Thin Layer Chromatography of Amino Acids
Introduction
The technique used for this experiment was thin layer chromatography (TLC). TLC is used to separate components of a mixture (Clark, 2016). The purpose of this experiment was to identify an unknown amino acid. The components will have different affinities to the stationary and mobile phases. The various components will travel at different rates based on their affinity to either the mobile or stationary phase (Clark). A polar stationary phase is used in TLC experiments and the mobile phase can be a combination of solvents or just one solvent (psu.edu, n.d.). TLC can also be used to monitor the progress of reactions and can tell if two compounds are identical (Padias, 2011). In general, more polar molecules will be attracted to the stationary phase and not move as far up the TLC plate (psu.edu, n.d.). Similarly, compounds that are less polar will be able to travel further up the plate because they have less attraction to the stationary phase.
Methods
A chromatography chamber was taken to the fume hood, and four mL of chromatography solvent was added with a pipette. The lid was placed on the chamber.
Handling the TLC slide by the edges, a horizontal line was placed 1 cm from the bottom edge of the plate.
Four hash marks were made on the line, equal distances apart. Under the drawn line the first mark was labeled “A” (for alanine). The second mark was labeled “G” (for glycine). The third mark was labeled “P” (for phenylalanine). The final mark was labeled “U” (for unknown).
With capillary tubes, one drop of each amino acid was placed on the TLC slide.
TLC plate was held vertically on the outside of the TLC chamber to make sure the line was higher than the solvent in the chamber.
With forceps, the TLC plate was quickly placed inside the chamber, and the lid was tightened.
The solvent was given time to move up the TLC plate until it was approximately 1 cm from the top. The TLC slide was removed from the chamber, and the solvent front was marked with a pencil. The slide was allowed to dry.
The TLC slide was placed in an over set at 800C for 15 minutes. This sped up the reaction between the ninhydrin and the amino acids, so the amino acids were visible.
A pencil was used to outline each TLC plate. The center of each spot was marked with a horizontal line.
Using a ruler, the distance from the origin to the solvent front was measured and recorded. The distance from the origin to each amino acid was recorded, and the Rf values were calculated.
Results
Figure 1
Amino Acid Structures
\s\s\s
Glycine Alanine Phenylalanine
Most polar Least polar
Data Table 1
TLC Measurements
Data Table 2
TLC Measurements for the unknown Amino Acid
Discussion
As seen in Data Table 1, the Rf value of Glycine was 0.528. Of the amino acids tested, glycine had the smallest Rf and was the most polar. Alanine had an Rf of 0.547, and the Rf of phenylalanine was calculated to be 0.708. Because phenylalanine had the largest Rf value, it was the least polar amino acid tested. The Rf of the unknown amino acid was 0.547. The surface of silica gel is very polar. The OH groups on both glycine and alanine could form hydrogen bonds with the silica gel thus, lowering their Rf values (Clark, 2016). Phenylalanine is a larger molecule, and its size prevented significant hydrogen bonding with the silica gel. The unknown amino acid was determined to be glycine because of the similarity in Rf values. I learned that TLC separates molecules based on polarity. Polar molecules do not travel as far as non-polar molecules. The importance of this experiment was that using this data, scientists can better understand how amino acids behave in the human body. Polar interactions between amino acids will affect the shapes of protein molecules. The most important part of amino acids is the R group (or side chain) (biochem.edu, n.d.). That part of the molecule determines whether it can pack in tight spaces or form bonds with other amino acids in proteins. Data from this TLC experiment sheds light on some properties of amino acids.
Conclusion
In conclusion, the unknown amino acid was found to be glycine. The Rf value of the unknown corresponded with the known sample of glycine, therefore the unknown is glycine.
References
Clark, J. (2016, June). Thin Layer Chromatography. Retrieved from
http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html
Padias, A. (2011). Making the Connections 2: A How-To Guide for Organic Chemistry Lab
Techniques, Second Edition. New York: Hayden-McNeil Publishing.
Sturm, N. (2015). Amino Acids/Proteins. Retrieved from
http://www.nbs.csudh.edu/chemistry/faculty/nsturm/CHE450/04_AminoAcidsProteins.ht
m
The Amino Acids. (n.d.) biochem.umd.edu. retrieved from
http://www.biochem.umd.edu/biochem/kahn/teach_res/amino_acids/
Thin-Layer Chromatography. (n.d.). psu.edu. retrieved from
http://courses.chem.psu.edu/chem36/Experiments/PDF's_for_techniques/TLC.pdf
