o-quinone structure on the benzene ring is not sufficient to produce fluorescence n-ith zinc acetate-0-benzoquinone with zinc acetate gave a nonfluorescerit green color. Iodine in the C-2 position decreased the production of fluorescence, although the presence of methyl had no effect. Because neither adrenochrome or any of its derivatives have yet been isolated from blood or urine, there is no absolute proof that this method measures adrenochrome. However, the method is accurate when adrenochrome is added to plasma or injected intravenously into blood. Plasma contains fluorescent factors which behave as if they n-ere adrenochrome. Therefore it is a working assumption that adrenochrome is being measured. However, proof must await isolation studies. After this manuscript was submitted, Szara, Axelrod, and Perlin (9) reported a sensitive specific method for measuring adrenochrome in plasma. They found less than 20 y equivalent of fluorescence which had no specific fluorescence peak. The two methods have been compared for sensitivity and are equally specific. Ten milligrams of authentic adrenochrome was injected intravenously into a subject. Ten minutes later blood was drawn from the other arm and placed in a flask containing heparin. This method showed 400 y per liter in the plasma. The Szara method yielded 76 y per liter. With this method, 1 y of authentic adrenochrome yielded 0.25 fluorescence unit compared to an increase of approximately 0.0335 unit (same scale) or one eighth the increase. Thus the method of Szara et al. is not sufficiently sensitive to settle the question as to whether or not adrenochrome is present in plasma. ACKNOWLEDGMENT
The authors thank R. Heacock for
S.F
Figure 3. Fluorescence spectra of plasma and spinal fluid acetone extracts before and after reaction with zinc acetate Plasma P. and spinal fluid S.F. acetone extracts Fluorescence measured a t 405-mp excitation 1. Before zinc acetate reaction 2. After zinc acetate reaction 3. After zinc acetate reaction with addition of 1 y of adrenochrome 4. Nonspecific light scattering peak
VI Y w P
3
300
400
500
600
&/ 3
300
400
W A V E LENGTH,
500
600
Mp
the preparation of crystalline 2-iodoadrenochrome and 5,6-dihydroxy-Nmethylindole and Burroughs Wellcome & Co. who supplied the 3,4-dihydroxyphenylethylmethylene hydrochloride (epinine). LITERATURE CITED
(1) Fischer, P., Derouaux, G., Lambot,
H., Lecomte, J., Bull. soc. chim. Belges
59,72 (1950). (2) Fischer, P., Lecomte, J., Ibid., 33, 569 (1951). (3) Harley-Mason, J., J. Chem. SOC. 1950,1276.
(4) Harley-Mason, J., Bu'Lock, J. D., Nature 166, 1037 (1950). (5) Heacock, R. A,, Nerenberg, C., Payza, A. N., Can. J . Chem. 36, 853 (1958). (6) Payza, A. N., Hoffer, A., to be published. ((7!,F&\ter, 7 ) Richter, D., Biochem. J . 3'1, 2022 \1aClr ). (1937). (8) Sprince, H., Rowley, G. R., Science 125,25 (1957). (9) Szara, S., Axelrod, J., Perlin, S., Am. J . Psychiat. 115, 162 (1958). RECEIVEDfor review June 23, 1958. Accepted February 3, 1959. Research supported by National Health Grants, Ottawa, The Rockefeller Foundation, New York, and The Saskatchewan Committee on Schizophrenia Research.
Spectroscopic Techniques for Identification of Organosilicon Compounds A. LEE SMITH and J. A. McHARD
Dow Corning Corp., Midland, Mich. ,Silicones may b e identified with the aid of chemical and Physical tests, but in many cases, simple spectroscopic examination, particularly by infrared, will satisfactorily characterize the material with a minimum of time and effort. Techniques for examining both organosilicon monomers and polymers are discussed. Some infrared
1174
ANALYTICAL CHEMISTRY
spectra of typical chlorosilanes and silicone polymers are shown, and the spectra are interpreted in terms of group frequencies. The applicability of ultraviolet, Raman# emission, and nuclear magnetic resonance spectroscopy to identification of silicones is reviewed.
A
of chemical and physical techniques may be applied to the problem of identifying organosilicon compounds (32). Although reliable chemical analyses are useful in characterizing these materials, supplementary structural information is often required which involves spectroscopic examination. I n many cases, too, satisfactory YARIETY
identifications can be made by spectroscopic means alone a t a fraction of the time and cost required for chemical analysis. Because of its applicability to samples in a n-ide variety of physical forms, and because of the specific nature of the absorption bands of organosilicon compounds, infrared is probably the most powerful of the spectroscopic methods. Therefore. this discussion is devoted largely to the use of infrared for the identification and characterization of silicones. Other spectroscopic techniques of more limited applicability are revien-ed briefly. INFRARED SPECTROSCOPY
Analysis of Monomers. Halogencontaining silanes present a special problem, because of their clifficult handling characteristics. T h e chlorosilancs. for example, hydrolyze readily in t h e picsence of moisture t o form siloxanes and hydrogen chloride. Consequently, sample manipulations involving chlorosilanes should be undertaken only in a well ventilated hood. The chlorosilanes are most convenientlx run as dilute solutions in carbon tetrachloride (2 to 7.5 microns) and carbon disulfide (7.5 to 25 microns). Silanes n hich boil belon- room temperature can be handled by this technique, if the sample is first cooled well below its boiling point and then introduced into a stoppered volumetric flask containing the solvent. Infrared cells used for running the chlorosilanes should be emptied and flushed immediately after use and checked periodically with pure solrents to monitor the inevitable build-up of hydrolysis products within the cell. Such deposits usually nianifest themselves by the appearance of a band in the siloxane region betn-een 9 and 10 microns and another band a t about 12 microns. h o m o - and iodosilanes are hydrolyzed even more readily than tlie chlorosilanes and may have to be handled in a dry box. Other substituted silane monomers are more stable toward hydrolysis and may be handled by conventional techniques. Silanes 11 hich h a w sufficiently high vapor pressure can also be run in the gas or vapor state. Here again chlorosilanes 13 ill react ith traces of moisture n hich remain adsorbcd on the walls of the gas-handling system even after thorough pumping out. This problem can be minimized by flushing the system with a suitable chlorosilane prior to insertion of the sample (26). Some of the nionosubstituted silanes as well as silane itself ignite spontaneously in the presence of oxygen, and suitable precautions must be taken if these eompounds arc encountered. Absorptions characteristic of the Si-Cl
bonds appear in the potassium broinide prism region of 16 to 25 microns (49). If more than one chlorine atom is present on silicon, two bands appear, corresponding to the symmetrical and asymmetrical stretching frequencies. In general, the asymmetrical stretch appears a t a shorter wave length and is the more intense of the two frequencies. The Si-Cl bands are the basis of a n analytical method for the determination of other chlorosilanes appearing as impurities in trimethyl chlorosilane (8). Spectra of some typical chlorosilanes are shown in Figures 1 to 5 . Frequency correlations which are discussed below for silicon containing polymers and silane monomers also apply to the chlorosilanes. However, electronegative substituents such 3s chlorine may shift some of these vibrational bands t o shorter wave lengths. The siloxane band in hexamethyl disiloxane, for example, is located a t 9.50 microns. As the methyl groups are replaced by chlorines, the wave length of the absorption gradually shifts until it is found a t 8.90 microns for hexachlorosiloxane. The shift of the Si--H stretching frequency in substituted silanes can be predicted precisely from the structure of the molecule (50). Analysis of Polymers. The classification of silicone polymers into resins, fluids, etc., has been discussed (36). The following remarks apply principally to pure silicone polymers. Separation of silicones from organic constituents will be discussed in a subsequent paper. Some polymers are soluble in carbon disulfide and carbon tetrachloride, and solution in these materials is a convenient method for obtaining the spectra for such polymers. However, high viscosity dimethylpolysiloxanes have limited solubility in carbon disulfide and it may be necessary, if a solution spectrum is desired, to use carbon tetrachloride throughout the entire spectral region or to use some solvent such as 2,2,4-trimethylpentane in the 11- to 16-micron region. Frequently, sufficiently good spectra may be obtained by simply casting a film of the polymer on a polished salt plate from a low boiling solvent such as chloroform. Hard, brittle materials such as cured resins can be run by the mineral oil mull ( 7 ) or potassium bromide pressed plate (SO, 44) techniques. I n cases lvhere a filler or a reinforcing agent is present, the pyrolysis technique of Harms (25) is very useful. Interpretation of Spectra. The infrared spc,ctra of silicones are notable for tn-o things: The characteristic vibrations of substituents vary only slightly in wave length, regardless of t h e compound in which they are found; and t h e intensity of t h e absorption bands lying beyond 7
Table 1.
Group Si-H Si-CH3 Si-CH&HS Si-C6HE Si-0-CH, Si-0-CH2-R Si-0-Aryl Si-C1 Si-OH Si-0-Si Si-CH=CH2
Absorption Data
Ahorption, Il'ave Lengths, Microns 4.4-4.8; 10 5-12.5 7.8-8 0 ; 11 6-13 1 8 0-8 1; 9 8-10.0; 10 3-10 6 7.0; 8 . 9 ; -13.7; 14.3-14.5 3.5; 8 . 4 8 . 4 8 . 8 ; 9.1-9.3; 10 1-10 6 10.3-10 9 16.5-24.0 2.7-3.1; 10.5-12.0 8.9-9.9 6.2-6.3; 9.9-10.0; 10,2-10.6
mivrons is 5 t o 10 times greater t h a n is normal with most organic compounds (55). These characteristics make silicones relatively easy to identify by their infrared spectra. Silicone polymers are distinguished by a strong absorption in the 9- to 10micron region arising from an Si-0 stretching vibration in the siloxane backbone of the polymer. Linear and cyclic polymers containing eight or fewer units can be characterized by the shape and position of the siloxane band (55). I n commercial polymers the silicon atoms are usually substituted by some combination of methyl, ethyl, phenyl, hydrogen, alkoxy, or hydroxyl groups. Figure 6 s h o w the spectrum of a dimethyl-substituted polysiloxane. This material is occasionally encountered in qualitative organic analysis, when inadvertent extraction of silicone stopcock grease by a n organic phase has occurred. The infrared spectrum of a methylsiloxane (hieHSiO), polymer is shown in Figure 7 . The strong bands a t 4.6 and 11.2 microns indicate the presence of Si-H, and the Si-Me group has a characteristic band a t 7.95 microns. Figure 8 shons the spectrum of a phenyl methyl-substituted siloxane polymer, and Figure 9 shon-s a copolymer of phenyl methyl and dimethyl-substituted polysiloxane. Silicone resins are differentiated from fluids in t h a t the degree of substitution of the resins is less than 2--that is, in the polymer (R,,Si04-,), - n < 2. This 2
means that a certain amount of cross linking occurs. An all-methyl silicone resin with a high (LIeSiOo,z)content is shown in Figure 10. The Si0312unit can be recognized by a strong band a t about 9 microns. Figure 11 shows a n ethyl-substituted resin which is essentially a silsesquioxane. The Et-Si group is characterized by absorptions a t 8.0, 9.9, and 10.3 microns. Figure 12 is the spectrum of a typical phenyl and methyl-containing silicone resin with mono- and disubstituted silicon. Figure 13 shows a silicone-modified alkyd resin VOL. 31, NO. 7,JULY 1959
1175
composed of 25% silicone, 25% fatty acid, and 50% glycerol phthalate. Silicones used to modify organic resins usually contain either the dimethylsiloxane (Me2SiO) group which absorbs a t 7.9 and 12.5 microns, or the phenylsilicon group, which shows a needlesharp band a t 7.0 microns, or both. A broad absorption in the 9- to 10-micron
region indicates the presence of siloxane. Table I gives a brief compilation of characteristic frequencies of substituted silicones (4, 20, 33). A more complete discussion of structure-spectra correlations in organosilicon compounds will be given in a subsequent paper. The spectra of other silicone polymers have beep discussed in greater detail by
Kright and Hunter (55), Young, Servais, Currie, and Hunter (58)) and Richards and Thompson (41). The dimethylpolysiloxane fluids have been studied by Smith, French, and O'Neill (52) and the identification of silicones in cosmetics was reported by Pozefsky and Grenoble (39). Infrared has been used for the identi-
Y C
P
U
I
3 3 N V U I W S N V I l I N 3 3 U3d
Figures 1 to 5. 1.
2. 3. 4. 5.
1176
Spectra of alkyl and aryl halogenated silanes
Methyltrichlorosilane 100 mg. p e r cc. in CCL, 2 to 7.5 p; 20 mg. p e r cc. in C S , 7.5 to 25 p Dimethyldichlororilane 100 mg. p e r cc. in CCl4, 2 to 7.5 p; 20 mg. p e r cc. in CSZ, 7.5 to 25 p Trimethylchlorosilane 100 mg. p e r cc. in CCId, 2 to 7.5 p; 20 mg. p e r cc. in CSz, 7.5 to 25 p Phenyltrichlorosilane 100 mg. p e r cc. in CClr, 2 to 7.5 p ; 100 mg. p e r cc. in CS, 7.5 to 16 p; 20 mg. p e r cc. in CSn, 16 to 25 fi Methylphenyldichlorosilane 100 mg. p e r cc. in CClr, 2 t o 7.5 p; 20 mg. p e r cc. in C&, 7.5 to 25 p
ANALYTICAL CHEMISTRY
fication of silicone elastomers (12, I S ) , wire enamels (28), and resins (19, 29, 31). The infrared spectra of some silicone resins and their thermal degradation products have also been published (51,57). I n spite of its great utility, the infrared method has certain limitations which should be recognized. First, additives or substituents which are present in small amounts may be overlooked, unless special effort is made to detect them by using difference spectra or concentrated samples over limited spectral regions. Second, infrared is not very sensitive to differences in molecular weight or molecular weight distribution, factors which have considerable effect on the properties of polymers.
ULTRAVIOLET SPECTROSCOPY
The ultraviolet region of the spectrum is of considerably less value in identifying organosilicon compounds than is the infrared. A relatively small number of publications show that, as expected, alkyl-substituted silanes and siloxanes are not ultraviolet absorbers. Although Burkhard and Winslow (9) found several absorption bands for methylsiloxanes, work in the authors' laboratory shows that these compounds do not absorb above 220 mp and the bands observed by Burkhard and Winslow must arise from aromatic impurities. This conclusion is confirmed by Eaborn (It?), who found no absorption for trimethylsilanol, hexamethyldisiloxane, or trimethylmethoxysilane.
Phenyl substitution on silicon, however, gives rise to a series of absorption peaks located a t 248, 254, 260, 264, 266, and 271 mp. These bands are common to triphenylsilane, triphenylethoxysilane, and triphenylsilanol ( $ I ) , and trimethylphenylsilane (6). Parasubstituted phenyl groups produce an intense absorption a t about 270 mp ($1) It is obvious that, a t its present stage of development, ultraviolet spectroscopy is not a very good tool for identifying silicones. It is, however, very useful in detecting trace amounts of aromatic contaminants in dimethylsiloxane fluids or determining traces of aromatic-substituted silicones in the presence of nonabsorbing materials (39). MASS SPECTROMETRY
The mass spectrometric method is generally applicable to the identification as well as the quantitative analysis of silicones, because of the distinctive fingerprint formed by the natural abundance of isotopes silicon-28, silicon-29, and silicon-30. An additional advantage of mass spectrometry for identification purposes is that it gives directly the molecular weight of a compound. Sampling is straightforward; the only restriction is that the sample must have a vapor pressure of a t least 1 mm. of mercury a t 225' C. This condition, of course, excludes most siloxane polymers, but the method is applicable to monomers, to low molecular weight polymers, and to decomposition products of polymers. Published mass spectra for organosilicon compounds include trimethylsilyl derivatives of aliphatic alcohols (47), tetramethylsilane (16), hexamethyldisiloxane and octamethyltrisiloxane (17), sym-tetramethyldiphenyldisiloxane ( 3 4 , and severaI alkyl and aryl chlorosilanes (53).
W A V E NUMBER, CM.-1
RAMAN SPECTROSCOPY
,
,I
/I
12
11
,1
11
I
I
1 W A V E LENGTH, MICRONS
Figures 6 to 9. Spectra of polysiloxanes Poly(dimethylsiloxane), trimethyl end blocked. 1 0 0 mg. per cc. in CCId, 2 to 7.5 fi; 20 mg. per cc. in C b . 7.5 to 16 u 7. Poly(methy1hydrogen siloxane), trimethyl end blocked. 100 mg. per cc. in CCla, 2 to 7.5 p ; 20 mg. per cc. in CS2, 7.5 to 1 6 p 8. Polybnethyl phenyl siloxane), trimethyl end blocked. 1 0 0 mg. per cc. in CC14, 2 to7.5 p; 20 mg. per cc. in C S , 7.5 to 1 6 p 9. Copolymerized methyl phenyl siloxane and dimethyl siloxane, trimethyl end blocked. 100 mg. per cc. in CClr, 2 to 7.5 p; 20 mg. per cc. in CSn, 7.5 to 1 6 fi
Generally speaking, one obtains the same sort of information from the Raman spectrum of a material as from its infrared spectrum. Thus, Raman spectra can also be used for identification of silicones. I n practice, however, restrictions on the type of sample which can be run severely limit the applicability of the method. It is not practical to examine solids, for example, by this technique. Nevertheless, Raman spectroscopy has some unique advantage which can be advantageously used in the analysis of certain types of silicones. The first advantage lies in the fact that vibrations of low frequency are easily observed. Consequently, the presence of Si-Br and Si-I, whose stretching vibrations fall in the range 150 to 450 cm.-l (14, 16), is easily VOL. 31, NO. 7, JULY 1959
1177
determined. A second advantage is t h a t Si-Si, which because of symmetry considerations gives very weak or no absorption in the infrared, can be detected (6). I n addition, double or triple bonds a t a center of symmetry give no characteristic infrared bands, but are very strong in the Raman spectrum (2). I n general, group frequency correlations hold for Raman spectra (64, 56). and because intensities are additive, quantitative group analyses can be carried out (2, 3). Some structures for which spectra are given in the literature include methylchlorosilanes (23),methylbromosilanes (%’), chlorosilanes (&), ethylchlorosilanes (1, 38, 49), other alkyl halosilanes (%), and siloxanes (10, 35, 48, 54). EMISSION SPECTROSCOPY
Mironov, V. F., Doklady Akad. Nauk S.S.S.R. 9 2 , 515 (1953). (4) Bellamy, L. J., “Infra-red Spectra of Complex Molecules,” Chap. 20, Methuen and Co., London, 1954. (5) Bethke, G. W.,Wilson, M. K., J .
many cases, the infrared spectrum will characterize the silicone without any further effort. If separations or extractions are indicated, their effectiveness may be followed by infrared examination of the fractions. If infrared alone is not sufficient to identify the silicone, elemental or group analysis by chemical means, possibly combined with examination by one or more spectroscopic techniques, will usually give a satisfactory answer.
Chem. Phys. 26,1107 (1957). (6) Bowden, K., Braude, E. -4., J . Chem. Soc. 1952, 1068. (7) Bradley, K. B., Potts, W. J., Jr., A p p l . Spectroscopy 12, 77 (1958). (8) Brovm, P., Smith, -4. L., A N A L . CHEX 30, 1016 (1958). (9) Burkhard, C. A., Winslow, E. H., 1. Am. Chem. SOC.’72, 3276 (1950). (10) Cerato, C. C., Lauer, J. L., Beachell, H. C., J. Chem. Phys. 2 2 , l (1954). (11) Currie, J. W., Harrison, G. W., Jr., J . Org. Chem. 23, 1219 (1958). (12) Davidson, W. H. T., Bates, G. R.,
LITERATURE CITED
(1) Batuev, M. I., Petrov, A. D., Pon-
amarenko, V. A., Matveeva, A. D., Proc. Rubber Technol. Conf., 3rd Conf. Izvest. Akad. Nauk S.S.S.R., Otdel. London 1954, 281. Khim. Nauk. 1956, 1070 (Consultant’s (13) Davidson, W. H. T., Bates, G. R., Bureau translation, p. 1087). Rubber Chem. and Technol. 30, 771 (2) Batuev, M. I., Ponomarenko, V. 9., (1957). Matveeva, A. D., Snegova, A, D., Zbid., (14) Delhaye-Buisset, M. B., Compt. rend. 1956, 1420 (Consultant’s Bureau trane244, 7i0 (1957). Iation, p. 1457). (15) Delwaulle, M. L., Buisset, 31. D., (3) Bazhulin, P. A,, Yegorov, Y. P.,
Because emission spectroscopy cannot distinguish the chemical state of the atoms, its main use in detecting silicones is to establish the presence or absence of silicon. Even if silicon is found, however, it does not follow that silicones are present, because silica fillers or contaminants will give a positive test for silicon. On the other hand, the absence of silicon lines is good evidence that no silicone is present in the sample. The low energy excitation of organosilicon compounds is not always successful in producing silicon lines (40). Organic materials may be prepared for arcing by a wet-ashing procedure (32), or if the sample is a resin or fluid of low volatility, it may be mixed with graphite and sparked directly from the surface of a flat electrode. OTHER SPECTROSCOPIC METHODS
The near infrared spectra of organosilicon compounds, to the author’s knowledge, have never been thoroughly studied. Cerato, Lauer, and Beachell (10) haye commented on the presence of a doublet at 5380 and 5415 em.-’ which may be indicative of the presence of Si-C bonds. h’uclear magnetic resonance promises to be a useful aid in the elucidation of the structure of silicon-containing molecules (11, 62). Chemical shifts have been given for proton (43,fluorine-19 (46, 46), and silicon-29 (67) resonances in organosilicon compounds. Proton shifts have also been used to obtain information about inolecular motions in liquid and solid methylchlorosilanes, polysiloxanes, and silicone rubber (42).
I
---
--I I
--
1
IO
--
>o-
-
-
13.--
-~
-
-
-
__
-
1
I
- _ _ _ _ .
i
L
I
CONCLUSION
The following procedure is recommended for examining a material suspected of containing silicone. First, a n infrared spectrum is obtained, using one of the techniques discussed. I n 1178
ANALYTICAL CHEMISTRY
WAVE LENGTH, MICRONS
Figures 10 to 13.
Spectra of resins
10. All methyl resin containing MeSiOa/t, MezSiO, and keaSiOl/t, 100 mg. per cc. in CClr, 2 to 7.5 pr 20 mg. per cc. in CSz, 7.5 to 16 p 1 1, (EtSiO:/,), type resin, film film 12. Pure silicone resin containing MezSiO, MeSiOa/g, #%Si0 and @SiOV2. 13. Silicone-modified alkyd resin, film
Delhaye, M., J . Am. Chem. SOC.74, 5768 (1952). (16) Dibeler, V. H., J. Research Natl. Bur. Standards 49, 235 (1952). (17) Dibeler, V. H., Mohler, F. L., Reese, R. M., J . Chem. Phys. 21, 180 (1953). (18) Eaborn, C., J. Chem. SOC. 1953, 3148. (19) Fishl, W., Young, I. G., Appl. Spectroscopy 10,213 (1956). (20) Gilman, H., ed., “Organic Chemistry,” Vol. 111, pp. 143-51, Wiley, New York, 1953. (21) Gilman, H., Dunn, G. E., J. Am. Chem. SOC.72, 2178 (1950). (22) Goodman, L., Silverstein, R. M., Shoolery, J. X., Jr., Ibid., 78, 4493 (1956). (23) Goubeau, J., Siebert, H., Winterwerb, bl., 2. anorg. Chem. 259, 240 (1949). (24) Goubeau, J., Warneke, R., Ibid., 259,233 (1949). 125) Harms. D. L., ANAL. CHEM. 25. . 1140 (1953). (26) Hawkins, J. A., Wilson, M. K., J . Chem. Phys. 21,360 (1953). (27) Holzman, G. R., Lauterbur, P. C., Anderson, J. H., Koth, W., Ibid., 25, 172 (1956). 128) Hummel, D.. Farbe u. Lack 62. 529 . (1956). (29) Hummel, D., Eunststoffe 46, 442 (1956). (30) Ingebrigtson, D. N., Smith, A. L., ANAL.CHEY.26, 1765 (1954). .
I
(31) Kagarise, R. E., Weinberger, L. A., U. S. Naval Research Laboratory, Office of Technical Services, Rept. PB 111438 (May 11, 1954). (32) Kline, G. M., ed., “Chemical Analysis of High Polymers,” Chap. 14, Interscience, New York, 1959. (33) Kreshkov, A. P., Mikhailenko, Yu. Ya., Yakimovich, G. F., Zhur. Anal. Khim. 9,208 (1954). (34) McLafferty, F. W., ANAL. CHEM. 28,306 (1956). (35) Murata, H., J . Chem. Phys. 19, 659 (1951). (36) Murata, H., Science & I d . (Osaka) 30. 164 (19561. (37) ’Murata, H., Hayashi, S., J. Chem. Phys. 19,1217 (1951). (38) hfurata, H., Okawara, R., Watase, T., Ibid., 18,1308 (1950). (39) Pozefsky, A., Grenoble, M. E., Drug & Cosmetic Ind. 80,752 (1957). (40) Radell, J., Hunt, P. D., Murray, E. C., Burrows, W. D., ANAL.CmM. 30, 1280 (1958). (41) Richards, R. E., Thompson, H. W., J . Chem. SOC.1949. 124. (12) Rochow, E. G.; LcClair, H. G., J . Inorg. & Nuclear Chem. 1, 92 (1955). (43) Savidan, L., Bull. SOC.chim. France 1953, 411. (44) Scheidt, U., Appl. Spectroscopy 7, 75 (1953). (45) Schnell, E., Rochow, E. G., J. Am. Chem. SOC.78, 4178 (1956). (46) Schnell, E., Rochow, E. G., J . Inorg. &Nuclear Chem. 6 , 303 (1958).
(47) Sharkey, A. G., Jr., Friedel, R. A., Langer, S. H., ANAL. CHEW 29, 770 (1957). (48) Slobodin, Ya. M., Shmulyakovskif, Ya. E., Rzhedzinskaya, K. A., Doklady Akad. Nauk S.S.S.R. 105, 958 (1955). 149) Smith. A. L.. J . Chem. Phvs. 21. 1997 (1953). (50) Smith, A. L., Angelotti, N. C., Spectrochim. Acta, in press. (51) Smith, A. L., Brown, L. H., Tyler, L. J., Hunter, M. J., Ind. Eng. Chem. 49, 1903 (1957). (52) Smith, D. C., French, J. hf., O’Neill, J. J., U. S. Naval Research Laboratory, Office of Technical Services, Rept. P-2746 (January 1946). (53) Sokolov, N. N., Andrianov, K. A., Akimova, S. M.,Zhur. Obshchei Khim. 25,675 (1955); J.Gen. Chem.(U.S.S.R.) 25. 647. (54) ’Ulbrich, R., 2. Naturforsch. 9b, 380 (1954). (55) Wright, X., Hunter, M. J., J . Am. Chem. SOC.69, 803 (1947). (56) Yegorov, Y . P., Bazhulin, P. A,, Doklady Akad. Nauk S.S.S.R. 88, 647 (1953). (57) Yonemoto, T., Senzok, K., Bull. Electrotech. Lab. (Tokyo) 18,428 (1954). (58) Youn C. TV., Servais, P. C., Currie, C., Hunter, M. J., J . Am. Chem. SOC.70, 3758 (1948). .
I
.
8.
RECEIVED for review December 22, 1958. Accepted March 16, 1959.
Spectrophotometric Determination of Amino Acids Alkaline Copper Salt Method Using Cuprizone, Biscyclohexanoneoxalyldihydrazone RAYMOND BORCHERS Department of Biochemisfry and Nutrition, University of Nebraska, Lincoln 3, Neb.
b The sensitivity of the alkaline copper salt method for amino acids has been increased b y the use of cuprizone, biscyclohexanoneoxalyldihydrazone, for the determination of solubilized copper. Amino acids may b e determined on a 1-ml. sample a t 0.1 to l.OmM concentration. Prior treatment of samples with nitrous acid provides a procedure for checking on nonamino acid interfering compounds. The method has been applied in following the course of proteolytic activity and to amino acid determinations on 0.2ml. blood sample.
T
alkaline copper salt method for the determination of amino acids of Pope and Stevens (8) as modified by Schroeder, Kay, and Mills (9) has been further modified. Modifications have been concerned with the estimation of copper brought into solution by the amino acids from a n alkaline suspension of washed copper phosphate. HE
The copper has been estimated by iodometric titration ( 9 ) , spectrophotometry of the copper-amino acid complex (3, 4, 10, l l ) , spectrophotometry involving diethyldithiocarbamate ( l a , I S ) , polarography ( 5 ) , and flame photometry ( 2 ) . A procedure for the application of cuprizone, biscyclohexanoneoxalyldihydrazone, to the determination of copper in the alkaline copper salt method for amino acids is presented. Cuprizone has been used as a chromogenic reagent for copper (6) and applied in the determination of serum copper (7). The comparative sensitivity of cuprizone for copper is attested by its molar absorbancy of 16,000 (7); 12,700 for diethyldithiocarbamate (7) ; and 6200 and 56 for the copper-amino acid complex a t 235 and 620 mp, respectively (11). REAGENTS
WASHED COPPER PHOSPHATE Sus-
Prepare as directed (9). Age 2 days before using. Prepare weekly. BORAXBUFFER. oH 9.1. Preoare as directed (9). CUPRIZONE, 0.27,. Dissolve 200 mg. of cuprizone, biscyclohexanoneoxalyldihydrazone (G. Frederick Smith Chemical Co.) in 100 ml. of 50% ethyl alcohol with gentle heating. It is stable indefinitely. AMINO ACIDS AXD RELATEDCOMPOUNDS. Make freshly prepared solutions in distilled water. These compounds were used as purchased. PEXSION.
I
_
PROCEDURE
Add one volume of sample (0.1 t o l.OmM amino acid concentration) &to one volume of copper phosphate suspension. Neutralize samples when necessary by adding one drop of phenolphthalein plus sodium hydroxide to a faint pink. Prepare a blank with one volume. of water plus one volume of suspension. Mix and let stand for 5 minutes. Centrifuge a t 2000 r.p.m. for 5 minutes. To 1 ml. of the superVOL. 31, NO. 7, JULY 1959
* 1179
