BCHM 221 Lab 10 Notebook (1)
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Lab 10: Analysis of Amino Acids using chemical reactivities and TLC
Introduction
Amino acids are the basic building blocks of proteins, and they serve as the nitrogenous
backbones for compounds like neurotransmitters and hormones
2
. Amino acids are important for
life and well-studied. Amino acids consist of a central carbon atom connected to a side chain, a
hydrogen, a nitrogen-containing amino group and a carboxylic acid group
1
. This experiment
aims to use methods of paper chromatography and chemical reactivity tests to determine the
identity of 4 amino acids. This is important and applicable to researchers and those in industry as
when an unidentified amino acid appears in solution it is important to identify it and come to a
clear conclusion. The hypothesis for this experiment was that the unknowns with similar results
to the amino acids on the TLC and chemical reactivity tests will lead to an approximation of the
unknown’s identities, was not supported by our data from this experiment
Materials and Methods
Paper Chromatography
The paper chromatography was completed as outlined in Lab 10 protocol with the exception that
we used unknowns provided by the TA instead of ours from last week.
Chemical Reactivity
The primary amines, amides, substituted benzene rings, and thiol group tests were completed as
outlined in Lab 10 protocol.
Results
The chemical reactivity of samples and the paper chromatography was performed on the
samples. Unfortunately, as seen in figure 5, no spots appeared on the TLC plate which prevents
us from calculating Rf values and determining the polarity of the samples compared to the
known amino acids. Figure 1 shows that all the samples were negative for benzene rings except
for the positive control which was tryptophan. Figure 2 shows that proline is an imino acid and
produces a yellow color. Leucine produced a yellow-ish purple color. Unknowns 244 and 2
produced the same color as leucine while 17 produced a light purple and 4 produced a deep
purple. Figure 3. shows that unknown 4 has a basic pH while the other unknowns have an acidic
pH. This was supported by the positive (glutamine) and negative (leucine) controls. Figure 4.
shows that all the unknowns were negative for thiol. Only the positive control cysteine turned a
positive yellow color.
Figure 1. Benzene ring chemical reactivity test. Samples from left to right are: unknowns 17, 2,
24, and 4 followed by tryptophan and leucine.
Figure 2. The primary amines chemical reactivity test. From left to right the samples are: Proline,
Lysine, Unknowns 24, 17, 4, and 2.
Figure 3. The amides chemical reactivity test. The samples from left to right are: Unknowns 17,
2, 24, and 4 followed by glutamine and leucine.
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Figure 4. The thiol chemical reactivity test. Samples from left to right are unknowns 2, 4, 17, and
24 followed by cysteine and leucine.
Figure 5. Paper Chromatography with the four unknowns and seven knowns. From left to right
the order is unknowns 17, 2, 24, and 4 followed by cysteine, glutamine, leucine, lysine, proline,
tryptophan, and asparagine.
Figure 6. Drawings of known amino acids tested.
Discussion
Our hypothesis, that the unknowns with similar results to the amino acids on the TLC and
chemical reactivity tests will lead to an approximation of the unknown’s identities, was not
supported by our data from this experiment. Due to the lack of results in the TLC, we were
unable to compare the Rf values. There could have been many reasons as to why we did not see
spots but it was likely because there was not enough ninhydrin on the chromatography before it
was placed into the oven. The rest of our results produced good results however they are less
helpful without the chromatography. Additionally, we used unknowns provided from the TA
rather than our own from the last experiment to hopefully improve our results. Should we have
seen two spots on the chromatography for cysteine I would assume it is related to the two
oxidation states of cysteine. I cannot discuss the relationship between Rf values and side chain
polarity as we did not have results but we can infer that if the unknown samples had the same or
similar Rf as the known amino acids, they are likely the same amino acid. I cannot determine the
identity of the amino acids without the TLC results however I can determine that none of the
samples contain thiols, therefore not cysteine, and none of the samples contain benzene rings
therefore they are not tryptophan. If I were to complete this experiment again I would run the
TLC while doing the chemical reactivities that way if we had no spots we could run it again.
References
[1]Libretexts. (2020, August 14).
6.2: Defining protein
. Medicine LibreTexts.
https://med.libretexts.org/Courses/American_River_College/General_Nutrition_Textbook_(not_
Plant-Based)-_reference_for_NUTRI_303_(Hagenburger)/06%3A_Proteins/6.2%3A_Defining_
Protein
[2]Lopez MJ, Mohiuddin SS. Biochemistry, Essential Amino Acids. [Updated 2023 Mar 13]. In:
StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK557845/
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