Thin layer chromatography CHE 303 L Joao Paulo Toledo

Abstract The purpose of this activity was to apply thin layer chromatography (TLC) to identify amino acids present in an unknown sample. This technique of separation needs a stationary phase immobilized, and an organic solvent. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. The constituents of a sample can be identified by simultaneously running standards with the unknown. The amino acids presented difference in the affinity with the solvent and the amino acids present in the unknown sample were found.

Introduction Amino acids are crystalline solids with surprisingly high melting points. Decomposition and melting tend to be in the 200 - 300C range. For the size of the molecules, this is very high. A general structure of an amino acid has both a basic amine group and an acidic carboxylic acid group [1].

There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group to leave an ion with both a negative charge and a positive charge. This is called a zwitterion.

A zwitterion is a compound with no overall electrical charge, but which contains separate parts which are positively and negatively charged. This is the form that amino acids exist even in the solid state. Instead of the weaker hydrogen bonds and other intermolecular forces that you might have expected, you actually have much stronger ionic attractions between one ion and its neighbors. These ionic attractions take more energy to break and so the amino acids have high melting points for the size of the molecules [1]. Amino acids are generally soluble in water and insoluble in non-polar organic solvents such as hydrocarbons. This reflects the presence of the zwitterions. In water, the ionic attractions between the ions in the solid amino acid are replaced by strong attractions between polar water molecules and the zwitterions. This is much the same as any other ionic substance dissolving in water. The extent of the solubility in water varies depending on the size and nature of the "R" group [1]. Thin layer chromatography (TLC) is a chromatography technique used to separate nonvolatile mixtures.[2] Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose. This layer of adsorbent is known as the stationary phase. First a sample must be applied on the plate, then a solvent or solvent mixture, the mobile phase, is drawn up the plate via capillary action. The separation is achieved as different analytes ascend the TLC plate at different rates [3]. Differences in their attraction to the stationary phase and differences in solubility in the solvent make different compounds in the sample mixture travel at different rates. By changing the solvent, or perhaps using a mixture, the separation of components, measured by the distance moved by compound/distance moved by solvent, can be adjusted [4]. Thin layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Some examples of these applications include: analyzing fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents [5]

Methodology The plates must be handled only by their edges because fingerprints can be detected by ninhydrin giving a false result of masking a true one. 1. By very gently using a pencil, (the resin rubs off the plate rather easily) mark a

baseline on the TLC plate 1.0cm from the bottom of the plate. Mark spots on this line 1.0cm apart (and 1.0cm from the plate edges). 2. Transfer 1.0L of amino acid solution onto the plate at a pencil mark and allow it

to dry. Repeat this once more for that same amino acid on the same spot (you're loading the spot with a reasonable amount of the amino acid). Repeat this for each of the remaining amino acids (including the unknown). Make note of which amino acid is at which spot. 3. Carefully place the spotted plate into the solvent chamber so that the solvent level

is just below your baseline. Close the lid on the chamber. 4. Allow the chromatogram to develop until the solvent front is 0.5cm from the top

edge of the plate (about 90 min). 5. Remove the chromatogram from the chamber and immediately mark the solvent

front all the way across the plate with a pencil. Allow the chromatogram to dry completely. 6. Spray the plate lightly but thoroughly with ninhydrin solution under a fume hood

and allow drying. If the spots don't develop, incubate at about 60 C for 5 min. 7. the spot. 8. Measure and record the distance from that line to the baseline. Measure and Note the color of each spot. Draw a small line across each spot in the middle of

record the distance of the solvent front line directly above the spot to the baseline. Determine the Rf values for each amino acid from the distance data. 9. Compare the spots and Rf values for the unknown amino acids with those for the

known amino acids to determine the identity of the unknown amino acid. Results As shown in figure 1, leucine is the amino acid with greater affinity for three different solvents, while arginine is the amino acid with lower affinity for the three different solvents. In the three solvents, the unknown sample presented to be composed by arginine and cysteine. The

composition of the unknown could be observed by the Rf and the color of the dot in the three plates, yellow for cysteine and purple for arginine.

Figure 1. TLC plate for 4 amino acids and one unknown sample in three different solvents. A= arginine, B= cysteine, U= unknown, C= glutamic acid and D= leucine. Dark dots represent purple and light dots represente yellow.

Discussion

The experiment proved a substitution in the polar solvent often results in a change in resolution, while a change in the less-polar solvent results primarily in a change in Rf of the sample components, which is used to distinguish if the analyte is more attracted to the solvent than the stationary phase. Controlled particle size can bring faster separations and improve resolution. A good buffer has a pH between its pKa -1 and pKa +1. So the rage for each amino acid to be a good buffer would be 1.48-3.48 and 10.51-12.51 for arginine, 0.96-2.96 and 7.18-9.18 for cysteine, 1.19-3.19, 3.25-5.25 and 8.67-10.67 for glutamic acid, and 1.36-3.36 and 8.6010.60 for leucine.

Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 (carbon dioxide) through a series of reactions; the net result is ninhydrin in a partially reduced form hydrindantin. Ninhydrin then condenses with ammonia and hydrindantin to produce a blueish-purple pigment. This result is usually associated with primary amino acids. In these amino acids, the N is free to react with ninhydrin. However, in proline, the N is not available for reaction as it is locked in the ring structure. Therefore no ammonia is produced, so no blue color is presented (figure 2).

Figure 2. Reaction between ninhydrin and proline

This is a good method to determine constituents of a mixture. The method was validated since the unknown could have its composition determined, when compared with the amino acids known. References 1. "An Introduction to Amino Acids." An Introduction to Amino Acids. Jim Clark, Aug. 2007. Web. 09 Dec. 2013 < http://www.chemguide.co.uk/organicprops/aminoacids/background.html >. 2. Harry W. Lewis and Christopher J. Moody (13 Jun 1989). Experimental Organic Chemistry: Principles and Practice (Illustrated ed.). WileyBlackwell. pp. 159173. 3. A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, and P.W.G. Smith. Vogel's Textbook of Practical Organic Chemistry (5th ed.). 4. Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54. 5. Reich, E.; Schibli A. High-performance thin-layer chromatography for the analysis of medicinal plants (Illustrated ed.). New York: Thieme, 2007.