Tests for the Presence of Various Organic Molecules

TITLE – Tests for the Presence of Various Organic Molecules

ABSTRACT
The purpose of this report is to discuss the results of multiple substances that were tested for
the presence of organic molecules, reducing sugars, starches, lipids, proteins, and DNA.

INTRODUCTION
Life on earth is based on the element carbon. Carbon is a versatile atom because it can form
four covalent bonds with itself or other atoms such as hydrogen, oxygen, etc. Organic
molecules are carbon based and have covalent bonds between carbon and hydrogen (Dameron
& Gollapudi, 27). These bonds store considerable amounts of energy. Organic molecules will
burn and blacken when exposed to heat due to these high-energy carbon and hydrogen bonds
(Dameron & Gollapudi, 28), whereas inorganic molecules will not. There are four basic kinds of
organic molecules: Carbohydrates, lipids, proteins, and nucleic acids (Dameron & Gollapudi,
27).
It is from organic molecules that life forms derive their energy and structure for living. This
study performed various tests to determine what (if any) organic molecules certain substances
contained.

The first kind of organic molecule is the carbohydrate. The monomer form of carbohydrates is
known as a monosaccharide (Dameron & Gollapudi, 29). Reducing sugars are mostly
monosaccharides (and sometimes disaccharides) that are able to reduce other molecules. A
molecule is said to be “reduced” if it gains an electron (or gains a hydrogen atom or loses an
oxygen atom). Benedicts reagent (CuSO4/citrate), blue in its oxidized state, will precipitate and
turn to a yellow-red color when it becomes reduced, and thus can be used to test for the
presence of a reducing sugar (Dameron & Gollapudi, 30, 31). Starch is a polysaccharide made
from three or more monosaccharides. Amylose, a type of starch, is a chain of hundreds of
glucose monomers joined by α 1-4 glycosidic linkages. It forms a coil in water which is
insoluble. Iodine is used to test for the presence of amylose. When dissolved in water and
potassium iodide, iodine will make a tri-iodide ion complex (I2KI), which is water soluble. When
the I2KI is added to a sample that contains amylose, this complex will slip into the amylose coil
resulting in an intense blue-black color. The tri-iodide ion complex will not react in the same
manner with other polysaccharides (Dameron & Gollapudi, 33).

The next kind of organic molecule is the lipid. Triglycerides (a type of lipid) consist of a glycerol
molecule backbone with three fatty acids attached via dehydration synthesis reactions. A
triglyceride contains a high proportion of carbon-hydrogen bonds that equally share electrons,
making the molecule nonpolar and water-insoluble (water is polar). Sudan IV is a nonpolar dye
that will dissolve in other nonpolar solvents and will not dissolve in polar solutions. Detergents
are amphipathic molecules that have a nonpolar region that will interact with nonpolar
triglycerides and a polar region that will interact with water. These properties of detergent are
why they are used to help dissolve oils in water. Samples containing triglycerides will also leave

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 Tests for the Presence of Various Organic Molecules

behind a wet-looking (sometimes translucent) residue long after they have come into significant
contact with paper, whereas an aqueous solution, after coming in contact with paper, will
evaporate, leaving the paper dry and opaque (Dameron & Gollapudi, 36).

The third kind of organic molecule is the protein. The monomer structure of a protein is the
amino acid. Amino acids are covalently bonded together in what are known as peptide bonds.
Biuret reagent, blue in color, is a solution that reacts with the peptide bonds in proteins, turning
purple, and thus is useful in the detection of proteins. The intensity of the color change is
directly related to the concentration of peptide bonds in the sample that contains protein
(Dameron & Gollapudi, 38).

The fourth kind of organic molecule is the nucleotide. Nucleotides are the monomers of RNA
and DNA. DNA can be identified and distinguished from RNA and other biological molecules
chemically by the Dische Diphenylamine test. DNA contains deoxyribose sugar that will bind to
Diphenylamine under acidic conditions, to form a molecule that is blue in color. The intensity
of the color produced is an indicator of the concentration of the DNA present in the solution
(Dameron & Gollapudi, 41).

MATERIALS AND METHODS

All Materials and Methods used are outlined in General Biology 1 (BIO 161) Laboratory Manual,
pp. 27-46 in the procedure sections of activities 1, 2A, 2B, 3A, 3B, 4 and 5 of Exercise 2.

RESULTS
Activity 1 tested substances to determine if they contained organic molecules. Gelatin, salt,
sugar, milk, water and an unknown substance (unknown #2) were all tested to determine
whether or not they were organic. Table 1 summarizes the results of Activity 1, Organic
Molecules Test (Dameron & Gollapudi, 29). Gelatin, sugar, milk and unknown #2 all burned
whereas the salt and water did not burn.

TABLE 1: Organic Molecules Test Results

Substance Tested Did it Burn/Blacken? Conclusion
Yes/No
Gelatin Yes Organic
Salt No Inorganic
Sugar Yes Organic
Milk Yes Organic
Water No Inorganic
Unknown #2 Yes Organic

Activity 2A tested substances to determine if they contained reducing sugars. Using water as
the negative control and reducing sugar solution as the positive control, apple juice, sucrose

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 Tests for the Presence of Various Organic Molecules

solution, onion juice, powdered cracker in water, chewed cracker in water and unknown #2
were all tested to determine if they contained reducing sugars. Table 2 summarizes the results
of Activity 2A, Benedicts Test for Reducing Sugars (Dameron & Gollapudi, 32). Apple juice,
sucrose solution, onion juice, chewed cracker in water and unknown #2 all underwent a yellow-
red color change like the positive control whereas the powdered cracker in water did not
experience a color change.

TABLE 2: Benedicts Test for Reducing Sugars Results

Test Material Color Change Observation Conclusion
Water No Change No Reducing Sugars
Reducing Sugar Solution Yellow-Red Reducing Sugar
Apple Juice Yellow-Red Reducing Sugar
Sucrose Solution Yellow-Red Reducing Sugar
Onion Juice Yellow-Red Reducing Sugar
Powdered Cracker in Water No Change No Reducing Sugars
Chewed Cracker in Water Yellow-Red Reducing Sugar
Unknown #2 Yellow-Red Reducing Sugar

Activity 2B tested for the presence of starch, a polysaccharide, specifically amylose. Using
water as the negative control and starch solution as the positive control, glucose solution,
sucrose solution, rough paper, smooth paper, potato, bread, cracker and unknown #2 were
tested for the presence of amylose starch. Table 3 summarizes the results of Activity 2B, Iodine
Test for the Presence of Starch (Dameron & Gollapudi, 34). Smooth paper, potato, bread and
cracker all changed blue to blue-black when reacted with the I2KI whereas the glucose solution,
sucrose solution, rough paper and unknown #2 did not change blue to blue-black when reacted
with the I2KI.

TABLE 3: Iodine Test for the Presence of Starch Results

Test Material Color Change Observation Conclusion
Water None Non-Amylose
Starch Solution Blue Amylose
Glucose Solution None Non-Amylose
Sucrose Solution None Non-Amylose
Rough Paper None Non-Amylose
Smooth Paper Blue-Black Amylose
Potato Blue-Black Amylose
Bread Blue-Black Amylose
Cracker Blue Amylose
Unknown #2 None Non-Amylose

Activity 3A tested for the presence of lipids, specifically triglycerides. Using water as the
negative control and oil as the positive control, oil, detergent and unknown #2 were tested for
the presence of triglycerides (Dameron & Gollapudi, 35). Table 4 summarizes the results of

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 Tests for the Presence of Various Organic Molecules

Activity 3A, Sudan IV Test for the Presence of Oils (Dameron & Gollapudi, 36). The oil and water
mixture and the oil, water and detergent mixture developed a reddish color at the top of the
test tube and a cloudy white color at the bottom during the final interaction whereas the
unknown #2, like the water, did not change color, but formed a black floating precipitate on the
top of the contents of the test tube.

TABLE 4: Sudan IV Test for the Presence of Oils Results

Test Material Color Changes/Observations Conclusion
Water No color change. Black Polar
precipitate floats to top of
test tube.
3ml Water + 1ml Oil Reddish color at top, cloudy Both
white at bottom.
0.5 ml Oil + 2ml Water + 15 Sudan Red precipitates to Both
drops of Detergent (well bottom of oil and foams; black
mixed) precipitate form on bottom of
tube fluid turned red with
white cloudy fluid below red
fluid.
Unknown #2 No color change. Black Polar
precipitate floats to top of
test tube.

Activity 3B was a simple activity that tested for the presence of triglycerides. Using water as
the negative control and vegetable oil as the positive control, sucrose solution, a potato chip,
peanuts and unknown solution #2 were all tested for the presence of triglycerides. Like the
negative control, the sucrose solution and unknown #2 both dried within a few moments,
whereas the potato chip residue and peanut residue remained wet-looking like the positive
control. Table 5 summarizes the results of Activity 3B, Greasy Spot Test for Triglycerides
(Dameron & Gollapudi, 37).

TABLE 5
Test Material Observation Conclusion
Water Clear/Dry Aqueous
Vegetable Oil Wet-Looking Lipid
Sucrose Solution Clear/Dry Aqueous
Potato Chip Wet-Looking Lipid
Peanuts Wet-Looking Lipid
Unknown #2 Clear/Dry Aqueous

Activity 4 tested for the presence of proteins. Using water as the negative control and protein
solution as the positive control, sucrose solution, milk, powdered cracker, crushed peanuts,
vegetable oil and unknown #2 were all tested for the presence of protein. Table 6 summarizes

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 Tests for the Presence of Various Organic Molecules

the results of Activity 4, Biuret Test for Protein (Dameron & Gollapudi, 39). Like the negative
control, the biuret reagent remained blue when exposed to the sucrose solution, powdered
cracker, vegetable oil and unknown #2, and turned purple like the protein solution when
exposed to the milk, and crushed peanuts.

TABLE 6: Biuret Test for Protein Results

Test Material Observation Conclusion
Water Blue No Proteins
Protein Solution Purple Proteins
Sucrose Solution Blue No Proteins
Milk Purple Proteins
Powdered Cracker Blue No Proteins
Crushed Peanuts Purple Proteins
Vegetable Oil Blue No Proteins
Unknown #2 Blue No Proteins

Activity 5 tested for the presence of Deoxyribonucleic Acid (DNA). Using water as the negative
control and DNA solution as the positive control, a DNA solution-water mixture and unknown
#2 were tested for the presence of DNA. Table 7 summarizes the results of Activity 5, Testing
for the Presence of DNA (Dameron & Gollapudi, 41). Similar to the water, unknown #2 did not
turn blue, whereas the DNA solution-water mixture did turn blue when reacted with the Dische
Diphenylamine reagent.

TABLE 7: Test for the Presence of Deoxyribonucleic Acid (DNA) Results

Solution Color Explanation
1ml of DNA solution + Clear Deep Blue Lower concentrated DNA
1ml of distilled Water
2 ml of DNA solution Opaque Deep Blue Higher concentrated DNA
2 ml of Distilled Water Slightly Cloudy with some Absence of DNA
white chunks
Unknown #2 Slightly cloudy yellow Absence of DNA

Activity 6 was simply a comparison of the results of the unknown #2 in activities 2A, 2B, 3A, 3B,
4 and 5 against the positive and negative controls of each activity. Table 8 summarizes the
contents of Unknown substance #2. This substance changed yellow-red in the Benedicts test,
did not change color in the starch test nor the Sudan IV lipids test. It remained blue when
reacted with the Biuret reagent in the proteins test and remained clear-cloudy when reacted
with the Dische Diphenylamine.
TABLE 8: Contents of Unknown #2 Results
Control Substances Results
Molecule Reagent Positive Negative Positive Negative Unknown
Tested Control Control #2
Reducing Benedicts Reducing Water Yellow-Red No Color Yellow-Red

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 Tests for the Presence of Various Organic Molecules

Sugars Sugar Color Change Color

Solution Change Change
Starch Iodine (I2KI) Starch Water Blue Color No Color No Color
Solution Change Change Change
Lipids Sudan IV 3 ml Water Water Reddish No Color No Color
Powder + 1 ml Oil Color at Change; Change;
Top; Black Black
Cloudy Precipitatio Precipitatio
White at n at Top n at Top
Bottom
Proteins Biuret Protein Water Purple Blue Color Blue Color
Solution Color
DNA Dische DNA Water Deep Clear- Slightly
Diphenylami Solution Opaque Cloudy cloudy
ne Blue Yellow

DISCUSSION
Following are the conclusions arrived at based on the results reported above:

1. In the organic molecules test, gelatin, sugar, milk and unknown #2 all burned, indicating
that they are organic, whereas the salt and water did not burn, indicating that they are
inorganic.

2. In the Benedicts test for reducing sugars, apple juice, sucrose solution, onion juice,
chewed cracker in water and unknown #2 all underwent a yellow-red color change like
the positive control, indicating that they all contained reducing sugars, whereas the
powdered cracker in water did not experience a color change like the negative control,
indicating it did not contain reducing sugar.

3. In the Iodine test for the presence of starch, smooth paper, potato, bread and cracker all
changed blue to blue-black, like the positive control, when reacted with the I 2KI,
indicating they all contained starch, whereas the glucose solution, sucrose solution,
rough paper and unknown #2 did not change blue to blue-black when reacted with the
I2KI like the negative control, indicating that they all did not contain starch.

4. In the Sudan IV test for the presence of oil, the oil and water mixture and the oil, water
and detergent mixture developed a reddish color at the top of the test tube and a
cloudy white color at the bottom during the final interaction, indicating that they both
contained lipids as well as polar substances, whereas the unknown #2, like the negative
control, did not change color, but formed a black floating precipitate on the top of the
contents of the test tube, indicating that it was polar and did not contain lipids.

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 Tests for the Presence of Various Organic Molecules

5. In the paper rub test for the presence of triglycerides, the sucrose solution and
unknown #2 both dried within a few moments, like the negative control, indicating that
they both did not contain triglycerides, whereas the potato chip residue and peanut
residue remained wet-looking like the positive control, indicating that they both
contained triglycerides.

6. In the Biuret test for proteins, like the negative control, the biuret reagent remained
blue when exposed to the sucrose solution, powdered cracker, vegetable oil and
unknown #2, indicating that protein was absent in all of them, and turned purple like
the positive control when exposed to the milk, and crushed peanuts, indicating that
protein was present in them both.

7. In the test for the presence of DNA, similar to the negative control, unknown #2 did not
turn blue, indicating the absence of DNA, whereas the DNA solution-water mixture did
turn blue when reacted with the Dische Diphenylamine reagent, like the positive
control, indicating the presence of DNA.

8. Finally, the unknown solution tested negative for everything except for the presence of
organic molecules and the presence of reducing sugar, indicating it is a reducing sugar.

WORKS CITED
Dameron, Kristina, Ph.D. & Gollapudi, Lakshmi, Ph.D. General Biology 1 (BIO 161) Laboratory
Manual. Grayslake, IL: College of Lake County Biological & Health Sciences Division