IDENTIFICATION OF CARBOHYDRATES,

PROTEINS, AND LIPIDS

Introduction
The scientific method has enabled scientists to unravel the mysteries of life through a
systematic process which might be defined simply as a common sense approach for
determining cause and effect. It is not a complicated process. It requires making
observations and using those observations to make a generalization which is called a
hypothesis. Forming a general hypothesis from specific observations is inductive
reasoning. Since a hypothesis is only one possible explanation for the observations, it
must be tested through experimentation. The results of the experiment, the data, are
used to form a conclusion. Data are observations themselves. Your conclusion from
these additional observations may be that your original hypothesis needs to be
adjusted. One way to test a hypothesis is to predict future outcomes. Using a general
hypothesis to predict specific events is called deductive reasoning. A hypothesis that
has been tested by many scientists and has an abundance of proof supporting it
becomes a scientific theory. While in everyday usage a theory is something that is not
proven, in scientific usage a theory represents an explanation of why something works
that is accepted by the scientific world. It is not a guess but the truth as science knows
it.
You probably utilize at least part of the scientific method to solve day-to-day problems in
your home without being aware of it. For example, if you discover that your refrigerator
is not working you try to determine the cause of the problem. You probably consider
several possible options such as checking to see if the refrigerator is plugged in,
whether the receptacle is hot, whether the fuse was blown or the breaker tripped, if the
electricity is off, or if something is wrong with the refrigerator motor. In other words, you
hypothesize possible causes based upon your observation that the refrigerator isnt
running. You test each possible cause and, if you were successful, you find that the
problem is due to one of them. This approach for solving the refrigerator problem is the
same approach that a scientist would use to determine cause and effect.
Let's examine a problem that a scientist may encounter and discuss the protocol used
to solve the problem. Suppose that a plant scientist develops a new strain of fruit and
desires to determine its nutritional value. The scientist may have tried to develop a fruit
with fewer carbohydrates, more protein, and no lipids. Remember, the scientist is trying
to do this. The nutritional content of the fruit has not yet been analyzed to determine
whether the scientist succeeded or not. The common sense approach for providing the
actual content of the fruit would be to test the fruit for carbohydrates, proteins, and
lipids. Based upon the results of the tests, one may be able to state the content (amount
and type) of the biologically important molecules found within the fruit. The scientist may
discover that the desired nutritional value was not obtained based upon the data
collected during the testing process. Perhaps, the amounts of carbohydrates were
lowered but the lipid concentrations were not. If this is the case, the scientist would
work further to develop the fruit based upon these findings.

In this lab you too will be testing for biologically important molecules. Most molecules
that are biologically important are organic. They always contain carbon. They also
always contain hydrogen and oxygen. There are four groups of organic molecules that
you will need to be familiar with in biology: carbohydrates, lipids, proteins, and nucleic
acids.
Carbohydrates include monosaccharides like glucose, fructose, dextrose, and
galactose. When two monosaccharides are bonded together they form disaccharides
like sucrose, maltose, and lactose. Polysaccharides are complex sugars that are
formed when many monosaccharides are bonded together. Complex sugars like starch,
cellulose, chitin, and glycogen fall in this category.
Both carbohydrates and lipids are your body's primary energy storage molecules.
Lipids are also used to insulate and cushion your internal organs. Like carbohydrates
lipids are also made of carbon, hydrogen, and oxygen. Some lipids may also contain
other elements like phosphorus. These are called phospholipids. Lipids are one of the
few substances that will not dissolve in water. Examples of lipids include fats, oils,
waxes, and steroids.
Proteins are organic molecules that contain nitrogen in the form of amine groups as
well as carbon, hydrogen, and oxygen. Fibrous proteins are important structural
molecules. Myosin and actin form muscle. Collegen is an important component of
almost all connective tissues. Globular proteins are important functional molecules in
your body. Enzymes and many hormones are globular proteins.
Nucleic acids, like proteins, always contain nitrogen but they also always contain
phosphorus. The DNA of your chromosomes is a nucleic acid. Another important
nucleic acid in your cells is RNA which is essential to the formation of proteins.
Testing is a very important tool for the scientist. Testing involves a definite set of precise
procedures just like a recipe for baking a cake. Today you are the scientist who
analyzes various substances for their organic molecules by using different testing
methods. You will follow precise testing procedures to ensure that your data is accurate.
Study each of the following testing methods thoroughly so that you understand how to
perform the test and how to read or interpret the results. You will test for two types of
carbohydrates (reducing sugars and starch), lipids, and proteins. You will also use a
control.
A controlled experiment provides baseline data to compare to the experiment in which
you change a variable. A variable is a factor in the experiment that changes. A control
provides known results. A control causes no change in the data or provides negative
results. In this experiment our control is a substance we know will not react in any of
the tests. It is water. Water is not a carbohydrate, lipid, or a protein so all the following
tests on water should yield negative results. If it doesnt yield negative results that
would indicate there is a problem with the test. Perhaps you didnt do the test correctly,
or a chemical was contaminated, or your glassware wasnt clean. In any case the

problem must be corrected before any more tests are done. That is why you should test
the water first. The purpose of this lab is to test various substances, including an
unknown, in order to determine whether they contain reducing sugars, starch, proteins,
and/or lipids.

Methods
General Instructions
Be sure you wash all your glassware and containers with soap and water before
beginning and after you are finished. Contaminated tools produce misleading results.
Use test tube racks for holding the test tubes. Use test tube holders to move test
tubes to and from the hot water bath. Alert your instructor about the presence of any
broken glass.
Cleaning Up Before Leaving (REQUIRED)
1. When finished put everything back where it belongs.
2. Turn off hot plates.
3. Put paper used in the sudan dye test in the trash.
4. Empty entire contents of other experiments into the plastic container located on
the counter by the sink.
5. Wash all used test tubes, porcelin plates, etc., with soap and water.
6. Place the used test tubes in the plastic container located on the counter by the
sink.
7. Wipe down the tables with damp sponges.
Test Substances
Test all of the following substances using each of the methods outlined below.
1.
water (control)
2.
1 % glucose
3.
5 % glucose
4.
10 % glucose
5.
10 % sucrose
6.
10 % starch
7.
10 % albumin
8.
50 % albumin
9.
salad oil
10. milk
11. soda (clear, not diet)
12. unknown
Benedict Test for Reducing Sugars
The benedict test is used to quantitatively measure the presence of reducing sugar. By
quantitative we mean that this test can tell you whether or not a reducing sugar is
present and give you an idea of what quantity is present. Accurate measurements are
critical in this test to get accurate results. This test is based on the capacity of a
reducing sugar to convert a blue cupric hydroxide solution to an orange-red cuprous
oxide precipitate.

Directions:
1. Obtain test tubes for each substance to be tested (see list above) and label each
test tube with the name of the substance to be tested.
2. Add 1 ml of benedict reagent to each test tube.
3. Add 8 drops of the substance to be tested to its test tube.
4. Mix each test tube.
5. Heat each test tube for 3 minutes in a beaker of water heated to boiling.
6. With a test tube holder, remove the test tubes from the beaker and put them in the
test tube rack.
7. If the benedict reagent remains blue there is no reducing sugar. If it changes color
(green to red or brown) then there is reducing sugar
present. To determine the amount of reducing sugar present refer to the following
table:
clear blue = negative (0 gm %)
cloudy green = less than 0.5 gm %
yellow green = 0.5-1.0 gm %
green yellow = 1.0-1.5 gm %
yellow = 1.5-2.5 gm %
orange = 2.5-4.0 gm %
red/brown = more than 4.0 gm %
8. Record the results in the table as a color and as positive or negative.
Iodine Test for Starch
The iodine test is a testing method to identify the presence of starch. Iodine turns dark
(blue/black) if starch is present. If no starch is present the iodine remains the same
color. This is a qualitative test. It can tell you whether or not starch is present but does
not indicate the amount of starch. Since it does not tell you the quantity of starch
present, measurements are not as critical in this test.
Directions:
1. Obtain two porcelain spot plates and label the depressions with the names of the
substances to be tested (see list above).
2. Place 5 drops of each substance to be tested in its labeled depression.
3. Place 5 drops of iodine onto each of the depressions.
4. Look carefully for the iodine to change to a blue/black color. If it changes color there
is starch present. This is a positive result. If the iodine remains the same color there
is no starch. This is a negative result.
5. Record the results in the table as color and positive or negative.
Biuret Test for Proteins
The biuret test is one of many tests used to identify the presence of protein in
foodstuffs. Proteins may be identified by a color reaction when combined with biuret
reagent. Biuret is a deep bluish purple. In the presence of protein it turns a pinkish
purple or lavender.

Directions:
1. Obtain a test tube for each substance to be tested (see list above) and label it using
a grease pencil.
2. Add 1 ml of biuret reagent into each of the test tubes.
3. Add 20 drops of the substance to be tested into its test tube and observe for 3
minutes.
4. If the biuret stays the same bluish purple there is no protein (negative result). If the
biuret changes color to a deeper purple or pinkish lavender then protein is present
(positive result). Do not confuse the color of the substance tested with a color
change in the biuret. This change is very subtle and easiest seen against a white
background like a sheet of paper. It also helps to compare each test tube to your
control (whose results should be negative) to determine whether they are different
from the control or not.
5. Record the results in the table as color and positive or negative.
Sudan Dye Test for Lipids
Two tests will be used to identify lipids: the sudan dye test and the grease spot test.
The sudan dye test takes advantage of two properties of lipids. One is that lipids are
insoluble in water and the other is the affinity sudan dye has for lipids.
Directions
1. Obtain a test tube for each substance to be tested (see list above) and label it using
a grease pencil.
2. Add 3 ml of water to each test tube.
3. Add 1 ml of the substance to be tested to its test tube.
4. Add 3 drops of sudan dye to each test tube.
5. Shake each test tube vigorously and allow it to stand for 5-10 minutes.
6. If the contents of the test tube form two distinct layers then the substance tested is a
lipid (positive results). If the contents do not separate into two distinct layers then
the substance is not a lipid (negative results).
7. Record the results in the table as positive or negative.
Grease Spot Test for Lipids
The easiest method to identify lipids is to take advantage of the fact that lipids make
translucent grease spots on unglazed paper. A translucent substance allows light
through it but you cannot see through it. Frosted glass is an example of a translucent
substance.

Directions
1.
Obtain a piece of unglazed brown paper for each of the substances to be tested (see list ab
2.
Put 1 drop of the substance to be tested on its piece of paper and allow it to soak in briefly.
3.
Shake the excess liquid off of the paper and allow it to dry. You can blow on it or fan it to he
4.
If it dries without leaving a translucent spot then it is not a lipid. If a translucent spot remains
5.
Record the results in the table as positive or negative.
6. Remember to throw the used pieces of paper away.

Student _____________________________

Date ______________________

Instructor Signature ____________________

Results
1. Record the results of all your tests in the following table:
No.
1
2
3
4
5
6
7
8
9
10
11
12

Substance

Benedict
Test
color
+/-

Iodine
Test
color
+/-

Biuret
Test
color
+/-

Sudan
Dye
+/-

Grease
Spot
+/-

water (control)
1 % glucose
5 % glucose
10 % glucose
10 % sucrose
10 % starch
10 % albumin
50 % albumin
salad oil
milk
regular soda
unknown

2. Fill out the table below for the results of the benedict test:
No.
1
2
3
4
5
6
7
8
9
10
11
12

Substance
water (control)
1 % glucose
5 % glucose
10 % glucose
10 % sucrose
10 % starch
10 % albumin
50% albumin
salad oil
milk
regular soda
unknown

Gm %

Discussion
3. You tested a clear regular (non-diet) soda. How much reducing sugar was present in
your sample? Is the reason it is sweet because it contains reducing sugars? How do
you know this?

4. Did the milk you tested contain protein? How do you know this? If it contained
protein can you determine how much? Why or why not?

5. Do you think the sudan dye test is a quantitative or qualitative test? Why or why not?

6. What is an indicator substance? Give an example.

7. Did you get the same results for the sudan dye and grease spot tests? Why or why
not?

8. Which tests gave you a positive result when you tested your unknown? What does
this tell you?

9. In your data you should have found that sucrose gave a negative result when you did
the benedict test since it is not a reducing sugar. What would you have to do to sucrose
in order the change it into a reducing sugar? How would you go about changing it into a
reducing sugar?

10. Outline the functions of the polysaccharides in the table below.

Polysaccharide
Glycogen
Chitin
Cellulose
Starch

Function

11. Outline the functions of the proteins in the table below:

Protein
Hemoglobin
Keratin
Myoglobin
Fibrin
Serum albumin
Insulin
Calmodulin
Immunoglobins
Cytochromes
Vasopressin
Pepsin
Ferritin

Function

12. Outline the functions of the lipids in the table below:

Lipid
Lecithin
Prostaglandins
Cholesterol
Estrogen
Carotene

Function

13. Which groups of organic molecules would you expect to find in the following:
a. corn oil
b. silk
c. rubber
d. soap
e. molasses