Identification of Some Macromolecules in Solutions: Starch, Glycogen,

Proteins and Reducing Sugars

Name: Taylor Meadus (20830157)

Partner: Jordan Salb

Teaching Assistants: SaraRae McLeod

Nursat Khan

Course Number: Biol 130L

Lab Section: 004

Lab Time: Monday 7:00 – 10:00

Lab Location: STC 4009

Date of Experiment: September 23, 2019

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Introduction

The purpose of this experiment was to identify some macromolecules in certain known

substances and one unknown substance, solution number 250. Three tests were conducted to see

the presence of proteins, reducing sugars, starch and glycogen in each substance. To test for

proteins, the biuret test was used, to test for reducing sugars, the benedict’s test was used, and to

test for starch and glycogen, the iodine test was used. With the use of controls for each test, the

colour of the solution after each test was observed to conclude whether the macromolecule tested

for was present in that solution.

The biuret test uses a biuret reagent mixed with the solution to show if proteins are

present in a substance. The biuret reagent consists of copper sulfate and sodium hydroxide which

makes the solution blue initially (Gornall, Bardawill & David, 1948). When the reagent is mixed

with a solution, the copper and low concentration of alkali causes the solution to turn violet in

colour when there is a presence of protein in the solution indicating a positive result (Gornall,

Bardawill & David, 1948).

The benedict’s test uses the benedict solution to show the presence of reducing sugars in

a solution. The benedict solution contains cupric ions and when combined with a substance and

heated, any reducing sugars present in the substance will reduce the cupric ions and produce a

coloured red or orange precipitate (Simoni, Hill & Vaughan, 2002). Therefore, if there are

reducing sugars present in a solution, the benedict’s test will produce a coloured solution as

apposed to an initial blue colour which indicates a positive test.

Lastly, the iodine test is used to test for the presence of starch and glycogen in solutions.

The initial colour of the iodine reagent is yellow. When it encounters starch, the solution will
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turn blue/black because of amylose in the starch reacting with the iodine indicating a positive test

for starch (Hollo & Szejtli, 1957). Glycogen on the other hand, contains amylopectin which has a

more highly branched structure which produces a different red/brown colour when it reacts with

the iodine indicating a positive test for glycogen (Hollo & Szejtli, 1957).

These experiments will help identify macromolecules present in the solutions being

tested. Most are already known and can be used as controls, whereas the unknown solution will

be discovered throughout the tests.

Materials and Methods

All procedures were carried out as outlined in Identification of Some Macromolecules, Biol 130

lab manual, pages 25-30 (Department of Biology, 2019). No deviations were made to these

protocols.

Results

The following tables show the results of each test and indicate which macromolecules are

present in each solution. Solution #1 is 1% glucose solution, #2 is 0.3% glucose1phosphate, #3 is

1% maltose solution, #4 is 5% honey solution, #5 is 1% sucrose solution, #6 is 1% lactose

solution, #7 is 1% glycogen solution, #8 is 1% starch solution, #9 is 1% protein solution, #10 is

beer, #11 is distilled water, and #12 is the unknown solution number 250.
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Table 1: Results from benedict’s test showing presence of reducing sugars in each solution
Solution # Colour of Solution Results
1 Red/Orange +
2 Blue -
3 Red/Orange +
4 Brownish +
5 Blue -
6 Red/Orange +
7 Blue -
8 Blue -
9 Blue -
10 Bright Orange +
11 Blue -
12 Blue -

Table 1 shows the results of the benedict’s test on each solution. Benedict’s solution was

added to each solution and heated for 5 minutes. The red/orange colour indicates a positive test

and the presence of reducing sugars in the solution and a blue colour indicates a negative test

since this is the initial colour of the benedict’s solution (Simoni, Hill & Vaughan, 2002).

Table 2: Results from biuret test showing presence of proteins in each solution
Solution # Colour of Solution Results (+/-)
1 Blue -
2 Blue -
3 Blue -
4 Green -
5 Blue -
6 Blue -
7 Blue -
8 Blue -
9 Purple +
10 Blue -
11 Blue -
12 Purple +
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Table 2 shows the results from the biuret test on each solution. Sodium hydroxide and

copper sulfate was added to each solution. A blue colour produced indicates a negative test since

this is the initial colour of the biuret solution whereas a purple colour indicates the presence of

proteins and a positive test (Gornall, Bardawill & David, 1948).

Table 3: Results from iodine test showing presence of starch or glycogen in each solution
Solution # Colour of Solution
1 Yellow
2 Yellow
3 Yellow
4 Yellow
5 Yellow
6 Yellow
7 Red/Brown
8 Blue/Black
9 Yellow
10 Pale Orange
11 Yellow
12 Yellow
Results Starch (+/-) Results Glycogen (+/-)
- -
- -
- -
- -
- -
- -
- +
+ -
- -
- -
- -
- -

Table 3 shows results from the iodine test on each solution. A drop of iodine was added

to each solution. A yellow colour indicates a negative test since this is the initial colour of the

solution. The red/brown colour indicates a positive test for glycogen and a blue/black colour

indicates a positive test for starch.

Discussion

The purpose of this experiment was to identify macromolecules in certain solutions

which was successfully achieved with the 3 experiments conducted. In the first experiment, the

benedict’s test, positive and negative controls were used to compare results to and make sure the
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experiment was working correctly. The positive control was solution #1, the 1% glucose

solution, since glucose is known to be a reducing sugar. The negative control was solution #11,

distilled water, since water is not a reducing sugar. Five solutions turned out to be positive

including the positive control, the 1% maltose solution, the 5% honey solution, the 1% lactose

solution, and the beer. These solutions are all reducing sugars. When the solution is heated with

the benedict’s solution, the sugar becomes fragmented (Simoni, Hill & Vaughan, 2002). The

sugars have an aldehyde group attached to them which reacts with the copper ions in the

benedict’s solution therefore becoming reduced and oxidizing the copper ions (Simoni, Hill &

Vaughan, 2002). This is what caused the colour change. All negative results were not reducing

sugars.

The second experiment, the biuret test, tested each solution for proteins. A positive

control used for this test was solution #9, the 1% protein solution. The sodium hydroxide and

copper sulfate added to each solution creates alkali conditions which allow peptide bonds in

proteins to react with the copper ions producing a violet colour (Lubran, 1978). This test

produced 2 positive results, the positive control and the unknown solution. These results indicate

that the unknown solution must be a protein. A negative control used was distilled water since

water is not a protein. All negative results are not proteins.

The third experiment, the iodine test, tested each substance for starch and glycogen.

There are two positive controls used for this experiment since there are two macromolecules

being tested for. Solution #7, the 1% glycogen solution, was used as a positive control for

glycogen and solution #8, the 1% starch solution, was used as a positive control for starch. One

component of glycogen is amylopectin which has a highly branch structure and produces a

red/brown colour when reacting with iodine (Hollo & Szejtli, 1957). Starch contains amylose
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which is an unbranched molecule and when reacting with iodine produces a blue/black colour

(Hollo & Szejtli, 1957). The experiment resulted in 2 positive results, one for starch and one for

glycogen. Both of which were the positive controls meaning none of the other substances were

starch or glycogen. A negative control for this test was distilled water since water does not

contain starch or glycogen.

In conclusion, most solutions were identified to be proteins, starch, glycogen, or reducing

sugars. The unknown solution was identified to be a protein since it tested positive in the biuret

test. Although these tests seemed to show correct results, there may have been sources of error.

One source of error could have been contamination of solutions or materials. This is hard to

avoid since you can not be sure where the materials have been, and the solutions were already

made. Another source of error could have been human error in recording data. The data could

have been interpreted or recorded wrong. Lastly, these tests were useful in observing certain

macromolecules in solutions and could be used in many ways. For example, the benedict’s test

could be used for detecting glucose in urine and blood. Overall, these tests were helpful in

identifying macromolecules in solutions.

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References

Department of Biology. (2019). Introductory cell biology laboratory. Waterloo, Canada:

University of Waterloo Media.Doc.

Gornall, A. G., Bardawill, C. J., & David, M. M. (1948). Determination of serum proteins by

means of the biuret reaction. The Journal of Biological Chemistry, 177(2), 751-766.

Holló, J., & Szejtli, J. (1957). The mechanism of starch-iodine reaction. Starch, 9(6), 106-110.

Lubran, M. M. (1978). The measurement of total serum proteins by the biuret method. Annals of

Clinical and Laboratory Science, 8(2), 106-110.

Simoni, R. D., Hill, R. L., & Vaughan, M. (2002). Benedict’s solution, a reagent for measuring

reducing sugars: The clinical chemistry of Stanley R. benedict. The Journal of Biological

Chemistry, 277(16), 10-11.