was added, followed by gentle heating in a Bunsen Burner or hot water bath.
Sample I gave a blue-green color while sample J gave a muddy-brown colour.

Questions
1. Use the results in all the 7 experiments to investigate the identities of chemicals in samples 1 to
7. State the name of the test, and present your results in tabular form, explaining the
mechanism(s) behind any colour development (e.g.).

Sample Constituent chemicals [10] Explanations and deductions [15]
A
B
C
D
E
F
G
H
I
J

1. Explain why B, C and D failed to produce the blue-black color after Step 4 of experiment 1. [2]
2 Identify Reagent X used in experiments 2 and 4. [2]
3. State and explain the results expected in step 5 of experiment 2, explaining the difference between
Reagent X and Barfoeds reagent. [4]
4. Explain the difference (in principle) between experiments 2 and 3. [2]
5. (a) In experiment 4, explain why Tube 2, gave an orange-red precipitate after acid treatment.
Hence state the name of the reaction of Sample G constituent with HCL. [5]
(b) Explain why this is important to neutralize HCl with NaOH, prior to addition of Reagent. [2]
6. Explain the difference between test in experiments 5 and 6. [2]
7. Explain why 1% albumin solution in Experiment 6 failed to give the positive Ninhydrin test. [2]
8. Explain, giving names, difference between samples B and H. [2]
9. Explain the mechanism(s) behind formation of blue-green and muddy-brown colours in Exp.8 [2]