relatively high temperature at low pressure in the microscope; consequently, loss of mass is possible by evaporation during the time required to obtain the photographs of the particles prior to the reaction. However, by keeping the beam intensity low, this effect is minimized. I n the second method. the chromium shadow must be cast a t a low angle to the plane of the specimen support and must be tenuous. Otherwise, the metallic coating will inhibit the reaction. Because the reaction may proceed a t more than one site on an individual crystal, in determining the length of the reaction product the summation oi the lengths of all individual filaments in a particular reaction site is used as the length of the reaction product. Consequently, if a particle is composed of more than one silver iodide crystal, it will be recorded as one partirle of niass equal to the summation of the masses of the individual particles. In certain applications--e.g., the identification of the nucleus of a n ice crystal-- it is necessary to prevent the
movement of the particle during thP identification reaction ; the procedure as described has been found to fulfill this requirement satisfactorily. However, in general, because of the impossibility of ascertaining the direction of the growth of the ribbons, it is impossible to establish the original location of the particle more accurately than within the region occupied by the silver filament. If the precise location is required, a thin shadow cast before the reaction as shown in Figure 3 will provide this information. RESVLTS
The results of tti!; application of this test to a replica of :$I] ice crystal in which crystallization w:is initiated by means of a silver iodide particle from an evaporation source and to the products from three forms of silver iodide aerosol generators are illustrated in Figures 4 through S. It. map be observrd that the percentage composition of silver iodide in the particles from each of these methods of aerosol product,ion is different.
The smallest identifiable silver fiiament is considered to be 250 A. in length, which corresponds to a silver iodide particle 200 A. in diameter or 3 X lo-’’ gram in mass if a spherics: partirle of normal density is assume(;. ACKNOWLEDGMENT
The author expresses appreciation tl. Barbara J. Tufts of this laboratorv for her assistance. LITERATURE CITED
(1) Fuquay. D. M., Wells, H. J., Pro!v$ Skyfire Cloud Seeding Generat.or, Finli. Rept. of Advisorv Committee o.~. Weather Control, Vol. 11, pp. 273-82; U. S. Government Printing Officr, Washington, n. C., 1957. (2) Mees, C. E. K., “Th: Theory of the. Photographic Process, pp, 305-31 I Macmillan, l T e wYork, 1942
RECEIVEDfor review March 9, 1956. Accepted July 10, 1959. Presented i n part before the 174t,h Meeting, American Meteorological Society, X;ew York, N.Y., January 1959. Research supported by Grant ll’SF-G4644 from t h r Sat.ionai Scienre Foundation.
CoIo rimetric Dete rmination of Dicarboxy1ic Acid Derivatives as Hydroxamic Acids VIVIAN FISHMAN-GOLDENBERG Department of Biochemistry, Hillside Hospital, Glen Oaks,
N. Y.
PAUL E. SPOERRI Department of Chemistry, Polytechnic lnstitute of Brooklyn, Brooklyn,
b This paper reports on an investigation of the reaction between dicarboxylic acid derivatives and aqueous alkaline hydroxylamine. Dicarboxylic acid esters and amides whose carboxy groups are separated by one or two carbon atoms react with atypical speeds and color yields; longer chain dicarboxylic acid amides and esters give normal results. The cyclic dicarboxylic acid imides tested react rapidly and yield one equivalent of hydroxamic acid. A mechanism is presented to explain the data.
A
micromethod for the determination of soluble carboxylic mid derivatives is based on their reaction with alkaline hydroxylamine in aqueous medium t o fom hydroxamic acids ( I , 6). The literature concerning this method was summarized in a recent paper (4) which deals with the influence of structure on the speed of reaction beCONVENIENT
N..Y.
tween monocarboxylic acid derivatives and aqueous alkaline hydroxylamine. It was found that electrondonating and vicinal bulky groups retard hydroxamic acid formation by monocarboxylic acid amides and benzoic acid esters. Although a-chloro, a-amino, and a-hydroxy substituents do not inhibit hydroxamic acid formation by aliphatic carboxylic acid esters, they exert a considerable hypochromic effect on the color reaction between ferric ion and the resultant hydroxamic acids. The present paper deals with a n extension of this study t o the reaction between dicarboxylic acid derivatives and aqueous alkaline hydroxylamine. EXPERIMENTAL
Alkaline Hydroxylamine Reaction.
The procedure and reagents have been described (4). Organic Preparations. Succinamide (6), dimethyl isophthalate (8), maleamide (7), and glutaramide (2) were syn-
thesized according to procedures described in the literature. All the other compounds tested were commercially obtainable at a high grade of purity. RESULTS
The data pertaining t o the reactions between dicarboxylic acid amides and esters with aqueous alkaline hydroxvlamine at room temperature are summarized in Tables I and I1 and Figures 1 and 2. Comparison with earlier studies (4) shows that dicarboxylic acid amides and esters whose carboxy groups are separated by one or two carbon atoms (excluding esters of fumaric acid) give atypical color yields and time-study curves : Phthalamide, malonamide, succinamide, and maleamide react with unusual velocities and produce roughly one half the color yields of comparable monocarboxylic acid derivatives ( 4 ) . Whereas acetamide and succinamic acid require ’approximately 6.5 hours VOL. 31, NO. 10, OCTOBER 1 9 5 9
1735
1
0.300
- -
3 4 c j
1
--
Figure 1 . Comparative rates of hydroxamic acid formation
Succinomide ( 2 5r10~’M) W L>
4 by dicarboxylic acid amides
z a
0201/
m E
0
m m n
Temperature, 28’
C., absarbance a t
TIME
‘6
20
24
0 2.5 X 10-’M acetanilide A 1.25 X lO-%I adiparnide Temperature, 25’
C., absorbance at 540 rnp
(mtn)
11) tlroxmiic acid formation, IJithalamid(~reacshes 93% mauimum color fornxition \\ ithin 2 minutes. m i ni:ileamidv, nialonamide, and \riccinnniidr r q u i r c , 1.O-2.3 hours for iiinwnum hydroxaniiv acid formation. Furtlicwuorc, plltlialaniide (0.0025M) ~irotlurc~sa n at)sorlxincc of 0.47 as roini):ircd to 0.45 for ethyl bcnzoatc, 10.0025M), and malonamide ancl succin,imide (0.0025-M) yield absorbanccs of 3.50 and 0.41, respectively, as (.oinpnrcd t o 0 38 for rthyl acetatc 0 0025M). Diethyl malc~iitc~h:iq approximatel?
koi niauniuni
oiic lialf the color yield found for a n tquimolar solution of the trans isomcar diethyl fumarate, and diethyl malonate, dimethyl succinate, and dimethyl ophthalate have considerably b s s than twice the absorbances found for comparable (equimolar) monocarboxylic :toid esters (Table I). The data of Goddu, Lo Blanc, and Wright (3) concerning the nicthanolic hydroxylamine rractions of esters at reflux temperature, contrary t o their report, also indicate that dimethyl malonate, dimethyl maleate, and di-
Hydroxylamine Reactions of Dicarboxylic Acid Esters (25’C.)
Reaction Time, Max HydroxL amic Acid Formation, Min.
s o . of
C’s between Max. Iron-Complex Carbonyls Molarityo -4bsorbance at 540 M p Diethyl malonrttc.’ 1 0.0025 0.58(0.37-0.39C) 8 2 0,00125 0.52(0.45-0.54c) Diethyl fumarate 2 2 Dicthyl fumarate 0.0025 1.05(0.45-0).54c) 2 2 Diethyl maleate O.OO25 0.61(0.45-0.54c) 2 2 Dimethyl phthalatc. 0.0025 0.41 (0.45-0).54c) 13 Dimethyi succinatr” 0.0025 0.65(0.37-0.35) 2 8 0.00125 0.41(0.37-0.39c) 2 Diethyl glutaratc, 3 2 Diethyl glutaratt, 0,0025 0.83(0.37-0.39c) 0 .W125 0.49(0.45-0.54~) 3 Dimethyi isopht1iiil:t t i , 4 0.00125 0.42(0.374.30~) Dimethyl adipatts 2 8 2 Diethyl sebacatc 0.00125 0.37(0.37-O.39C) Molarity of eat ei solutions before treatment with hydroxylamine reagent ’’ Hestrin obtained similar values for these compounds (6). Ma\imum iroii-c*omplex ahsorhances of comparable 0.0025111 monocnrboxylic acid iitbrivatives a t 540 nip ( 4 ) .
-
?
I
I
(25”C.) Xeaction Time for Max. Hydroxamic h i d Formation, Min. 2*
Hydroxylamine Reactions of Dicarboxylic Acid Amides
Y o , of C’,. between Carboxy
h‘lax. Iron-Complex Absorbance a t 540 M p 0.47
Groups Molarityo Phthalamidc 0.0025 Malonamide 0.0025 0.50 90 Maleamide 0.0025 0 .25c 60 Succinamide 0.0025 0.41 140 qlutaramide 0.00125 0.27d 320 .idipamide 0.00125 0.24c 540 Molarity ot amide solution before mixing with hydroxylamine reagent. 93% of maximum reaction completed (Figure 1). Lon absorbance probahly due to instability of starting material and reaction product in alkaline medium. Monocarboxylic acid amides (0.0025111) which require 6-6.5 hours for maximum hydroxamic acid formation have absorbances in range 0.25-0.36 ( 4 ) . e Time-study ciirvc of 0.00125M adipamide is ~imilarto that of 0.0025M acetanilide (Fig. 2).
1734
12
Figure 2. Comparative rates of hydroxamic acid foimation
00 + 7 I I 1 I I 0 60 I20 I80 240 300 360
Table II.
8
A
TIME ( h r )
0 10
Table I.
0
540 m p
ANALYTICAL CHEMISTRY
nic.tliyl eplithalate yield less than two equivalents of hydroxamic acid.
On the other hand diethyl funiarate and dicarboxylic acid amides and esters that have more than ti\o carbon atoms between carboxy groups react with nornial speeds and produce more than one equivalent of hydroxamic acid. For example, dimethyl ndipate, dimethyl isophthaiate, diethyl glutarak. diethyl fumarate, and diethyi sthacato \-ield approximately t x o equivalents of hydroxamic acid (Table I), and glutaramide and adipamidr react with nornial speeds for amides and produce more than one equivalent of hydroxamic acid (Table 11 and Figure 2). I n viex of the anomalous color yields and atypical time-study CUNCS found for specific dicarboxylic acid esters and amides, these observations shouid be taken into account when procedures are being established t o dctwmine dicarboxylic acid derivatives as hvdroxamic acids. The results of the hydroxylamine reactions of cyclic imides are listed in Table 111. The cyclic: imides tested yield approximately onr equivalent of hydroxamic acid and react rapidly. Because succinimide, phthalimide, and .V-phthalyldz-phenylalanine mdergo hydroxamic acid formation at considerably greater speeds than the corresponding monoamido acids (Figure 3), it is improbable that they react with the hydroxylamine reagent via the monoamido acid intermediate ( 1 ) :
+
-
Succininlide NaOH NaOOC-CH2--CHz-- -CUNH? (1)
-
NaOOC-CX-CH2-CONWp 4NH&H NaOM--CHZ--CH2-CONHONa (2)
In view of the fact that phthaiimide and 1%’-phthalyl-&phenylalanine react cornpietely within 2 minutes, whereas phthalaniidic acid and A;-(o-carboxybenzoyl)-dl-phenylalaninc d o not give a n y appreciable reaction a t the end of 24 hours, it should be possible t o estimate these cyclic imides in the presence of their monoamido arid analogs.
50%
-
rivatives of fumaric acid and derivatives of dicarboxylic acids, whose carbox? functions are separated by more than two carbon atoms, undergo the monohydroxamic acid reaction. The carboxy functions are too widely separated from one another for the cyclization step (described above) to occur without great, difficulty. A mechanism for the hydroxylamini reaction of dicarboxylic acid irnideh which is similar to the monohydrommk acid reaction described aimve and is i r i accordance with the ( I n k niay i w outlined as follows:
0
w 35
-
N ph fhalyl- d l - phenylolontne
\\
m o - c a r b o x y b e i z o y l - d/- p F e x a l o n t i e 1 1-
\ I
TIME ( H R
I
Figure 3. Comparative rates of hydroxamic acid formation by cyclic imides and their acid amide analogs at concentrations of 2.5 X 10-3M Temperature, 25' C., absorbance a t 540 mN
Table 111. Hydroxylamine Reactions of Dicarboxylic Acid Imides (25" C.) (0.OO25M)"
Reaction Time for Max. HYdroxamic Max. Acid ForIron-Complex Absorbance at mation, 540 Mfi Min. Phthalimide 0.50 ( 0 . 4 M . M b ) Phthalylglycine 0.45(0.45-O.5Pb) Phthalyldmethionine 0.48(0.45-0.54b) Phthalyl-dc phenyl-
w e Succmmude
2
amic acid formation via more than one pathway:
0
Dihydroxamic Acid Reaction
0
0
+
M-UH-L-Li-M "*OH
NaOH A
(M
=
OR or "2)
NaONHCo-LH-LH-
co--NHoNa (3)
Monohydroxamic Acid Reaction
2 2
0.42(0.454.54b) 2 0.45(0,37-0.39b) 1206
&H-AH
o=cI I
hf
"Molarity of imide solution before
mixing with 2 volumes of hydroxylamine
rerrgent. * Maximum iron-mmplex absorbancesof comparable 0.0025M monocarboxylic acid derivatives at 540 m p (4). c Teat repeated many times; invariably it took 2 hours a t 25.5" instead of 60" aa reported by Bergmann (1).
I I
C=O
+ "?OH
M
I
I
o=L
c=o
CH-CH X:&H
I
LITERATURE CITED ___t
( 1 ) Bergmann, F.,
cussion. Theoretically it is possible for dicarboxylic acid derivatives whose carboxy groups are separated by no more than two carbon atoms t o undergo hydrox-
CHEM. 24,
(2).Bunbury, H. Xi., Heilbron, I., "Dictionary of Organic Compounds," Vol. 2, p. 606, Eyre and Spotteswoode, London, 1953. (3) Goddu, R. F., Le Blanc, N. F., Wright, C. M., ANAL. C n m . 27,
NaOOCdH~EI-CONHONa The same type of reaction is possible for malonic ester.
1251 (1955). (4)Goldenberg, V., Spoerri, P. E., Ibid., 30, 1327 (1958). ( 5 ) Hestrin, S., J . B i d . Che7n. 18G, 249 (1949). ( 6 ) Morrell, G. F., J . Chem. Soc. 105, 2704 (1914). ( 7 ) Rinkes, I. J., Rec. trav. chin. 46, 272 (1927). (8) Weith, W., Landolt,, A., Ber. 8, 722 (1875).
The two reactions occur simultaneously and the relative proportions of the reaction products formed depend on the extent t o which one reaction predominates over the other. This in turn depends on the structure of the particular ester or amide under consideration. Conversely, it is unlikely that de-
RECEIVEDfor review February 4, 1959. Accepted June 11, 1959. Meetin in Miniature, Metropolitan Long Isfind Subsection, New York Section, ACS, Brooklyn. N. Y., February 15, 1957. Based on a thesis submitted by Vivian Goldenberg in February 1957, In partial fulfiliment of the requirements for the degree of doctor of philosophy in the Polytechnic Institute of Rrooklyri.
DISCUSSION
The data indicate that whereas diethyl fumarate and dicarboxylic acid esters and amides whose carboxy groups are separated by more than two carbon atoms react with the hydroxylamine reagent with normal speeds and color yields, shorter chain dicarboxylic acid derivativea give anomalous results. A possible explanation for this phenomenon, which is in agreement with the data, is presented in the following dis-
ANAT..
13G7 (1953).
VOL. 31, NO. 10, OCTOBER 1 9 5 9
1737
