(10) D. F. S. Natusch, J. Am. Chem. Soc., 03,2566 (1971). (11) 0. A. Gansow, A. R. Burke, and G. N. LaMar, Chem. Commun., 456 (1972). (12) G.C. Levy and J. D. Cargioll, J. Magn. Reson., 10, 231 (1973). (13) R. Freeman, H. D. W. Hill, and R. Kaptein, J. Magn. Reson., 7, 327 (1972). (14) J. J. Klrkland, "Modern Practice of Liquid Chromatography", Wiley-lnterscience, New York, 1971, p 208. (15) J. T. Swanslger, F. E. Dickson, and H. T. Best, Anal. Chem., 48, 730 (1974). (16) M. Orthin, C. Golumbic, S.T. Anderson, and H. H. Storch, U.S. Bur. Mines Bull., 505 (1951).
(17) 0. C. Levy, U. Edlund, and J. G. Hexem, J. Magn. Reson., 10, 259 (1975). (18) D. L. Wooton, H. C. Dorn, L. T. Taylor, and W. M. Coleman, Fuel(London), 55, 224 (1976). (19) T. D. Alger, D. M. Grant, and E. G. Paul, J. Am. Chem. SOC.,88, 5397 (1966).
RECEIVEDfor review February 18,1976. Accepted August 16, lg76The support of'the vpl I% Su Research Division is gratefully appreciated.
Spectrophotometric Microdetermination of Amines and Sulfamates with 1,4-Benzoquinone G. Anthony Benson and Wllllam J. Spillane" Chemistry Department, University College, Galway, Ireland
Twenty allphatlc, prlmary and secondary, allcycllc and heterocyclic amines In chloroform have been determined spectrophotometrically by measurement of the absorbance (A, 478 to 510 nm) of the reaction product from their reactlon at 60 'C wlth I% ethanollc l,4-benroqulnone. Cyclopentyl- and cycloheptylamlnesand the artlflclalsweeteners cyclopentyl-, cycloheptyl-, and cyclooctylsulamailes have been determined after extraction from urine. The mean per cent recoveries f average devlatlon for cyclopentylamine and cycloheptylsulfamate In urlne are 97 f 1.99 and 103.61 f 4.42, respectively. Beer's law was followed In all cases over an approximate range of 5-100 pg/ml. The sensitlvity of the method (using cyclopentylamlne) was 3 pg/ml (2 ppm). A preclsion study showed that the relative standard davlatlon of absorbance readings was 0.6 % for n-ectylaaine.
Folin and Wu (!) established that sodium 1,2-naphthoquinone-4-sulfonate was the most suitable o-quinone to employ as a reagent for the determination of amino acids. The reaction has been modified for the determination of ammonia, aliphatic and aromatic amines, sulfonamides and some alkaloids ( 2 4 ) .Schmidt (2) investigated the reaction and showed that a slightly basic medium was necessary for reaction to take place. He found that many amines, e.g., higher aliphatic and aromatic, gave insoluble orange-red precipitates which could not be used for determinations and that some, possibly sterically hindered, amines, e.g., diisopropyl- and ethylenediamines failed to react. Another problem has been interference from the colored unreacted reagent and there are many descriptions of the use of decolorizing mixtures to discharge the color of the unreacted reagent ( 4 ) .Rosenblatt et al. ( 5 ) have shown, provided that the amine reacts, that at least for some amines these difficulties can be overcome by extracting the insoluble dye with a suitable organic solvent, which removes the colored product from excess reagent and decomposition products and also increases the sensitivity of the reaction. This method has been applied by others (4,6, 7). A Japanese group (8,Q) have used either 1,4-benzoquinone or quinhydrone, instead of 1,2-naphthoquinone-4-sulfonate, to couple with cyclohexylamine probably giving rise to the colored products, 1 or 2 depending on the procedures used.
2
1
This reaction has been used successfullyfor the determination of the artificial sweetener, sodium N-cyclohexylsulfamate (sodium cyclamate) (8-10). In our studies on related sulfamate sweeteners, we have adopted this method for their determination (first, by hydrolysis of the sulfamate and then coupling of the amine thus produced) (11, 12). Simultaneously, we have examined the scope and usefulness of the coupling reaction using nearly 30 different aliphatic, alicyclic, and aromatic amines. In this paper, we present results which show that reaction with 1,4-benzoquinone can be used for the determination of about 25 of these amines, in the approximate range of 5-100 gg/ml with a high precision and sensitivity.
EXPERIMENTAL Apparatus. Absorbance readings were made at 20 O C on a Perkin-Elmer uv 124 spectrophotometer with 10-mmquartz cells. Centrifugation was carried out on a MSE Centrifuge. Reagents and Chemicals. Generally amines were used as obtained but the following amines were redistilled before use: aniline, N methylaniline,_cyclopentyl-,cycloheptyl-,cyclooctyl-, n-octyl- and dicyclohexylamines. 1,4-Benzoquinonewas freshly sublimed before use. Chloroform,ethanol, 1,4-dioxane(reagentgrade),chlorosulfonic acid, sulfosalicylic acid (AnalaR), and perchloric acid were used without further purification. Sodium N-cyclopentyl, -heptyl and -octylsulfamates were made by previously reported methods (13, 14).
Procedures. All coupling reactions with 1,4-benzoquinonewere carried out in a darkened room in order to obtain stable absorbance readings and to avoid photodecomposition of the yellow ethanolic 1,4-benzoquinoneA,, 435 nm (10). Reaction was indicated by the development of a red color. Ethanolic 1,4-benzoquinonewas freshly prepared before each experiment and the same solution was used throughout that experiment. Determination of the Optimum Concentration of 1,4-Benzoquinone. Twenty-five ml of chloroform containing 51.6 pg/ml of cyclopentylamine was added to eight 60-1111 volumetric flasks. Ten ml of ethanolic 1,4-benzoquinone(varyingin concentration from 0.1 to 1.3% w/v) was added to each flask. The flasks were immersed in a waterbath at 60 "C for 2 h. The absorbanceof each solution w a then ~ read against
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Table I. Effect of Concentration of l,4-Benzoquinone on Reaction with Cyclopentylaminea 0.9 0.778
0.2 0.3 0.655 0.580 g/100 ml ethanol. 51.6 yg/ml cyclopentylamine. Concnb Absorbance
a
-
0.1 0.400
1.0 0.805
1.2 0.800
1.1
0.801
1.3 0.799
Table 11. Effect of Temperature and Reaction Time on the Reaction of 1% 1,4-Benzoquinone and Cyclopentylaminea 40 "C Time, min.
15
30
45
60
70
80
90
100
110
120
Absorbance
0.628
0.824
0.901
0.980
1.00
1.02
1.04
1.079
1.080
1.090
110 1.142
50 O
a
C
Time, min. Absorbance
30 0.745
45 0.905
60 0.990
70 1.107 60 "C
80 1.100
90 1.130
100 1.142
Time, min. Absorbance
15 0.640
30 0.942
45 1.104
60 1.120
70 1.150
80 1.130
90 1.130
51.6 yg/ml cyclopentylamine.
Table 111. Reaction of 1% 1,4-Benzoquinone with Amines at 60 "C Amine n -Butylamine n -Hexylamine n-Octylamine n -Dodecylamine Ethanolamine Ethylenediamine Isobutylamine Isoamylaminea tert- Octylamine Dimethylamine Diethylamine Di-n-propylamine Di-iso-propylamine Di-n-butylamine Dicyclohexylamine N-Methylcyclohexylamine Tri-n -butylamine Morpholine 4-Methylpiperidine N -Aminohomopiperidine Cyclopropylamine Cyclobut ylamine Cyclopentylamine Cycloheptylamine Cyclooctylamine Cyclododecylamine Aniline N -Methylaniline
Amam
nm
Time, h
Beer's law range, Fg/ml
490 0.75 485 2.0 483 1.75 487 2.0 484 1.0 478 1.33 490 1.33 488 2.50 490 Reaction slow, only gained in intensity after 24 h 500 2.0 506 12.0 500 24.0 No reaction, after 24 h 510 24.0 No reaction, after 24 h 506 Reaction slow, only gained in intensity after 24 h No reaction, after 24 h 488 3.0 510 1.5 510 Reaction very slow even after 24 h 481 2.0 488 1.33 490 1.25 489 2.0 488 2.17 490 2.0 No reaction, after 24 h No reaction, after 36 h
14.8-59.2 10.9-65.4 11.1-66.6 5.0-44.16 11.56-43.37 7.19-53.94 10.0-35.4 8.5-37.55 7.5-22.6 10.0-50.2 10.54-63.25 10.95-87.65
14.0-48.5 12.38-42.11 16.4-65.0 8.32-81.2 6.25-100.0 15.36-92.16 12.9-64.5 24.8-124.37
a With this compound, carbon tetrachloride, benzene, and ethanol were additionally used and a red color developed in each case with, , ,A = 482,485, and 486 nm, respectively.
a set of blanks each of which contained 25 ml chloroform and 1,4benzoquinone (0.1 to 1.3 w/v) in 10 ml ethanol (Table I). Determination of the Optimum Temperature for Reaction of 1,4-Benzoquinonewith Cyclopentylamine. Fifty ml of 1%ethanolic 1,4-benzoquinonesolution was preheated to the chosen temperature (40,50, or 60 O C ) in a constant temperature water bath. Two 10-ml aliquots of this solution were transferred to 50-ml volumetric flasks containing cyclopentylamine (51.5 yglml) in 25 ml chloroform and 25 ml chloroform, respectively. Suitable aliquots of these solutions were transferred to quartz cells which were thermostated at the de2150
sired temperature. Absorbances were then read at various times (Table 11). Determination of the Time Required t o Reach ,A, in the Reaction of 1 % Ethanolic 1,4-Benzoquinone at 60 "C with Various Amines. Establishment of Beer's Law. For liquid amines a concentration of 1 ~1/25 ml chloroform was used and for solid amines, 50 pg/ml chloroform. The procedure was identical t o that described above. Beer's law was checked for most of the amines which underwent reaction. The ranges covered are indicated in Table 111. An average of six different concentrations of amine were used in these determinations.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
Table V. Percent Recovery of Cyclopentylamine from Urine
Table IV. Percent Recovery of Sodium Gycloheptylsulfamate from Urine
a
mg/mmla
Recovery, %
mg/mla
Recovery, %
2.91 2.91 5.09 5.09 6.38 6.38
98.2 100.3
4.3 4.3 4.3 5.1 5.1 5.1 6.8 6.8 6.8
95.34 98.83 97.67 90.60 96.89 95.44 96.80 99.56 101.74 Mean f average deviation = 96.98 f 1.99
111.1
109.0 99.1 103.2 Mean f average deviation = 103.61 f 4.2
Amount of sodium salt per ml urine.
Determination of Sulfamates in Urine. Quantitiesranging from 3-33, 2-16, and 2-12 mg of cyclopentyl-, cycloheptyl-, and cyclooctylsulfamates,respectively, were dissolved in 1-ml samples of urine contained in 10-ml volumetric flasks. Four ml of 1,4-dioxane and 0.5 rnl of 5 M perchloric acid were added to each sample. The flasks were tightly stoppered and placed in an oil-bath at 95 "C for
3.5,4.5, and 4 h, respectively.Two-ml samples were pipetted from the flasks and extracted with 2 X 10 ml chloroform. The extracts were made up to 25 ml chloroform and transferred to 50-ml volumetric flasks. Ten ml of 1%benzoquinone was added to each flask and the solutionswere then heated at 60 "C for 1.25,2,and 2.17 h, respectively, and the absorbances read at A, = 490,489, and 488 nm, respectively. Plots of absorbance vs. concentration were linear over the concentration ranges employed. During the extraction procedure, emulsions occasionally formed and then a separation of the urine and chloroform layers was achieved by centrifugation at 3000 rpm for 30 min. A per cent recovery was established for cycloheptylsulfamate using this procedure (Table IV). Determination of Cyclopentylamine and Cycloheptylamine i n Urine. Quantitiesfrom 0.6-4.5 and 1-5 p1 of cyclopentyl- and cycloheptylamines,respectively, were added to 5-ml samples of urine. Five ml of 20% (w/v) aqueous sulfosalicylic acid was added and the mixtures were basified with 1.5 ml of 10 M sodium hydroxide. Each sample was extracted with chloroform and the remaining procedure was identical with that described above for the determination of sulfamates in urine. Standard curves were obtained over the ranges covered. A per cent recovery was established for cyclopentylamine (Table V) using the same procedure. RESULTS A N D DISCUSSION Effect of l,4-Benzoquinone Concentration. From Table I, the concentration of 1,4-benzoquinonegiving the maximum absorbance in its reaction with cyclopentylamine is seen to be 1.0%. This result is in agreement with the findings of the Japanese workers (8) who used a closely similar concentration of cyclohexylamine (50 pg/ml) in their study. One percent ethanolic solutions of 1,4-benzoquinone were therefore used subsequently in this study. Effect of T e m p e r a t u r e a n d Heating Time. The effects of temperature and of heating time on absorbance in the reaction of 1,4-benzoquinone with cyclopentylamine is shown in Table 11.A temperature of 60 OC was used subsequently in this work. N a t u r e of the Product. Scope of the Reaction. Beer's Law. Under the conditions used in this study, the product = 495 nm (Method B formed is thought to be 1 @-IO), ,A, of Ref. 8) and not 2, A,, = 355 nm (Method A of Ref. 8), which has been prepared independently (9,15). The reaction is successful for a wide number of amines and Beer's law has been established for these. The range of absorbances covered in typical Beer law calibration curves was about 0.2 to 0.9, but sometimes as wide as 0.1 to 1.4. Table I11 gives for each amine, A,, for the product of the reaction, the time taken to develop the maximum absorbance, i.e., to reach a plateau in a plot of absorbance vs. time and the range over which Beer's law was established. The reaction can be used for the determination of the artificial sweeteners cyclopentyl-, cycloheptyl-, and cyclooctylsulfamates in urine. They are first hydrolyzed to amine in a dioxan-acid medium, using a procedure (11, 16)
Amount of cvclouentvlamineDer ml urine. which avoids the necessity for high pressures and temperatures to achieve quantitative hydrolysis. The amine is then extracted into chloroform for determination. A percent recovery for cycloheptylsulfamate gave a mean f average deviation of 103.61 f 4.42 (Table IV). Cyclopentyl- and cycloheptylamines have also been determined in urine by extraction into chloroform and subsequent reaction with 1,4-benzoquinone. A percent recovery of cyclopentylamine from urine gave a mean f average deviation of 97 f 1.99 (Table V). Sensitivity, Precision, a n d Stability of Solutions. A sensitivity study was carried out using cyclopentylamine; 10.37, 6.88, 3.44, 1.14, 0.58, 0.27, and 0.135 pg/ml of cyclopentylamine in chloroform were reacted with 1,4-benzoquinone. The first three solutions produced a color within 1.25 h and, after 19 h, the same three samples had absorbances of 0.278, 0.166, and 0.072, respectively. The fourth sample had developed a slight color (absorbance, 0.025) and the remaining three samples gave no absorbances. Therefore for cyclopentylamine the lower limit of detection is about 3 pg/ml after a reaction period of 1.25 h. (Table 111);if the reaction period is prolonged for 19 h, the sensitivity is greater and about 1.1 pg/ml can be detected. A precision study was carried out by taking 10 replicate measurements on 31.08 pg/mE solutions of n-octylamine after 1.75 h. The mean absorbance, standard deviation, and relative standard deviation were 0.699,0.0045, and 0.006, respectively. In general, solutions appeared to be stable for some hours when kept in the dark. The solutions from the above precision study were kept in the dark for an additional 2 and 16 h (after reaction) and the mean absorbances were 0.689 and 0.676, respectively. The solutions were then placed in daylight for a further 2 h and the mean absorbance fell to 0.638. Comparison of t h e 1,2-Naphthoquinone-4-sulfonate a n d l,4-Benzoquinone Methods f o r Amines. The reaction 3 and which occurs between 1,2-naphthoquinone-4-sulfonate amines is given (3,4 ) as: 0
@+
0
HLNR
@"
S03Na
fiR
3
4
Schmidt showed that there are difficulties due to the failure of some amines to react, interference due to the absorbance of unreacted 3, formation of insoluble colored products, and, since the products possess indicator properties, a dependence of ,A,, on the pH of the solution. These difficulties have been got over for some amines by extracting the colored product
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