(3) Th. Schmidhofer, H. R. Egli, E. Hauser, and W. Kunzler. Alimenta. 12, 145 (1973). (4) M. J. Brennan, FoOdEng., 46, 102 (1974). (5) S. J. Toma and S. Nakai, J. FoodSci., 35, 507 (1971). (6) R. S. White, N. R. Ghandi, and G. H. Richardson, J. Dairy Sci., 56, 630 (1973). (Abstr). (7) G. E. Schman, M. A. Stanley, and D. Knudsen, Soil Sci. SOC. Amer. Proc., 37, 480 (1973). (8) D. G. Vakaleris and W. V. Price, J. Dairy Sci., 42, 264 (1959). (9) C. E. Childs and E. 6. Henner, Microchem. J., 15, 590 (1970). (10) R. Fiedler, G. Proksch, and A. Koepf, Anal. Chim. Acta, 63, 435 (1973). (11) W. T. Greenway, CerealChem, 49, 609 (1972). (12) Technicon Corporation, Tarrytown, N.Y., Industrial Method 31-69A (1969). (13) J. G. Brisson, "Determination of Total Nitrogen in Milk Using a Techni-
con Autoanalyzer System," presented at the Technicon Symposium, "Automation in Analytical Chemistry," Sept. 9. 1965. (14) W. M. Gantenbein, J. Ass. Offic. Anal. Chem., 56, 31 (1973). (15) S. C. Jacobs, J. Clin. Pathol., 21, 218(1968). (16) J. L. Cox and B. G. Harmon, Automat. Anal. Chem. Technicon Symp. 1, 149 (1967). (17)
Technicon Corporation, Tarrytown, N.Y., industrial Method 103-70A/ Preiiminary (1972).
Kramme, R. H. Griffen, C. G. Hartford, and J. A. Corrado, Anal. Chem., 45, 405 (1973). H. G. Lento and C. E. Daugherty, Food Prod. Develop., 5,86 (1971). Technicon Corporation, Tarrytown, N.Y., Industrial Method 146-71A/ D. G.
Preliminary (1971). (21) E. L. Quarne, W. A. Larson, and N. F. Olson, J. Dairy Sci., 51, 527 (1968). (22) R. Aschaffenburg and J. Drewry, Proc. X V lnt. Dairy Congr., 3, 1631 (1959). (23) W. Horwitz, Ed., "Official Methods of Analysis of the Association of Official Analytical Chemists." 1 Ith ed.. The Collegiate Press, Menasha. Wis., 1970, p 858. (24) I. W. Burr and L. A. Foster, "A Test for Equality of Variances." Purdue Univ. and Valparaiso Univ., Dept. of Statistics, Div. of Math. Sci., Mimeograph Series No. 282, April 1972.
RECEIVEDfor review July 24, 1974. Accepted November 1, 1974. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by the Cooperative State Research Service, U.S. Department of Agriculture.
Pyridine Catalyzed Reaction of Volatile N- Nitrosamines with Heptafluorobutyric Anhydride Terry A. Gough, Keith Sugden, and Kenneth S. Webb Laboratory of the Government Chemist, Cornwall House, Stamford Street, London SE 1 9N0, England
A procedure has recently been devised for the direct conversion of trace quantities of volatile N-nitrosamines to fluorinated anhydride derivatives via a pyridine catalyzed reaction with heptafluorobutyric anhydride (HFBA). The objective of the present study is to investigate the nature of these derivatives by use of combined gas-liquid chromatography and mass spectrometry. For this purpose, a series of dialkyl and heterocyclic nltrosamlnes were treated with HFBA In the manner previously described. The derivatives were formed in hexane solution, separated on a 2 % OV-1 gas chromatographlc column at 135 O C and detected by flame ionization. Kovats retention indices are presented. The gas chromatograph was linked to the mass spectrometer via a silicone membrane separator. Low resolution mass spectra were obtained for the derivatives, and parent and other characteristic ions were accurately mass-measured at resolution 18,000. Structures for the derivatives are postulated. The N-nitrosodimethylamine-HFBA reaction is shown to be unique.
The carcinogenic properties of many of the N - nitrosamines are well known, and sensitive methods for the detection of the steam volatile nitrosamines are available ( I ) . Most procedures rely on mass spectral confirmation after preliminary separation and detection by gas chromatography (2). A frequently adopted means to quantitatively determine any trace constituent is to prepare a derivative which can be separated from interferants by gas chromatography and detected by electron capture. This approach has been used indirectly for nitrosamine determination ( 3 ) . Heptafluorobutyric anydride (HFBA) is a well known acylating agent and has been employed to form derivatives with amines (4, 5 1, N - methylcarbamate insecticides (6) and various drugs found in biological samples ( 7 ) . A method (8) has been described whereby volatile N - nitrosamines are converted into electron capturing derivatives by a pyridine catalyzed reaction with HFBA.
The mechanism for the reaction of HFBA and pyridine with nitrosamines has not been established. There is little information in the literature regarding the nature of the reaction products, and only a N - nitrosodimethylamine derivative was studied by mass spectrometry (8). Furthermore the HFBA derivatives of other nitrosamines were identified by gas-liquid chromatography only, and the results were reported to be tentative. It is the aim of this work to investigate the nature of these other derivatives by combined gas chromatography and mass spectrometry (GC-MS) and to describe a second N - nitrosodimethylamine derivative. EXPERIMENTAL Apparatus. A Pye 104 chromatograph fitted with a flame ionization detector was used. The mass spectrometer was an AEI Model MS902 double focusing instrument fitted with peak matching facilities. The GC-MS interface was a silicone membrane separator (9) housed in the GC oven between the column exit and the detector. The separator was connected to the mass spectrometer via a heated 0.5-mm i.d. stainless steel line. The operating conditions are given in Table I. Reagents. N-Nitrosodimethyl-, diethyl-, dipropyl-, and dibutylamines and N-nitrosopiperidine (NDMA, NDEA, NDPA, NDBA, and NPIP) were purchased from Eastman Chemical Company; N-nitrosopyrrolidine (NPYR) from K and K Laboratories, Inc.; heptafluorobutyric anhydride (HFBA) from Pierce Chemical Company; pyridine (Spectrograde) and chloroform (Analytical Reagent grade) from Fisons, and hexane (Puriss) from Kochlight. Procedure. The 50 J/ml solutions of NDMA, NDEA, NDPA, NPIP, and NPYR in chloroform were prepared, and 60-pl aliquots of these solutions were treated with pyridine and HFBA. The procedure employed was identical to that in the literature (8). To minimize losses due to evaporation, hexane (200 ~ 1 was ) used as the final solvent instead of diethyl ether although it is recognized that the derivatives are more soluble in the latter. Five-pl portions of the hexane solutions were injected onto the chromatograph for analysis.
RESULTS AND DISCUSSION Preliminary gas chromatography demonstrated that the nitrosamine-HFBA derivatives are completely and irreversibly adsorbed by stainless steel. Quantitative passage
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Table I. Operating Conditions for the GC-MS Analysis of Nitrosamine-HFBA Derivatives Gas chromatograph Pye 104 Detector Flame ionization Helium Carrier gas Flow rates (ml min-') 11 Helium 35 Hydrogen Air 500 Temperatures, "C: Injection port 135 Column 135 Detector 2 50 135 Membrane separator 170 Transfer line 2.7 m X 4 mm i.d. glass, Column 2% OV-1 on 100-120 BS mesh AW-DMCS chromosorb G Sample size 5 lil MS902 Mass spectrometer Accelerating voltaqe 8 kV Trap current 100 pA Electron beam voltaqe 70 eV Multiplier voltage 2.5 kV 5 X Torr Ion source pressure 100 "C Ion source temperature 18,000 (for accurate mass Resolution (10% valley) measurement)
100 7
I
c
40-
*
20-
1
Figure 2a.
Mass spectrum of NDMA-HFBA derivative
% a.
ii
20'
m
m L
Table 11. Retention D a t a for Nitrosamine-HFBA Derivatives HFBA derivative
Retention Time, min
Retention Index
NDMA
2.5 1 8
NDEA NDPA NDBA NPIP NPYR
7
790 1335 1100 1190 1290 1410 1100
9 14 25
7
loo
,
1
1
1
.
.
1
t.
1
250
Figure 2b.
300
350
am
4m
mia
Mass spectrum of NDEA-HFBA derivative
1.31 1.2 "HDM
100
;
BO
qr r n 3 4 c
*
20 27 I.
0
0.5
I
I
11 12 N U M B E R O F C A R B O N ATOMS PER MOLECULEOF H F B A O E R I V A T I V E
Figure 1.
18
m
1-
1
Retention data for nitrosamine-HFBA derivatives
of the derivatives was achieved after treatment of the metal parts of the system with bis(trimethy1 silyl) acetamide. Kovats retention indices were determined for eac'h of the derivatives, and are listed in Table 11. I t will be noted that two retention times are quoted for the nitrosodimethylamine derivative, one corresponding to the compound origi510
'm
I .
,
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
Figure 2c. Mass spectrum
of NPIP-HFBA derivative
L " 2m
I
,
m
Table 111. Molecular Weights of Nitrosamines and Their HFBA Derivatives Nitrosamine
Molecular weight of nitrosamine
NDMA NDEA NDPA NDBA NPIP NPYR
74 102 130 158
114 100
Table V. Proposed Structures of Nitrosamine-HFBA Derivatives
'Molecular weight of derivative
STRUCTURE
522 476 504 532 488 474
Table IV. Mass Spectral Fragmentation D a t a of the NDEA-HFBA Derivative Mass of fragment, measured
476.0195 439.0119 429.0263 428.9919 289.0221 278.9995 239.0224 211.0100
Assigned formula
Fxmr of mass measurement
C12H6N202F,, C12H4N20Fi3 CiiH,N20F,, CioH2N202F1, CSH4N2OF: C7H2N202F7 C,H,N,OF, CgH,N202F,
0.0002 0.0002 0.0002 0.0003 0.0004
0.0003 0.0008 0.0014
A N-N(CCC~.j-
NDMA
*N-K
NDEA
(cH,=cH)~N-N(coc,F,)~
NDPA
(c,H, ),N-N
NDBA
(CIH,)~N-N(COC,F,)~
NPIP
G - S i
u
(COC, F , ) ~
COC F 1
NPYR
Source of fragment
Molecular ion (M) M-F-HZO M-F-CO M-F-CzH, M-C,FT-H20 M-C,FT-C2Hd M-C,F,-H,O-CF, M-C,F,-CF,-C2H3
nally reported by Brooks ( B ) , and another of much longer retention time, the subject of the present study. A graph of log retention time us. carbon number (Figure 1)indicates that all the dialkyl nitrosamines except NDMA form a homologous series. Mass spectra were obtained by scanning a t 8 seconds per decade mass, in the region of the GC peak maxima. As examples, spectra of the derivatives of NDMA, NDEA, and NPIP are shown in Figures 2a, b, and c, respectively. All the derivatives in this study give molecular ions. To confirm that the derivatives did not undergo decomposition at the GC-MS interface, mass spectra were also obtained directly with a heated direct insertion glass probe. The molecular weights of the derivatives and the corresponding nitrosamines are listed in Table I11 from which it can be seen that with the exception of NDMA, the difference in molecular weight of any two of the derivatives is the same as that of the corresponding nitrosamines. The above observations indicate that all the nitrosamines studied except NDMA, react with HFBA in a similar mode. T o aid structural elucidation, the NDEA derivative was studied by high resolution mass spectrometry and the masses of the major fragments were measured by reference to the appropriate fragment ion of perfluoro-tri-n- butylamine. The data are listed in Table IV, and Table V gives the proposed structure of the derivative. The structures of the higher dialkyl derivatives and the heterocyclic nitrosamines were subsequently deduced from accurate mass measurements of the parent ions and low resolution spectra. The NDMA derivative has been shown to be atypical by gas chromatographic and low resolution mass spectral data presented above. It is not possible for NDMA to form a stable adduct with HFBA in the same manner as the other dialkyl nitrosamines. High resolution mass measurements of the characteristic fragments are listed in Table VI, and the proposed structure arising from interaction of two molecules of NDMA and HFBA is given in Table V. Determination of the position of the C-C double bonds in the dipropyl, dibutyl, and piperidyl derivatives was attempted using nuclear magnetic resonance spectrometry after trapping of the GC effluent, but was inconclusive.
Table VI. Mass Spectral Fragmentation D a t a of the NDMA-HFBA Derivative Mass of fragment measured
Assigned formula
Fmor of mass measurement
522.0373 448.9979 403.0458 353.0489 311.0501 283.0317
Ci2H8N40,F,, 0.0004 0.0012 C,,H,N202Fi4 C,,H8N403F, 0.0007 CgHEN,03F: 0.0006 0.0002 CEH,N,02F, CTHGN202F7 0.0002
281.0514
CEHEN20FT
Source of fragment
Molecular ion (M)
M-CZH,-N,O M-CzF, M-C,F, M-C,F,-CNO M-C,F:-C*H,-N20 or M-C,F,-C&CH2-N2 0.0001 M-C,F,-C@N20
The reaction of NDPA and HFBA in the presence of pyridine-db was followed in an NMR tube. There was no rapid change in the spectrum, although after several hours the presence of olefinic groups was observed. Gross interference throughout the reaction made further interpretation difficult. It was deemed undesirable to carry out any larger scale preparations and purifications in view of the carcinogenic nature of the nitrosamines. Bromination of the dialkyl-HFBA adducts gave yellow precipitates except in the case of NDMA derivative. Chromatography of the reaction mixture showed an absence of the nitrosamineHFBA derivatives, which were present prior to bromination. The precipitate from the diethyl derivative was subjected to mass spectral analysis via the direct insertion probe, and was found to contain four bromine atoms. The product was, however, thermally unstable and pyrolysis occurred prior to electron bombardment. In contrast, the NDMA-HFBA derivative did not react with bromine, and comparison of the chromatograms before and after the addition of bromine showed no change. Various experiments in which the derivatives were subjected to hydrogenation were undertaken. A glass hydrogenation chamber consisting of activated neutral palladium on glass beads was inserted between the column exit and the membrane separator. Details of the preparation of the catalyst have been published previously (10). Hydrogen was used as carrier gas. Provision was made to heat the hydrogenation chamber independently of the column. Low resolution spectra were run under the same operating conditions as those listed in Table I where appropriate, and although the majority of each derivative passed through the chamber unchanged a t temperatures up to 180 "C, an increasing proportion of material did undergo reaction as the temperature was raised. The study was repeated under high resolution conditions and the significant fragments arising from the hydrogenation of the NDEA derivative are given in Table VII. No fragments were observed correA N A L Y T I C A L C H E M I S T R Y , VOL. 47, NO. 3, M A R C H 1975
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~
Table VII. Mass Spectral Fragmentation D a t a of the Hydrogenation Product of NDEA-HFBA Derivative (M) Mass of fragment, measured
Assigned formula
C12H,N,0,Fl, Cj?H,N,OF,, C,H,N?OF,
478.0358 441.0240 291.0377
Error of mass measurement
0.0012 0.0029 0.0009
Table VIII. Hydrogenated Products of Nitrosamine-HFBA Derivatives NDEA and NPYR
E-\~L.(
NPIP
M + 2H M-F-0 M-CsF7-0
sponding to the addition of more than two hydrogen atoms, indicating that the divinyl moiety did not give rise to a diethyl product. On this evidence and by comparison with the data presented in Table IV, it is concluded that a saturated cyclic structure had been formed (see Table VIII). Mass measurements on the hydrogenation products of the MDPA and NDBA derivatives gave similar results. The possibility of ion molecule reactions in the mass spectrometer was ruled out, since the proportion of M 2H produced from any of the above derivatives could be varied merely by changing the temperature of the hydrogenation chamber. At temperatures below about 150 “C, no hydrogenated material was detected. The temperature a t which the maximum proportion of hydrogenation occurred, increased as the series was ascended, but over 230 O C , pyrolysis occurred. Hydrogenation products of the heterocyclic nitrosamine derivatives were also studied under high resolution and both the NPIP and NPYR derivatives gave rise to some M 2H and M 4H, the structures being given in Table VIII. The result of saturating the ring in the case of the NPYR derivative is to produce a compound having the same structure as the hydrogenated NDEA derivative. The effect of using hydrogen as carrier gas was particularly interesting in the case of the dimethyl derivative, since one product was eluted a t the retention time of the NDEA de-
+
+
F
Source of fragment c-\(C‘OC
F
I
rivative and behaved in the same manner in the hydrogenation chamber. Somc unchanged NDMA derivative, together with a mixture of pyrolysis products, was eluted at the expected retention time. The exceptional behavior of the reaction between nitrosodimethylamine and HFBA is worthy of further study. The hydrogenation of the long retention dimethyl, and the diethyl and pyrrolidyl nitrosamine derivatives to give a common product is of potential analytical value. LITERATURE CITED (1) A. E. Wasserrnan, “N-Nitroso Compounds, Analysis and Formation,” International Agency for Research on Cancer, Publication No. 3, Lyon, 1972, p 10. (2) T. A. Gough and K. S. Webb, J. Chromatogr., 79, 57, (1973). (3) T. G. Alliston, G. B. Cox, and R. S. Kirk, Analyst (London). 97, 915 (1972). (4) D. D. Clarke, S. Wilk, and E. S. Gitiow, J. Gas Chromatogr., 4, 310 (1966). (5)J. B. Brooks, W. B. Cherry, L. Thacker, and C. C. Alley, J. hfec. Dis., 126, 143 1972). (6) J. N. Seiber, J. Agr. FoodChem., 20, 443 (1972). (7) J. July/August W. Blake, 1973. R . Huffman, J. Noonan. and R. Ray, Int. Lab., pp 57-61,
+
(8) J. B. Brooks, C. C. Alley, and R. Jones, Anal. Chem., 44, 1881 (1972) (9) T. A. Gough and K. S.Webb, J. Chromatogr., 64, 201 (1972). (10) M. Beroza and R. Sarmiento, Anal. Chem., 35, 1353 (1963).
RECEIVEDfor review May 20, 1974. Accepted November 1, 1974. Published by permission of The Government Chemist.
Selective and Sensitive Complexometric Determination of Calcium and Magnesium in Dolomites Using “Palladiazo” as a Metallochromic and an Adsorption Indicator Simultaneously M. D. Alvarez Jimenez, J. A.
Perez-Bustamante, and F. Burriel Marti
Departamento de Quhica Analhica, Faculad de Ciencias y C.S.I.C., Universidad Complufense, Ciudad Universitaria, Madrid-3, Spain
The suitability of the palladiazo reagent for the complexometric (EDTA) titration of Ca( II) and Mg(ll) in dolomite has been demonstrated on the basis of the statistical evaluation of the experimental results obtained for the analysis of a certified standard dolomite sample. A curious and original feature of the method derives from the fact that the palladiazo reagent can act both as a metallochromic reagent (titration of Ca(ll) alone) or as an adsorption indicator (Ca Mg sum), thereby bullding a Ca-Mg-palladiazo blue ternary lake on the MZI(OH)~precipitate which turns purple upon the equivalent titration of Ca(ll) which is liberated from the lake while a binary purple Mg(OH)2-palladiazo lake remains undissolved. One to ten mg Ca(ll) can be determined directly in the presence of up to 10 mg Mg(ll) in 0.1 M NaOH
+
512
+
medium while the Ca( II) Mg( II) sum can be determined in a separate aliquot in a NH3-NH4+ medium (pH 10 f 1). The method shows an average standard deviation of f0.4% for Ca(ll) while for the indirect subtractive Mg( II) determinatlon, a maximum f0.6% value has been established.
1,8-Dihydroxy-3,6-disulfonic-2,7-bis(azophenyl-p -arsonic)acid, trivially known as “palladiazo” ( I ) , has proved to be a useful reagent for the spectrophotometric determination of Pd(I1) (2, 3 ) , although no suitable application has been shown so far in connection with the complexometric determination of metal cations. Its structural isomer arsenazo I11 is far more useful in this respect, acting as a valuable metallochromic indicator in the complexometric de-
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