In the Laboratory
Determination of the Enantiomeric Purity of Commercial L -[U-14C]Valine An Experiment Utilizing Reversed-Phase Thin-Layer Chromatography and Liquid Scintillation Counting Joseph W. LeFevre Chemistry Department, State University of New York (SUNY) College at Oswego, Oswego, NY 13126
A survey of this Journal shows that only a few experiments involving radioactive organic compounds have been published in the last 30 years (1–9). References 10 and 11 cite numerous experiments utilizing radioactive inorganic elements. The experiment described here analyzes a commercial uniformly carbon-14 labeled L-valine sample (L-[U14 C]Val) for its enantiomeric purity. This type of information is crucial to researchers who require enantiomerically pure radioactive amino acids for investigating stereochemical aspects of biosynthetic pathways (12, 13). The experiment utilizes a reverse-isotope dilution technique that was performed on the microscale level in three threehour periods by pairs of students in an advanced chemistry laboratory at SUNY Oswego. The experiment involves the following steps: 1. Diluting 0.5–1 µCi of L-[U-14C]Val with nonradioactive racemic DL-valine,1 followed by conversion to the highly fluorescent 5-dimethylamino-1-naphthalenesulfonyl (dansyl, DNS) derivatives using DNS-chloride (14). 2. Resolving the enantiomeric DNS-DL-Val by reversedphase thin-layer chromatography (TLC) via diastereomeric inclusion complexes with β-cyclodextrin (β-CD) as a mobile-phase additive (15, 16). 3. Scraping the pure DNS-D- and -L-Val from the plate and subjecting each sample to liquid scintillation counting (LSC) to determine the relative amounts of radioactivity present in each.
N(CH3)2
N(CH3)2 H3C
NH2 CH3
DL-valine
1) 0.2 M NaHCO3 2) 1 N HCl (to pH = 2 - 4)
+ S O Cl O
DNS-C l
CO2H H3C
NH
S O O
CH3
DNS-DL-valine
Safety Ample time should be spent with the students discussing the necessary safety precautions when working with radioactive materials. Carbon-14 is a weak β-emitter with a half-life of 5730 years. Although working with less than 1 µCi poses no threat to students, all of the radioactivity must be contained. Lab coats, safety goggles, and disposable plastic gloves should be worn at all times. All work with carbon-14 should be performed in a tray lined with plastic-backed absorbent paper in a well-ventilated area. Avoid pipetting radioactive solutions by mouth. The work area should be monitored for contamination before leaving the laboratory. A complete list of precautions has recently appeared in this Journal (9). A useful text on radiotracer methods is reference 17. Compliance with all local and federal regulations is important. Materials
To make the correct stereochemical identification of the DNS-D- and -L-Val on reversed-phase TLC, samples of each enantiomer are prepared in nonradioactive form from D- and L-Val. This enables each student to do the reaction once before the radioactive sample is synthesized. The reaction is illustrated in eq 1. CO2H
The experiment introduces students to a variety of techniques, including microscale synthesis, normal and reversedphase TLC, handling of radioisotopes, and LSC. It also reinforces important stereochemical concepts such as enantiomers, diastereomers, and resolution. The experiment is suitable for laboratories in organic chemistry, biochemistry, and analytical chemistry provided the safety precautions listed below are followed.
(1)
Unlabeled D-, L-, and DL-Val, L-[U-14C]Val, DNS-DL-Val, and DNS-Cl are available from Sigma Chemical Company, P. O. Box 14508, St. Louis, MO 63178. Normal-phase silica gel TLC plates with fluorescent indicator, #13181, were purchased from Eastman Kodak Company, Rochester, NY 14650. β-CD can be obtained from CERESTAR, USA, Inc., 1100 Indianapolis Blvd., Hammond, IN 46320-1094. Whatman reversed-phase KC18 TLC plates, 1 × 3′′, 200 µm thickness, without fluorescent indicator, are available by special order from Whatman, Inc., 9 Bridewell Place, Clifton, NJ 07014. Alternatively, Whatman reversed-phase LKC18 plates, 5 × 20 cm, 200 µm thickness, also without fluorescent indicator, can be used. They are available from Alltech Associates, Inc., 2051 Waukegon Continued on page 1288
JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education
1287
In the Laboratory
Road, Deerfield, IL 60015-1899. ULTIMA GOLD LS cocktail was purchased from Packard Instrument Company, 800 Research Parkway, Meriden, CT 06450. Experimental Procedures
Week 1. Synthesis of Nonradioactive DNS-D- and-L-Val (Requires approximately 2 h) To a 5-mL conical vial, add 100 µL of a 1 mM solution of D-Val in 0.2 M NaHCO3 (pH = 9.5)2 via a 10–100-µL adjustable Eppendorf pipet. Dispense 100 µL of a 10 mM solution of DNS-Cl in acetone to the vial, cap tightly, and shake for a few seconds. Let the vial stand at room temperature for one hour. Adjust the pH of the solution to between 2 and 4 by adding 1 N HCl dropwise.3 Extract the aqueous solution three times (1.0 mL each) with diethyl ether. Combine the ether extracts, dry with 200 mg of anhydrous MgSO4 and filter through a Pasteur pipet containing cotton into a small labeled vial. Allow the ether to evaporate. Repeat the procedure on a sample of L-Val. Week 2. Synthesis of DNS-L-[U-14C]Val (Requires approximately 2 h) CAUTION: Wear disposable gloves and do all work in a tray lined with plastic-backed absorbent paper. Do not pipet by mouth! Always keep the 1-mL conical vial in a 50-mL beaker to avoid spillage. To a 1-mL conical vial, add 0.5–1 µCi of L-[U-14C]Val and 100 µL of a 1 mM solution of nonradioactive DL-Val in 0.2 M NaHCO3 (pH = 9.5). Add 100 µL of 10 mM DNSCl in acetone, cap tightly, and shake gently for a few seconds. Let the reaction stand for 1 hour at room temperature. While the reaction is proceeding, go to the next section entitled “Identification of the DNS-D- and -L-Val by NormalPhase TLC.” After 1 hour, add 30 µL of 1 N HCl to the radioactive sample and leave the labeled vial uncapped in a locked fume hood until the next laboratory period so that the solvent can evaporate. Week 2. Identification of the DNS-D- and -L-Val by Normal-Phase TLC (Requires approximately 1 h) Dissolve the nonradioactive DNS-D- and -L-Val in 200 µL each of wet ethyl acetate (EtOAc).4 This is prepared by shaking 2 mL each of water and EtOAc for 1 min in a 5-mL conical vial. After the layers separate, use the top layer. Using a micropipet, spot a 3.5 × 6.5-mm plastic-backed TLC plate at the center of a very light pencil line drawn horizontally 1 cm from the bottom edge. Use a solution containing approximately 1 mg of a commercial sample of DNS-DL-Val dissolved in 4–5 mL of methanol (MeOH). On one side of the DNSDL-racemate spot the DNS-D-Val, and on the other side DNSL-Val, placing each spot 5 mm from the edge. Develop the plate in a solvent composed of toluene5:pyridine:acetic acid (AcOH)/30:10:1 (v/v/v) (18). Calculate and compare the retention factors (R f ’s) of the three spots. Repeat the separation on a new TLC plate in a solvent composed of n-heptane: n-butanol (BuOH):AcOH/20:8:3 (v/v/v) (19). Calculate and compare the R f ’s. Week 3. Reversed-Phase TLC Analysis (Requires approximately 2–3 h) Dissolve the radioactive DNS-Val in exactly 200 µL of wet EtOAc using a 100–1000-µL adjustable Eppendorf pipet. 1288
Cap the vial tightly and shake for 1 min. Remove exactly 5 µL of the solution using a 0.2–10-µL adjustable Eppendorf pipet and place in a clean, dry 100-µL conical vial. Using a micropipet, spot all of the sample on a 1 × 3′′ reversed-phase TLC plate in a series of very small, closely spaced spots, beginning and ending 6 mm from each side on a pencil line drawn very lightly across the plate 8 mm from the bottom edge. Spot the nonradioactive DNS-D-Val one time as a single spot on the line 3 mm from the left-hand edge of the plate, and spot the nonradioactive DNS-L-Val once on the line 3 mm from the right-hand edge. Develop the plate for approximately 1 hour in 4–5 mL of a solvent composed of acetonitrile (CH3CN):0.2 M aqueous β-CD/3:7 (v/v) in a 4-oz. screw-cap jar. The aqueous portion is also 0.6 M in NaCl and is saturated with urea. Without NaCl, mobile phases containing more than 50% water will dissolve the binder that attaches the stationary phase to the glass plate. The urea increases the solubility of β-CD in water more than tenfold (15). If you use 5 × 20-cm plates full development will take several hours, but complete separation of the D- and Lbands should be apparent in 1–2 hours. When the plate has fully developed, mark the solvent front and allow the solvent to evaporate for at least 30 minutes in a fume hood. Visualize the spots by longwave UV light (366 nm) and carefully outline the two strongly fluorescent green bands lightly with a pencil, being careful 1 2 3 4 not to disturb the silica gel. Using the DFigure 1. Reversedand L-isomers, identify the corresponding bands in the radioactive sample. Calcu- phase TLC separalate the R f ’s and α, the separation factor.6 tion of DNS-D- and -L-val. 1 = DNS-DA typical separation appears in Figure 1. val; 2→3 = DNSIn a fume hood, using a 2-mm-wide [U- 14 C]val; 4 = TLC scraper, scrape the upper D-isomer DNS- L -val; solvent band first and transfer the silica gel quan- = CH3CN: 0.2 M titatively to an LS vial using a folded piece β-CD/3:7. of weighing paper. Rinse the scraper with 2 mL of LS cocktail using a Pasteur pipet, collecting the rinse in the LS vial. Repeat the process on the lower L-isomer band using a second LS vial. Add another 8 mL of LS cocktail to each vial, cap, shake well for at least 1 min, and count the samples for at least 3 min each.7 Also, in order to calculate the percent yield, count an equivalent aliquot of the L-[U14 C]Val that was given to each pair of students. An average sample gives 6.01 × 105 counts/min (cpm), which corresponds to 0.64 µCi.8 Typical R f ’s for the various solvent systems appear in Table 1. Table 1. Average TLC Retention Factors Compound
Normal Phase
Reversed Phase
A
B
C
DNS-D-val
0.60
0.49
0.50
DNS-L-val
0.60
0.49
0.40
NOTE: R f’s may vary slightly depending on the activity of the silica gel. A = Toluene:pyridine:AcOH/30:10:1 (v/v/v). B = n -heptane: n- BuOH:AcOH/20:8:3 (v/v/v). C = CH3CN:0.2 M β-CD/3:7 (v/v).
Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu
In the Laboratory
Discussion The experiment provides experience with both normal and reversed-phase TLC as well as the reinforcement of important stereochemical principles. For example, as is evident from Table 1, students can see that with normal-phase TLC, the DNS-D- and -L-Val have the same R f . However, with the introduction of the chiral β-CD in the reversed-phase system, the R f ’s are quite different for the two isomers (Fig. 1). These results reinforce the fact that enantiomers have identical physical properties and that they cannot be separated unless a diastereomeric relationship is introduced. β-CD makes the separation possible. It is composed of seven α-D-(+)-glucose units bonded through α-(1,4)-glycosidic linkages and resembles a truncated cone (20). The larger mouth is lined with secondary hydroxyl groups, while primary hydroxyl groups are located at the smaller base. The relatively nonpolar cavity is the correct size to accommodate the nonpolar naphthalene ring of the DNS-D- and -L-Val and thus allows diastereomeric inclusion complexes to form. Enantioselection occurs by Hbonding interactions that differ slightly in energy between the DNA-D- and -L-amino acids. In reversed-phase TLC systems such as this one, the D-isomers always elute ahead of the L-isomers (15, 16). The experiment offers valuable experience in radioisotope methodology. The procedure was carefully designed so that no extractions were necessary for the isolation of the radioactive products, in order to minimize handling. It took about three days for the water–acetone solvent to evaporate to dryness in a fume hood at room temperature. If desired, ether extractions can be used to isolate the DNS-[U-14C]Val. After acidifying the aqueous layer to pH = 2–4, extract three times (200 µL each) with diethyl ether, which removes 70% of the radioactivity. The use of extractions causes a reduction in the calculated percent yield of the reaction because the extraction efficiency is not 100%, but no change in the relative portions of the DNS-D- and -L-Val because each enantiomer is extracted with the same efficiency. Hence, the enantiomeric purity calculations are identical with or without extractions. Accurate results depend upon the fact that no detectable racemization occurs in the dansylation reaction. If any were to occur, information concerning the enantiomeric composition of the original L-[U-14C]Val would be lost. High-performance liquid chromatographic (HPLC) analysis showed that no detectable racemization (
