A Project Lab for an Advanced General Chemistry
Course Featuring the Amino Acid, Glycine. Emily P. Dudek Brandeis University, Waltham, MA 02254
Student interest in a laboratory course can be stimulated by a coordinated set of experiments in aproject-type investigation. Projects are particularly attractive if they focus on a theme closely related to everyday life. A classical example has been the aspirin study ( I ) . In this paper, another relevant compound, the amino acid, glycine, is suggested as the focal point of a study suitable for an advanced general chemistry lab course. Actually this glycine investigation goes beyond the synthesis, potentiometric titration, and spectronhotometric analvsis of asnirin. and encomnasses the measurement of the hitrogen'content by eudiometry and the e elvcine estimation of the R ~ v a l u of . as compared with other amino acids in apaper chromatography experiment. Glvcine mav be svnthesized bv reacting aqueous ammonia withan aqueous s&tion of m o ~ o c h l o r o ~ c eacid ~ i c a t a pH established with ammonium carbonate (2). The first step in the synthesis involves obtaining 30 mL of a nearly saturated solution of ammonium carbonate (35 g). Patience and a water bath no higher than 60 "C are required. Ten grams of chloroacetic acid are dissolved in 15 mL of water. Students are warned of the corrosive nature of this acid. T o avoid student contact with the pure CICHzCOOH, the aqueous solutioncould be prepared in advance for thestudents. After the addition of excess aqueous ammonia and chloroacetic acid to the ammonium carbonate solution, the flask containing the reaction mixture is stoppered and set aside for the next lab neriod. In the second period, the solution of 110 mL must be evaporated down to 30 mL using a warm water bath of 75 OC and a reduced pressure provided by an aspirator. Addition of 100 mL of methanol and chilling the solution results in the precipitation of glycine. The product isolated by filtration and washed with methanol may be contaminated with ammonium chloride. Students test their products for NHX1 bv dissolvine averv small samnle in water, addine a drop of nitric acid an2 then adding aqieous silver n i t r a c Usually enough AgCl solid is produced to warrant a recrystallization of the glycine. The recrystallization is accomplished by dissolving the crude glycine in a minimum amount of room-temperature water and then adding methanol and chilling the mixture. The recrvstallized material generally gives negative chloride test.- he average yield has been 50% for the crude sample, 75% recovery from one recrystallization, and an overall yield of 40% or 3 g. The synthesis does require two lab periods, but there are idle moments in which students may check in, if the course starts with the synthesis, or may clean and calibrate glassware or, in the second period, may make the glass bends for the nitrogen gas collection. The final product prepared by the students is usually more than 95% pure, but students are to check the purity by three quantitative experiments and one qualitative one. First, the weight percent of nitrogen in glycine is measured by dissolving a 0.1-g sample of glycine in 7 mL of water, adding 0.5 mL of glacial acetic acid, and then introducing 2 mL of 6 M sodium nitrite (2). The acetic acid and sodium nitrite yield nitrous acid which in turn reacts with glycine to yield nitrogen gas,
a
The Nz gas is collected in an inverted buret over a column of water. A drying tube of FeS04. 7Hz0 must be inserted in the line between the closed reaction flask and the U-shaped glass tubeleading to the buret because nitrous acid alone can decompose to yield nitrogen oxide gas. The NO gas flow is stopped by binding the NO to the Fe(I1) ion while the Np gas passes through the drying tube. Commonly found in a general chemistry lab course is an experiment involving the collection of a gas at measured temperature, volume, and pressure values in order to determine the number of moles of gas, assuming ideal gas behavior. This analysis of glycine for its nitrogen content has the added attractions of (1)analysis of a material that students have ~ r e n a r e dand (2) comparison of the results with cornparahie quantitative experiments, titration. and s ~ e c t r o ~ h o t o m e t wStudents' . results have been precise to about 3%,but the error in accuracy was closer to 8%with most students obtaining too large a weight percent of nitrogen. Most likely the FeSOa . 7H20 did not absorb completely the NO gas. Large-size drying tubes were used, and the tubes were replenished for each lab period. The spectrophotometric analysis of glycine is based upon the intense purple color (ahsorptivity of 2.5 X lo3 M-' cm-' at 570 nm) produced upon reacting glycine with an excess of aqueous ninhydrin buffered at pH 4.7 with an acetic acidsodium acetate mixture. The standard used to verify Beer's Law and to give the ahsorntivitv value was commercial glycine. ~ r o d u c t i o nof the &ple color requires heating t h e solution. Unfortunately the intensity of the color for a given concentration of glycine depends upon the temperature and duration of the heating. T o standardize the heating step, a specifiedvolume of gly&ne in aqueous solution was added to the buffered ninhydrin solution only after the solution was boiling. Then the heating was continued for just 20 min more, followed by a rapid chilling of the solution. The solution was then transferred to a volumetric flask and diluted with water. Despite the regulation of the heating, the ahsorotivitv " values were nrecise to onlv 20%. and eenerallv led to too low percent purity values. he uncertznty was reduced to 10% and the accuracy was better than 5% if the analysis of the glycine's purity was based upon parallel treatment of a commercial sample and a student-svnthesized sample of glycine with the absorbances of the two samples being compared. I t is unfortunate that the critical heating step reduced the precision of the spectrophotometric analysis; however, the experiment does introduce Beer's Law, involves measuring the absorbance of a colored solution of large absorptivity with an absorbance maximum near the miadle of the visible region, and the color-producing reaction is utilized again in the chromatographic analysis of amino acid mixtures. The third quantitative analysis of glycine involves potentiometric titrations of glycine both as an acid and as a base (3). The experiment not only gives the percent purity of
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Number 10
October 1967
899
glycine, hut also its K. and Kb values along with its isoelectric point. Samples of student-svnthesized elycine are titrated &th a standard HCI solution, and then other samples are titrated with a standard NaOH solution. The data from these titrations lead to the equilibrium constant values. To determine the equivalence point and actual concentration of glycine in solution, a third gmup of glycine samples are titrated with the standard NaOH solution after formaldehyde has been added to the glycine solution. The formaldehyde reacts with the glycinate ion, 2HC(O)H(aq)+ H,NCH,CO;(aq)
-
(HOCH,),NCH,CO,(aq)
Thus, the formaldehyde in effect enhances the acid strength of glycine by shifting the titration reaction,
toward comoletion. Ninetv nercent of the students found the purity oE their glycine t o b e within 2% of loo%, hut with about twice as manv reportine a percent ereater than 100% rather than less than 160%. The percent deviation was less than 1%.The DK. values were reoroducihle to better than 1% and accurateto within 5% of the literature values (for the glycinium ion pK.1 = 2.34, pKaz = 9.60, and the isoelectric point of glycine is 5.97). The estimation of DK., . ... was also incor~oratedinto a voltair cell exprrinwnt. Halt cdls are set up of buttered solutions Therell wtential of with ~\~inh\.drone-vlatinumelectrodes. any two haif dells a i 25 "C is equal to 0.059 ( p ~ , - -pH2) (4). The value of pH1 may he set by a pHl = 7.00 buffer solution. The value of pH2 for solutions of glycine-hydrochloride and glycine of ratios 10:1, 1:1, and 1:10 may then be established by measuring voltages, the anticipated values being 0.33, 0.27, and 0.22 V, respectively. For the students, the error in accuracy of the recorded voltages was about 10%. The qualitative characterization of the student-synthesized glycine involves checking its purity by paper chromatography. Only one spot should he seen, and its Rf value should equal that of commercial elvcine. Actuallv. .. onlv . amino arid impurities cnn be detected since the color-developinp. rrnrent is ninh\rdrin.'l'hes~)lventmixtureused isethvl acetate-f&ic acid (90%)-water in a volume ratio of 7:21 (5). Students are also eiven mixtures of amino acids to he analyzed by paper chr&natography. The amino acid solutions are 0.01 M in 20% isopropyl alcohol, and the amino acids studied are lysine, aspartic acid, glycine, valine, and
900
Journal of Chemical Education
leucine with Rr values of 0.14, 0.28, 0.36, 0.67, and 0.82, respectively. The spots are located with a 0.02% solution of ninhydrin in 95% ethanol. For good resolution the spots must he small (melting point capillaries are not narrow enough), and students must avoid getting their fingerprints on the paper. Thissequence of glycine experiments has been carried out by three classes of more than 150 students in the introductory chemistry lab course. The glycine project has been discontinued now that i t has become clear that the patience in the svnthesis. the care in sample the soectro. .ore~arationfor . photometric analysis, and the theory of polyprotic acid titrations are too advanced for the averaee student in the eeneral chemistry lab course. Currently, ;carbonate sequence of complementary analytical methods has been instituted as the opening of the course (details to he published). On the other hand, the glycine project has proven to he an attractive, instructive study f i r advanced-general chemistry students. For such students, the investigation could he extended in several ways. With some investment in highly accurate calorimeters, the enthalpies of the titration reactions, that is, the glycine proton transfer enthalpies, may he determined (6).The glycine in dietetic beverages can be estimated hv simple fluorimetric analvsis (7). Just as the aspirin study has been extended to lnclude preparation and characli of the material ( 8 1 .the t e r i ~ a t ~ uofn the c o ~ n ~ n lcomolex copper(I1) complex bf glycine may he prepared in one lab neriod, hut, unlike the aspirin comolex, the his(elvcinato) copper(11) complex exists i s two isomers. The cis-isomer is first synthesized and then converted into the trans form (9). If a lab course were to end with special projects, students could investigate the physical and chemical properties of the stable trans isomer aspart of an integrated analytical, and inorganic chemistry project (10). Upon request, the author will provide detailed instructions for the synthesis of glycine, the three quantitative analyses, and the paper chromatography experiment. Llieraiure Clied
A.
W. J.Chrrn.E&~. l984.61.76. 7. Coppols. E. 0.;Hanna, J. G. J. C h e m Edut. 1976.53.322. R. Dudek , *1 - P. J. Chern.Edv?. 1911.64 829. 9. O'R~ien.P. J. Chsm. ~dui.~~i&.Js, 1052. LO. Farrel1,d.J.J Cham. Educ.,I977.54445. 6. Rametfe.
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