Mark M. J o n e s a n d Gwendalyn R. Champion Vanderbilt University Nashville, Tennessee
A Simple Liquid-Liquid Extraction Experiment for Freshmen
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T h e fact that an oreanic comnound distributes itself between two immiscible liquids is an important one and has manv nractical conseauences. The w a t e r h i d distrihution ratios bf drugs are important in understanding their distribution in the bodv. their mode of action on it. and their excretion from it. (1;~).The determination of such distrihution ratios often involves techniques beyond the abilities of beginning students as well as compounds whose toxicity is too high to make them a .. ~ ~ r. o o r i amaterials te for such students tohandle. For an experiment of this sort to he appropriate for a freshman laboratory it must also use only the simplest and cheapest of rquipment. The procedure described here ran be carried our with the equipment normally present in a typical freshman laboratory. Because of the restrictions above which must he met, the experiment turns out to be a very good test of the students' technique and understanding. The system selected is the distrihution of salicylic acid between water and n-smvl , alcohol. Salicvlieacid is the ~harmacoloeicallv .. , effrctiveslweiesderived from rheauet,lialicylicarid i n aspirin tablets. The coneentratmn of salicylic ariu in each iayrr is drtrrminrrl via titration with standard (1.1 M xdium hydroxide solution, using phenolphthalein as an indicator. For a compound like salicylic acid the distribution ratio will he dependent on the pH of the solution, because the acid and its anion will have a different eauilibrium constant for arti it ion between water and a lipid-like solvent. In such cases the ion~rforms nre generally murh moreaoluhle in watpr than the neutral mo.ecules.'l'he presrnt erp~rimentis run using diqtillrd water and no sprcial controls ovrr the p H of the system. ~
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Procedure Take a clean dry 400-ml beaker. To it add 50 ml of n-amyl alcohol, 50 ml of distilled water measured out with a graduated cylinder, and 0.5 g of salicylic acid. Mix these well, using a stirring rod. Try to breathe as little of the n-amvl alcohol fumes as vou can. Pour the mixture into a 100-mlgraduated flmk and set it aside in the hood. Allow i t to srttle for 20-30 min. At the end uf this time, the liquid should have separated into two clear layers. Tramfer the top layer to a clean beaker, using a pipet or medicine dropper. ~~~
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Determination of Salicylic Acid in Water Pipet 20 ml of the water layer into a clean Erlenmeyer flask, and add 4 droos of ohenolohthalein indicator solution. Titrate this drcpeiep with 0.1 Y scdium hydroxide r h t i u n ~ n t i the l indicator rhnnxcs color. Only n very small amwnt ofrdium hydroxide solution wll h~ required. Repeat this determination on a second 20-ml portion of the water layer. Determination of Salicylic Acid in n-Amy1 Alcohol Pipet a 20-ml portion of the alcohol layer into a clean Erlenmeyer flaskand add 4 drops of phenolphthalein indicator solution. Now titrnte this slowlv with 0.1 N sodium hvdroaide solution. Make sure that y m swirl ;he airohul well 3s you &Idthrsodium hydroxide 30lution as the 4icyiic acid is eatrnrtpd frum thp alcuhol layer and neutralized in this tirration. When t h p watprlnyer in the titrationlayer turns pink permanently you have reached the endpoint. Determination of Blank Titration A hlank titration is carried out to determine the amount of sodium hvdmxide ,~~ solution which is neededto chanee the color of the indicator in theahzenceoiany arid. To find your hlank titration value, take a clean Errnmeyrr tlnsk, add 20 ml uf distilled w a w and 4 drops of the phendphrhnlein indicator wlution. Titrare this dropwrw u,ith 0.1 N sodium hydroxide solution and determine the volume of base needed to change the indicator color. ~~~
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Background Information When a soluhlr material is present in a system whirh contains two immisciblesolvents. A and B. it will distrit~uteitself between them. Such a system reaches equilibrium; the equation written to represent this process is solute(^) solute(^^
For this system we can write the equilihrium constant as
.-when [Solute]~is the concentration of the solute dissolved in solvent B and lSolutela is the concentration of the solute dissolved in solvent A,'. For the system investigated in this experiment, the equilibrium is Salicylic Acid (in water) +Salicylic Acid (in n-amyl alcohol) and the equilibrium constant is [SalicylicAcid]., ,,I .-,I,.,M K = [SalicylicA C ~ I H ~ The concentration of the salicylic acid in each layer can he calculated from the titration data and, from these values, values for K can be easily found. This exneriment was used in a lahoratorv course for nonmajors (nurses). In a section of 16 students the average value of K found was 43 hut the standard deviation was 17. The poorest student in the class contributed more than 50%of the standard deviation. The verv small amount of salicylic acid which remains in the water layer r ~ s u l t sin a range ofstudent values for it; concentration and hence for the value of K also. In this respect the experimental value obtained is a rather effective measure of the student's technique. The value of K determined by us in a seljarate experiment, urinn the same equipment as was availatde to the students. was i 0 . An attemptwas made to carry out this experiment with the more water soluble gallic acid rather than salycilic acid. The titration curve of gallic acid however was accompanied by intrinsic color changes which made it unsuitable for a freshman experiment. Toxicological Data. A comparison of the toxicities of the compounds used in this study with some other common materials is shown in the table (3). 'Goth, A,, "Medical Pharmacology," 8th. Ed., The C. V. Moshy Company, St. Louis, Mo., 1976, pp. 245-246. XaDu, B. N., Maudel, H. G., and Way, E. L., "Fundamentals of Drug Metabolism and Drug Disposition," The Williams and Wilkins Co.,Baltimore, Md., 1971,pp. 34,93. Christensen, H. E., and Luginhyhl, T. T., (Editors), "TheToxic Substances List 1974 Edition," US.Dept. of Health, Education and Welfare, NIOSH, Rockville, Md. 20852. Toxlcltles of Compounds Used Compound ~ m yalcohal l ~ m yalcohol l
ethanol salicylic acid sodium benzoate benzoic acid gailic acid
LDso (mglkg)
Conditions
3030 1390 5560 89 1 4100 2370 5000
oral, rat oral. dog oral, guinea pig oral, rat oral, rat (faad additive) oral, mouse oral, rat
Volume 55, Number 2, February 1978 1 119
