Table 111. Total Phosphorus Recovery Substance, 50 nmole
Recovery, 2
AEP N-Methyl-AEP N,N-Dimethyl-AEP 2A 3PPA Phosphoserine a Average of six determinations.
101
97 97 95 97
Relative standard deviation, 1.7 1.4 2.4 I .4 1.5
phorus compounds. Table I11 shows total phosphorus recovered from the organophosphorus compounds relative to total phosphorus recovered from orthophosphate standard along with relative standard deviations of the replicates. DISCUSSION
The residual acid from the digestion procedure must be controlled for subsequent orthophosphate determination by Bartlett’s ultramicro method. The digestion mixture is designed to provide 0.8 ml of concentrated nitric acid, 0.1 ml of concentrated perchloric acid, and 3.0 meq of sulfuric acid to each tube. Nitric acid provides the reserve of oxidizing capacity to oxidize 0.040 gram of organic material without exhausting the oxidizing capacity of the perchloric acid. Perchloric acid provides the oxidizing potential necessary to break the C-P bond of organophosphonates and neutral-
izes residual cations. Larger amounts (than 0.6 meq) of cation can be handled by increasing the amount of perchloric acid and the digestion time. By digesting at a temperature significantly above the boiling point of nitric and perchloric acids but well below that of sulfuric acid, excess nitric and perchloric acids are volatilized from the tubes during the digestion period. The remaining sulfuric acid reproducibly provides 3.0 meq of free acid residue over wide ranges of operating conditions and sample tontents, thereby eliminating adjustment of acidity prior to analysis. The digestion procedure presented here has been applied in this laboratory to measure total phosphorus in amino acid analysis chromatograms containing between 2 and 1000 nmole/ml each of various organophosphates and aminophosphonic acids. Effluent collected from the amino acid analy7er contained between 0.0196 and 0.0343 gram of Na,. citrate 2 H s 0 per 1.O ml fraction ( 4 ) . Although our primary concern is with phosphorus analysis, the digestion procedure may find wider application in the preparation of biological and environmental samples for many analytical procedures.
RECEIVED for review April 30, 1971. Accepted June 8, 1971. Supported by Grant 4294 from the Robert A. Welch Foundation. (4) D. H. Spackman, “Methods of Enzymology,” C. H. W. Hirs, Ed., Vol. XI, Academic Press, Inc., New York, N. Y . , 1967, p 3.
Analysis of Insect Chemosterilants Action of Phosphate Buffers on Aziridine J. George Pomonis, Ray F. Severson,’ Patricia A. Hermes, Richard G. Zaylskie, and Andrew C. Terranova Metabolism and Radiation Research Laborator),, Agricultural Research Serrice, U.S . Department of Agriculture, Fargo, N . D . 58102 WHENCHEMICALS, especially alkylating agents, are to be used to produce sterile insects, it is essential to measure the period of persistence of these substances and their decomposition products. Several methods of analysis for chemosterilants such as aziridine-containing compounds (1-5) and alkylating agents such as alkyl-halides and sulfonates (6, 7) have been reported and have been widely used in degradation and metabolic studies. These kinetic studies were performed in conjunction with those used in the development of an auto-
mated analytical procedure for insect chemosterilants (8, 9). Aziridine, a product of hydrolysis of tris(1-aziridiny1)phosphine oxide (tepa) (9,was selected for extensive study. We report here the kinetic effect of phosphate buffers on aziridine and show that a modification of the standard colorimetric method of assay (1) for aziridine is suitable for the collection of kinetic data.
* Predoctoral assistant, Entomology Research Division, Agricul-
Apparatus. All measurements of absorbance were made with a Cary Model 14 spectrometer at ambient temperature. All measurements of pH were made at a temperature of 27 =t 0.1 “C on a Radiometer Model 22 pH meter equipped with a Model PHA 630 Pa scale expander and a combined glass and calomel electrode assembly (Radiometer GK 2021C). A 10gallon constant temperature bath was maintained at 27 + 0.05 “C with a Bronwill Thermo-regulator-stirrer for the hydrolyses. Materials. Aziridine from commercial sources was purified by distillation from NaOH and stored in dark bottles
tural Research Service, USDA. North Dakota State University. Fargo, N. D. 58102. (1) D. H. Rosenblatt, P. Hlinka, and J. Epstein, ANAL. CHEM., 27, 1291 (1955). (2) R. M. V. James and H. Jackson, Bioclient. Pharmacal., 14, 1947 (1965). (3) J. Epstein, R. W. Rosenthal, and R . J. Ess, ANAL. CHEM., 27, 1435 (1955). (4) A. W. Craig, H. Jackson, and R. M. V. James, Brit. J. Plrarrnnco(., 21, 590 (1963). (5) M. Beroza and A. B. Borkovec, J . Med. Clwm., 7,44 (1964). (6) E. Sawicki, D. F. Bender, T. R. Hauser, R . M. Wilson, Jr., and J. E. Meeker, ANAL.CHEM., 35, 1479 (1963). (7) T. A. Connors, L. A. Elson, and C. L. Leese, Biochem. Pharmacol., 13,963 (I 964).
EXPERIMENTAL
(8) A. C. Terranova, J. G. Pomonis, R. F. Severson, and P. A. Hermes, Proc. Techiiicoii N . Y . Symp. Automrrtion in Anal. Cliem., I, 501 (1967). (9) A. C. Terranova, J . A g r . Fond Clien?.,17,1047 (1969).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
1709
w1.0V
z a
m
a
0 v)
90.5
-
1
0
100
200
300
400
0
TIME (MIN.)
Figure 1. Influence of pH on rate of aziridine hydrolysis Total concentration of phosphate buffer was 0.05M, and temperature was 27°C
I
I
100
I
200 TIME (MIN.)
I
I
300
400
Figure 2. Effect of concentration of phosphate buffer on rate of aziridine hydrolysis pH of the solutions was 7, and the temperature was 2 7 - C
at a 45" angle for 1 minute. The resulting mixture was allowed to stand for 5 minutes, and the organic layer was separated from the aqueous layer and dried over Na2S04. The chloroform solution was then transferred to a cuvette, and the absorbance determined. Blanks (containing no aziridine) were treated in the same manner.
over KOH. Sodium 1,2-naphthoquinone-4-sulfonate was purified by the method of Horning ( I O ) . ACS grade potassium dihydrogen phosphate and sodium hydroxide were used to prepare the buffer solutions. Chloroform suitabIe for the assay was prepared by passing it through alumina. 4-(l-Aziridinyl)-1,2-Naphthoquinone. To a solution of 0.52 gram of sodium 1,2-naphthoquinone-4-sulfonate in 50 ml of pH 8 phosphate buffer (0.05M, presaturated with N?), we added 1 ml of aziridine dropwise and continued stirring for 5 minutes. The mixture was cooled, and the solid was collected by filtration. The product was recrystallized twice from C H C k M e O H and dried in cacuo at 30 "C, mp 174-175 "C (dec),,,,A, 4240 nm; E,,, 3000 Limo1 cm; Anal. (Analysis was performed by the Huffmann Microanalytical Laboratories, Wheatridge, Colo.). Calcd for C1?HgNO?:C, 72.35; H , 4.56; N , 7.03. Found: C, 72.16; H , 4.78; N, 6.96. Kinetics. All kinetic measurements were made at 27 0.05 "C. The disappearance of aziridine with time was followed spectrophotometrically by recording the decrease in absorbance at 4220 nm. Standard curves were constructed from CHCls solutions of 4-(l-aziridinyl)-l,2-naphthoquinone and by extraction into CHCI, of the product of the reaction of aziridine with sodium 1,2-naphthoquinone-4-sulfonate.The pH of the hydrolysis solution was determined at the beginning and end of each test, and additional checks were made periodically during the test. I n a typical experiment to determine the initial rates, about 100 mg of aziridine (about 0.09 ml) was transferred to a tared 50-ml volumetric flask, and the volume was adjusted to the mark with one of the following phosphate buffers: pH, 6. 7, 8 of 0.05, 0.1, 0.15, or 0.2M concentration. The solution was allowed to equilibrate to the bath temperature (27 "C) for 30 minutes, and then 1 ml was removed for the assay; the remainder was transferred to a serum bottle that was sealed and immersed in the water bath. Thereafter, samples (ca. 1.5 mi) were withdrawn by a hypodermic syringe about every 5 minutes for 100 minutes, and 1 ml of the withdrawn solution was pipetted onto a solution of 5 ml, 0.25M pH 9 carbonate buffer and 1 ml of 1% aqueous naphthoquinone reagent (prepared fresh daily) contained in a 125-ml separatory funnel. The funnel was swirled to produce good mixing and was allowed to stand for 5 minutes. Next, 10 ml of chloroform was delivered to the separatory funnel ria an automatic pipet, and the funnel was stoppered and shaken
Hydrolysis of Aziridine. Aiiridine, a weak organic base, reacts with sodium 1,2-naphthoquinone-4-suifonateto yield a chromogen that is extractcd into chloroform. The absorbance of the chloroform solution is determined spectrophotometrically and related to the concentration of aziridine ( I ) . Because the structure of the chromogenic substance had not been described and because knowledge of the structure would aid in kinetic studies, we undertook the task of isolating and synthesizing the compound, which is identified as 4-(1aziridinyl)-l,2-naphthoquinone (I I ) by elemental analysis, proton magnetic resonance, and infrared spectrometry. A peak in the absorption spectrum at A,,,, = 4240 nm was used in the colorimetric assay ( I ) . The E,,,;,,(molar absorbance) was calculated as 3000 I./mol cm. and the solutions obeyed Beer's law over a 100-fold range of concentration as determined in 0.1-, 1-. and IO-cm quartz cells. At ambient temperature, the rate of reaction of aziridine with sodium 1,2-naphthoquinone-4-sulfonate was so fast at the conditions of testing that we could not measure it. The stability of the chromogenic substance in chloroform was excellent for 1.5 hours. A comparison of the absorbance of chloroform solutions of synthetic 4-(l-aziridinyl)-l,2-naphthoquinonewith the absorbance obtained by mixing measured quantities of ,aziridine with sodium 1.2-naphthoquinone-4-sulfonate indicated nearly complete reaction of the aziridine with the reagent and nearly quantitative extraction of the reaction product into chloroform. The efficiency of extraction of the product from water into chloroform was checked further by extracting water solutions of varying concentrations of 4-(l-aziridinyl)-l,2naphthoquinone into measured volumes of chloroform and determining the absorbance spectrophotometrically. The results of these experiments indicated a 99+ % efficiency of extraction of the aziridinyl napthoquinone into chloroform.
(IO) E. C . Horning, "Organic Synthesis. Coll. Vol. 111," Wile), New York, N.Y., 1955, p 633.
( 1 1 ) To Farbenfabriken Bah-er, A. G., British Patent 806.079 (1958); Cliein. A h t i , . . 53, P12301 (1959).
*
1710
e
RESULTS AND DISCUSSION
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
a m
lY
II 9 0.5-
0
1
I
100
J
I
200
400
300
0
1
2
3
4
5
6
CONCENTRATION OF HPO;'
TIME (MIN.) Figure 3. Effect of concentration of phosphate buffer on rate of aziridine hydrolysis pH of the solution was 7, and the temperature was 27°C. The break in the curve probably resulted from the large increase in ethanolamine concentration which was shown to be significant at higher buffer concentrations (5). At lower buffer concentrations, ethanolamine is not formed as rapidly The Influence of p H . The rate of aziridine disappearance in 0.5M phosphate buffers at p H 6, 7, and 8 a t a temperature of 27 "C (the average normal temperature of insect hemolymph) was determined over a period of 440 minutes. The results observed with a solution of aziridine (1.8 X lod4 gram/ml) are shown in Figure 1. The p H of each solution, determined at the beginning and end of each experiment, decreased slightly, and the data suggested that the rate of disappearance increased as the p H of the buffered solution was decreased a t a constant molar concentration of buffer. An opposite result has been reported for aziridine in saturated phosphate buffer, where the rate of disappearance of aziridines increased as the p H of the buffered solution was increased (5). Althollgh these differences in rates first appeared t o be contradictory, it will be shown that there is a consistency of facts to explain these differences. The Influence of Buffer Concentration. To study the effect of increased ion concentration, we determined the rate of aziridine disappearance in solutions of pH 7 phosphate buffers in each of the following concentrations: 0.05, 0.10, 0.15, 0.20, and 0.25M for 400 minutes a t 27 "C. The results are
Figure 5.
x IO'
7
8
LITER
Plot of -d'AZ1'dz us. [HP04-2].
[AZI [H+1
Points on the plot were obtained from total concentration of buffer shown
shown in Figures 2 and 3. These data suggested that an increase in the rate of disappearance occurred with an increase in the molar concentration of the phosphate buffer, but again, it should be noted that the small decrease in pH was observed during the course of hydrolysis, which indicated that the buffering capacity of the solution was maintained. Therefore, t o establish more clearly the possible mechanism of the hydrolysis in phosphate buffer, we measured initial rates at four concentrations of buffer at 27 "C (Figure 4). When the rate constant was determined, it was found to fit the following mechanism if we assumed a steady-state concentration for the aziridinium ion in Equation 1 :
0 A
(3)
0 HzN-CH~--CH-O-P-OH
.t. J-
L
a100
5 90-
. - --
.
.
. .
.
.
.
0.1M
-
. . *
.
. . .
. .
..
. I
,
,
0.2M,
+ H zPO4 '-
HzN-CH2-CH2-0H HzP04-l
90
4
0
._
a
$100
+ HzO
e HP04'- +
+ H
(4) (5)
However, steps 4 and 5 are only speculative since we did not analyze for the product but only for aziridine disappearance. The rate equation derived on the basis of the proposed mechanism is shown in Equations 6 and 7 in which [AZ] = concentration of aziridine and kwl = kw[HnO]:
After factoring and rearranging, Equation 6 can be written in slope intercept form as:
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
1711
Then when the experimental values of - d[AZ]/dt/[AZ][H+] were plotted as a function of [HP04-*], a linear correlation was obtained that indicated that the kinetics were consistent with the suggested mechanism (Figure 5; each point is the average value of three to four tests lasting 100 minutes with samples withdrawn from the solution every 5 minutes). Then, the evaluation of the constants in Equation 7 (from Figure 5) resulted in the following relationship :
k-i k? = slope - = slope (&) ki from Figure 5,
slope = 8.25 X lo4 l./mol min
(9)
This value of kz for HP04-' is in the range of other nucleophiles that attack the aziridinium ion (13, 14). The differences of the rates of disappearance of aziridine in our work and in that reported by other workers ( 5 ) can now be explained. At the higher p H of the saturated phosphate buffer system used by these investigators, the high concentration of the HP04-2 ion (acting as a stronger nucleophile than water) will attack the protonated aziridine at a faster rate, even though the concentration of protonated aziridine (aziridinium ion) is low. At the more acidic pH, the concentration of the HP04-* ion is low and probably makes the hydrolysis of aziridine the predominant mode of ring opening.
(10)
ACKNOWLEDGMENT
Therefore, when the value of K, for the aziridinium ion (12) is taken at 22 "C (assuming that this value holds at 27 "C and at high ionic strength), Equation 9 becomes: k2
=
8.25 X lo4]./mol min)
x
I
60 sec min-1
1
x 0.955 x 10-8 ~
(11) which can be reduced to :
k2 = 14.3 X (12) G. J. Buist and H. J. Lucas, (1957).
The authors acknowledge the technical assistance of Carol Guilbert, Parnell Freeman, Barbara Weir, and also thank T. S. A d a m for his statistical analysis.
l./mol sec
(12)
J. Amer. Chem. Soc., 79,
6157
RECEIVEDfor review February 2, 1970. Resubmitted and accepted June 24,1971. Mention of a proprietary product or company name in this paper does not constitute a n endorsement by the USDA. (13) B. Cohen, E. R. Van Arstdalen, and J. Harris, ibid., 74, 1878 (1952). (14) J. E. Early, C. E. ORourke, L. B. Capp, J. 0. Edwards, and B. C. Lewis, ibid., 80, 3458 (1958).
Tape-Controlled Gradient Elution Chromatography System for Steroid Analysis David F. Johnson,' Nancy S. Lamontagne,' Grant C. Riggle,2 and Frank 0. Anderson2 National Institutes of Health, U. S . Department of' Health, Education, and Welfare, Bethesda, Md. AN AUTOMATED PUNCHED-TAPE system for gradient elution chromatography ( I ) , developed in our laboratories, has been used successfully for the analysis of adrenocortical steroids in biological mixtures (2-6). Application of the method to analysis of the residues from incubation of tissue with radioactive steroids and precursors revealed a need for further refinement in the apparatus. Radioactive compounds from such incubations were, in many cases, so similar in polarity that even the use of polarity reversal was not effective in resolving certain 1 Steroid Section, Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases. * Instrument Engineering and Development Branch, Division of
closely associated peaks. The refinement described in this paper includes an improved pumping system for the accurate delivery (0.2 %) of preselected volumes of eluting solvents, and a new solvent mixing chamber design which minimizes volume carry-over when solvent ratios are changed. These improvements have resulted in: a greater resolution of individual peaks in our chromatographic method for separating adrenocortical steroids on water-impregnated silicic acid columns ( I ) using petroleum ether (PE) and dichloromethane (DCM); and the development of a new combined method for analysis of adrenocortical and ketosteroids (7). EXPERIMENTAL
Research Services. ( I ) D. F. Johnson, D. Francois, G. C. Riggle, and C. I. Ramsden, Ann. N . Y . Acad. Sci., 130,792 (1965). (2) D. Francois, D. F. Johnson, and H. Y . C. Wong, Steroids, 7, 297 (1966). (3) D. Francois, H. Y . C. Wong, and D. F. Johnson, ibid., 8, 289 ( 1966). (4) Ibid., 9, 1 5 (1967). ( 5 ) Ibid., 10, 115 (1967). (6) D. Francois, R. W. Bates, and D. F. Johnson, Endocrinology, 81, 246 (1967).
1712
Apparatus. The complete apparatus is shown in Figure 1 . The main units, some of which are described in detail under procedure, consist of: water-jacketed solvent reservoirs, 1 and 2; chromatographic column, 4; tape punch and readout, 10 and 1 1 ; tape control electronic system, 12; and syringe pumps, 13. I n addition to the reservoirs and column, the outflow lines to the mixing chamber, 3, are also water-jacketed with 19.5 "C water from the cooling unit, 5. (7) N. S. Lamontagne and D. F. Johnson, Steroids, 17, 365 (1971)
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
