Anal. Chem. 1986, 58,3184-3188
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Registry No. NH,Cl, 12125-02-9;HzO, 7732-18-5;hydroxylammonium chloride, 5470-11-1;guanidinium chloride, 50-01-1; maleic acid, 110-16-7;fumaric acid, 110-17-8;imidazole,288-32-4. LITERATURE CITED (1) Jesson, J. P.; Meakin. P.; Kniessel, G. J. Am. Chem. SOC.1973, 9 5 , 618-620. (2) Campbell, I.D.; Dobson, C. M.; Jeminet, G.; Williams, R. J. P. FEBS Left. 1974, 4 9 , 115-119. (3) Pan, S L.; Sykes, B. D. J. Chem. Phys. 1972, 5 6 , 3182-3184. (4) Benz. F. W.; Fenney, J.; Roberts, G. C. K. J. Magn. Reson. 1972, 8, 114- 12 1, (5) Redfield. A. G., Kunz, S. D.; Ralph, E. K. J. Magn. Reson. 1975, 79 114-117. (6) Rabenstein. D. L.; Isab, A. A. J. Magn. Reson. 1979, 3 6 , 281-286. (7) Plateau, P.; Gireron, M. J. Am. Chem. SOC. 1982, 104, 7310-7311. (8) Hore, P. J. J. Magn. Reson. 1983, 54, 539-542. (9) Clore. G. M.; Kimber, 6.J.; Gronenborn, A. M. J. Magn. Reson. 1983, 54, 170-173. (IO) Hore, P. J. J. Magn. Reson. 1983 5 5 , 283-300. (11) Bryant, R.; Eads, T. M. J. Magn. Reson. 1985, 6 4 , 312-315. (12) Rabenstein, D.L.; Fan, S.;Nakashima, T. T. J. Magn. Reson. 1985, 64, 541-546. (13) Eads, T. M.; Kennedy, S. D.;Bryant, R. G. Anal. Chem. 1986, 58, 1752-1756.
(14) Freeman, R.; Hill, H. D. W. I n Dynamic Nuclear Magnetic Resonance Spectroscopy; Jackman, L. M., Conon, F. A., Eds.; Academic Press: New York, 1975: pp 131-162. (15) Carr. H. Y.; Purcell, E. M. Phys. Rev. 1954, 9 4 , 630-638. (16) Meiboom, S.;Gill, D.Rev. Sci. Instrum. 1958, 2 9 , 688-691. (17) Rabenstein, D.L. J. Biochem. Biophys. Methods 1984, 9 , 277-306. (18) Eigen, M.; DeMaeyer. L. I n Techniques of Organic Chemistry; Friess, S. L., Lewis, E. S.,Weissberger, A., Eds.; Interscience: New York, 1963; pp 1031-1050. (19) Cramer, J. A.; Prestegard, J. H. Biochem. Biophys. Res. Commun. 1977, 75, 295-301. (20) Rabenstein, D.L.; Isab, A. A. Anal. Biochem. 1982, 121, 423-432. (21) Nicholson, J. K.; O'Flynn, M. P.; Sadler, P. J.; Macleod, A. F.; Juui, S. M.; Soinksen, P. H. Biochem. J. 1984, 217, 365-375. (22) Brown, F. F.; Campbell, I.D.; Kuchel, P. W.; Rabenstein, D. L. FEBS Lett. 1977, 8 2 , 12-16.
RECEIVED for review April 14,1986. Accepted August 18,1986. This work was supported in part by the University of California, Riverside. The NMR instrumentation was supported in part by BRSG 2 SO7 RR07010-20 awarded by the Biomedical Research Support Grant Program, Division of Fksearch Resources, National Institutes of Health, and by Standard Oil Co. (OH).
Identification and Colorimetric Determination of Organic Cyanates in Nanomolar Quantities Joachim Kohn,'s2 Elizabeth C. Albert,' Meir Wilchek,' and Robert Langer*' Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Biophysics, Weizmann Institute of Science, Rehouot, 76100, Israel
A color reaction for the quaHtatlve and quantlatlve determlnation of organic cyanates (R-O-C=N) was developed. Upon treatment of an organic cyanate wlth color reagent (0.75 g of 1,3-dlmethylbarblurlc acid In 50 mL of pyrldlne/ water (455)) a purple dye formed. The mechanism of thls reaction was Investigated, and the purple dye was Identifled as a pentamethlne oxonol, 5-[5-(hexahydro-1,3-dlmethyl-
reactive enough to be valuable intermediates in organic synthesis (6). Cyanates were used as activating agents for polysaccharide resins for the immobilization of biologically active ethyl ester molecules (7,8);N-acetyl-p-cyanatophenylalanine was proposed as a selective protein-modifying agent (9), and cyanylated steroids were explored as contraceptives in women (IO). Aromatic cyanates are also frequently used in polymer chemistry where they serve as chain extenders, cross-linkers, 2,4,6-trloxo-5-pyrlmidlnyl)-2,4-pentadienylldene]-l,3-dlor monomers for the synthesis of several classes of polymers, methyl-2,4,6( lH,3H,5H)-pyrtmldInetrione. The spot test such as poly(triazines) (II), poly(iminocarbonates) (12,13), procedure had an ldentlflcation limit of 60 ng and a dllution or poly(isoureas) (14). Although organic cyanates can be limit of 1:833000 for phenyl cyanate. On TLC plates as little detected by various physical methods (NMR, IR, UV) (15), as 20 pmol of organic cyanate could be detected. The color for many of the above applications the availability of a sereaction was hlghly selectlve; isocyanates (R-N=C=O) lective and sensitive chemical method for the detection and/or and other CN-containing functional groups were unreactive. determination of organic cyanates would be desirable. The colorimetric procedure for the quantltatlve determination The formation of colored products from the reaction of of organic cyanates was used In the range of 0.04-200 hmol cyanogen bromide with pyridine was first observed by Konig of organic cyanate. The standard deviation for repetitive (16). This reaction was then investigated in detail by determinations ( n = 8 ) was 2%. Schwarzenbach and Weber (17). With barbituric acid or its derivatives as color-forming condensing agents, variations of Konig's reaction were later developed for the detection of nicotinic acid ( I @ , cyanide ion (19),carbodiimides (20,211, The publication of generally applicable procedures (1-3) and aliphatic, polysaccharide-bound cyanates (22). However, for the synthesis of organic cyanates (R-O-C=N) resulted no evidence has so far been presented in support of the rein an intense investigation of these compounds ( 4 ) . As a action mechanism and the suggested structure (19,22) of the general rule, cyanates are less reactive than the corresponding colored product. Likewise, it has not been examined whether isocyanates (R-N=C=O) ( 5 ) . In particular, the aromatic this reaction would be generally applicable to organic cyanates. cyanates (e.g., phenyl cyanate) are rather stable compounds Consequently, we report here on our work, aimed at the that are convenient to work with, while at the same time being elucidation of the mechanism of this reaction and the development of analytical procedures for the qualitative deMassachusetts Institute of Technology. * Weizmann Institute of Science. tection (spot test and TLC spray) and the quantitative de0003-2700/86/0358-3184$01.50/0 LC1986 American Chemical Society
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Scheme I
r N
L CSNJ
L
1-
J CH-CH=
CH-CH=CH
J
( I)
(2)
(3)
organic cyanate
glutoconate ion
bis(l,3-dimethyl barbituric a c i d ) pentamethine oxonol
termination of organic cyanates in nanomolar quantities.
EXPERIMENTAL SECTION Deionized water was used. All solvents were analytical grade. 1,3-Dimethylbarbituricacid (DBA) was from Chemical Dynamics Corp. (South Plainfield, NJ); 4-nitrophenyl cyanate was from Sigma (St. Louis, MO); potassium glutaconate was prepared according to Becher (23);phenyl cyanate was prepared by the cyanogen bromide technique (3);2,2-bis(4'-cyanatophenyl)propane (6) (I3),and compounds 7 and 8 (24)were prepared as described previously. HPLC was performed on a Perkin-Elmer Series 10 liquid chromatograph, equipped with a Vydac C18silica column, using for elution acetonitrile/water (82) at a flow rate of 1 mL/min. Field desorption mass spectrometry was performed by using benzonitrile-activatedcarbon emitters in a MAT 731 instrument, equipped with a combined EI/FI/FD ion source. Preparation of Compound 3. Method I . DBA (4 g, 0.022 mol) was dissolved in 20 mL of dimethylformamide (DMF), followed by the addition of water (2 mL). This solution was added all at once to a cooled (4 "C), stirred suspension of potassium glutaconate (1.36 g, 0.01 mol) in 50 mL of DMF. The mixture was kept with stirring at 4 "C overnight, during which time an intense purple color developed. Then 1L of anhydrous ether was added to the reaction mixture; a black powder precipitated. This precipitate was collected, washed 3 times with ether, and dried over P205in vacuo: yield, 4 g of compound 3 (80%). The crude product was dissolved in water (25 g/L) and reprecipitated by the addition of an equal volume of a saturated, aqueous solution of ammonium acetate. The precipitate was collected and dried at 0.01 mmHg for 72 h, and a small amount used for IR, NMR, and MS was further purified by reverse-phase HPLC: IR (KBr pellet, cm-') 3430 (br, w), 2955 (br, w), 1690 (sharp, m), 1650, 1630 (br, s, doublet), 1480,1469, 1451 (br, m, triplet), 1405 (sharp, m), 1335 (br, s), 1305 (sharp, s), 1230 (sharp, w), 1280-960 (unresolved broad absorptions, s), 785,776 (sharp, m, doublet), 650 (sharp, m); 'H NMR (300 MHz, dimethyl-d6sulfoxide, 6) 2.71 (12 H, s, 4CH3),6.42 (1H, s, broad, OH), 6.87 (3 H, m, 3CH), 7.09 (2 H, m, 2CH); field desorption MS. Found: M" m / z 374 at 26 mA emitter current. Calcd for C,,H,,N,06: M , = 374. mp: dec without melting. Method 11. DBA (4 g, 0.022 mol) was dissolved in 50 mL of pyridine/H20 (455). After cooling to 4 "C, phenyl cyanate (1.19 g, 0.01 mol) was added dropwise with stirring. The mixture was kept at 25 "C for 20 min. Then the purple dye was precipitated by the addition of ethyl acetate, followed by extensive washing (3 X 500 mL) with ethyl acetate: IR and 'H NMR as described for method I. Elemental Anal. Found C, 56.95%; H, 5.06%; N, 15.77%. Calcd. for C22H23N506 (salt with pyridine): C, 58.27%; H, 5.11%; N, 15.44%. mp: dec without melting. Color Reagent for Spot Tests and Quantitative Determinations. DBA (0.75 g) was dissolved in 45 mL of pyridine, followed by addition of 5 mL of water. A slightly yellow, clear solution was obtained. Spray Reagent for TLC Plates. DBA (0.5 g) was dissolved in 9 mL of pyridine, followed by addition of 1 mL of water. A yellow solution was obtained. Spot Test for Organic Cyanates. Color reagent (150 pL) was placed into a micro test tube, followed by 50 pL (1drop) of test solution. The presence of cyanates was detected by the formation of a purple color within 5 min.
Detection of Organic Cyanates on TLC Plates. The developed, dried plate was sprayed until saturation with freshly prepared spray reagent. Alternatively, the plate was quickly dipped into spray reagent. Immediate formation of blue spots on a white background indicated the presence of organic cyanates. Quantitative Determination of Organic Cyanates. The test sample (1mL) containing 0.04-200 pmol of cyanate in any inert organic solvent was placed into a 15-mL test tube. Then the freshly prepared color reagent (6 mL) was added rapidly. The mixture was immediately gently shaken and stoppered. After standing at 25 "C for exactly 10 min, the purple solution was transferred into a volumetric flask (volume, 100 mL for samples containing 0.04-1 pmol of cyanate or 1 L for samples containing 1-200 pmol of cyanate). Acetone was used liberally to rinse the test tube. After thorough mixing, the flask was filled to the mark with water. Depending on the amount of cyanates present in the sample, the purple solution was further diluted with water (up to 40-fold for samples containing 200 pmol of cyanate) in order to decrease the absorbance below 1. Absorbance was measured at 588 nm. The amount of cyanates in the sample was determined from a calibration curve or calculated from Beer's law, using the experimentally determined average molar absorptivity of 157000 L mol-' cm-'. Recrystsllization of DBA. Crude DBA (10 g) was dissolved in 100 mL of boiling carbon tetrachloride/chloroform (8:2). This solution was treated with 1 g of activated charcoal, filtered, and cooled to 25 "C. DBA precipitated as fine, colorless needles, which were collected and dried in vacuo to constant weight. RESULTS AND DISCUSSION The proposed mechanism (19, 22) of the color reaction (Scheme I) stipulates the glutaconate ion (compound 2) as an intermediate. Consequently, it should be possible to obtain compound 3 directly from glutaconate ion. In order to test this hypothesis, we prepared potassium glutaconate by an independent method (23)and reacted it with 2 mol of DBA. An intensely colored dye (method I, see Experimental Section) was obtained, which was compared to the dye obtained from the reaction of phenyl cyanate with the pyridine-DBA reagent, using the conditions employed for the determination of organic cyanates (method 11). When dissolved in water, the dyes obtained from both methods had identical absorption spectra in the range from 230 to 650 nm with peaks a t 361 and 588 nm (Figure 1). Both dyes had identical IR (KBr pellet) and 'H NMR spectra, and both materials charred without melting above 250 "C. We therefore concluded that by both reaction pathways (methods I and 11), the same compound was obtained, which was identified as a pentamethine oxonol by 'H NMR, elemental analysis, and field desorption mass spectroscopy. These findings support the reaction mechanism as outlined in Scheme I. Oxonol dyes of this type have previously been suggested for dye laser applications (25),in photographic processes (26),and as indicators for membrane potentials (27). Next we investigated the selectivity of the color reaction, using the spot test procedure. A variety of compounds were tested (Table I), and with the known exception of the carbodiimides (20),organic cyanates were found to be the only compounds able to efficiently yield the formation of the ob-
3186
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
r
O
"t c=o I
0 1
(71 R z e t h y l (81 R = h e a y l
Wavelength ( n m ) Figure 1. Visible spectrum from 230 to 650 nm of compound 3 prepared by method I (-) and by method I I (- - -).
Flgure 2. Structures of organlc cyanates employed as model compounds: phenyl cyanate (4), 4-nltrophenyl cyanate (J), 2,2-bis(4'cyanatopheny1)propane (e), N-[N-benzyloxycarbonyC3-@-cyanatophenyl)-~-alanyl]-3-@ -cyanatophenyl)-L-alanine ethyl ester (T), N - [ N -
Table I. Selectivity of the Pyridine-DBA Reaction
benzyloxycarbonyl-3-@-cyanatophenyl)- alanyl] -3-(p -cyanatephenyl)-L-alaninehexyl ester (8).
compd tested phenyl cyanate chlorosulfonyl isocyanate phenyl isocyanate cyanogen bromide cyanamide potassium cyanate potassium cyanide potassium thiocyanate acetonitrile dihexylcarbodiimide thionyl chloride diphenyl chlorophosphate benzoyl chloride benzyl chloroformate benzenesulfonyl chloride
functional group present -O-C=N -S(=O)2-Cl -N=C=O Br-C=N NH,-CsN OCNCNSCN-CN -N=C=NC1-S(=O)-C1 -P(=O)-CI -C(=O)-Cl
-o-c(=o)-c1
result"
+ and -N=C=O
Ob Ob
Table 11. Slope of Calibration Curves Obtained from Structurally Different Organic Cyanates compd
name
slope: L mol-' cm-'
-
-
4
-
6 7
-
-
+
-
8
phenyl cyanate 2,2-bis(4'-cyanatophenyl)propane N-[N-benzyloxycarbonyl-3-(p-cyanatophenyl)-~-alanyl]-3-(p-cyanatophenyl)L-alanine ethyl ester N-[N-benzyloxycarbonyl-3-(-p-cyanatophenyl)-~-aIanyl]-3-(p-~yanatophenyl)L-alanine hexyl ester
155 000 156OOOb 158OOOb 160 OOOb
turning purple only after several minutes.
The slope was obtained by linear regression from curves based on five data points. Each listed value represents the average slope of four repetitive curves. The observed range was +5000 L mol-' cm-' (n = 4). bFor compounds containing more than one OCN mouD Der molecule, calculations are based on "moles of OCN".
served purple color. Noteworthy is the fact that isocyanates, inorganic cyanates, and thiocyanates were unreactive. An apparent exception to this rule is the slight reactivity of chlorosulfonyl isocyanate. However, since benzenesulfonyl chloride was also slightly reactive, we concluded that the observed reactivity of chlorosulfonyl isocyanate was due to the presence of the chlorosulfonyl function rather than the isocyanate function. The spot test procedure was also used to investigate the sensitivity of the color reaction, employing increasingly diluted solutions of phenyl cyanate in ethyl acetate. The identification limit for phenyl cyanate was 0.06 pg (0.5 nmol) corresponding to a dilution limit of 1:833000 (28). Identical values were obtained in the presence of other commonly used solvents (ethanol, DMF, acetone, chloroform, toluene). In molar terms, structurally different cyanates (Figure 2, compounds 4-8) had identical identification limits (0.5 nmol). Hence, the color reaction seems to be generally applicable to the detection of compounds containing the R-OCN moiety. Next we investigated the applicability of the pyridine-DBA color reaction for the detection of organic cyanates on TLC plates by spotting small quantities of compound 6 (Figure 2) onto TLC plates, developing the plates, and testing for the presence of organic cyanates by spraying the plates with various mixtures of DBA in pyridine/water. With increasing concentration of DBA, the reagent became more sensitive
(data not shown). Maximum sensitivity was obtained when the reagent mixture contained 0.5 g of DBA in 10 mL of pyridine/water (9:l). Under these conditions, as little as 2.8 ng of compound 6 (20 pmol of R-OCN) gave a clearly visible, purple spot on a white background. Depending on the amount of cyanate present in the spot, the purple color faded within 5 min to 3 days. Only carbodiimides interfered. The quantitative determination of organic cyanates was investigated by establishing calibration curves for a variety of structurally different cyanates (Figure 2). In molar terms, all cyanates tested had nearly identical calibration curves, with an average slope of 157 000 f 5000 L mol-' cm-' (Table 11). The precision and accuracy of the color reaction was investigated using compound 6. Samples ranging from 5 to 25 mg were repeatedly analyzed (large samples were used to maximize weighing accuracy). The results of these experiments (Table 111) confirmed the linearity of the calibration curve. Repeated analyses (n = 8) had a relative standard deviation of 2 70,and the experimental values were accurate to within 1% (percent accuracy of the mean). The procedure for the quantitative determination of organic cyanates was employed for monitoring the polymerization reaction of compound 8 (Scheme 11) (24). During this reaction the cyanate end groups were converted to iminocarbonate bonds. Determination of the amount of residual cyanate end groups allows the calculation of the degree of polymerization
-S(=O),-Cl
0
(-) No reaction, (0) detection limit > 1 @mol(slightly reactive), (+) detection limit < 1 pmol. bColor changes to orange first,
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Table 111. Precision and Accuracy of the Quantitative Determination of 2,2-Bis(I'-cyanatophenyl)propane(CPP) by the Pyridine-DBA Color Reaction
amt of CPP added, mg
[OCNI present, pmol
mean net absorbance (n = 8) in 20 L
re1 std dev (n = 8), %
calcd amt of [OCN]," pmol
re1 accuracy of the mean, %
5.14 8.73 9.90 14.23 19.68 25.17
36.94 62.74 71.14 102.26 141.42 180.88
0.289 0.489 0.560 0.798 1.104 1.406
1.9 1.7 1.6 1.8 1.5 1.6
36.81 62.29 71.34 101.66 140.64 179.12
-0.35 -0.12 +0.28 -0.59 -0.55 -0.97
'Based on an average molar absorptivity of 157000 L mol-' cm-' for the purple dye. Scheme I1 OH
0-CEN
0-CiN
OH
CH2 0 I I1 I 1 I --C 2 - N H - C H - C - N H - C H - C - 0 - Y CH2 0
CH2 0 CH2 0 I II I II Z-NH-CH-C-NH-CH-C-0-Y
Z- Tyr-Tyr-Y-dicyanate
2 - Tyr - T y r -Y
(8)
t 0
II CH2 - CH - C- NH - CH - CHz-@-
I
I
NH
c=o I Y
I Z
poly (Z-Tyr-Tyr
NH I1
0- C- 0
-Y-iminocarbonate)
Table IV. Polymer End-Group Analysis during the Polymerization of Compound 8' by the Pyridine-DBA Reaction conversion of cyanates,
reaction time, min 0 2 5 20 40 60 100
0.850 0.814 0.697 0.392 0.231 0.160 0.054
%
calcd DP'
found DPd
0 4 18 54 73 81 94
1 1.04 1.22 2.11 3.70 5.26 16.67
1
2.3 5.6 15.4
'Compound 8 was polymerized according to Scheme I1 (ref 24). At the indicated intervals a 10-pL aliquot was removed from the reaction and treated with 5 mL of pyridine-DBA reagent in the usual way. Final volume after dilution with water: 1 L. Degree of polymerization (DP)= 1/(1- conversion) (ref 29). dDetermined by size exclusion chromatography relative to polystyrene standards (ref 24).
Z = benzyloxycorbonyl Y = hexyl
(DP) (29). The calculated values were in agreement with molecular weight determinations based on size exclusion chromatography (24)relative to polystyrene standards (Table
IV).
The reported use of oxonol dyes in photography (25) suggested the possibility of compound 3 being light sensitive. Therefore, we investigated the stability of compound 3 in diluted, aqueous solution (pH 7, 25 "C)under various conditions of exposure to white light (Figure 3). No attempt was made to quantitatively correlate dye degradation to the intensity or wavelength of the light. Our data indicate that the purple dye is photosensitive, and in the dark, dye solutions could be stored for several days without noticeable degradation. Hence, after completion of the quantitative test procedure, there was no need to accurately time the reading of the absorbance, as long as the solutions were protected from exposure to light. Finally, we investigated the stability of the pyridine-DBA reagent. As supplied, DBA is contaminated by a yellow impurity. Reagent prepared from such impure DBA initially appeared slightly yellow, but turned dark yellow to brown within a few hours a t room temperature. Even when stored at -15 "C under nitrogen, the reagent turned deeply yellow within 1 day. This coloration did not interfere with the formation of purple dye, but reduced the sensitivity of the test due to the masking effect of the yellow color. Reagent prepared from recrystallized DBA was initially colorless but turned yellow after a few days, even when stored at -15 OC. We therefore found it more convenient to prepare fresh aliquots of the color reagent prior to we, employing DBA without
.65
\
\
0
40
\
I20 T i m e (h)
80
I60
200
Figure 3. Stability of bis(l,3dimethylbarbituric acid) pentamethine oxonoi (compound 3) in diluted, aqueous solution (pH 7, 25 O C ) at various levels of illumination by white light: (0)stored in the dark; (A) stored at ambient, laboratory lighting conditions; (U) stored at ambient, laboratory lighting conditions with additional illumination from a 75-W light bulb. recrystallization.
CONCLUSIONS Due to the high molar absorptivity of the purple pentamethine oxonol dye, the color reaction is exceptionally sensitive and represents one of the few known colorimetric reactions capable of detecting less than 1 nmol of sample. We found the color reaction to be remarkably free of interferences, and, considering its high accuracy and precision, the pyridine-DBA reaction could be of value in a variety of applications requiring the qualitative detection or quantitative determination of organic cyanates. Since all tested cyanates gave
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nearly identical calibration curves, a single calibration curve can be used for the determination of structurally different organic cyanates. ACKNOWLEDGMENT We thank J. Davis for his help with the ‘HNMR spectra and S. D. Maleknia for providing the field desorption mass spectrometry. We also acknowledge the advice and help of M. Marletta and C. E. Costello in preparing this manuscript. Registry No. 2, 50962-50-0;3, 104549-45-3;4, 1122-85-6;6, 1156-51-0;7,93174-01-7;8, 104549-44-2;DBA, 769-42-6;pyridine, 110-86-1. LITERATURE C I T E D Martin, Dieter Angew. Chem. 1964, 7 6 , 303 (or Angew. Chem., I n t . Ed. Engl. 1964, 3 , 311). Jensen. Kai A,: Due, Marie; Holm, Arne Acta Chem. Scand. 1965, 19. 438-442.
&gatlErns< Putter, Rolf Chem. Ber. 1964, 9 7 , 3012-3017. The Chemistry of Cyanates and Their Thio Derivatives; Patai, S . , Ed.: Wiley: New York, 1977. Hedayatullah, Mir; Denivelle, Leon C . R . Acad. Sci., Ser C 1969, 268, 427-429. Martin, Dieter; Bacaloglu, Radu Organische Synfhesen mit Cyansaureestern: Akademie-Verlag: Berlin (East), 1980. Kagedai, Lennart: Akerstrom. Stig Acta Chem. Scand. 1970, 2 4 , 160 1-1 608. Kohn, Joachim, Lenger, Reuben: Wilchek. Meir Appl, Biochem . Biofechnol. 1983, 8 , 277-235. Powers, James C.; Tuhy, Peter M.: Witter. Frank Biochim. Biophys Acta 1976, 445, 426-436.
Martin, Dieter: Bacaloglu, Radu Organische Synthesen mif Cyans&reestern ; Akademie-Verlag: Berlin (East), 1980; p 25. Minnesota Mining and Manufacturing Co. Br. Patent 1 305 762, 1973. , Farbenwerke Bayer (West Germany). US. Patent 3491 060, 1970. (13) Kohn, Joachim; Langer, Robert Biomaterials 1986, 7 , 176-182. 114) Farbenwerke Baver (West Germanv). Ger. Patent 1220 132. 1964. Ben-Efraim, Davib A,’ I n The Chem/stiy of Cyanates and Their Thio Derivatives; Patai, S . , Ed.; Wiley: New York, 1977; Chapter 5. Konig, W. J . Prakt. Chem. 1904, 69, 105-110. Schwarzenbach, G.; Weber, R. Helv. Chim. Acta 1942, 2 5 , 1628-1639. Meites. Louis Handbook of Analytical Chemistry. 1st ed.; McGraw-Hill: New York, 1963; pp 6-67. Lambert, Jack L.; Ramasamy, Jothi; Paukstelis, Joseph V. Anal. Chem. 1975, 4 7 , 916-918. Wilchek, Meir; Miron, Talia; Kohn, Joachim Anal. Biochem. 1981, 774, 419-421. Kohn. Joachim; Wilchek, Meir J . Chromatogr. 1982, 240, 262-263. Kohn, Joachim; Wiichek, Meir Anal. Biochem. 1981, 715, 375-382. Becher, Jan Org. Synth. 1980, 5 9 , 79-84. Kohn, Joachim: Langer, Robert J . Am. Chem. Soc., in press. Eastman Kodak Co. U.S. Patent 3 879 678, 1975. Minnesota Mining and Manufacturing Co. Br. Patent 2 136 590, 1984. Waggoner, A. S. Ann. Rev. Biophys. Bioeng. 1979, 8 , 47-68. Feigl, Fritz; Anger, Vinzenz Spot Tests in Inorganic Chemistry, 6th ed.; Elsevier: New York, 1972; p 4. Odian, George Principles of Polymerization, 2nd ed.; Wiley-Interscience: New York. 1981; p 55.
RECEIVED for review May 20,1986. Accepted July 17,1986. This work was supported by NIH Grant GM 26698. The MIT Mass Spectrometry Facility is supported by NIH Grant RR 00317 (to K. Biemann).
Identification of Organic Additives in Rubber Vulcanizates Using Mass Spectrometry R. P. Lattimer,* R. E. H a r r i s , a n d C. K. Rhee T h e BFGoodrich Research and Development Center, Brecksville, Ohio 44141 H.-R. S c h u l t e n Fachhochschule Fresenius, 6200 Wiesbaden, Federal Republic of Germany The identification of the Ingredients in a compounded polymer is often a complex task for the analytical chemist. A wlde variety of components are involved-polymers, fliiers, soivents, and organic and inorganic addltives. This report considers the IdentHicatlon of organic additives in rubber vulcanizates by various mass spectrometric methods. Direct thermal desorption has been used with three different ionization methods (electron impact, EI; chemical ionlzatlon, CI; fleld ionization, FI). The vukanlzates were also examlned by direct fast atom bombardment mass spectrometry (FARMS) as a means for surface desorptkn/lonitatkn. Rubber extracts were examined directly by four ionization methods (EI,CI, FD, FAB). Of the various vaporlzatlon/ionizatlon methods, It appears that field desorption/ionizatlon (FD/FI) is the most efficient for identifying typical organic additives in rubber vuicanirates. Other Ionization methods may be requlred, however, for detection of speclfic types of additives. I n this study there was no clear advantage for dlrect analysis as compared to extract analysis. Dhect analysis Is a Httle faster, but extraction methods are more versatile. Mass spectrometry is a very effective means for the rapid Identification of organic additives in rubber vuicanizates.
The identification of the ingredients in a compounded polymer is a difficult task for the analytical chemist. A wide 0003-2700/86/0358-31S8$01.50/0
variety of components are involved-polymers, fillers, solvents, and organic and inorganic additives. Various methods have been developed to separate, identify, and quantify the numerous ingredients. Our laboratory is in the process of evaluating new or improved techniques for analysis of compounded polymers. Considerable effort in this regard is being focused on mass spectrometry as a means to supplement and extend our current analytical methods. We are particularly interested in direct methods of analysis, i.e., examining the polymer with no or minimal pretreatment of the sample ( 1 ) . In this report we will consider the identification of organic additives in rubber vulcanizates by various mass spectrometric methods. This general topic has been the subject of a recent review (2). The identification of additives in compounded polymers is made complex by a number of factors (2): (1) There are a wide variety of additive types; many compound classes are represented. Additives differ greatly in molecular weight, volatility, and polarity. Some additives are pure compounds, while others are complex oligomeric mixtures. (2) Many additives are labile. Accelerators and other curatives decompose during processing of the polymer. Various stabilizers are designed to be reactive during the useful lifetime of the polymer. (3) Complex mixtures of additives will normally be present in a compounded polymer. Some additives may interact chemically with each other and/or with the polymeric components. (4)Organic additive concentrations may be quite low (
