Detection and Determination of Some Carboxylic Acids in Aq ueous Solution by Nickel(I I)-Cata Iyzed Hydroxamic Acid Formation Kenneth A. Connors and James W. Munson School of Pharmacy, University of Wisconsin, Madison, Wis. 53706 Carboxylic acids react directly with hydroxylamine in aqueous solution in the presence of nickel(l1) to form hydroxamic acids. Detailed studies of the kinetics and equilibrium of this process have led to an extension of the familiar ferric hydroxamate method for carboxylic acid derivatives. A simple spot test gives positive results for most aliphatic acids and negative responses for aromatic and a-amino acids. An initial rate assay for aliphatic acids is applicable to 10-4 to 10-1M solutions. Acetic acid has been the most fully studied acid. The distinctive advantage of these new methods is their direct applicability to aqueous solutions of the carboxylic acids or their salts.
THE"FERRIC HYDROXAMATE" method, which is a general approach to the colorimetric determination of carboxylic acid derivatives, is based on Equation 1. RCOX
+ NHzOH e RCONHOH + HX
(1)
The carboxylic acid derivative RCOX is converted to the corresponding hydroxamic acid RCONHOH by reaction with hydroxylamine. Addition of iron(II1) results in complex formation (1) and a color development suitable for spectrophotometric measurement. Esters, amides, lactones, imides, lactams, anhydrides, and thiolesters are some of the important carboxylic acid derivatives that can be determined in this way (2-6). As usually carried out, the reaction is catalyzed by base, though some very labile compounds such as anhydrides (9, thiolesters (7), and penicillin (8) undergo hydroxamic acid formation under neutral conditions. Under the usual analytical conditions, carboxylic acids do not interfere in this method for carboxylic acid derivatives, which as presently described in the literature cannot be applied to carboxylic acids. It is known, however, that acids react with hydroxylamine in acidic and neutral solution to yield hydroxamic acids (9), and it has been shown that acetohydroxamic acid formation from acetic acid is catalyzed by nickel(I1) in neutral solutions (10). A recent detailed kinetic study (11) of Equation 2, the formation of acetohydroxamic acid from acetic acid and hydroxylamine in aqueous solution as catalyzed by acid and by nickel(II), has resulted in the pHrate profile shown in Figure 1.
k
CHICOOH
+ NHzOH $ CH3CONHOH + Hz0 k,
(2)
In this figure k , is the second-order rate constant, in units M-I-sec-l, at 90.5 "C, the pH being measured at 90.5 "C. The kinetic analysis of these curves (1 2 , 22) reveals that the substrate in the acid-catalyzed reaction is acetic acid, with rate terms of the form ~[CHEOOH][NHZOH][H+]~, where n can be 0, 1, and 2. The nickel(I1)-catalyzed reaction has a that rate term of the form ~N~[CH~COO-][NH?OH][N~*+], is, acetate ion is the substrate. The position of equilibrium of Equation 2 is pH-dependent, with decreasing pH giving a smaller equilibrium constant for acetohydroxamic acid production (7, 11). For this reason the acid-catalyzed reaction is not analytically attractive. This paper shows how analytical use can be made of the nickel (11) catalysis of hydroxamic acid formation demonstrated in Figure 1. EXPERIMENTAL
Materials. Unless otherwise stated, chemicals were analytical reagent grade and were used directly. Ferric perchlorate hexahydrate, Fe(CIO&. 6H20, and nickel perchlorate hexahydrate, Ni(C104)z.6Hz0(G. F. Smith Co.) were used directly. Hydroxylammonium perchlorate was prepared in situ by mixing equimolar quantities, in aqueous solutions, of silver perchlorate (G. F. Smith Co.) and hydroxylamine hydrochloride (Mallinckrodt Chemical Co.), and removing the precipitated silver chloride by filtration. The aqueous solution of hydroxylammonium perchlorate was used immediately. Benzohydroxamic acid (Eastman Organic Chemicals) was recrystallized from water; mp 127-129 "C [lit. 124-125 "C (23)J. Acetohydroxamic acid was prepared by reacting hydroxylamine with ethyl acetate in basic solution (14); it was recrystallized twice from 1O:l ethyl acetate:methanol; mp 89 "C [lit. 89-91 "C (15)]. Phenylacetic acid (Eastman Organic Chemicals) was recrystallized from Skellysolve B; mp 74-75 "C [lit, 76-76.5 "C (16)]. p-Methoxybenzoic acid (Merck Chemical Co.) was recrystallized from aqueous methanol; mp 181-182 "C [lit. 184 "C (17)]. Diphenylacetic acid (Eastman Organic Chemicals) was recrystallized from aqueous methanol; mp 145-146 "C [lit. 144-145 "C (18)l. 3,5-Dinitrobenzoic acid (Fisher Scientific Co.) was recrystallized from aqueous methanol; mp 204.5-206 "C [lit. 205-207
(1) G. Aksnes, Acta Chem. Scand., 11,710 (1957). (2) U. T. Hill, IND.ENG.CHEM.,ANAL.ED., 18,317 (1946). (3) Ibid., 19,932 (1947). CHEM., 24,1367(1952). (4) F. Bergman, ANAL. ( 5 ) R. F. Goddu, N. F. LeBlanc, and C . M. Wright, ibid., 27, 1251 ( 1955). (6) V. Goldenberg and P. E. Spoerri, ibid., 30,1327 (1958). (7) W. P. Jencks, S. Cordes, and J. Carriuolo, J. Biol. Chem., 235, 3608 (1960). (8) J. H. Ford, IND.ENG.CHEM., ANAL.ED.,19,1004(1947). (9) W. P. Jencks, M. Caplow, M. Gilchrist, and R. G. Kallen, Biochemistry, 2, 1313 (1963). (10) J. M. Lawlor, Chem. Commun., 1967,404. ( 1 1 ) J. W. Munson and K. A. Connors, J. Amer. Chem. Soc., in
press. 336
(12) J. W. Munson, Ph.D. Dissertation, University of Wisconsin, Madison, 1971. (13) L. W. Jones and C. D. Hurd, J. Amer. Chem. SOC.,43, 2422 (1921). (14) W. M. Wise and W. W. Brandt, ibid., 77, 1058 (1955). (15) W. N. Fishbein, J. Daly, and C. L. Streeter, Anal. Biochem., 28, 13 (1969). (16) R . Adams and A. F. Thal, "Organic Syntheses," Coll. Vol. I, J. Wiley and Sons, New York, N. Y., 1941, pp 436-437. (17) S. Kanno, J . Phnrm. Soc. Jup., 72,1193 (1952). (18) C. S. Marvel, F. D. Hager, and E. C. Candle, "Organic Syntheses," Coll. Vol. I, J. Wiley and Sons, New York, N. Y., 1941, p 224.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
-4'0
in water to make 100 ml. This stable solution is 0.8M in hydroxylamine and 0.04M in nickel(I1). NICKEL-HYDROXYLAMINE ASSAYREAGENTSOLUTIONwas prepared by dissolving 6.95 grams of hydroxylamine hydrochloride and, 2.38 grams of nickel chloride hexahydrate in 25 ml of water; 12.5 ml of dioxane was added, and the pH was adjusted to 6.3 with saturated sodium hydroxide. The volume was brought to 50.0 ml and the final pH adjusted to 6.2. This solution, which should be used within 3 hr, is 2 M in hydroxylamine, 0.2M in nickel(II), and 25 (v/v) in dioxane. Procedures. KINETIC MEASUREMENTS. Typical initial concentrations were 0.8M hydroxylamine hydrochloride, 0.04M nickel chloride, and 0.04M carboxylic acid. Three-milliliter portions of the reaction mixture were transferred to 5-ml glass ampoules with a 10-ml glass syringe fitted with a 15-gauge needle. The ampoules were sealed, and the reaction was initiated by placing them in a 90.5 "C water bath. Ampoules were removed at recorded times and immersed in a 2-propanol-dry ice bath to quench the reaction. The ampoules were then stored at - 5 "C to -10 "C until all samples had been collected. They were brought to 25 "C. One milliliter of the contents of an ampoule was added to 20 rnl of ferric perchlorate reagent solution contained in a foil-wrapped flask. After 1 hr, the absorbance of the solution at 530 nm was measured. [It has been shown by Notari and Munson (23) that the stability of the color of the ferric hydroxamate solution is a function of the iron (III)/hydroxylamine ratio, which should be at least 5 for optimum stability. In these studies this ratio was 4, with the result that an initial period of instability is followed by a fairly stable color, hence the 1-hr waiting period.] Rate constants were calculated from initial velocities, initial concentrations, and the rate equation. The initial velocity dC/dt was obtained from the initial slope dAS30/dtwith the equation dC/dt = (l/be)(dAs30/dt, where b is path length and e is the molar absorptivity of the ferric hydroxamate solution under the analytical conditions. Molar absorptivities obtained from authentic samples of hydroxamic acids were 1.17 X l o 3 for acetohydroxamic acid and 1.56 X l o 3 for benzohydroxamic acid. For reactions in which the product hydroxamic acid was different from these, the molar absorptivity of acetohydroxamic acid was used for aliphatics and that of benzohydroxamic acid for aromatics. DETECTION OF CARBOXYLIC ACIDS. One milliliter of approximately 0.1M aqueous solution of the sample compound was added to 5 ml of nickel-hydroxylamine spot test reagent solution. The resulting solution was adjusted to about pH 6 with concentrated hydrochloric acid or saturated sodium hydroxide solution; the pH adjustment may be made with the aid of pH indicator paper, such as pHydrion paper The reaction mixture was placed in a boiling water bath and 3-drop samples were removed at 30-min intervals for testing. A 3drop sample was transferred to a spot plate depression. Two drops of concentrated hydrochloric acid followed by 2 drops of ferric chloride reagent solution were added, A positive test is indicated by the immediate appearance of a reddish brown or reddish violet color. See Table I for the scope of this test. DETERMINATION OF CARBOXYLIC ACIDS. An aqueous solution about 0.01M in the sample acid was adjusted to pH 6.2. Five milliliters of this solution and 5.0 ml of the nickel-hydroxylamine assay reagent solution were sealed in a 20-ml glass ampoule. This procedure was repeated with a standard solution of the same carboxylic acid having approximately the same concentration as the sample solution. Both ampoules, standard and unknown, were placed in a boiling water bath. After 30 min, both ampoules were removed and immediately placed in an ice-water bath to quench the reac-
9 fl t t
-6.0 I 0
o\ I
I
I
I
2
3
4
5
\I
I 6
PH Figure 1. pH-rate profiles for the acid-catalyzed (open circles) and nickel(I1)-catalyzed (full circles) formation of acetohydroxamic acid from acetic acid at 90.5 "C in aqueous solution "C (19)]. Benzoic acid (Mallinckrodt Chemical Co.) was recrystallized twice from methanol; mp 121-122 "C [lit. 122 "C (20)]. Glycine (Fisher Scientific Co.) was recrystallized from aqueous methanol; mp 225 "C dec [lit. 225-230 "C dec (21)l. All water was redistilled from alkaline permanganate. Acetic acid stock solutions were prepared by diluting glacial acetic acid (Du Pont Chemical Co.) with water and standardizing by titration with standard sodium hydroxide. Standard pH buffer solutions were prepared according to Bates (22). Apparatus. Spectral measurements were made with either a Cary Model 14 or Model 16 spectrophotometer fitted with thermostated cell compartments maintained at 25 "C. pH measurements were made with either a Radiometer pH Meter Model 25 with scale expander and equipped with a Sargent combination electrode S-30072-15, or an Orion Model 801 pH meter with a Fisher high-temperature combination electrode 13-639-90. Thermometers were calibrated against thermometers carrying an NBS or an ASTM certificate. Reagent Solutions. Reaction mixtures for kinetic studies were prepared by mixing appropriate aliquots of reactant stock solutions and adjusting pH with concentrated sodium hydroxide or hydrochloric acid. Reagent solutions for analytical use were prepared as follows: FERRIC PERCHLORATE STOCKSOLUTION (1.5M) was prepared by dissolving 693.45 grams of ferric perchlorate hexahydrate and 217 ml of 60 perchloric acid in enough water to make 1 liter. This solution was stored in the dark. FERRIC PERCHLORATE REAGENT SOLUTION was prepared by diluting 100.0 ml of ferric perchlorate stock solution to 1 1. with methanol. FERRIC CHLORIDE REAGENT SOLUTION was made by dissolving 1 gram of ferric chloride hexahydrate in water to make 100 ml and adding 1 ml of concentrated hydrochloric acid. NICKEL-HYDROXYLAMINE SPOT TEST REAGENT SOLUTION was prepared by dissolving 5.56 grams of hydroxylamine hydrochloride and 0.95 gram of nickel chloride hexahydrate (19) R. Q.Brewster and B. Williams, "Organic Syntheses," Coll. Vol. 111, J . Wiley and Sons, New York, N. Y., 1955, p 337. (20) R . S. Jessup and C. B. Green, J . Res, Nat. Bur. Std., 13, 469 ( 1934). (21) H. T. Clarke and E. R. Taylor, "Organic Syntheses," Coll. Vol. I, J. Wiley and Sons, New York, N. Y., 1941, p 298. (22) R. G. Bates, J . Res. Nat. Bur. Std., 66A, 179 (1962).
(23) R. E. Notari and J. W. Munson, J . Pharm. Sci., 58, 1060 ( 1969).
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Table I. Results with the Nickel(I1)-Catalyzed Ferric Hydroxamate Spot Testa Reaction time Compound 30 min 60 min A. Simple aliphatic carboxylic acids 1. acetic 2. propionic 3. n-butyric 4. chloroacetic 5. malonic 6. succinic 7. glutaric 8. phenylacetic 9. hydrocinnamicb B. Aromatic carboxylic acids 1. benzoic 2. p-nitrobenzoicb 3. phthalic 4. isonicotinic C. a-Aminocarboxylic acids 1. glycine 2. leucine 3. proline 4. glutamic acid D. Miscellaneous 1. Unsaturated acids a. maleic b. cinnamicb c. fumaric d. crotonic 2. a-Hydroxy acids a. citric b. methyllactic c. tartaric d. lactic E. Other carboxylic acid derivatives 1. methyl acetate 2. methyl benzoate 3. acetamide + indicates positive test; - indicates negative test. Compounds giving a positive test at 30 minutes were not tested at 60 minutes. Acid was not fully soluble at 0.1M .
++ + ++ ++
+
+ ++*
tion. The ampoules were brought t o 25 "C and 1.0 ml of the contents of each was added t o 20.0 ml of ferric perchlorate reagent solution contained in separate foil-wrapped flasks. After 1 hr the absorbance of each solution was measured a t 530 nm against a common blank. The concentration of the unknown sample solution was calculated with Equation 3,
cu
=
cs
($)
(3)
where C, and A,, are the concentration and absorbance of the unknown sample solution and C, and A , are the concentration and absorbance of the standard solution. Both concentrations are on the basis of C,, so a correction may be necessary to account for dilution of the original sample in achieving the desired pH. RESULTS AND DISCUSSION Rate Studies. Figure 1 (full circles) presents the dependence on p H of the nickel(I1)-catalyzed reaction of acetic acid t o form acetohydroxamic acid in fully aqueous solution, the counter-ion being chloride (added as hydroxylamine hydrochloride, nickel chloride, and potassium chloride). Some variants in the solvent composition were studied in attempts to achieve faster rates. These are reported in Table 11. A modest rate enhancement is observed with all additives. The effect of perchlorate, which is a less effective ligand than chloride, may be a consequence of decreased competition 338
Table 11. Effect of Solvent Additives on Rate of Nickel (11)Catalyzed Acetohydroxamic Acid Formation5 Additive Relative Additive molarity rateb None 0 1.00 Methanol (25W v/v) 6.14 1.48 t-Butanol(25 v/v) 4.68 1.59 Dioxane (25 v/v) 2.92 1.91 Pyridine (25 v/v) 3.09 1.12 Perchloratec 1.o 2.44 a At 90.5 OC,pH 6.15 (25 "C), ionic strength 1.0; initial concentrations 0.80M hydroxylamine, 0.04M nickel(II), 0.045M acetic acid. Rate constant relative to that in fully aqueous solution, which has the value 2.8 X 10-6M-1-sec-1. 0 CIOa- replacing C1- in fully aqueous medium. Table 111. Relative Rates of Nickel(I1)-Catalyzed Hydroxamic Acid Formation from Carboxylic Acidsa Acid Relative rate Acetic acid 1.oo Chloroacetic acid 1.20 Phenylacetic acid 0.58 Diphenylacetic acid 0.06 Benzoic acid 0.02 p-Methoxybenzoic acid 0.02 o-Methoxybenzoic acid 0.003 3,s-Dinitrobenzoic acid 0.03 Glycine nil At 90.5 "C, pH 6.00 (25 "C), in 25z (v/v) dioxane; initial concentrations 0.8M hydroxylamine, 0.04M nickel, 0.045M carboxylic acid.
for the nickel ion, which is believed to exert its catalytic activity by coordination with acetate (11, 12). The solvents may exert a direct kinetic effect on the reaction rate, but could also affect the acid-base dissociation equilibria of acetic acid and hydroxylamine ; the data are too sparse t o justify further interpretation. On the basis of these results, the rate assay conditions were selected, a 25 (v/v) aqueous dioxane medium being used. It would be desirable t o replace chloride with perchlorate in the analytical medium, but the necessity of preparing hydroxylammonium perchlorate is a disadvantage. Table I11 gives relative rate data for hydroxamic acid formation from several carboxylic acids under a common set of conditions. Aromatic acids react much slower than d o aliphatic acids. Glycine is a special type of aliphatic acid because of its capability for chelation with metal ions, and no detectable hydroxamic acid formation occurred. Detection of Carboxylic Acids. Lawlor (10) had suggested that Ni(I1)-catalyzed hydroxamic acid formation might provide a general spot test for carboxylic acids, but he did not describe suitable conditions or investigate the scope of such a test. Table I gives the results of the spot test described above (see Experimentao when applied to numerous acids. The spot test usually gives positive results for aliphatic acids and negative results for aromatic acids and a-amino acids; this is, at least for the aromatic acids, a kinetically controlled selectivity. Esters and amides give positive tests, but these substances can easily be distinguished from acids because they also give positive tests with a n alkaline ferric hydroxamate procedure whereas acids d o not (24). The minimum (24) F. Feigl, "Spot Tests in Organic Analysis," 6th ed., Elsevier Publishing Co., Amsterdam, 1960, pp 249-254.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
0.8
0.4
1PR
0.2
IO-^
TIME (SEC)
Figure 2. Absorbance-time plots for acetohydroxamic acid formation from acetic acid Conditions: 90.5 “C, pH 6.2, 1.OM hydroxylamine, 0.1M nickel (II), 25 (v/v) dioxane. Initial concentrations of acetic acid, top to bottom, M X lo*: 4.41, 2.20, 0.441, 0.044
amount of acetic acid giving a positive test in 15-min reaction time was 30 micrograms. This spot test has the advantage over others for carboxylic acids in being directly applicable to aqueous solutions of carboxylic acids or carboxylate salts. Kinetic Determination of Carboxylic Acids. Despite the catalytic effect of nickel(I1) and a high reaction temperature, the absolute rate of hydroxamic acid formation from carboxylic acids is low. The extent of reaction is, moreover, equilibrium-limited. For these reasons a “total change” method of analysis is not feasible, and instead an initial rate
(25) assay was developed. The procedure described above M solution of acetic is planned for an approximately acid, but by suitably modifying the reaction time and spectrophotometer path length a wide variation in concentrations and sample acids could be accommodated. The technique is probably limited to aliphatic acids. The relative rate data of Table I11 suggest that some selectivity or freedom from interference may be achieved in mixtures of aliphatic and aromatic acids. Figure 2 shows absorbance-time data for several concentrations of acetic acid covering a 100-fold range. The initial slope is a linear function of concentration, and this relationship provides a working curve alternative to the “onepoint” procedure that is described. The reproducibility of these measurements is indicated by replicate determinations of initial slope dAsoo/dt for 5 solutions containing 2.20 X 10-2M acetic acid; the mean slope value was 3.15 X sec-l, with standard deviation 0.026 X sec-l and range 0.09 X sec-l. Though it is clearly limited by slow reaction rates and by interference (for example, from carboxylic acid derivatives), the method suggested here has the desirable feature of being a spectrophotometric procedure for aliphatic carboxylic acids that is directly applicable to their aqueous solutions, in the to 10-1M range, regardless of ionic state. RECEIVED for review August 12, 1971. Accepted September 29, 1971. Financial support by the National Institute of Dental Research Training Program (DE-171) and National Institutes of Health General Research Support Grant R R 05456 is gratefully acknowledged. (25) H. B. Mark, Jr., and G. A. Rechnitz, “Kinetics in Analytical Chemistry,” Interscience Publishers, New York, N. Y., 1968,
Chap. 3.4.
LABTRAN-A Language and System for Programming Chemical Experiments E. C. Toren,”Jr., R. N. Carey,’ A. E. Sherry, and J. E. Davis2 Departments of Medicine and Pathology, University of Wisconsin, Madison, Wis. 53706 A new interpretive language demonstrates the principles of the manipulation of laboratory instrumentation in a real-time, on-line system. Although specifically developed to carry out chemical experiments using a p-LINC computer and specially designed hardware, these principles can be extended to other types of experiments and computers. Each one-letter code either calls a calculation routine or operates a specific device (pipet, valve, or rate measuring interface). Using the interactive editor, programs can be written by laboratory personnel totally unfamiliar with computers and the usual programming languages. Operating programs are stored on magnetic tape and can be readily recalled for execution. A test result exceeding some threshold value can cause immediate, further, on-line testing of the same sample for different constituents.
COMPUTERIZED AUTOMATION in chemistry and clinical laboratories is developing at a rapid rate; the recent reports are I n absentia from Department of Chemistry, Duke University, Durham, N.C. Present address, Department of Chemistry, Purdue University, Lafayetie, Ind.
becoming too numerous to cite completely. From this laboratory, we have reported the development of a computer interface ( I ) based on the double-integration principle of Cordos, Crouch, and Malmstadt ( 2 ) and the development of a system, ELLA (3),to acquire, reduce, and analyze laboratory data. Many on-line and off-line plotting and numerical analysis features were added and, finally, the resulting ELLA system ( 4 ) was used in a closed-loop control situation t o perform the kinetic characterization of enzymes (with respect to K.M,the Michaelis constant, and Vw,the maximumvelocity). In the ELLA system, operator control was used only to load the turntable and control the plotter operation after completion of the experiment. Deming and Pardue (5), however, (1) E. C. Toren, Jr., A. A. Eggert, A. E. Sherry, and G. P. Hicks, Clin. Clzem., 16, 215 (1970). (2) E. M. Cordos, S . R. Crouch, and H. V. Malmstadt, ANAL. CHEM.. 40.796 (1968). (3) G. P: Hicks, A. A.’ Eggert, and E. C. Toren, Jr., ibid., 42, 729 (1970). ( 4 ) A. A. Eggert, G. P. Hicks, and J. E. Davis, ibid., 43,736 (1971). ( 5 ) S. N. Derning and H. L. Pardue, ibid., p 192.
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