Atomic Absorption Method for Determining Micromolar Quantities of 1,2-Diols Philip J. Oles and Sidney Siggia Department of Chemistry, University of Massachusetts, Amherst, Mass. 0 1002
The determination of compounds containing adjacent hydroxyl groups has been accomplished by means of atomic absorption spectrophotometry. Use is made of the oxidation of adjacent hydroxyl groups with periodic acid. The iodate formed in the reaction is separated from the reaction medium by precipitation as silver iodate. After removal of excess silver from the reagent by filtering, the silver iodate is dissolved in ammonium hydroxide and the resultant solution is analyzed for silver content by means of atomic absorption spectrophotometry. The phenomena of overoxidation is studied and useful data are presented for the determination of d-tartaric acid. The precision of the method is f0.95.0% in the range of 0.3-4.0 prnol/ml of 1,2-diol. Determinations of less than 1 % of 1,2-diol in the presence of a 1,3-diol are readily accomplished because of the specific behavior of periodic acid as an oxidant. The practical limit of detection is 0.2 pmol/ml of 1,Z-diol and will be lower in cases where more than one mole of periodic acid is consumed per mole of functional group.
The oxidation of the adjacent hydroxyl functional group with periodic acid is, by far, the most widely applied reaction for determining this functional group. HOCH,(CHOH),CH,OH
(>z
+ 1)HI03
+
(n
+
+
2HCHO
l)HIO,
+
-
nHCOOH
+
H,O
(1) Periodic acid is also known to oxidize a-hydroxy ketones, a-hydroxy aldehydes, a-amino alcohols, a-hydroxy carboxylic acids, a,@-diketones,a-amino aldehydes, and some activated methylene groups. The majority of procedures described for any of the functional groups above involve the determination of unconsumed periodate after reaction or of iodate produced in the reaction. Therefore, any of these procedures may be adapted to the determination of compounds containing the adjacent hydroxyl functional group and this review will deal with analytical applications of periodic acid oxidations in general. The amount of periodate consumed in the reaction may be determined iodometrically by making use of the fact that periodate and iodate liberate different amounts of iodine from iodide. A titrimetric procedure has been described ( I ) HI04
+
7HI
+
41,
+ 4H20
HI03
1
5HI
---+
31,
+
3H,O
(2 1
whereby the liberated iodine is titrated with standardized sodium thiosulfate. However, since the liberated iodine may react with the aldehydes present from the periodate oxidation reaction, a preferred procedure is to add a known excess of sodium arsenite in the presence of sodium bicarbonate and determine the excess sodium arsenite by backtitration with standard iodine solution ( 2 , 3 ) . W. D. Pohle, V. C. Mehlenbacher. and J. H. Cook, Oil Soap (Chicago), 22, 115 (1945).
HIO,
+
Na,AsO,
+ NaHCO,
-+
Na,AsO,
+
H,C03
+
NaIO, (3)
Residual periodate may also be determined by potentiometric titration with hydrazine sulfate ( 4 ) . Since glycerol releases formic acid in the oxidation reaction, but ethylene glycol does not, a procedure has been reported ( 5 ) for determining glycerol in the presence of ethylene glycol and other 1,2-diols by titrating the formic acid produced in the reaction with sodium hydroxide. The periodate anion has an ultraviolet absorption maximum a t 223 nm, and the amount of adjacent hydroxyl functional group in a sample may be determined by monitoring the decrease in absorption of periodate (6, 7 ) . However, nitrate, sulfate, carbonate, and carboxylate are reported to interfere (as would any organic ultraviolet absorbing species in a sample) and the molar absorptivity of periodate may be seriously affected by smal! changes in PH. The determination of 1,2-propylene glycol in the presence of ethylene glycol has been reported by Dal Nogare et al. (8). The acetaldehyde produced from the former compound in the periodate reaction reacts with hypoiodite to form iodoform which is determined spectrophotometrically. A gas chromatographic procedure has been described for the determination of lactic acid in which a large excess of periodic acid is introduced with the sample into the injection port maintained a t 100 "C (9). The acetaldehyde formed in the reaction is then determined quantitatively. CH,CHOHCOOH
+ HIO,
-
CH,CHO
+
CO,
+
H,O
+
HIO, (4)
A general application employing periodic acid as oxidant for several a-hydroxy acids has been reported by Hoffmann et al. (10) using this same approach. This procedure may be applied to the determination of compounds containing adjacent hydroxyl groups; however, mixtures in some cases would be impossible to determine when various amounts of the same product are released from each compound in the mixture. Several polarographic procedures have been reported in which formaldehyde, if produced in the periodate oxidation, is separated from the reaction medium and determined polarographically (11, 12). Zuman e t al. (13) reports that excess periodate may be destroyed by the addition of (2) N. D. Cheronis and T. S. Ma, "Organic Functional Group Analysis." Interscience, New York. N.Y., 1964, p 507. (3) R. J. B. Reddaway, Analyst(London). 82, 506 (1957). (4) A. Berka and J. Zyka, Cesk. Farm., 8, 136 (1959). (5) P. Bradford, W. D. Pohle, J. K. Gunther, and V. C. Mehlenbacher, Oil Soap (Chicago). 19, 189 (1942). (6) C. E. Crouthamel, H. V. Meek, D. S. Martin, and C. V. Banks, J. Amer. Chern. Soc., 71,3031 (1949). (7) J. S. Dixon and D. Lipkin, Anal. Chem.. 26, 1092 (1954). (8) S. Dal Nogare, T. 0. Norris, and J. Mitchell, Jr.. Anal. Chem., 23, 1473 (1951). (9) N. E. Hoffmann, J. J. Barboriak, and H. F. Hardman, Anal. Biochem., 9, 175 (1964). (10) N. E. Hoffmann and P. J. Conigliaro. Develop. Appl. Spechosc., 4, 299 (1965).
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 14, DECEMBER 1974
2197
~
Table I. Determination of Adjacent Hydroxyl Groups by Atomic Absorption Spectrophotometry
',":ECompound
umoll ml
Concentration, bmol/ml a
Micromoles
Taken Found
Recovery,
3.53 3.53 3.46 9 8 . 0 i 1.1 (5) Glycerol 1 . 9 3 1.93 1.90 98.4 i 4.4 ( 4 ) 1,2,6-Hexanetriol 7 -(2.3 -Dihydroxypropyl)- 1.66 1.66 1.64 9 8 . 8 2.4 ( 5 ) theophylline 3.39 3.39 3.28 96.8 i 0.9 ( 4 ) 1 , 2- P r o p a n e d i o l 1 . 8 5 1.85 1 . 7 5 94.6 f 5 . 0 (5) / ~ Z - 1S, 2 - C y c l o h e x a n e diol 3 . 5 5 3.55 3 . 5 1 98.9 1 . 1 ( 5 ) 3 -Chloro -1,2 - p r o p a n e diol 1.42 1.42 1.39 9 7 . 9 i 2 . 5 ( 5 ) 3-Piperidino-1,2propanediol 1 . 6 1 1.61 1 . 5 8 98.1 i 1.1 ( 5 ) 1-Phenyl-1.2-ethanediol Figures in parentheses indicate number of determinations.
*
*
arsenite, and the f o r m a l d e h y d e d e t e r m i n e d directly in the reaction m e d i u m , or since f o r m a l d e h y d e may not be one of the p r o d u c t s of the p e r i o d a t e reaction, a more general app r o a c h is to d e t e r m i n e the p e r i o d a t e itself polarographically. T h i s m e t h o d is suitable f o r d e t e r m i n i n g micromolar q u a n t i t i e s of 1,2-diols; however, o t h e r reducible species may interfere i n the p e r i o d a t e d e t e r m i n a t i o n , and large quantities of other organic species in a s a m p l e would p r o b ably decrease the precision of the method. The present work makes use of the q u a l i t a t i v e t e s t ( 1 4 ) for a n y f u n c t i o n a l g r o u p which reduces p e r i o d a t e to iodate. Addition of silver n i t r a t e solution to a sample which has been treated w i t h periodic acid will r e s u l t in the precipitat i o n f r o m solution of silver i o d a t e if a n y reactive f u n c t i o n a l g r o u p is present. After s e p a r a t i o n f r o m excess silver rea g e n t by filtration, the silver i o d a t e is dissolved i n concentrated a m m o n i u m hydroxide. The silver c o n c e n t r a t i o n of the resultant solution is then d e t e r m i n e d by a t o m i c abs o r p t i o n s p e c t r o p h o t o m e t r y . The sensitivity of the procedure described is equal to or g r e a t e r than existing approaches and will be useful f o r d e t e r m i n i n g s u b - m i c r o m o l a r a m o u n t s of a n y f u n c t i o n a l g r o u p which reacts q u a n t i t a tively with periodate. The c o m p l e t e d e t e r m i n a t i o n of six s a m p l e s may be accomplished in n i n e t y m i n u t e s or less.
EXPERIMENTAL Reagents. Paraperiodic acid (HjIOs) was purchased from G. Frederick Smith Chemical Company, Columbus, Ohio, and was used without further treatment. Standard solutions of this reagent were contained in glass stoppered volumetric flasks and stored in the dark. Solutions of periodic acid are reported (15) to decompose slowly even when briefly exposed to sunlight and were, therefore, discarded after one month. The exact concentration of this reagent does not need to be determined and concentrations of between 11 and 22 pmol/ml. were used successfully except where otherwise noted. Nitric acid reagent was prepared by mixing 3 volumes of concentrated nitric acid (Baker Analyzed Reagent) and 1 volume of distilled deionized water. Silver nitrate reagent, 2M, was prepared by dissolving silver nitrate (Fisher Chemical) in distilled, deionized water and was protected from light. Glycerol (Baker Grade) was purchased from Baker Chemical Company, and was 1,2-propanediol, 3-piused as received. 3-Chloro-1,2-propanediol, peridino-1,2-propanediol, 1-phenyl-1,Z-ethanediol,7-(2,3-dihy(11) B. Warshowsky and P. J. Elving, hd. Eng. Chem., Anal. M., 18, 253 ( 1946). (12) P. J. Elvina. B. Warshowskv. E. Shoemaker, and J. Maraolit, Anal. Chem., 20,-25 (1948). (13) P. Zuman and J. Krupicka, Collect. Czech. Chem. Commun., 23, 598 (1958). (14) R. Shriner, R. Fuson. and D. Curtin. "The Systematic Identification of Organic Compounds," Wiley, New York, N.Y., 1967, p 145. (15) G. Dryhurst, "Periodate Oxidation of Diol and Other Functional Groups." Permagon Press, New York. N.Y., 1966, p 20.
2198
Table 11. Determination of &Tartaric Acid by Periodic Acid Oxidation Tartaric acid
Periodic acid
Temp
Reaction tiFe, Iodate found, mm il mol
Recovery, 9; b s c
1.05 11.5 20 90 1.49 142 i 4.0 (5) 1 75 1.21 115 i 4.4 (5) 1.05 5.7 1 60 0.554 114 f 4 . 6 (4) 0.243' 1.0 0.243' 1.0 1 25 0.356 73.4 i 11 (4) 1.22 13.4 100 45 3.47 94.8 i 0.9 (8) a Concentrations listed are those of tartaric acid and periodic acid before mixing. Figures in parentheses indicate number of determinations. Recoveries based on l mole of iodate produced per mole of tartaric acid present except for results obtained at 100 "C. d Two-milliliter samples taken.
droxypropy1)-theophylline (diphylline), l,Z,6-hexanetriol, cis- and trans- 1,2-cyclohexanediol, trans- 1,2-cyclohexanediol and 1,3-butanediol were purchased from Aldrich Chemical Company and all were the best grade available. All liquid diols were purified by vacuum distillation with the exception of 1,3-butanediol(Aldrich analyzed, 99+%) which was used as received. Standard solutions were prepared by weighing out freshly distilled diol (0.1-0.4 g), transferring to a 1-1. volumetric flask and diluting to the mark with distilled deionized water. All solid diols were purified by crystallization from acetone or benzene and were dried in a vacuum desiccator for 48 hr. Standard solutions were prepared in the same manner as described above for liquid diols. Aqueous standard solutions of diols were stable for at least 3 weeks when stored in the dark a t room temperature. A stock solution of silver nitrate (Fisher) was prepared by weighing out 0.10-0.25 g of AgN03 (dried at 100°C for 1 hr), transferring to a 250.0-ml volumetric flask and diluting t o volume with 1:l concentrated HN03-HzO. Standard solutions were then prepared by diluting appropriate aliquots of this stock solution. The stock silver nitrate solution was stable for about 1 week and the standard solutions were prepared just prior to each analysis. Apparatus. Absorbances were measured at 328.1 nm with a Perkin-Elmer 403 Atomic Absorption Spectrophotometer. A single element silver hollow cathode lamp operated a t 24 mA was used as a source of radiation. An air-acetylene flame was used for all determinations and operating conditions for the instrument were as outlined in reference (16). Eppendorf pipets (Brinkmann Instruments) were used to pipet all aqueous samples. A fine frit (4-5.5 pm) Pyrex glass funnel was used for all filtering. Procedure. A 1.00-2.00 ml sample of diol in water is added to a 6-inch test tube. The concentration of the diol should be between 0.10-4.00 pmol/ml. A volume of periodic acid reagent in water equal to the volume of the sample is added to the test tube. The contents of the test tube are mixed thoroughly and the test tube (protected from light) is placed on a mechanical shaker for 10-30 min. At this time, a volume of nitric acid reagent which is exactly one-half the volume of the original sample (0.50-1.00 ml) is added to the test tube. The contents of the test tube are again mixed thoroughly and a volume of silver nitrate equal to the volume of nitric acid reagent (0.50-1.00 ml) is added to the test tube. (Note: the volume of reagents used is critical and will he discussed in a later section.) The test tube is again placed on the mechanical shaker for 10-30 minutes to allow the AgIOz to flocculate. The test tube is then cooled to -10 to -15 "C in a mixture of ice-acetone (or placed in a freezer) to suppress the solubility of the AgI03. The contents of the test tube are transferred to a fine frit (4-5.5 pm) Pyrex glass funnel and suction is applied. The test tube is rinsed three times with 4-5 ml portions of acetone-water ( l : l , v/v), containing 0.2% (v/v) concentrated nitric acid maintained at approximately -15 "C, each time allowing the rinsings to pass through the filter. The test tube containing the acetone-water mixture is placed in crushed ice between rinsings. Three ml of concentrated ammonium hydroxide are then added to the test tube, a clean 125-ml suction flask is placed below the filter, and the ammonium hydroxide is added to the filter. Suction is applied and the test tube and filter are rinsed with two portions of water. The contents of the flask are then transferred to a 25.0-ml (0.1-1.0 pmol 1,2(16) "Analytical Methods for Atomic Absorption Spectrophotometry," Perkin-Elmer Corporation, Norwalk, Conn., 1968.
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 1 4 , DECEMBER 1974
Table 111. Determination of Total 1,2-Diol Content in a Mixture
?$-,:
Micromoles umol Found 1,2-diol/ml Taken
Mixnue
Recovery, C/ra
1,2 -Propanediol, 0 . 5 7 2 pmol I
7 -(2,3-dihydroxypropyl) -the0 -
0.908 0.908 0.869
95.7 + 1.9 (5)
phylline. 0.336 kmol 1.3 -Butanediol.
131.1 pmol 1 . 2 -Propanediol. 0.9698 pmol
+ 1.61 1.61 1 . 5 6 96.9 r 2.1 ( 5 ) 3 -Piperidine-1.2propanediol, 0.6449 pmol 0 Figures in parentheses indicate number of determinations.
allow cooling of the wash solution to -15 OC without freezing and it would reduce the surface tension of the wash solution. The reduction in surface tension accounts for a more efficient rinsing of the test tube and results in a small and reproducible blank value of 0.35-0.44 ppm Ag+ in 50.0 ml with three rinses and 0.09-0.12 ppm Ag+ in 50.0 ml with four rinses. The addition of the nitric acid to the wash solution prevented peptization of the precipitate since the same results were obtained for a number of compounds whether precipitates were washed three times or four times. Overoxidation Phenomena. The oxidation of some functional groups with periodic acid may result in the formation of products which are susceptible to further, and in many cases, slow oxidation by periodic acid. This phenomenon causes overconsumption of periodic acid and can result in recoveries significantly greater than 100%when the percent recovery is based only upon the first oxidation step. An example of this phenomenon which was encountered in this work is the oxidation of tartaric acid with periodic acid. HOOCCHOHCHOHCOOH A HIO, +
diol), 50.0-ml (1.0-2.0 firno1 1,2-diol) or 100-ml (2.0-4.0 pmol 1,2diol) volumetric flask and diluted to the mark with rinsings from the suction flask. This solution is then analyzed for silver content by means of Atomic Absorption Spectrophotometry.
RESULTS AND DISCUSSION
2COOH
4
H,O
+ HIO, (5)
CHO The glyoxylic acid produced in the reaction is subject to further oxidation by periodic acid, however, a t a much slower rate.
Optimum Concentration of Reagents. The concentration of nitric acid and silver nitrate reagents is critical in this procedure. The addition of nitric acid is necessary t o prevent the precipitation of silver periodate; however, an excess must be avoided because the solubility of silver iodate increases with increasing nitric acid concentration. Final concentrations of nitric acid and silver ion of 2.OM and 0.34M, respectively, after addition of all reagents to a sample were found to produce satisfactory results as indicated in Table I. Samples which are very acidic or basic should be adjusted to a p H of approximately 7 with dilute nitric acid or sodium hydroxide before addition of periodic acid. T h e optimum concentrations of reagents given may be varied by a t least 10% without affecting recoveries, and the concentration of periodic acid may be varied from approximately 0.34-7.4 pmol/ml (final concentration after addition of all reagents). Solubility of Silver Iodate. The solubility of AgI03 a t 100 "C in pure water is 30 mg/l. (17).This corresponds to approximately 0.11 pmol AgIO,/ml; however, the solubility is greatly suppressed by the high concentration of Ag+ and by maintaining samples and wash solution a t -10 to -15 "C. I-Jnder the conditions given in the experimental section the quantitative recovery of as little as 0.4 Kmol of AgI03 precipitated from 6.0 ml of solution may be obtained. The Source of the Blank. In the present study, blank values were of the order of 0.5-1.0 ppm Ag+ in 50.0 ml when precipitates were washed three times with pure distilled, deionized water (at 0 "C). T o reduce the blank value, precipitates were washed four times, which reduced the blank value but caused low recoveries due to peptization of the Ag1O:i precipitate. Peptization was observed to occur only when the precipitate was washed four times with pure water or pure water-acetone. T o circumvent this problem, a wash solution was prepared containing equal volumes of water and acetone to which had been added concentrated "03 (0.2% v/v). Acetone was chosen because it would
Two means are available for controlling this phenomenon so that results may become analytically useful. Conditions may be chosen for the reaction which are extremely mild, for example, low concentrations of reactant and periodic acid and low temperature. If the reaction rates of the two oxidation steps are significantly different, these mild conditions should enable a differentiation to be made so that essentially only iodate produced in the first and faster reaction will be determined (in this case 1 mole of iodate produced per mole of tartaric acid). Alternately, conditions for the oxidation may be made relatively severe, by heating for example, thereby forcing both reactions to completion (in this case 3 moles of iodate produced per mole of tartaric acid present). Although the latter approach results in a threefold increase in sensitivity, the former procedure is preferred since heating solutions of periodic acid causes the selectivity of the reagent to be lost ( 1 8 ) .Table I1 illustrates the results obtained from both approaches for the determination of tartaric acid. The most precise and accurate results are those that result from heating. However, this procedure is not recommended in general for all cases where glyoxylic acid may be an oxidation product since other products (or compounds in a sample) may undergo partial and irreproducible oxidation when heated with periodic acid. Determination of Total 1,2-Diol Content in a Mixture. Determinations of total 1,2-diol content in mixtures were made and the results appear in Table 111. The specific behavior of periodic acid toward oxidation of 1,2-diols but not 1,3-diols may be noted from the results illustrated in this table. This procedure should be particularly useful for determining 1,2-diol impurities in compounds containing non-adjacent hydroxyl groups.
(17) R. C. Weast, Ed., "Chemical Rubber Company Handbook of Chemistry and Physics," The Chemical Rubber Company, Cleveland, Ohio, 1965, p 8-219.
(18) G. Dryhurst, "Periodate Oxidation of Diol and Other Functional Groups," Permagon Press, New York. N.Y., 1966, p 72.
COOH
HIO,
+
HCOOH
CO,
+
HIO,
(6)
CHO
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 1 4 , DECEMBER 1974
2199
Table IV. Determination of 1,P-Diols at the ppm Level Compound
1,2-Propanediol 3-Piperidino1.2 -propane -
concentration, umol/ml
Taken
Micromoles
0.339 0.285
0.339 0.570
Found
0.349 0.575
Recovery, % a
103 * 5.0 (5) 101 i 3 . 8 (7)
diol 0 Figures in parentheses indicate number of determinations.
Concentration Range. The upper limit of concentration of a reactive functional group may be extended by choosing less sensitive analytical lines or by diluting solutions to larger volumes when the final measuring approach is atomic absorption spectrophotometry. Lower limits of detection have been determined in previous work (19, 2 0 ) , however, by the reactivity of the particular functional group and this in turn may be greatly altered by the presence of various substituents. In the case of the periodate oxidation of 1,2-diols, it has been reported (21) that the reactivity increases from ethylene glycol to 1,2-propanediol to 2,3-butanediol because of the inductive electron release by the alkyl groups which stabilizes an intermediate in the reaction. Under the experimental conditions employed in this work (0.2-4.0 bmol/ml 1,2-diol) very little difference in reactivity of the various compounds studied was encountered. Reactions in all cases proceeded to completion within 45 minutes in the presence of electron donating groups (alkyl) or electron withdrawing groups (chloro). However, the time required for the AgI03 to precipitate from solution increased significantly when the concentration of iodate (and hence, 1,2-diol) was less than 0.07 pmol/ml. When the volumes of reagents employed in the experimental procedure are used, this corresponds to approximately 0.20 pmol/ml of 1,2-diol in the original sample. The time required for the precipitate to become visible a t these concentrations is approximately 30-45 min. This time interval has been used as a means of determining ethylene glycol ( 2 2 ) in dilute aqueous solutions. The time required for the formation of the precipitate is proportional to the concentration of ethylene glycol under critical experimental con(19) P. J. Oles and s. Siggia. Anal. Chem., 45, 2150 (1973). (20) P. J. Oles and S. Siggia, Anal. Chem., 46, 91 1 (1974). (21) G. Dryhurst, "Periodate Oxidation of Diol and Other Functional Groups." Permagon Press, New York, N.Y., 1966, p 20. (22) E. R. Hess, C. 6 . Jordan, and H. K. Ross, Anal. Chem., 28, 134 (1956).
2200
ditions. In the present work, about 2 hr are required t o flocculate precipitates when the concentration of 1,2-diol in the original sample is 0.12 pmol/ml. However, the precipitate did not always appear in all samples and special techniques ( i e . , scratching the walls of the test tube) to promote crystallization of the precipitate must be employed. Also, filters with smaller pore sizes than 4-5.5 pm would probably be required to obtain quantitative recoveries of AgI03. For these reasons, a detection limit of the order of 0.20 pmol/ml of 1,2-diol may be realized under the experimental conditions of this procedure. This detection limit may be divided by n (when n is not zero), the coefficient which appears in Equation 1. The results illustrated in Table IV are indicative of the precision and accuracy that may be expected for determinations of 1,2-diols a t the ppm level of concentration. CONCLUSION The determination of compounds containing the adjacent hydroxyl functional group may be accomplished by means of Atomic Absorption Spectrophotometry. Significant advantages with respect to speed of analysis, sensitivity, and detection limit are offered by this approach as compared to the most sensitive methods available. The problems presented by overoxidation phenomena may be overcome in some cases so that analytically useful data may be obtained. The specific behavior of periodic acid toward 1,2-diols is shown to be of particular value for analyzing compounds containing non-adjacent hydroxyl groups for 1,2-diol impurities. Although the majority of the compounds studied in this work contain the 1,2-diol functional group, this procedure may be adaptable to any of the many functional groups with which periodic acid is known to react quantitatively. In addition to the reactive functional groups mentioned in the preceeding discussions, it has also been reported (23) that the sulfide function of penicillin is quantitatively oxidized by periodic acid (to sulfoxide) and the determination of this compound should be possible by means of this procedure. RECEIVEDfor review May 20, 1974. Accepted August 26, 1974. This work was supported by Grant No. GP 37493X from the National Science Foundation. (23) L. Mazor and M. K. Papay, Acta Chim. Acad. Sci. Hung., 26, 473 (196 1).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 14, DECEMBER 1974
