from the calibration curve by using either procedure described above after adjusting the dilution of the sample so that the absorbance fell in the range of the calibration curve.
tures of pyridoxal and these compounds yielded quantitative recovery for pyridoxal. The data are recorded in Table 1.
The proposed procedure proved useful in studying nonenzymatic transamination with vitamin BBof the amino acids and keto acids listed above.
DISCUSSION
The absorption spectrum for the colored condensation product formed when pyridoxal is treated with acetone in the presence of base was obtained with a recording Beckman DU spectrophotometer and is shown in Figure 1. An absorption peak occurs at about 420 mp, Optimum color intensity develops rapidly and increases slo~vlyover a long period of time. Figure 2 shows the effect of time on color intensity over a 3-hour period. Fifteen minutes was selected as convenient for this method. Samples of pure pyridoxal hydrochloride were assayed by the proposed procedure. The recovery of pyridoxal was quantita-
LITERATURE CITED
O01i l
(1) Hochberg, T l m e in M i n u t e s
Figure 2. Effect of time on absorbance of colored condensation product
tive. Alanine, glutamic acid, pyruvic acid, a-ketoglutaric, other similar amino and keto acids, pyridoxine, and pyridoxamine do not produce color with the reagents employed in this assay. Mix-
IT., Melnick, D., Oser, B. L.. J . Bio7. Chem. 155. 109 11914). (2) Metzler, D. E., Snell, E. E.; J . Am. Chem. SOC.74,979 (1952). (3)_Scudi, J. V., J . Biol. Chem. 139, 107 (1941). ( 4 ) Swaminathan, M., Suture 145, 790 (1940). ( 5 ) Sweeney, J. P., Hall, W.L., J . Assoc. Ofic. Agr. Chemzsts 35, 479 (1952). (6) Wada, H., Snell, E. E., J . Biol. Chem. 236, 2089 (1961). RECEIVED for review September 25, 1961. Accepted December 14, 1961.
Determination of Aliphatic Aldehydes by Spectrophotometry ALBERTA M. ALBRECHT,' WILLIAM 1. SCHER, Jr.,2 and HENRY J. VOGEL lnstitute of Microbiology, Rutgers, The State University, New Brunswick, N. J.
b Aliphatic aldehydes can b e determined b y reaction with methylamine ( 1 .OM, used as hydrochloride) and o-aminobenzaldehyde (0.004M) in aqueous (or aqueous-ethanolic) solution at pH 8.4. The yellow products, obtained within 10 minutes at 25" C., presumably are lf2-dihydroquinazolinium compounds. With acetaldehyde, the absorbance of the reaction mixtures, measured at 440 mp, is directly proportional to the concentration of this aldehyde up to at least 0.0004Mf a t which the absorbance is approximately 1.0 (light path, 1 cm.). Quantitatively comparable responses are obtained with formaldehyde, propionaldehyde, butyraldehyde, n-valeraldehyde, or sodium glyoxylate. Inappreciable or slight responses are given b y chloral hydrate, glucose, acetone, a-ketog lutarate, pyruvate, or benzaldehyde.
D
AN enzymological study, it became desirable to find a method to determine aliphatic aldehydes with the aid of relatively mild reagents (adapted for use in aqueous solution a t a p H of approximately 8). I n the method developed, o-aminobenzaldehyde is employed. This com-
398
URISG
ANALYTICAL CHEMISTRY
pound was investigated extensively by Schopf and collaborators (1-3) and was applied by Vogel and Davis (5) to the detection of a n aminoaldehyde, glutamic ysemialdehyde, or its cyclized form, AI-pyrroline-5-carboxylate. It is, however, not necessary for the (aliphatic) amino and aldehyde groups to be part of the same molecule; o-aminobenzaldehyde, under suitable p H conditions, reacts in aqueous (or aqueousethanolic) solution when mixed with any one of several aliphatic primary amines plus a n aliphatic aldehyde. Yellow products are formed which, in line with the work of Schopf and collaborators, appear to be dihydroquinazolinium compounds. The accompanying schematic equation illustrates, a n instance of the presumable reaction involved, namely the formation of a 2 - alkyl - 3 - methyl - 1,2 - dihydroquinazolinium compound from oaminobenzaldehyde, methylamine (used as hydrochloride), and a n aliphatic aldehyde, R ,CHO; no specific reaction mechanism is implied : IVH,
+ 0HC.R
-H20
d
H
H OHEXPERIMENTAL
Reagents. Reagent A is a n aqueous solution, 2.0M in methylamine hydrochloride and 0.20il.1 in sodium pyrophosphate. This solution, which has a p H of approximately 8.4 (without adjustment) remains usable for at least 1 month when stored a t 3" C. It is convenient first to dissolve the requisite amount of finely ground sodium pyrophosphate (Na4P207. 10H20,analytical reagent, Mallinckrodt Chemical Works) and then to dissolve the methylamine hydrochloride (reagent grade, Fisher Scientific Co.). Reagent B is aqueous 0.04M o-aminobenzaldehyde which is dissolved with gentle warming. This reagent is prepared fresh daily and kept a t 3" C. until used. The c-aminobenzaldehyde is readily prepared by the following modification 1 Present address, Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, N. Y. 2 Present address, Kings County Hospital, Brooklyn, N. Y.
UC
0.24-
0.20-
A 1 .6 M 0 0.8M
A/A-A-A-A nco-o-o-o-
0 2c
4
015
z
Figure 1 . Effect of hydrogen ion concentration on reaction given by acetaldehyde
4
m
a
0 v)
m 4
OIC
0 0'
of the method of Smith and Opie (4). I n a 2-liter round-bottomed flask, 100 ml. of distilled water and 100 grams of ferrous sulfate heptahydrate are brought to a boil. The flask is removed to a fume hood and 5 grams of o-nitrobenzaldehyde (Dajac Laboratories, The Borden Chemical Co., Philadelphia 24, Pa.) are promptly introduced. Fifty milliliters of ammonium hydroxide (specific gravity, 0.90) are added in three approximately equal portions, over a period of 2 minutes, with manual shaking; some boiling results upon each addition. The mixture, which contains a copious black precipitate, is allowed to stand for about 5 minutes with occasional swirling, and 200 ml. of boiling distilled water are added. The flask is immediately mounted for distillation and connected to a watercooled condenser. The reaction mixture is distilled over a Meeker-type burner, and the flow of the cooling water is adjusted to avoid solidification of the product, o-aminobenzaldehyde, in the condenser. hpproximately 150 ml. of distillate are collected over a period of 15 minutes in a receiver chilled in an ice bath. iifter standing in the cold for an additional 15 minutes, the product, which crystallizes as water-repellent platelets, is filtered on a chilled Buchner funnel, washed with 25 ml. of ice-cold distilled water, and rapidly sucked as dry as possible. The platelets are transferred, without further drying, to a chilled container and stored a t -20' C. Under these conditions, the product appears to be stable for a t least several months; a t 3" C., the product is usable for a t least several weeks. The yield is 1.5 grams of low-melting crystals. Care is taken not to allow the crystals to melt before use, since the material
that solidifies after melting seems to be relatively unstable. Procedure. T o 0.4 ml. of a n aqueous or aqueous-ethanolic solution of the aliphatic aldehyde are added 0.5 ml. of reagent A s n d 0.1 ml. of reagent B. After agitation, the reaction mixture is allowed to stand a t about 25" C. for at least 10 minutes; standing for considerably longer periods usually is permissible. T h e absorbance of the reaction mixture is then read a t 440 mp (in a Beckman D U spectrophotometer) against a reagent blank that has the composition of the reaction mixture, except for omission of the aliphatic aldehyde. As a standard, acetaldehyde may be used. RESULTS AND DISCUSSION
Acetaldehyde served as a reference compound in the development of the above procedure. This aldehyde, a t a n initial concentration of 0.4 pmole per ml. of reaction mixture, gives a n absorbance of approximately 1.0 (light path, 1 em.). The absorbance is directly proportional to the concentration of this aldehyde up to a t least 0.0004J1. Reasons for the choice of various features of the procedure are given below, as are the response characteristics of some illustrative compounds. Effect of p H . The reaction velocity was strongly pH-dependent. The effect of hydrogen ion concentration was studied in the region of p H 6 to 9. For these studies, sodium pyrophosphate mas omitted from the reaction mixtures. The hydrogen ion Concentration was varied by suitable Linear Response.
additions of potassium hydroxide to the methylamine hydrochloride solutions, prior to volume adjustment with distilled water. Some of the results obtained are represented in Figure 1. Throughout the range tested, the initial reaction velocity increased with pH, but a t p H 9, the stability of the yellow product formed began to decrease noticeably. Consequently, a p H of 8.4 was chosen as a compromise between reaction velocity and product stability. A reagent containing 0.20M sodium pyrophosphate and 2.0-lf methylamine hydrochloride provided the desired p H (without adjustment). When this p H was used, the same results were obtained whether the pyrophosphate was present or not. Effect of Reagent Concentrations. The concentrations of both methylamine hydrochloride and o-aminobenzaldehyde affected the reaction velocity as well as the ultimate extent of reaction. Preliminary experiments indicated t h a t the use of rather high concentrations of the primary aliphatic amino compound was desirable. Accordingly, methylamine hydrochloride was em0.24r 8 mM 4 mM 2mu
~
2
4
6
8
1
0
TIME, MINUTES
Figure 3. Effect of o-aminobenzaldehyde concentration on reaction given b y acetaldehyde VOL. 34, NO. 3, MARCH 1962
0
399
relatively slight reactions (which may,
at least partly, be attributable to
Table I. Relative Response of Various Compounds on Molar Basis
Compound Formaldehyde Acetaldehyde Propionaldehyde Butyraldehyde n-Valeraldehyde Glyoxylate Chloral hydrate Glucose Acetone Pyruvate a-Ketoglutarate Benzaldehyde
aliphatic-aldehyde impurities). Spectra of Products. T h e absorption spectra of t h e reaction products derived from glyoxylate and the first five straight-chain aldehydes, Figure 4, exhibit a single peak in t h e visible region, with a maximum between 430 and 435 mp. The similarity of these spectra suggests that the various products are structurally similar; additionally, the spectra closely resemble the one given by the product of the reaction of o-aminobenzaldehyde with glutamic 7-semialdehyde ( 5 ) . The wavelength (440 mp) recommended for the present procedure gives adequately low reagent blanks, and yet is not too far removed from the absorption masima of the products.
Absorbance 0.96 1.00 0.92 0.99 0.81 0.88 0.02 0.01
0.01 0.09 0.05
0.09
ployed because it is readily water soluble, available a t reagent grade purity, and relatively inexpensive. The effect of methylamine hydrochloride concentration on the course of the reaction is illustrated in Figure 2. I n this experiment, 0.10M sodium pyrophosphate was included in the reaction mixtures. Since the concentration of methylamine hydrochloride has a n effect on the pH, adjustments to p H 8.4 were made. On the basis of the data obtained, a concentration of 1.0-11 methylamine hydrochloride in the final reaction mixture was selected as adequately effective. The effect of o-aminobenzaldehyde concentration on the reaction can be seen in Figure 3. These results led to the choice of a Concentration of 0.004M o-aminobenzaldehyde in the final reaction mixture. Although higher concentrations of this compound can yield increased reaction rates and extents of reaction, the concentration chosen gives satisfactorily low blank readings, and moreover is not wasteful of the material. Response of Illustrative Compounds. The relative response of
LITERATURE CITED 400
450
WAVE
500
550
L E N G T H , rnp
Figure 4. Absorption spectra of products derived from various aldehydes used at appropriately staggered concentrations 1. 2. 3.
Sodium glyoxylate Formaldehyde Acetaldehyde
4. 5. 6.
(1) Schopf, C., Komzak, $., Braun, F., Jacobi, E., Ann. 559, l ( 1 9 4 8 ) . (2) Schopf, C., Oechler, F., Ibid., 523, 1 i1936).
i..
558.
Propianaldehyde Butyraldehyde n-Valeraldehyde
several aliphatic aldehydes having free functional groups and t h a t of certain other carbonyl compounds or their derivatives are listed in Table I. The straight-chain aldehydes from CI to Ca and also glyoxylate respond comparably. Chloral hydrate or glucose, in which the aldehyde function is not free, or acetone, the lowest aliphatic ketone, is virtually inert. Benzaldehyde, as a representative of the aromatic series, and the keto acids, pyruvate and a-ketoglutarate, give
RECEIVED for review November 28, 1961. Accepted January 10, 1962. Division of Analytical Chemistry, 139th Meeting, ACS, St. Louis, Mo., March 1961. Based on material from a dissertation submitted by Alberta M. Albrecht in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Rutgers, The State University, June 1961. Alberta Albrecht acknowledges a predoctoral traineeship under U. S. Public Health Service Training Grant 20-507. Work was aided by a contract between the Office of Naval Research, Department of the Tavy, and Rutgers, The State University, and by research grants from the Damon Runyon Memorial Fund and the U. S. Public Health Service.
Evaluation of Amides and Other Very Weak Bases in Acetic Acid TAKERU HIGUCHI, CHARLES H. BARNSTEIN, HOSSEIN GHASSEMI,
and WALDO E. PEREZ
School of Pharmacy, University o f Wisconsin, Madison, Wis.
A modified version of the Type II plot proposed earlier has been developed for bases so weak that they exist to a significant extent in their free forms in the presence of excess perchloric acid in acetic acid. The relative basicities of compounds studied ranged over five orders of magnitude with dimethyl acetamide as the strongest and diethyl ether as the weakest measurable. Extremely precise titrations are possible for the more basic
400
ANALYTICAL CHEMISTRY
amides. Less basic compounds such as acetanilide are, however, determinable with less precision. Basicity data are presented for 40 different compounds.
D
that titration of various weak bases in acetic acid has been studied extensively for a number of years, its application to systems essentially nonbasic in water has been relatively limited. This communicaESPITE THE FACT
tion is concerned with results of an investigation designed to show some of the practical and theoretical limitations of this technique. Although the applicability of the method to both qualitative and quantitative determination of amides has been particularly stressed, a number of other functional groups have also been investigated as a part of this study. The basic relationships governing interactions between acids and bases
