Sherrington ( 4 ) . Magnesium is not extracted from the acetate buffer solution. The absorbance of the complex in chloroform is measured at 390 mp on a Carey Model 14 recording spectrophotometer. The reference solution is 1% 8-quinolinol in chloroform. A standard working curve is used to determine the concentration of aluminum. The working curve is linear from 5 to 68 pg. A typical aliquot for aluminum analysis contains about 20 to 30 pg. of aluminum. RESULTS
The following analysis is obtained by the above methods for whiskers grown from various commercial grades of alumina: Whisker Analysis
%
Alto,
Si02 Fez08 MgO
%
Absolute error
90 9 1.3 0.4
1 2 i l 10.2 f O .1
Some whiskers contained a higher iron content. Microscopic examination revealed a red-brown coating on the surface of these whiskers. The analysis obtained was different from the above:
Whisker Analvsis 70
AlZOS
SiOz Fez03 MgO
70
Absolute error
85 10 5 0.1
A2 &l zk0.5
...
Considering the difficulty of working with very small amounts of refractory oxides, these results are believed to be reasonable. The errors in typical analyses were obtained by triplicate analyses of the 250-ml. filtrate from the silica determination. A large batch of about 5 mg. of whiskers was divided to run duplicate SiOz determinations, the process repeated on another batch of whiskers, and the error estimated. Incomplete fusion is a serious problem and a very large excess of fusing agent is necessary. The MgO determination has a large error because of the lack of a sharp end point, which is not entirely overcome by the addition of more magnesium to the unknown solution. The color-matching standards for iron are satisfactory to the nearest 0.5 pg., which is from 10 to 20% of the iron concentration of a typical aliquot. LITERATURE CITED
(1) Corey, R. B., Jackson, M. L., ANAL. CHEM.25, 624 (1953). (2) Cunningham, A. L., Beasley, R. M.,
Kainer, E., Horizons, Inc., Final Rept., ONR, Contract Nonr-2619 (00) (April 1960); ASTIA No. AD 240892. (3) Dienert, F., Wandenbulcke, F., Compt. Rend. 176,1478 (1923). (4) Gentry, C. H. R., Sherrington, L. G., Analyst 71, 432 (1949). (5) Gysling, H., Schwarzenbach, G., Helo. Chim. Acta 32, 1484 (1949). (6) Hedin, R., “Colorimetric Methods for Rapid Analysis of Silicate Materials,” Swedish Cement and Concrete Research Institute, Royal Institute of Technology, Stockholm, 1947. ( 7 ) Jander, G., Wendt, H., “Einfuehrung in das anorganisch-chemische Praktikum” (Introduction to Inorganic Practice), pp. 168-9, S. Hireel Verlag, Leipzig, 1961 (8) Kolthoff, I. M., Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” 3rd ed., pp. 391-4, Macmillan, New York, 1952. (9) Sandell, E. B., “Colorimetric,, Determination of Traces of Metals, 3rd ed., pp. 251-5, Interscience, New York, 1959. (10) Sears, G. W., DeVries, R. C., G. E. Rept. 60-RL-2377M (March 1960); presented in part, Division of Inorganic Chemistry, 139th Meeting, ACS, BOBton, bIass., 1959. (11) Schwarzenbach, G., Gysling, H., Helv. Chim.Acta 32, 1314 (1949). (12) Yoe, J. H., Jones, A. L., IAD.ENG. CHEM., ANAL. ED. 16, 111 (1944). F. W. VAHLDIEK C. T. LYNCH
L. B. ROBINSON Metals and Ceramics Laboratory Aeronautical Systems Division Wright-Patterson -4ir Force Base Dayton, Ohio
High Precision Nonaqueous Titration of Monosodium Glutamate Sir: A rapid titration method for monosodium glutamate monohydrate (MSG), a flavor enhancement agent, was desired, capable of at least *O.l% precision to control routinely production grade material at a 99%+ purity level. An aqueous acid-base titration, such as the pH 7 to 3.2 titration used for U.S.Government specifications ( 7 ) , for example, would not be expected to yield suitable precision because of the low slope a t the end point [glutamic acid, pK1, 2.19; pKz, 4.32; pK,, 9.94 ( 9 ) ] , and would require an additional titration to correct for free glutamic acid. The usual nonaqueous titration procedures for amino acids (1, 4) or carboxylic acid salts (3), although offering a sharper end point, also are not sufficiently precise, primarily because of temperature effects. The titrants ordinarily used in the nonaqueous titration of bases, perchloric acid in acetic acid or in dioxane, possess the disadvantage of an approximately fourfold greater coefficient of thermal expansion than water. [Sakurai and Awada (6) list the coefficient, (Y x lo4, over the tempera-
1668
ANALYTICAL CHEMISTRY
ture range 20” to 40” C., as: HzO, 3.02; AcOH, 9.95; dioxane, 9.74. Pifer and Wollish ( 5 ) , on the other hand, claim a ratio of about five for AcOH/HzO, and that dioxane/AcOH>l.] The precision of nonaqueous titrations is thus considerably more sensitive t o temperature fluctuations (O.l%/’ C.) than is that of aqueous titrations (0.02 to 0.03%/” C.). Accuracy may suffer even if precision is attained, if the samples are titrated a t a temperature differing from that prevailing during the standardization of the titrant. Nadeau and Branchen (4), recognizing this effect, were able to achieve precision of =k0.03% by using a weight buret. For a complete potentiometric titration, however, use of a weight buret is neither convenient nor rapid. Subsequent workers have, therefore, tended to use volumetric burets, accepting the lower precision [about d ~ 0 . 2 7( ~I ) ] and the risk to accuracy thus incurred. A suitable compromise between precision and speed may be achieved, however, by adding the bulk a of the titrant-Le., about 9O%-by single weight addition, and then completing the titration in the conventional
volumetric manner. The temperature effect is reduced to a negligible magnitude, ~O.O10j,/”C., by this approach. As shown in the present work, this technique consistently yields precision better than =k0.05% for a nonaqueous titration with a sharp end point. EXPERIMENTAL
Assay Method. B y titrating t h e sodium carboxylate group of MSG, rather t h a n t h e primary amine group, interference from glutamic acid, a potential impurity, is eliminated. T h e sample is brought into solution, and the amine group is rendered effectively neutral b y acetylation (8),by boiling with acetic anhydride. Acetic anhydride is then used as the primary solvent, as a sharper end point is obtained in this solvent than in acetic acid. Dioxane is added further to sharpen the end point ( 5 ) . Solvent ratios and volumes were selected experimentally to optimize the end point break. Apparatus. Titrations were performed potentiometrically, using a Beckman H-2 pH meter, a Beckman No. 8990-71 glass indicator electrode
2 drous of water. Reflux for 2 minutei and cool. Using a weighing buret, accurately add about 11 grams (10 ml.) of 1N HC10, to the solution. Add 100 ml. of dioxane. Titrate potentiometrically with 0.1N HClOb. Obtain the end point from a plot of ApH/AV os. V .
Table
Titration of
=t
-
Rel. std. dev.,
%
1
.?
99 69
0.04
2
3
0.03 0.03
5
4 3 3
99.7s 99.99 99.79 99.98
3 4
Comparison of the normalized slopes of the end point regions of the non-
t
-1 00
MSG
Sample Replicates MSG, %
DISCUSSION AND RESULTS I
High Precision Nonaqueous
0.05 0 01
t
*
\
-2.00 -
98
97
99
100
Table 11. 101
Figure 1. Comparison of normalized end point regions for monosodium glutamate titrations
0
MSG
MSG,
’?$ EQUIVALENCE
0
Comparison of Assay Methods for
102
Sample
Acid-base Nonaqueous
99 .. n
99.85 =k 0.01
...
99
99.81 =t
n
Aqueous
02
1
2 3
0.07
_______
Enzymatic decarboxylase
JIicrobial
...
100
99.2
97.2
99.0
Aqueous Nonaqueous
and a concentric double electrolytic junction calomel reference electrode. T h e latter consisted of a Beckman sleeve-type aqueous potassium chloride calomel electrode, No. 1170-71, suspended in a tube closed a t t h e bottom with a porous frit. A saturated solution of potassium perchlorate in acetic acid was used as the electrolyte in the outer tube. Both the weighing and the volumetric burets were of 10-ml. capacity and were fitted with Teflon plug stopcocks. The volumetric buret was calibrated in 0.02-nil. increments, and was connected to a gravity feed titrant reservoir with Teflon tubing. Chemicals. Dioxane was dried with anhydrous sodium sulfate, passed through a charcoal column, and further purified b y repeated fractional crystallizations (usually 2 or 3) until a negligible blank was obtained. Acetic acid, when containing acidic traces, was purified with charcoal. All other chemicals, reagent grade when available, were used as received. Solutions. -4nhydrous acetous perchloric acid, I S , was prepared b y diluting 90 ml. of 70% perchloric acid a n d 225 ml. of acetic anhydride t o 1 liter with acetic acid. T h e components were mixed slowly while keeping the mixture a t or below ambient temperature. hnhydrous acetous perchloric acid, 0.1N, was prepared b y diluting 100 ml. of In’ acid plus 5 ml. of acetic anhydride to 1 liter with acetie acid. Both acid titrants were standardized against primary standard grade potaqsium acid phthalate, using the gravimetric-volumetric technique for the 1.V acid. The laYacid continuously darkens on standing, b u t maintains a constant titer for a t least a month. Procedure. A\ccurately weigh about 2 grams of sample into a 250-ml. beaker and add 50 nil. of acetic anhydride. Cover with a watch glass, then bring to a boil on a h o t plate, stirring magnetically. Carefully add
aqueous titration and the conventional aqueous titration ( T ) , as shown in Figure 1, clearly indicates the superiority of the nonaqueous approach. Precision. Typical results b y the weight-volume nonaqueous titration for several randomly selected production lots of MSG are shown in Table I. T h e relative standard deviation for three replicates is a b o u t +0.03%, with t h e upper limit of t h e precision range rarely exceeding =tO.O5%. Day to d a y reproducibility is shown b y t h e following results. Sample 5, titrated once on each of 3 days over a 9-day period, gave on d a y 1, 99.99%; d a y 3, 99.98y0; d a y 9, 99.97%. Accuracy. Accuracy was not explicitly checked, although the method is believed t o be fully accurate from general acid-base theory and t h e observation t h a t 20 samples, from diverse sources and believed t o be of high purity from other tests, clustered in t h e 99.5 t o 100.0% range, I n addition, comparison of t h e results to those obtained by other LISG assay methods (Table 11), indicates no significant error. The existing differences are attributed to the lower precision of the other methods, Specificity. Free carboxylic acids, such as glutamic and pyrrolidone carboxylic acids, d o not interfere. Sodium salts of carboxylic acids are titrated quantitatively, however, and must be corrected for b y independent assays ( 2 ) . Primary and secondary amine compounds (other amino acids, glutamine) will usually be acetylated and hence will not interfere. Inorganic contaminants must be considered on an individual basis. Sodium sulfate does not interfere. Sodium chloride, occasionally used in conjunction with MSG, interferes t o a variable b u t
slight extent. It is insoluble on the alkaline side of the titration, provided the ratio of acetic anhydride to acetic acid exceeds about two. I n the end point region, however, as local excesses of acid occur, its solubility increases, presumably because of the volatilization of hydrochloric acid. When hydrochloric acid is lost, the potential of the eolution drifts back to the alkaline side, thereby arresting the rate of solubilization of sodium chloride. The net effect is to lead t o drifting end points, poorer precision, and slightly high results. Large amounts of sodium chloride may be removed by filtration. ACKNOWLEDGMENT
The author acknowledges with appreciation the assistance of D. R. Gaskill in obtaining some of the data. LITERATURE CITED
S.,“Acid-Base Titrations in Sonaqueous Solvents,” pp. 12-23, The G. Frederick Smith Chemical Co., Columbus, Ohio, 1952. ( 2 ) Mahdi, A. A., Rice, A. C., Wechel, K. C., J . Agr. Food Chem. 7 , 712 (1959). (3) bfarkunas, P. C., Riddick, J. A., ANAL.CHEW23, 337 (1951). ( 4 ) Nadeau, G. F., Branchen. L. E., J . Am. Chem. SOC.57, 1363 (1935). (5) Pifer, C. W.,Wollish, E. G., ANAL. CHEM.24, 300 (1Y52). (6) Sakurai, H., Awada, E., J . Pharm. SOC.Japan, 76, 1026 (1956). ( 7 ) United States Government, Federal Specification EE-11-591, Oct. 10, 1952. (8) Wagner, C. D., Brown, R. H., Peters, E. D.. J . A m . Chem. SOC.69, 2609 (1947). (9) Wilson, H., Csnnan, R. IC., J . Biol. Chem. 119,309 (1937). (1) Fritz, J.
SORMAX ADLER
Chemical Division Rahway, Merck & K. Co.,J.Inc. VOL. 34, NO. 12, NOVEMBER 1962
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