The Resolution of DL-Histidine Arthur J. Bosch Central College Pella, Iowa 50219
An organic chemistry experiment
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using a n ion exchange resin
A n experiment involving the resolution of a racemic mixture is frequently performed in the beginning organic chemistry course. This article describes a new resolution experiment which gives a product of high optical purity and, in addition, demonstrates the use of an ion exchange resin and mixed solvent recrystallization. It is an inexpensive experiment, requires no elaborate equipment, and gives good results in the students' hands. The experiment involves the isolation of the amino acid, D-histidine, from DL-histidine.l The procedure is adapted from that of Pyman2 as modified by Kolenbrander and Berg.3 D-Histidine-D.-hydrogen tartrate4 precipitates readily from an aqueous solution in which L-histidine-hi hydrogen tartrate remains soluble. The tartrate is removed from the D-histidine-D,-hydrogen tartrate by a weak base anion exchange resin and the histidine is recrystallized from 80% ethanol. The chemicals used are DL-histidine,hi tartaric acid, and a weak base anion exchange resin. The cost per student for chemicals is $0.45, excluding the initial cost of the resin.s The results given in the experimental section are the averages of those obtained by a class of beginning organic chemistry students. The Experiment Into a 50-ml flask place 2.33 g (0.015 mole) of nl-histidine, 2.25 g (0.015 mole) of ~ ~ t a r t s racid, i e and 10 ml of water. Heat until solution is complete, stopper the flask, and set aside until the next laboratory period during which time precipitation of D-histidine-D,-hydrogen tartrate occurs. Pulverize the solid and collect it by suction filtration. Wash it with severd milliliters of water, air-dry, and weigh. Yield: 1.77 g, 77%. Prepare s, chromatography eolumn of a weak base anion exchange resin. A 50-ml Mohr buret, with a short piece of rubber tubing and a screw clamp, makes a good column. Tamp a plug of glass wool in the constricted end of the buret, add 25-30 ml
required. ~ P Y M AF. N ,L., J. Chcm. Soc., 99, 1386 (1911). ~ O L T ~ : N B ~ A N D E H. E . M.. A N D BERG.C. P.. Arch. Biochcm. B ~ O P ~ Y S 119,435 ., (1967). J. N., J. CHISM. EDUC.,41, 619 (1964), for a dis'See BAXTER, cussion on the nomenclature of tartario acids. The resin need be purchased only the first time the experiment is done since the combined used resin from a class can be regenerated for use by a subsequent elms. Slowly wash the used resin with 10% NaOH and then remove the excess base by washing with distilled water. 6 KNOOP, F.,Beitr. Chem. Physiol. Pathol., 11, 356 (1908). ' WEAST, R. C., (Editor), "Handbook of Chemistry and Physics," (47th Ed.), The Chemied Rubber Co., Cleveland, Ohio, 1966, p. C-365.
of resin as a water slurry, and tamp another plug of glass wool on top of the resin. The following resins have worked satisfactorily: REXYN 203 (OH-), 16-50 mesh, Fisher Scientific Co., and DOWEX 3 (OH-), 20-50 mesh, Dow Chemical Co. Amberlite IR-4B (OH-), Rohm & Haas Co., dso worked; however, when a. coarse grade of resin wm u d , some tartrate initidly passed through the resin. Dissolve 1.00 g of the isolated product in 20 ml of water, heating to dissolve the solid. Place the Dhistidine-D,-hydrogen tartrate solution onto the column without disturbing the surface of the resin. Allow the solution to enter the column slowly (0.3-0.5 ml/min), collecting the eluate in a 100-ml beaker. Periodiedy (after approximately 1, 10, and 20 ml of the solution have been eluted) collect a. drop of eluate in a test tuhe and test for the presence of the tartrate ion by adding a drop of 10% lead acetate solution. The absence of a white precipitate indlcates the desired absence of tartrate. The resin must remove all of the tartrate from the solution. If tartrate should pass through the resin (probably because the flow rate was initially too fast), return the tartrate-containing eluate onto the column until a test drop of eluate is tartrate-free. After 20 ml h w e been eluted also collect one or two drops of eluate to test for the presence of histidine. Into the test solution, carefully pour bromine vapors from a battle of bromine (HOOD!). Mix and add more bromine until the solution is yellow indicating its saturation with bromine. Heat the solution, driving off excess bromine. If histidine is present, a red color will appear immediately after the excess bromine has been driven off. The intensity of the red color is dependent on the histidine concentration. A drop or two of the initial eluate diluted to 1 ml with water should give a deep red color. This test is an adaptation of that described by Knoop.' When all of the histidine tartrate solution hm entered the resin column, place 40 ml of water onto the column to wash the residual histidine from the resin. Check the last ml of wash eluate for the presence of histidine. The last ml of wash eluate should be colorless or only faintly red indicating that all or most of the histidine has been removed from the resin. Place the eluted solution into a n evaporating dish and e w p e rate to dryness on a. steam bath. Air-dry and weigh the crude histidine. Yield: 0.42 g, 83%. Recrystallize all of the crude histidine as fallows: Add 10 ml of water per 0.28 g of solid, heat to dissolve the solid, and then add 4 volumes of ethanol per 1 volume of aqueous solution. Set aside overnieht for ervstalliziltion to occur. Collect the ~ r o d u c t by suction fitration, &ash with several ml of 80% ethanol-20% water (v/v), air-dry, and weigh. Yield: 0.27 g, 53%. Determine the optical purity of the D-histidine as follows: Dissolve 0.14 g of the dried solid in water to give 5.0 ml solution and determine the optical rotation of this solution in a 4 mm X 20 em semi-micro polarimeter tuhe. [a]o at room temperature: +40.0. (Lit. [*]a;: +40.2).'
Alternate Procedure for the Removal of Tartrate A batch technique, req~"ring less resin, can he used to remove the tartrate from the D-histidine- hydrogen tartrate. Add a solution of 1.00 g of n-histidine- hydrogen tartrate in 20 ml of water to 10 ml of wet weak base anion exchange resin in a flask. Stir, stopper the flask, and let the mixture stand for a day. Prepare a chromatography column containing approximately 2 ml
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of the same wet resin. Pour the mixture of resin and solution onto the column and allow the resin to settle. Collect the eluate (0.5-1.0 ml/min), testing for tartrate and histidine. Wash the resin with 30 ml of water to remove the residual histidine.
The author wishes to acknowledge a grant from the
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Journal o f Chemical Education
Small Grants Program of the Division of Chemical I R~~~ lB ioni exchange t ~ ~ d ~and ~ gift~of ~ t ~ i b ~~ resin from Rohm & Haas Co. A special thanks is due Dr. Harold M. Kolenbrander for his many helpful suggestions.
