Color Reactions and Thin-Layer Chromatography of Amino Acids An Undergraduate Organic Chemistry Experiment for Students in the Allied Health Sciences Michael 0. Hurst and D. Keith Cobb Georgia Southern University. Statesboro, GA 30460 Organic chemistry in chemistry courses for . experiments . alliedhealth sciences are usually simple illustrations of reactions and solubility tests and are often perceived by the students to be boring, weakening a n already low motivation level. W e have developed a T L C experiment for students in these courses t h a t we believe will be a n alternative. Two thin-layer chromatograms are run, each containing amino acid standards and an unknown. One chromatoeram is sprayed with ninhydrin to determine the R;s of a i of t h e aminb acids. A second chromatogram is sprayed with specific color reagents for arginine and histidine. B y heing ahle t o use both the R, values and t h e color tests the students can reliably identify their unknowns. Reagents Plastie-backed cellulose TLC sheets were from Eastman. The fluorescent arginine spots were identified with a hand-held UV lamp from Fisher. Five-microliter Microcap capillary tubes from Fisher were used for the spotting of samples. Spraying of the TLC sheets was done with a Sigma Spray Unit aerosol propellant system from Sigma. All other reagents were of reagent grade. Thin-Layer Chromatography (1) The 20- X 20-em TLC sheets were cut into eight 5- X 10-cm sheets. two for each student. Five microliters each of three 5-me/mL amino arid standards. leucine. arginine, and histidine, were ~pottpd un each TLC sheet along with an unknown chvien from the thrpe standards. Drying the spots with hair dryers greatly speeded up the spotting process. Each student prepared two identical TLC's in this manner. 400-mL beakers are used as a chromatography chamber. The solvent system was 60% n-hutanol/20% glacial acetic acidRO% water. The chambers wereequilibrated withsolvent by the students at the beginning of the lab. One beaker held two TLC sheets. The solvent reached 2.2 cm from the top of the sheet in 30 min. The sheets were then removed from the solvent and dried. Development with Nlnhydrln (2) One sheet war used to rdentrfy the unknown hy R. d u e . Thm one was sprayed with 03'7 ninhydrin in n.hutanol. The shpets were dried, placed in a 105-C o w n for 5 min, and the Riralue of each spot was determined (see table) Development with Specific Color Reagents The TLC sheet not sprayed for ninhydrin was instead developed with an arginine-specificreagent, a dione that reacts with the guani-
978
Journal of Chemical Education
R, Values of AmlnO Aclds on C e l l u l TLC ~ Sheets In Bulanol
Solvent Syslem amlnoacids
Ws
arginine hlstldine iem>cine
0.17 0.14 n an
din0 group (3). Two solutions were prepared for this, one being 0.02% phenanthrenequinone in absolute ethanol (stable at 4 OC in the dark for months) and the other heing 5 g of NaOH in 50 mL of 60%ethanol (stable for ahout 1month). Equal volumes of these two reagents were mixed together on the day of the experiment and used to spray the nonninhydrin chromatogram. After drying, the TLC sheets were viewed with a hand-held UV lamp (warn students that UVlight is damaging to the retina), the arginine spots showing up as greenish fluorescent spots. The same TLC sheet was then tested for histidine (4). Again, two reagents were combined, one being cold 5%sodium nitrite in water and the other cold 1%sulfanilic acid in 1M HCI. Equal volumes of the two reagents were mixed and left on ice for 10 min, then the TLC sheets were sprayed. The sulfanilic acid and sodium nitrite form diazosulfanilicacid, which reacts with imidazole compounds to give diazonium dyes (5). Histidine samples give a cherry-red or orange spot. The NaOH from the first test for arginine should render the sheet basic enough for this reaction; if no color develops, the sheets can be sprayed with 15%Na2C03. Discussion This experiment is a cood illustration of biochemical chromatographic techniques and of the polarity and organic chemistry of some amino acid side chains. T h e compatibilitv of the arginine and histidine tests allows t h e chromatography and color tests to all he done in the same lab period. By heing ahle t o use both Rf values and color tests t o identify their unknowns, the students can do so with confidence, which is important in lower level chemistry courses where morale is often a problem.
Literature Cited 1. Allen, G. Sequencing of RotainsondPeptide~:Elsevier: New York. 1981;p 126. 2. Rohyt, J.: White.B.Biochemico1 Techniques: ThaoryondPractire:BrookslCole:Monterey, CA, 1987:p 227. 3. Ysmads. S.: ltsno, H. Biochim. Biophys. Aclo 1%66,130.53&540. 4. Zweig. G.; Whitaker. J. Paper Chramofogrophy and Eirclrophoreaia: Academic: Now Yark, 1967: 1, p 96. 5. Amen.B.: Mifehe1l.H. J. Chem.Soc. 1952.74.2522553,
