Noor Hammoud
Fo7981
Abby Wicks
Wed. 8:30-11:30 Sec. 501
Amino Acids: Final Lab Report
Introduction
Amino acids are an important group of macromolecules. Like other macromolecules,
they contain carbon, hydrogen, and oxygen. However, they also include nitrogen (Wikipedia).
Nitrogen is found in the amino acid's special amine group (NH2). Along with the amine group,
amino acids contain a carboxyl group (COOH) and a variable "R" group. These three groups are
attached the central carbon (Van Thiel 21). The "R" group is especially important in the amino
acid. It is a side-chain that gives each amino acid its unique properties. Amino acids are
classified by the properties of the side-chain (Wikipedia).
An amino acid comes together with other amino acids to combine in unbranched, linear
fashions to form polypeptides (Van Thiel 21). They bond this way with the carboxyl carbon of
one amino acid attaching to the amine nitrogen of the next amino acid. These polypeptides form
peptide bonds, which are also known as C-N bonds. Peptide bonds grow and an actual protein
may contain thousands of amino acids connected this way (Van Thiel 21). Polypeptides also
have different structures. The primary structure of a polypeptide is a simple string of amino
acids. The next secondary structure consists of hydrogen bonds causing the string to wrap into a
coil and fold into sheets. Next, tertiary structures include the polypeptide reacting with water to
form globular packets. Lastly, quaternary structures are composed of the combination of these
globular packets. These structures help create the structure of a protein.
With amino acids, polypeptides, and proteins, testing can be done to detect amino acids
and several peptide bonds. In detecting simple amino acids, the ninhydrin test is used. Ninhydrin
is a chemical that causes amino acids to turn a specific color when heated, causing a reaction.
Basically, the nitrogen of the amino acid combines with two ninhydrin molecules to produce
color (Van Thiel 21). Amino acids that have an alpha-amino group turn shades of purple from
pink to deep violet. The only exception to this is proline, which turns bright yellow because of a
substitution of the alpha-amino group (Van Thiel 21-22). Amino acids actually do not have their
own unique colors and therefore turn orange, blue, and so forth, so ninhydrin is combined with
thin layer chromatography. The experiment that will be discussed further into this report consists
of this chromatography technique. TLC is a sheet containing a glass microfiber matrix
impregnated with polysilicic acid gel (Van Thiel 22). Depending on the "R" groups of different
amino acids, the amino acids will migrate along the TLC to different locations. By using
ninhydrin in the chromatography solvent, the positions and Rf values of the migrated amino
acids will identify them. Then, heat will be applied to the sheet to produce the amino acidninhydrin color change.
Other color changes can be used to test amino acids, specifically testing peptide bonds,
and this is done in the second experiment of this lab. This is the biuret test, in which the reagent
is an alakaline solution of copper sulfate that will react with C-N bonds to form a purple color
(Van Thiel 22). More intense positive reactions correlate with more peptide bonds and this
ranges from light purple to deep purple. Solutions with no peptide bonds, like water or individual
amino acids, will remain the clue color of the copper solution. The one amino acid that is an
exception is cysteine which has the presence of free sulfide groups. In the copper solution, few
sulfide groups will cause a yellow color. More sulfide groups will cause a green color, and
ultimately, many sulfide groups create a black color. This will be seen in the experiment.
However, in peptide bond reactions, positive results mean that there are several peptide bonds,
not exactly polypeptides or proteins. For example, a nitrogenous compound derived from urea
will give a positive biuret test even though it is not a polypeptide or a protein. The urea is an
analog that will react negatively with the test, but when heated will produce four C-N bonds and
react like a polypeptide; this is called biuret (Van Thiel 22). In the experiment that will soon be
discussed, biuret and ninhydrin testing will be done with different solutions and two unknowns
(unknown A and unknown B). With the ninhydrin-TLC test, the unknowns will not be amino
acids, but could be proteins. Their complex make-up will probably include peptide bonds that
will be detected in the biuret test.
Materials and Methods
The procedures and materials for both tests are found on pages 24-26 in "Experiments in
Biology: From Chemistry to Sex". There are a few procedure changes, which are found under C
on the Lab #3 handout. Follow those instructions instead. Also beef broth is changed to unknown
A: yogurt, and milk is changed to unknown B: cat food.
Results
The results are indicated on chart 3.1 (Van Thiel 27) as well as on tables 3.1 and 3.2
(Van Thiel 28-29).

Ninhydrin test - TLC test
Sample
Reaction
Color
Origin to
Spot
Glutamic acid
Brick red,
pink orange
Yellow
Proline
Rf Ratio
Rf Value
1 cm
Origin to
Solvent
Front
4.8 cm
1 / 4.8
0.208
1 cm
4.8 cm
1 / 4.8
0.208
Tyrosine
Cysteine
Alanine
Arginine
Glycine
Urea
Biuret
Casein
Hydrolyzed
casein
Yogurt
Catfood

Peach
Peachyellow-red
Red - fades
Red - fades
Yelloworange to
light pink
No change
No change
No change
Red to pink to
orange-brown
Purple stain at
origin, faintly
fades up pink
Pink to
yellow to
light pink
0.5 cm
2.8 cm
4.8 cm
4.8 cm
0.5 / 4.8
2.8 / 4.8
0.104
0.583
1.5 cm
0.3 cm
1 cm
4.8 cm
4.8 cm
4.8 cm
1.5 / 4.8
0.3 / 4.8
1 / 4.8
0.313
0.063
0.208
0 cm
0 cm
0 cm
0.5 cm
4.8 cm
4.8 cm
4.8 cm
4.8 cm
0 / 4.8
0 / 4.8
0 / 4.8
0.5 / 4.8
0
0
0
0.104
1 cm
4.8 cm
1 / 4.8
0.208
2.3 cm
4.8 cm
2.3 / 4.8
0.479
Biuret Test
Sample
Glutamic acid
Proline
Tyrosine
Cysteine
Alanine
Arginine
Glycine
Urea
Biuret
Casein
Hydrolyzed Casein
Yogurt
Catfood
Water
Expected
Results (CN Bonds)
Few
Few
Few
Few
Few
Many
Few
Few
Many
Few
Few
Few
Few
None
Color (-, +, ++)
++
+
+
++
++
-
Actual
Results (CN Bonds)
None
None
None
Many
None
None
None
None
Few
None
Few
Many
Many
None
Discussion
With the ninhydrin-TLC test, each sample changed color and reacted except for urea,
biuret, and casein. These three samples also did not migrate. With regards to urea, urea is a not a
true, simple amino acid and probably did not migrate or change colors because of this. Biuret did
not migrate or change colors because its make-up is not that of an amino acid; it contains four
peptide bonds and it reacts like a polypeptide. For casein, as far as the TLC test goes, it is not an
amino acid due to the results. As for the rest of the samples, they migrated and changed colors,
indicating that they are amino acids. As mentioned before, proline turns yellow and it did in fact
turn yellow on the TLC because of the substitution of the alpha-amino group (Van Thiel 22).
Another different reaction on the TLC sheet is unknown A, or yogurt, which stained at the origin
and very faintly dissolved in the solvent. This most likely indicates that there is probably a low
concentration of amino acids in the solution and that yogurt isn’t very attracted to the TLC.
However, in contrast to this, the most migration occurred with cysteine, cat food, and alanine.
Cysteine’s Rf value on the TLC sheet is 0.583, cat food’s is 0.479, and alanine’s is 0.313,
indicating that these samples are the most attracted to the TLC. It also seems that some high Rf
values indicate CN bonds as well which will be discussed with the biuret test. The least
migration occurred with arginine, which proposed an Rf value of 0.063, again indicating that
there is probably a low concentration of amino acids in the solution.
Regarding the biuret test, blue color results in no CN bonds. With that being said,
glutamic acid, proline, tyrosine, alanine, arginine, glycine, urea, casein, and water gave a blue
color. The water is used as a control. These solutions that indicated no CN bonds indicate no
peptide bonds present, and therefore, no polypeptides or proteins present. Despite blue, a violet
color results in few CN bonds. Biuret and hydrolyzed casein resulted in a slight purple-violet,
translucent color, indicated few CN bonds. These solutions could be proteins or polypeptides.
Hydrolyzed casein did in fact migrate on the TLC sheet, however, so it is slightly positive for
both tests. It is strange that casein did not exhibit change in both tests, however, while
hydrolyzed casein did. Contamination could have been involved here and hydrolyzed casein
probably just contains more peptide bonds. Next, deep purple reactions indicate many CN bonds,
and cysteine, yogurt, and cat food turned deep purple. These solutions have many peptide bonds,
so they are potential polypeptides or proteins. Yogurt and cat food do in fact contain many
proteins, so it is no surprise that they tested for many peptide bonds. This proved the hypothesis
in the introduction to be true. However, while yogurt barely migrated or showed color change at
all regarding the TLC test, cat food greatly migrated. Since this sample is positive for both tests,
cat food contains both peptide bonds and single amino acids. In this way, the biuret test and the
ninhydrin-TLC test could be correlated. Regarding the biuret test, expected results did not really
match actual results. Samples such as glutamic acid, proline, tyrosine, cysteine, alanine, and
glycine were expected to give at least a few CN bonds because of the way their molecular makeups appeared. They appeared to share some carbon and nitrogen bonds. However, they don't
have enough peptide bonds to be considered polypeptides or proteins. Despite that fact, their Rf
values were precise and clear with no mistakes or Rf values greater than the value of one; that
would pass the solvent front.
Conclusion
In conclusion, most of the ninhydrin-TLC and biuret tests were accurate. As predicted,
unknown A and unknown B (yogurt and cat food) contained many peptide bonds, making them
either polypeptides or proteins. However, replicating the cat food and yogurt on the TLC sheet
would help to confirm cat food migrating and yogurt staying at the origin. In turn, that would
confirm if there are also single amino acids present as well as peptide bonds. Other results were
precise. As stated before, urea is only an analog, so it is clear why urea did not migrate on the
TLC sheet and turned blue for the biuret test. It is not a true amino acid and it doesn't have
peptide bonds. Again, peptide bonds are formations of CN bonds and polypeptides. Amino acids
combine to form those polypeptides. Also, amino acids are tested using ninhydrin and a TLC
sheet while peptide bonds are tested using the biuret test. Overall, amino acids and peptide bonds
are vital macromolecules that are needed for growth and life.
References


Van Thiel, Linda. Experiments in Biology: From Chemistry to Sex. Dubuque:
Kendall/Hunt, 1985.
"Amino Acid." Wikipedia. Wikimedia Foundation, 27 Sept. 2014. Web. 28 Sept. 2014.
http://en.wikipedia.org/wiki/Amino_acid.