Table 111. Recovery of 1 pg. of 5,6Dihydro 5 (2 imidazolin 2 ylmethyl)-morphanthridine from 5 MI. of Blood
- - -
Sample“
- -
Found, p g . 0.87
Difference, p g . -0.13
1.05 1.00
+0.05 0.00 -0.07
2-B
0.93 1.04
4-B 5-E3
0.85
-0.07 +0.04 -0.08 -0.15 -0.05
1-A 2-A 3-A 4-A
5-A
I-B 3-B
0.93 0.97 -0.03 Mean 0.96 Std. dev. 0.08 0.92
0.95 Mean 0.94 Std. dev. 0.07 Samples 1-A to 5-A were determined using blood from Patient A; samples 1-B to 5-B represent blood from Patient B. 5
attributed to the ring methylene group
\
/ \
connected to the nitrogen (CH2-N),
showed up as a sharp signal when spectra were obtained a t higher temperatures. (A more detailed discussion will be presented in the future describing this effect together with an explanation of the various shifts of the methylene groups.) Final proof that the carbonyl group was adjacent t o the nitrogen of the seven-membered ring was obtained from the spectra of the hydrolyzed product of Compound I--namely, Compound 10. This compound exhibited normal ultraviolet (Figure 3) and nuclear magnetic resonance spectra as well as a well-defined oxidation wave.
Based on all of the above data, it can now be inferred that the isolated compound has the structure assigned to Compound VI11 in Figure 1. Compound VI11 is probably formed in a manner as represented by scheme 1 in Figure 1. A species such as that given by Compound IV would probably be responsible for the fluorescent characteristic of Compound I. A similar mechanism can be applied to Compound 6 (Table I), thereby explaining its fluorescent properties. Since this mechanism can also be applied to Compounds 2 and 9, it can be seen that a second prerequisite is needednamely, a dihydromorphanthridie nucleus. A plot of fluorescent intensity us. time indicated that optimum fluorescence was obtained when the compound was heated with acetic anhydride a t 100” C. for 45 minutes. At temperatures lower than 100” C., the reaction proceeded very slowly. A plot of fluorescent intensity us. concentration of an extracted standard was found to be linear up to a t least 5 pg. per 25 ml. of final solution. Since this would encounter the expected concentration in blood, higher concentrations were not studied. Standards, blood samples, and blood blanks were always run in duplicate when possible, and duplicate intensity readings never differed by more than 5%. At all levels studied the “noise” of the instrument was of no significance. Recoveries of 85 to 105% were obtained from blood a t the level of 0.2 pg. per ml. Table I11 summarizes the recovery of Compound I a t this level.
A blood blank, although not necessary most of the time, was run in the same manner as the sampb, since it was found that the blood of several patients and animals contained compounds which tended to give higher results. The interference could clearly be seen by comparing the fluorescent spectra of the standard and sample on the oscilloscope. The highest blood blank observed would be equivalent to 0.2 pg. of drug per ml. of blood. However, since the fluorescent intensity reading is sufficiently high for acceptable precision and accuracy at this low level, it is felt that such a high blank is no drawback in this procedure. ACKNOWLEDGMENT
The authors thank Keville Finch,
L. H. Werner, and W. E. Rosen for their advice and interest in this work and Louis Dorfman and his group for their assistance in interpreting the nuclear magnetic resonance spectra. LITERATURE CITED
(1) Hofman, K., “Chemistry of Hetero-
cyclic Compounds, Imidazole and Its Derivatives,” Part I, p. 221, Interscience, New York, 1953. (2) Kamlet, M. ,!., “Organic Electronic Spectral Data, Vol. I, p. 216, Interscience, New York, 1960. (3) Korzun, B. P., Dorfman, L,, Brody, S. M., ANAL.CHEM.35, 950 (1963). (4) Mader, W. J., “Organic Analysis,” Vol. 2, p. 253, Interscience, New York, 1954. (5) Rehm, C. R., Smith, J. B., CIBA Pharmaceutical Co., Summit, N. J., unpublished work, July 1963. (6) Tishler, F., Brody, S. M., J . Pharm. Sci. 53, 161 (1964). RECEIVED for review November 3, 1964. Accepted April 12, 1965.
A Study of the Effect of Tetracyanoethylene, TrinitrofIuorenone, and Other Pi Acceptors on the Fluorescence of Aromatic Carbons GEORGE H. SCHENK and NORMAN RADKE Department of Chemistry, Wayne State University, Detroit,
b Tetracyanoethylene (TCNE), 2,4,7trinitrofluorenone (TNF), 7,7,8,8-tetracyanoquinodimethane (TCNQ), and other 7r acids or acceptors were studied to determine their relative quenching efficiency and their effect on the fluorescence excitation and emission maxima of aromatic hydrocarbons such as anthracene and pyrene. TCNE does not eliminate the fluorescence of anthracene via the Diels-Alder reaction since fluorometric excitation wavelengths at 365 mp cause photodecomposition of the TCNE-anthracene adduct. Both TCNQ and TNF are more effective 910
ANALYTICAL CHEMISTRY
Mich. 48202
quenchers of pyrene than TCNE; this correlates with the magnitude of their ground state formation constants. TCNQ, TCNE, and TNF appear to exert a small effect on the fluorescence excitation and emission maxima of the aromatic hydrocarbons themselves.
P
ACCEPTORS or acids such as tetracyanoethylene (TCNE) (16) and 2,4,7-trinitrofluorenone(TNF) (16) have been studied for their potential use in spectrophotometric methods for n donors or bases. Another potential I
analytical use for these compounds is that of quenching or modifying the fluorescence of aromatic hydrocarbons. It is our object to summarize the pertinent literature and present analytical studies. A distinction must be made between the action of the A acceptor in quenching the fluorescence emission of the n donor itself, and that of the 7r acceptor in forming a n complex which also emits fluorescent light, but a t longer wavelengths. If the R acceptor is strong enough, the fluorescence emission of the P complex w ill occur far enough away
from that of the donor so that only the quenching effect will be observed. Therefore the analytical chemist will be chiefly interested in the quenching ability of various R acceptors or R acids. Usually solvents or solutes containing nitro, carbonyl, or cyano groups are the most effective in quenching fluorescence although carbon tetrachloride is known to quench the fluorescence emission of terphenyl dissolved in xylene (4). A common example of solvent quenching is the quenching of the fluorescence emission of aromatic hydrocarbons by nitromethane or nitroethane. This appears to be universal except for fluoranthene-type hydrocarbons, and is the basis for a selective fluorometric method for this type of aromatic hydrocarbon (13). The stronger a R acceptor toward a given donor in the ground state, the lower the concentration of R acceptor needed for quenching the fluorescence emission of the donor (6). A 1M maleic anhydride solution is required to quench anthracene effectively (17), but much less s-trinitrobenzene is required to quench anthracene (IO). The mechanism for quenching of the fluorescence emission of the R donor appears t o be well established (6, 10). It involves (Equation 1) first the rapid formation of a R complex ('BA)
1B
+A
$
1BA $ 3BA $ A
+ 3B (+ B + h r )
(1)
from a R base or donor in an excited singlet state ('B) with a R acceptor ( A ) in the ground state. [Apparently the excited singlet state has a high probability of forming such a complex before fluorescence emission can occur ( S ) . ] Then an extremely fast intersystem crossing occurs to give a T complex (3BA) containing the R base in a lower energy triplet state. Finally, the complex dissociates to the R acceptor in the ground state and the lowest excited triplet state of the R base (3B) or donor. This state reverts to the ground state ( B ) with phosphorescence emission. This has a theoretical basis in the calculations of Murrell (12) which suggest that there may be appreciable interaction of the charge transfer state with ?r donor excited states such as 'B, even when there is no stability of the complex in the ground state. Another area of interest is the fluorescence of complexes in the solid state. Thus a residue deposited from a TCNEbenzene solution has been shown to fluoresce (21). I n addition, the absorption and fluorescence emission maxima of frozen dilute solutions of aromatic hydrocarbons and s-trinitrobenzene have also been measured ( Z ) , as have the reflectance fluorescence spectra of aromatic hydrocarbons and s-trinitro-
benzene in potassium bromide pellets (20).
There were three experimental objectives of this study: to determine the effect of TCNE on the fluorescence of anthracene, to investigate the quenching abilities of various R acids or acceptors, and to determine the fluorescence excitation and emission maxima of various R bases or donors in the presence of R acids. The fluorescence emission maxima of various other complexes have already been reported (19). EXPERIMENTAL
Instrumentation. Fluorometric measurements were made with a G. K. Turner Model 110 filter fluorometer. A 7-60 (365 mp) primary filter and a 2A (415-mp sharp cut) secondary filter were used for anthracene: the 7-60 primary filter and 2A plus 2ND (reduction) secondary filter with a 3X intensity setting were used for pyrene. The fluorescence and excitation spectra were obtained with a Farrand automatic recording spectrofluorometer equipped with a high intensity 150-watt zenon arc lamp. Slit widths were 5 mp, and a 1P28 photomultiplier tube was used. Cuvettes (10 X 20 X 50 mm.) were of fused quartz. A primary filter excluding radiation above 410 mp and a secondary filter excluding radiation below 410 mp were used at all times. Reagents. Tetracyanoethylene (TCNE), Eastman White Label grade, was sublimed to a white solid and was stored over sodium hydroxide in a desiccator. Methylene chloride and p-dioxane were distilled, the latter over potassium hydroxide. Anthracene was purified by sublimation; pyrene was Eastman White Label grade. The Diels-Alder adduct of anthracene and TCNE was made by adding 0.72 gram of TCNE to 1 gram of anthracene in benzene. The solution was heated to 60" C. and cooled to room temperature. The resulting precipitate was washed with benzene and recrystallized from benzene and acetone (9). 2,4,7-Trinitrofluorenone (TNF) was purified by recrystallization from 3 :1 nitric acid-water (16). Maleic anhydride was purified by sublimation. 2,3,5,6Tetrachlorobenzoquinone (Matheson, Coleman, and Bell grade) and 7,7,8,8 - tetracyanoquinodimethane (TCNQ) were used as received. The latter was supplied as a sample by E. I. du Pont de Nemours and Co. The R acids were used as 0.01M solutions in distilled methylene chloride. The Diels-Alder adduct of TCNE and anthracene was dissolved in dioxane and in methylene chloride. RESULTS AND DISCUSSION
Diels-Alder Reaction. It was thought t h a t trace amounts of T C N E could be added to a mixture of aromatic hydrocarbons to react with anthracene via the Diels-Alder reac-
Table 1. Photodecomposition of TCNEAnthracene Diels-Alder Adduct Adduct concn. : 20 p.p.m. in CHICl?, Excitation wavelength: 365 mp (primary
filter) Emission wavelength: 415 mp (filter) F in Turner units" Samples F-first F-second of excitation excitation same (min. after (5 min. after solution dissolution) first excitation) 1 46 (7 min.) 57 2 51 (14 min.) 62 3 57 (22 min.) 70 4 62 (28 rnin.) 75 5 67 (34 min.) 80 6 73 (4U rnin.) 91 Measurements of F were made abso- . lutely, that is, using a black rod t o set the zero reading rather than using a solvent blank.
tion. This would remove its fluorescence and permit selective fluorometric determination of other aromatics. It had already been established that anthracene and TCNE react quantitatively a t levels as low as 10-4M after 40 minutes (14). However, the addition of equal and 10-41W amounts of TCNE t o anthracene did not completely quench its fluorescence. This indicated that the 365-mp excitation light was able to supply enough energy to preclude the Diels-Alder reaction or to cause photodecomposition of the Diels-Alder adduct. To test the latter hypothesis, the adduct was synthesized and used to prepare a 20-p.p.m. solution under ordinary room light. Seven minutes after dissolution (Table I), a sample of this solution was excited a t 365 mp. A fluorescence emission ( F ) of 46 units was observed, indicating the probable presence of anthracene. Five minutes later, the same sample was again excited, and F was found to be 57 units. Two minutes later (14 minutes after dissolution) a second sample had a fluorescence emission reading of only 51 units after only one excitation. These and other measurements in Table I indicate a continuous photodecomposition of the adduct under room light, a decomposition which is accelerated by irradiation a t 365 mp. The decomposition is probably a reverse Diels-Alder reaction, giving anthracene and TCNE. This is not inconsistent with the reported thermal instability of adducts of maleic anhydride and anthracene derivatives such as 9methylcholanthrene (1). It also explains why equimolar amounts of T C N E will not quench the fluorescence emission of anthracene. If any adduct is formed, it is readily photodecomposed to anthracene and TCNE, in all probability. VOL. 37, NO. 7, JUNE 1965
91 1
Table II, Quenching of Pyrene in Methylene Chloride Pyrene, 5 x l o - 4 ~ F in Turner units T Acid 2x 1x
or
acceptor
TCNQ TNF TCNE Tetrachloro-
Kin lO-4M CH2C12" Acid8 78.4* 6.0 73c 17.5 29.5d 56.5
lO-Vd
Acid' 1.0 1.0 35.0
benzo~~
quinone Maleic anhydride Sone
29.5d
58.5
36.0
17.6d
58.5 61.5
41.0 58.5g
-
Formation constants for 1 : l complexes (8). Melby, Harder, Hertler, Mahler, Benson, and Mochel ( 7 ) . Schenk. Vance. Pietrandrea. and Moizis (16). Merrifield and Phillips (8). e 0.1 ml. of 0.01M acid added t o 5 ml. of pyrene solution. 1.0 ml. of 0.01M acid added t o 10 ml. of pyrene solution. After dilution with 1.0 ml. of CH2C12, F was 55.0 units. f
Q
Table 111.
Quenching of Pyrene in Dioxane
2.5 X
M Pyrene F in Turner units 2 x 1 0 - 4 ~4 x 1 0 - 4 ~ Acid" Acidc 4.0 0.5 16.0 5.5 48.5 42.0
T Acid TCNQ TNF TCNE Tetrachlorobenzoquinone Maleic anhydride Xone
40.0
29.0
53.0
48.0 59.56
59.P
5 0.10 ml. of 0.01M acid added to 5 ml. of Dvrene solution. $ . h e r dilution with 0.10 ml. of dioxane,
F was 57.0 units. 0.20 ml. of 0.01M acid added t o 5 ml. of pyrene solution. After dilution with 0.20 ml. of dioxane, F was 55.0 units.
Table IV. Variation of Fluorescence Emission (F)with Quencher Concentration 2 ml. of 1 X 10-5M anthracene in dioxane
365 mp.(filter) excitation, 415 emission 0 ,O l M F"/F TCNE Trial 1 added. ( F " = 97.5 ml. units)"
0.50
mp (filter)
4.0
Trial 2 (F" =
94. O)a
ANALYTICAL CHEMISTRY
In this equation, (C) is the molarity of the complexed pyrene, ( B ) - (C) is the molarity of the uncomplexed pyrene, K is the constant for 1: 1 complex formation, [TI is the initial mole fraction of r acceptor, and [C], which is negligible in this case, is the mole fraction of the complex at equilibrium. [Jurinski and de Maine (5) have recently found evidence for 2: 1 and higher order complexes, but these should be minimal a t this dilution.] Using a value of 6 X 10-6 for the mole fraction corresponding to 1 X M R acceptor, Equation 2 for TCNQ or T N F is:
6.3
Readings are uncorrected for dilution. Initial fluorescence emission readings ( F ' ) vary somewhat because of the time lapse between readings.
91 2
Quenching by R Acceptors. When T C N E was added in excess to anthracene, the fluorescence of anthracene was effectively quenched. This suggested a study of the relative quenching abilities of other A acceptors to determine whether a better quenching agent could be found. The quenching of pyrene was studied since the formation constants for many of its complexes in the ground state are known. This also avoided the possibility of the Diels-Alder reaction. Small amounts of R acceptors dissolved in methylene chloride were added to pyrene dissolved in that solvent or in dioxane. As shown in Table 11, the quenching abilities of the various A acceptors roughly parallels their strengths in forming ground state complexes with pyrene in methylene chloride. This is consistent with the findings of McCartin (6) for the quenching of zinc phthalocyanine by nitrosubstituted A acceptors. Both T N F and 7,7,8,8-tetracyanoquinodimethane (TCNQ) appear to be more effective quenching agents than tetranitromethane. Thus, 10-4M tetranitromethane has been reported to quench 4 X 109M benzo(a)pyrene completely (11), compared to the amost complete quenching of pyrene by only a twofold excess of either TCKQ or TNF. As in the case of T N F ground state complexes (16), it appears that the greater overlap of R orbitals possible with large A acceptors makes them more effective quenching agents than small R acceptors, such as TCNE. A simple calculation with the following equation (15) permits an evaluation of the relative amounts of complexed and uncomplexed pyrene in the ground state :
Thus for TCNQ or TNF, the complexed pyrene in the ground state is less than 0.5% of the uncomplexed pyrene. For TCNE, the complexed pyrene is calculated to be less than 0.2y0of the un-
complexed. It does not appear likely that such weak ground state complexation could be responsible for almost complete quenching. Certainly, complexation in the excited state (6, IO) is a more attractive mechanism. The data in Table I11 for quenching in dioxane indicate that the solvent does influence quenching efficiency as might be expected if complexation were involved. TCKQ and tetrachlorobenzoquinone, both quinoid structures, are better quenching agents in dioxane than in methylene chloride. TCNE is less efficient in dioxane than in methylene chloride. Since the formation constants in Table I1 do not hold for dioxane, there is no correlation possible between the constants and the results in dioxane. The concentration of pyrene was kept a t the 10-4M level to avoid excited state dimerization (19). Quencher Concentration Effects. McCartin (6) has noted that the ratio of fluorescenceyields ( F o / F ) ,in the absence and presence of a quenching agent, varies linearly with the quencher concentration for weaker quenchers. For the strongest quenchers, this ratio is given by a quadratic equation in (T), the molarity of the quencher. Table IV contains data showing the variation in the fluorescence yield of anthracene with changes in concentration of TCNE. Xeglecting any correction for dilution, both sets of data give fairly linear plots of F o / F V S . concentration of TCKE until dilution errors become severe. A possible conclusion is that TCNE is not one of the strongest quenchers of anthracene. Certainly its smaller R orbital system overlaps much less with that of anthracene than do the large R orbitals of TCYQ or TXF. Excitation and Emission Spectra. Studies have been made of the fluorescence emission spectra of R complexes resulting from fluorescence excitation a t wavelengths where the complexes themselves absorb light. For example, fluorescence excitation of the phenanthrene-tetrachlorobenzoquinone complex a t 467 mp results in a fluorescent emission peak a t 676 mp (19). Our investigation was not concerned with this phenomenon, but instead with the effect of a R acceptor on the fluorescence excitation and emission of the aromatic donor itself. The fluorescence excitation of pyrene (Table V) was not carried beyond 410 mp since the primary filter in the Farrand spectrofluorometer cuts off excitation light beyond this wavelength. Since 410 mp is well short of the first pyrene-TCXE complex absorption peak a t 495 mp ( 8 ) , this seems t o indicate the measurements in Table V are essentially the fluorescence emission of pyrene, not the A complex. The excitation peak used for pyrene was the one normally employed (18).
Table V. Effect of A Acids on Excitation and Emission Peaks of Pyrene
1.5 X 10-6M Pyrene Excitation Emission maxima, maxima, Solvent-Acid mp mC1 CHpClz-none 370 450 CHpClz0.002M TCNQ 340,345 465 CHpClp0,002M TCNE 350 430 Dioxane-none 390 455 Dioxane0,002M TNF 370 430 Dioxane0.002hl T C S E 350 500 The data in Table V reveal an apparent effect of the K acceptor on the fluorescence excitation and emission peaks of the K donor itself. All of the acceptors appear to cause a decrease in the fluorescence excitation maxima of pyrene. [This is in direct contrast to the fluorescence excitation maxima of K complexes which shift to longer wavelengths with respect to the same bands for the donor (19).] However, other
measurements involving TCKE and anthracene or benzo(a)pyrenein dioxane showed that T C K E caused an increase of 20 to 30 mp in the fluorescence excitation peaks of these donors. The effect of the K acceptors on the fluorescence emission of pyrene itself is not uniform; again T C S E caused an increase of 20 to 30 mp in the fluorescence emission maxima of anthracene and benzo(a)pyrene in dioxane. KO explanation for these effects is offered a t this time. LITERATURE CITED
(1) Bachman, W. E., Kloetzel, M. C., J . Am. Chem. SOC.60, 481 (1938). (2) Czekalla, J., Briegleb, G., Herre, W., 2. Elektrochem. 63, 712 (1959). (3) Gouterman, hl., J . Chem. Phys. 37,
2266 (1962). (4) Gusynin, V. I., Tal’roze, T. L., Doklady. Akad. Nauk. SSSR. 135, 1160 (1960), CA 56, 207%. (5) Jurinski, N. B., de Maine, P. A. D., J . Am. Chem. SOC.86, 3217 (1964). (6) McCartin, P. J., Ibid., 85,2021 (1963). 17) Melbv. L. R.. Harder. R. J.. Hertler. W. R.Y’Mahler, W., Benson, R. E.; Mochel, W. E., Ibid., 84, 3374 (1962). (8) hlerrifield, R. E., Phillips, W. D., Zbid., 80, 2278 (1958). ~
(9) Middleton, W. J., Heckert, R. E., Little, E. L., Krespan, C. G., Zbid., 80, 2783 (1958). (10) McGlynn, S. P., Boggus, J. D., Zbid., 80, 5096 (1958). (11) Miller, J. A., Baumann, C. A,, Cancer Res. 3, 217 (1943). (12) hlurrell, J. Pi., J . Am. Chem. Soc. 81, ,5037 f 19.59’i. (13)Sawicki, E., Stanley, T. W., Elbert, W. C., Talanta 11, 1433 (1964). (14) Schenk, G. H., Ozolins, M., Zbid., 8, 109 (1961). (15) Schenk. G. H.. Santiago. M.,Wines. P., ANAL’cHEM.’35, 16771963j. (16) Schenk, G. H., Yance, P. W., Pietrandrea, J., Mojzis, C., Zbid., 37, 372 (1965). (17) Simons, J. P., Trans. Faraday SOC.56, 391 (1960). (18) Tan Duuren, B. L., AKAL.CHEM.32, 1436 (1960). (19) Tan Duuren, B. L., Chem. Revs. 63, 325 (1963). (20) Tan Ihuren, B. L., Bardi, C. E., ANAL.CHEM.35, 2198 (1963). (21) Wvant. K. E.. Poziomek. E. J.. Poire;, R.’H., A n d . Chim. Actb 28, 496 (1963). \ - - - - I
~
RECEIVED for review February 1, 1965. Accepted April 19, 1965. Work supported by Public Health Research Grant GM07760 from t)he National Institutes of Health.
Determination of 2-Methyl-6-tert-Butyl-p-Cresol in Gasoline BERNARD BRAITHWAITE, GEORGE PENKETH, and LlLlAN UNDERWOOD lmperial Chemical lndustries limited, Heavy Organic Chemicals Division, Research Department; Billingham, County Durham, England
b A method for the determination of 2-methyl-6-tert-butyl-p-cresol (24M6B) in gasoline is described. The antioxidant is chromatographically separated b y absorption on alumina followed by elution of the gasoline with chloroform and pentane. The pentane removes the last traces of gasoline and chloroform, and a final elution with ethanol removes the 24M6B from the column. The antioxidant in the eluate is then determined colorimetrically by oxidizing with potassium ferricyanide and coupling with diazotized p-nitroaniline.
T
to protect modern gasolines against aerial oxidation has led to the development of two main types of antioxidants based on either aromatic amines or hindered phenols. The effectiveness of these antioxidants, normally added to gasoline at the 0.01 to O.lyolevel, can be assessed by some form of accelerated oxidation test ( 1 , 2 ) in which the sample-pressurized with oxygen in a bomb-is HE NEED
heated at 100” C. until degradative oxidation occurs. However, although they give an indication of antioxidant efficiency, these tests do not simulate actual conditions of use or storage and their precision leaves much to be desired. I n an attempt to devise a more rapid test, Hamilton and Tappel (8) developed a polarographic technique based on measurement of available oxygen. For many purposes however, a direct determination of antioxidant content of a gasoline would be preferable. Considering that the petroleum industry now uses about 4000 tons of costly phenolic and amine antioxidants per annum, it is surprising that so few methods for their determination have appeared in the literature. This is probably due to the difficulties associated with the isolation of small quantities of antioxidants from such a complex substrate as gasoline. Williams and Strickland (11) determined amine antioxidants by reaction with phosphotungstic acid after HCI extraction, while Gaylor, Conrad, and Lander ( 7 )used a polarographic method.
As far as we are aware no specific methods have been published for the determination of phenolic gasoline antioxidants. One of these-widely used in Korth America-is 2,6-di-tertbutyl p-cresol (4M26I3). This could probably be determined by an adaptation of the methods used by us for the determination of this antiosidant in turbine oils ( 5 ) . 2-Methyl-6-tert-butyl p-cresol (24M613), however, finds considerable application in Europe and the Middle East and it is with thedetermination of this compound that we have been concerned recently. Analytically the problem is complicated by the fact that 24M613 is often used in admixture with other alkylated phenols, h-hich may amount to as much as 20 to 25y0 of the total. With this in mind the best chance of success was considered to be in developing a specific method for 2411613, that could be used for either the pure compound or miytures. Infrared spectrometric method5 based on measurement of hydroxyl absorption a t 2.87 microns (6) could obviously be discounted, while other absorption VOL. 37, NO. 7, JUNE 1965
913
