Titration of Alanine Monitored by NMR Spectroscopy
Francis J. Waller,' lclal S. Hartman, and Shirley 1.Kwong Simmons College Boston, Massachusetts 021 15
I
A biochemistry laboratory experiment
During t h e last several years, chemistry departments of small educational institutions have been acquiring extensive instrumentation including modest nuclear magnetic resonance (nmr) facilities. I n order to demonstrate a t t h e undergraduate level t h e application of proton n m r spectroscopy to t h e study of biological problems, i t s theory a n d techniques were introduced a s p a r t of our biochemistry course a n d laboratory. The experiment described here involves simultaneous monitoring of pH and nmr chemical shifts during an aqueous titration of a- and 8-alanine. The data obtained far titrations from p H 7-14 permit the calculation of ~ K + Nvalues H ~ for the two amino acids. This approach has been described el~ewhere.~.3 Alanine was chosen for this eaperiment hecause of its high solubilit9 and the opportunity to observe haw the relative positions of the carhmyl and amine functions influence ~ K + N H , . The chemical shifts of the non-exchangeable protons of the amino acid move upfield with increasing pH.3 This is due to the increased negative charge density in the molecule as the equilibrium (eqn. (1)) shifts to the right. The effect is greatest for protons nearer the earhoxvlate anion. In acid. deshieldine is often sufficient to cause the a-p;otons to overlap with the wate;signal. The total titration of a-alanine is shown in eqn. (1) CH, + NH,-H-C02H
concentration (1 M )of aminoacid is employed in the experiment. This necessitates an even higher base concentration (9M )so that dilution is kept to a minimum. Even though these high concentrations were used the titration curves of u,,b versus p H gave a reasonable and reproducible ~ K + Nvalue H ~ for a- and 0-alanine. The midpoint of the S-shaped titration curve gives thepK, value since p H is equal topK. when stationary concentrations are equal. Therefore, eqn. (2) reduces to eqn. (3) and vz, VA are experimentally determined at the isoeleetric p H and strongly alkaline conditions, respectively. "oh
1 =2 ("2 + "A)
(3)
Figure 1illustrates the chemical shift change for the methyl protons7 of a-alanine as a function of pH. It is more difficult to follow the a-proton7in a-alanine. However, 8-alanine has two sets of methylene protons7 each equally detectable. The data shown in the table for o-alanine is graphically displayed in Figure 2. T~~PK.NH valuer , ohtnmrd by ourstudentsrsnge from9.7-10.4 for u - s h i n e and 10.4-10.5 for R-alanine. These values are uncorrected for ionic strength effects dccurring a t the high concentrations Titration Data for a-Alinine
CH,
I
OH-
+
I
* NH,-CH-C0,-
OH-
+
(Z) CH3
I
NH2-CH-C02-
(1)
(A) Going from neutral to alkaline conditions, the zwitterions (2) are deprotonated a t the ammonium group to generate anions (A). The observed chemical shifts are averaaes af those for each soeeies as expressed by eqn. (2)2 where ws = [Zlv, + [ A h (2) shifts and [Z], [A] are the stationary concentrations5 of the pure zwitterion and anion, respectively. At this point, it should be noted that v,,b does not change appreciably. The progress of the titration is monitored by simultaneously measuring the p H of thesolution and the nmr chemicalshifts of the protonsaandlor 0 to the *NHz moiety with respeet to an external tatramethylsilane (TMS) standard.Vhe chemical shifts far the Pprotons should he read with great accuracy. In order to ensure reasonahle proton signals a high
uz. YA are the chemical
'Present address: E. I. du Pont de Nemours and Company, Inc., Plastics Department. Wilmindon. Delaware 19898. 2Handloier, C. S., ~hakrabart;, M. R., and Mosher, M. W., J. CHEM. EDUC.. 50.510 (1973. 3Bovey, rank A:, "High ~esolutionNMR of Macromolecules," Academic Press, New York, 1972, p. 247. 4DL-alanine, 16.6g/100ml @ 25'C; 8-alanine is very soluble. 5The stationary concentrations of [Z] and [A] are standardized as 171 1 1,. 6. 1 ,-, ,= 1 I
I.
6The external standard is constructed by pulling the heated end of a disposable pipet to uniform hore (0.d. 1 1 mm). One end of a uniform 4-in. section is sealed with a flame. While cwling this end in a Dry-lcelacetone bath, TMS is delivered until the tube is half-filled and the open end is carefully sealed with a flame. 'In a-alanine, the methyl protons appear as a doublet and the a proton is a quartet. In 8-alanine, both sets of methylene protons are triplets.
-
100
110
. 120
2.0 1.0 S PPM HZ. Figure 1. (lefl) NMR specba of the methyl group of a-alanine as a function of pH.
Figure 2. (right)Plot of pH versus chemical shift fathe memy1 protons in aalanine. Volume 54. Number 7, July 1977 1 447
of reagents used in this experiment. With mare sensitivenmr facilities, it might be possible to perform this experiment a t several initial concentrations to assess the influence of ionic strength. However, with only one determination the student is able to obtain results close to the reoorted values of 9.7 and 10.2. In addition. the student is able to observe that the pcaition of the neighboring carboxyl group influences the acidity of the +NH3. This laboratory experiment can be performed individually or in pairs. A total of six laboratory hours is sufficient time for the student to prepare solutions, TMS capillarv, gain operatine . . and . . - experience with a-60 MHz nmr spectrometer.
Experimental Equipment Mini-combination electrode, p H meter, micro-buret, disposable pipets, nmr tube, capillary TMS, 60-MHz nmr spectrometer.
Reagents Ten milliliters of 9 M NaOH, 25 ml of 1 M o-alanine andlor 0-ala-
448 1 J w r ~of l Chemlcal Education
nine solutions, buffer solutions a t p H of the p H meter.
-
7 and 10 for standardization
Procedure 1 Insert the capillary TMS into nmr tube. 2 Take an nmr spectrum of -0.5ml of 1 M amino acid solution. 3 Calibrate p H meter with the two buffer solutions. 4 Titrate 15 ml of the 1M amino acid solution with the below listed increments of 9 M NaOH using the micro-buret. After addition of each increment of base, determine the resultingpH and the nmr spectrum using 4.5-ml aliquot. This and all subsequent aliquots used to measure the chemical shift must be returned to the titrationvessel before adding base increment. Take nmr spectra at following increments of NaOH (the figures in parentheses are total NaOH added)
