ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
319
Spectrophotometric Determination of Secondary Amines Dale H. Karweik" and Carl
H. Meyers'
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
A rapid, selective method for the trace analysis of secondary alkyl amines has been sought since the early 1930's. Primary interest in these years was directed toward the determination of secondary amines in pharmaceutical mixtures (1-3). Recently it has been noted that nitrates found in food, either added as a preservative or from nitrate rich soils, can be reduced by bacteria t o nitrite ( 4 ) . T h e nitrite anion in the presence of p p m amounts of dimethylamine in foods such as spinach or fresh fish can react t o form dimethyl nitrosamine ( 5 ) . Similar reactions can take place with the secondary amines found in tobacco smoke (6-7). Because of t h e carcenogenic properties of nitrosamines, it would be desirable to be able to determine not only trace amounts of nitrosamines b u t also t h e unconverted secondary amines. T h e reaction of carbon disulfide with dimethylamine was first investigated by Dowden (8) as a possible method for the detection of small amounts of secondary amines in biological fluids. T h e reaction results in the formation of a dialkyl dithiocarbamate complex which forms a stable complex with metal ions. T h e reaction illustrated in Equation 1 requires t h e presence of a base (ammonia or pyridine) t o force the reaction t o completion. T h e copper bis(dithi0carbamate) complex ( C U ( D T C ) ~can ) then be extracted into a suitable organic solvent such as chloroform or benzene and the concentration of t h e amine determined directly from t h e C U ( D T C )complex ~ or indirectly from t h e amount of copper remaining in t h e aqueous phase.
Table I. Beer's Law Behavior of Copper Bis(dithi0carbamate) Complexes concn amine range, PPm t slopea diethylamine 0-3 1.14 10' 0.076 0.043 dibutylamine 0-12 1.16 x 10' dibcnzylaminc 0-4 1 . 0 9 r 10' 0.025 morpholine 0- 7 1.26 'K 10' 0.064 a Slope is equal t o absorbance divided by ppm concen. frat ion. Table 11. Summary of Results of t h e Direct
Determination of Secondary no. of secondary samamines ples ppm diethyl6 2.46 amine dibutyl7 6.14 amine dibenzyl6 3.69 amine 6 8.00 morpholine
detection abs., limit, 434 n m abs. SD ppm 0.187 ~ 0 . 0 0 3 0.02 0.276
*0.003
0.07
0.097
iO.003
0.04
0.516
+0.011
0.01
solution for dibenzylamine. Following neutralization with 2 N hydrochloric acid, the solutions are diluted to 1.00 L with saturated ether/water solution or water for dibenzylamine. Stock solutions to deliver approximately 100 pg of copper can be prepared by dissolving copper shot (99.99%) in nitric acid or by dissolving CuCl2.2H2Oin water. Solutions of 97.0 Mg/mL and 105.8 p g l 5 . 0 mL were prepared by the respective methods. Analyzed spectral grade chloroform (Baker) and reagent grade carbon disulfide (Fisher) were used without further purification. Recommended Procedure. Transfer 10.0 mL of the secondary amine sample to a 125-mL separatory funnel; followed by 5.0 mL of 1.2 M, pH 9.4 ammonia-ammonium chloride buffer; and 100 Fg of copper(I1) stock solution. Add 0.5 mL of carbon disulfide, followed by 10.0 mL chloroform, shake for 60 s , and allow the layers to separate. Add 1.0 mL of 25% (v/v) acetic acid, shake for 60 s, and allow the two layers to completely separate. For the direct method, transfer the chloroform extract to a 25-mL volumetric flask and wash the aqueous layer with a second 10-mL portion of chloroform, combining the two extracts before dilution to volume. A reference blank is prepared by substituting an equivalent amount of water for the secondary amine solution. The absorbance of the copper complex in chloroform is measured a t 434 nm vs. the reference blank. For the indirect method, the aqueous layer, following extraction and removal of the organic solvent traces (91, is diluted to 25.0 mL with distilled water. The excess copper can be analyzed using AA and a standard curve or spectrophotometrically using Gahler's neocuproine method (10).
T h e direct determination has been used in principle by several authors for t h e determination of dimethylamine (2, 3, 8 ) and more recently for several alkyl and aryl amines ( I ) . Improvements in t h e direct determination and the development of an indirect determination are reported in this paper for a variety of symmetric secondary amines.
EXPERIMENTAL Apparatus. Spectrophotometric measurements were made using 1.00-cm matched cells on a Cary 14 spectrophotometer. A Perkin-Elmer 137 infrared spectrophotometer was used to assay the n-methylaniline. Atomic absorption measurements were made using a Model 1301 Beckman Atomic Absorption system with a DB-G spectrophotometer and recorder. A Model WL-22603A Westinghouse copper hollow cathode lamp was used for all measurements. Excess organic solvents were removed from the aqueous layer prior to AA analysis using a simple vacuum method. Reagents. Stock solutions of the secondary amines were prepared from freshly opened bottles of diethylamine (Fisher), dibutylamine (Kodak), morpholine (MCB), rz-butylaniline (Aldrich), and dibenzylamine (Kodak). The stock solutions are prepared by dissolving 1.00 mL of the secondary amine in 400.0 mL of saturated ether/water or 400 mL of (7:l) waterethanol
RESULTS AND DISCUSSION T h e visible absorption spectra of various concentrations of copper(I1) bis(dibuty1dithiocarbamate) in chloroform are shown in Figure 1. These spectra are typical for all t h e complexes reported. In all cases the copper:amine ratio was found t o be 1:2. Beer's law plots of all the solutions produced linear plots with zero intercepts. T h e concentration range studied, t h e molar absorbtivity at 434 nm, and the slope of the Beer's law plot are given in Table I for t h e four amines studied.
IPermanent address: Pall Corporation, 30 Sea Cliff Avenue, Glen
Cove, N.Y. 11542.
0003-2700/79/035 1-0319$01 0010
Amines
c
1979 American Chemical Society
320
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
I
Table 111. Summary of Results of t h e Indirect Determination of Secondary Amines no. of sam% absolute secondary amine ples ppm recovery SD die thy lamine" 6 2.46 97.6 0.08 dibutylamineb 7 6.14 94.7 i1.8 dibenzylamine" 6 3.70 95.5 t2.8 morpholine" 6 8.0 89.5 12.2 0.0 ---n-methylaniline" 6 --n-bu tylaniline" 6 _ _ . 0.0 ---a Visible spectrophotometry. Atomic absorption spectrometry.
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0.4 W
V
z
g0.3 Q
0
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$0.2
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0.0 400
,
450 500 WAVELENGTH, nm
Figure 1. Absorption spectra of Cu-bis(dibuty1dithiocarbamate). Concentrations of Cu(DBDTC), in CHCI,. (1) blank, (2) 3 00 ppm, (3) 6.1 p p m , (4) 9 . 2 ppm, (5) 12.3 p p m
T h e slope of the plot is given by the absorbance divided by the concentration in ppm. T h e precision of the direct determination was determined via replicate determinations on a known "unknown". The results of the precision study are summarized in Table 11. T h e detection limit is defined as the concentration which produces an absorbance equal to three times the standard deviation of the blank. The indirect method for analysis of the secondary amines is based on the determination of the copper(I1) ions remaining in the aqueous solution. T h e neocuproine spectrophotometric method (10) was used for all the amines except dibutylamine which was determined indirectly by atomic absorption spectrometry. The precision of the method was studied in two ways. First, multiple samples of "known unknowns" were analyzed and the percent recovery was determined. Second, the amines were analyzed by both the direct and indirect methods. The results of these studies are summarized in Table 111. The concentrations listed in Tables I1 and I11 are the concentrations expected in t h e extract, based on the known concentrations of the corresponding secondary amines. In all cases the slope of the plot of the amine concentration determined by the direct method vs. the concentration found by the indirect method was found to be equal to the previously determined percent recovery. Additionally the standard deviation of the slope was found to he nearly equal to the absolute standard deviation determined in the recovery experiments. The slopes and standard deviations were calculated using a standard linear curve fitting method (11). The calculated intercept in all cases was equal to or less than the probably error calculated for the indirect method. Using either method of analysis, 6 samples plus blank can be completed within 1h. The solutions of copper(I1) dithiocarbamates show no change in absorbance over a period of several days. Therefore the spectrophotometric procedure need not he performed immediately.
T h e method reported here offers several advantages over previously reported determinations ( I , 8) which result from changes in the solvent system. The basic changes result from the substitution of ammonia for pyridine as the required base and from t h e substitution of chloroform for the mixture of isopropanol and benzene as the extraction solvent. In addition to the replacement of toxic solvents, these changes result in a decrease in t h e color development time. Umbreit reports that color development required 20 min for N-methylaniline and 180 min for diethylamine (1). By replacing pyridine with ammonia, this was reduced t o less than 15 min for all the amines reported in this paper. However, it should be noted t h a t with this solvent system the N-alkyl anilines do not produce complexes which are extractable in chloroform. No absorbances were detected for Cu(I1) his(dithi0carbamate) complexes formed from N-methyl-, N-ethyl-, or N-butylaniline. This may result from the substitution of ammonia for pyridine (12, 13). A further advantage is derived from the development of the indirect determination. The indirect method can be used to either corroborate the results of the direct determination or as the primary method for samples which contain spectral interferences. This would include interferences caused by scattering and other overlapping absorbance bands.
ACKNOWLEDGMENT The authors acknowledge the late David F. Boltz for the initial suggestion of the project and for early direction of one of the authors (C.H.M.).
LITERATURE CITED (1) (2) (31 (4)
G. R. Umbreit, Anal. Chem., 33, 1572 (1961). L. Nebbia and F. Guerrieri, Chim. Ind. (Milan),35, 896 (1953). E. L. Stanlev. H. Baum. and J. L. Gove, Anal. Chem.. 23, 1779-82 (1951). J Weisburger and R Raineri, Tox/col Appl Pharmacol , 31, 369 (1975) (5) S Hypta, Nafta, 28, 3 1 (1972) (6) M Pailer. W J Hubsch, and H Kuhn, Fachhche Mnt Osterr Tabakregie. 7 109 (1967) (7) M.Paile;, J. Vollmen, C. Karmencic, and H. Kuhn. fachliche Min. Osterr. Tabakregie. 10, 165 (1970). (8) H. C. Dowden, Biochem. J . , 32, 455 (1938). (9) J. A. Bowman and J. B. Willis. Anal. Chem.. 39, 1210 (1967). (10) A . R. Gahler, Anal. Chem., 26, 577 (1954). (11) P. R. Bevington, "Data Reduction and Error Analysis for the Physical Sciences", McGraw Hill, New York, 1969, pp 92-118. (12) S. J. Snedker, J . SOC.Chem. Ind., 44, 74T (1925). (13) H. S. Fry and 6.S. Farguhar. Red. Trav. Chim. Pays-Bas, 57, 1223 ( 1930),
RECEIVED for review August 8, 1978. Accepted October 27, 1978.
