ANALYTICAL CHEMISTRY
248 Table 11.
Replicate Data Obtained with Nitrogen and Helium
Kitrogen Containing 0.00083 Vol. '70 0 2 " Dew Dew point Ozr iioint, H:, vel. 0 F. F. 7%
-
+
- 60 -GO -GO
- 5G - 59
-$;
0 0008
0.0011
-aa
-56 -52
0.0008 0.0010 0.0009 0.0008
__
-0a
-57 AI'. 0.0009 .\lean dev. &0.0001 Clieniical analysis ( 3 ) .
-61
0
Helium Containing 0 00113 Vol. % Oaa Dew Dew point Oa. point, Hz, vol.
F.
- 54 - 54 - 54 -51 - 02 - 50
+
' F.
%
-50 -49
0.0011 0.0014
0.0011 0.0013 0.0010 0.0011 0.0012
-50 -47 -49 -47
4v. Mean dev. =kO.OOOI
I n high prescure gas cylinders, there is almost always stratification, which, if not recognized, will produce erroneous and discouraging resultq. This is almost always true of the first samples of gas removed. These replicate runs were made on gases fram cylinders which were halt emptied in the course of the preliminary experiments. In addition a number of cylinders contained free water and the moisture conteiit of the gas increased as the cylinder was depleted; this should be taken into account if the moisture content as well w the oxygen content must be controlled or avoided. The contribution in terms of total oxygen (H20 0 2 ) of the added hydrogen, is negligible. The hydrogen flow is 5 cc. per minute while the test gas flows a t -2800 cc. per minute. The 02) in the electrolytic hydrogen used was total osygen (H,O 0.04 volume %. Thus the positive error from this source is only 0.00007%. Because of this negligible effect, there is no need to use any special grade of hydrogen nor to resort to elaborate
+
+
means of cleanup, such as diffusion through palladium. Stoichiometrically 5 cc. per minute of hydrogen are in excess of the usual oxygen concentrations in gases. This rate is 0.22 millimole per minute, which would be equivalent to 0.0892 volume yo of oxygen in the test gas a t a flow rate of 2.8 liters per minute. There are no interfering compounds in the common gases such as nitrogen, argon, hydrogen, helium, or carbon dioxide. The only care required is to exclude particulate matter and oil vapors which may deposit on the mirror and necessitate cleaning. ,4 simple glass wool filter in the gas supply will usually eliminate such interference. As the freezing point of carbon dioxide is - 110" F., and it can be liquefied only under pressure, there is no interference from carbon dioxide. ACKNOWLEDGMENT
The author is particularly indebted to William N o a k of this laboratory for his valuable assistance in fabricating the prototype instrument and obtaining most of the data, and Florence Blinn of this laboratory for performing the chemical analyses for oxygen in the test gases. LITERATURE CITED
Y. "General Electric Dewpoint Recorder Instructions," GIE-40444. ( 2 ) Ilixson. A. W., and White, C. E., IND. ENG.CHEM...LS.AL. ED., ( I ) General Electric Co., Schenectady, S .
10, 235 (1938). (3) Pepkowita, L. P., and Shirley, E. L., .IN.AL. CHEM.,25, 1718 (1953). RECEIVED f o r review July 31, 1954. Accepted October 18, 1954. T h e Knolls Atomic Power Laboratory is operated by t h e General Electric Co. for the Atomic Energy CommisSion. Work carried ont rinder Contract So. W-31109 Eng-52.
Determination of Benzo[~]pyrene in Complex Mixtures Use of Catalytic Iodination on Activated Alumina RUSSEL TYE, MARY JANE GRAF, and A. WESLEY HORTON Kettering Laboratory, University of Cincinnati, Cincinnati, O h i o
This paper descrihes the isolation and identification of the polyc>clic h?drocarhon, benzo[a]pqrene, in a residual product of the cata1)tic cracking of petroleum, together with the anal) tical method developed for the estimation of the concentration of this compound in products of refining operations. 4 selected fraction of a given sample is obtained by the use of a standardized chromatograph). Two equal portions of the fraction are taken, and one of them is subjected to a catalj tic iodination on a column of actitated alumina. Spectrophotometric measurement of the difference in absorbance hetween the iodinated portion and its mate is then used to determine the concentration of benzo[a]p?rene present.
A
HIGHLIGHT of the many years of effort by numerous investigators to identifl- the carcinogenic constituents in certain tars produced from coal was the isolation and synthetic proof of structure of benzojalpyrene by Hieger (1'0)and Cook and Hewett (6). .\]though there has been no direct proof of the relationship of the pure h j drocarbon to human cancer, such effects on certain experimental animals have been well documented (9). I n the course of the current investigation of the possible carcinogenic properties of high boiling products from petroleum
refining operations, it was observed that the ultraviolet absorption spectrum of a distilled fraction of a catalyticallj cracked residuum showed maxima indicative of the presenre of benzopyrene in significant concentration. The compound u as isolated by the follom-ingprocedure in a sufficient state of puiity for positive identification As a matter of conservation of time, no attempt was made a t quantitative separation. Fourteen hundred grams of the oil R ere subjected to a simple vacuum distillation. A fraction, boiling a t 205" to 275" C. a t 0.5 mm. of mercury and weighing 175 grams, was subjected to chromatography on alumina, and fractions enriched in benzopyrene \$ere selected spectrophotometrically. The chromatographic procedure was repeated five times, i~esultingin 6.5 grams of a red semisolid, the ultraviolet absorption spectrum of a-hich indicated a content of benzopyrene between 0.8 and 2.5 grams (curve I, Figure 1). The formation of iodine complexes (4,1 4 ) folloa-ed by filtration was used to remove pcrylene and other unideptified compounds, reducing the weight of the concentrate to 5 grams. This was dissolved in benzene and extracted with cold, concentrated sulfuric acid. The acid was diluted with ice, and the resulting precipitate was dissolved in benzene (for spectrum, see curve 11, Figure 1). Further fractionation by chromatography and fractional crystallization from n-heptane produced 0.116 gram of (#rudebenzopyrene, having substantially the same ultraviolet absorption as that of a n authentic sample. Further recrystallizations from n-heptane and from eth? I alcohol produced 12 mg. of crystals, melting point 175-176.5' C. (corrected). The melting point of a mixture with synthetic benzo-
V O L U M E 27, NO. 2, F E B R U A R Y 1 9 5 5
249
pyrene was 176-17io C. (corrected), a s compared with that of the pure material, 178-178.5" C. (corrected). The spectrum of the isolated compound is shown a s curve 111, Figure 1. The niethod of Goulden and Tipler ( 8 ) , as adapted by Waller ( 1 7 ) ,for the determination of benzopyrene in soot and city dusts utilizes chromatography and fluorescence spectroscopy, the final estimation of concentration being made by visual comparison of the spectrum of the u n k n o n with that of a known sample composed of benzopyrene and suitable impurities. Falk et al. (6) and Wedgwood and Cooper (18) have used chromatography and ultraviolet absorption spectrophotometry t o separate and identify polynuclear aromatic hydrocarbons from complex mi.ctui,es. Fieser and Campbell ( 7 ) have suggested that a more selective analytical method for benzopyrene might be developed if the hydrocarbon were first converted to the highly colored derivative resulting from its reaction with p-nitrobenzenediazonium chloride. The method described in this paper attempts to utilize the chromatographic technique not only t o concentrate the benzopyrene but t o aid in identifying it by its rate of movement along
a carefully standardized column. Secondly, a selection is made by catalytic iodination, and finally, identification and measurement are accomplished on the basis of the change in the ultraviolet absorption spectrum caused by the iodination. The use oi difference spectra, such as that of Figure 2, furnishes a double qualitative confirmation that benzopyrene was actually the compound iodinated, as the curve not only shows the maximum a t 405 mp due to the derivative, but also negative peaks or inflections at approximately 368 and 389 mp due to the hydrocarbon. The method is relatively fast, requiring about 3 hours per analysis, or less if multiple analyses are made simultaneously. CATALYTIC IODINATIOY
I n the case of certain severely cracked materials, such as some tars from by-product coke ovens, it is possible to obtain a reasonable estimate of the content of benzopyrene from the absorption spectrum of a suitably beleeted chromatographic fraction. However, for the majority of the mixtures examined by the authors, a considerable improvement in accuracy is obtained by converting the benzop) rene to monoiodo derivative before spectrophotometric analysis. b n y interference due t o conipounds which do not react with iodine under the experimental conditions may then he essentially eliminated by use of the technique of difference spectra for analysis of the derivative.
~~
2400
2600
2800
3000
3200
34KJ
3600
3800
4000
4200
4400
Wave Length, Angstroms
Figure 1. Ultraviolet absorption spectra of three stages i n isolation of benzo[a]pyrene
Wave Length, Angstroms
Figure 3. Ultraviolet absorption spectrum of 6-iodobenzo[a]pyrene in iso-octane
I_
OAO 7 0.35
convenient method of accomplishing the desired reaction is t o pass a solution of benzopyrene and iodine in suitable concentration in benzene through a short column of activated alumina in the manner of elution chromatography. Under the conditions of the procedure used in this analysis, 6-iodobenzopyrene is produced in about 65% yield. (In the formulas below, t h e authors prefer the use of the symbolic ?r electron in presenting a resonance hybrid such as benzopyrene.)
I
I Benzo[a]pyrene
Wove
Figure 2.
Length ,m)c
Spectra involved in analysis for benzo[a]pyrene
6-Iodobenzo [alpyrene
The iodo compound has been purified by chromatography and recrystallization. Its melting point was 214' to 215" C. (corrected); its iodine content (calculated as 33.6% for C20H,,I) was 33.1%. The ultraviolet absorption spectrum is shown in
ANALYTICAL CHEMISTRY
250 Figure 3; it is noteworthy that the compound does not have any visibly perceptible fluorescence. The position of substitution was indicated by conversion of the iodo derivative to the nitrile by heating with a small excess of copper(1) cyanide [ C U ~ C N )in~ a' wiled glass tube a t 306" C. for 1.5 hours. .4 yield of 97% of cwde product was obtained. This product, when purified, mrlted a t 239.0-239.5" C. (corrected j . 6-Cyanobenzo[a!p!-re1ie, prepared by the method of W n d a u s and Raichle (19j, melted a t 238-239" C. (corrected), and, in a mixture with the first cyano compound, melted a t 238.5-239.0" C. (corrected). The ultraviolet spectra of the two samples were in agreement anti corresponded with the spectrum oht:ainetl by ,Jones ( 1I ).
\ i
highly fused structures, such as benzopyrene, and that of other components seems to be the important characteristic which makes it possible in this analysis to eliminate many potential interferences by the iodination step. The solubility of various polycyclic aromatics in cold, concentrated sulfuric acid (4)without sulfonation is presumably a function of the basic properties of the hydrocarbons. These solutions have vivid colors ranging from yellow to wine-red, suggesting the formation of onium-ion complexes (3, 13). Perylene, :3-methylcholanthrene, benzopyrene, pyrene, and anthracene can be estrarted from benzene with varying degrees of completeness by an equal volume of concentrated sulfuric acid a t 10" C. Under the conditions of the analytical procedure, the same compounds react with iodine to similar relative extents. Benz [ a ] anthracene can be extracted by sulfuric acid but reacts negligibly ivith iodine. Chrysene, benzo[c]phenanthrene, dibenz [a,h]anthracene, and phenanthrene cannot be extracted by cold concentrated sulfuric acid, and are not iodinated by the analytical procedure. .I moi'e generalized check on the apparent rule that polycyclic, aromatic hydrocarbons not soluble in cold, concentrated sulfuric acid are not substituted by iodine under the conditions of this arialytical method, has been made. The cracked residuum from wliirh benzopyrene vas isolated, believed to contain also a wide variet). of other polycyclic aromatics including alkylated and hydrogenated derivatives, was exhaustively extracted by cold concentrat,ed sulfuric acid, about 50% of the aromatic compounds being thus removed. Two equal portions of the nonextract (raffinate) were taken, one portion iodinated, and the difference spectrum measured according to the standard analytical procedure. S o apparent reaction had occurred. APPAR-hTUS
The chromatugraphic colurnn whirh is used to prepare a suitable fraction for the analysis is composed essentially of a reservoir and two separate sections of adsorbent, as shown in Figure 4. The spectrophotomet,er employed in the development of the method was the Beckman Model DU equipped with 1-em. rclle and a tungsten lamp. It is assumed that any comparable instrument, including the recording models, could be used equally well. Figure 4.
Apparatus
Other activated adsorbents also catalyze the iodination of benzopyrene. The p H of the adsorbent appears to be important. Under ronditions similar to those of the analysis, t,he use of .ittapulgus fuller's earth of Ion- activity and of a lower pH than grade F-20 alumina (as measured by p H meter on aqueous extracts) produces roughly equal yield8 of 6-iodobenzopyrene and an additional product, probably an iodo- or diiodobenzopyrene. h similar mixture of products is obtained from the alumina catalyzed reaction if the acidity is raised by a large increase in thr, relative quantity of iodine used. Activated magnesia, having a pH much higher than grade F-20 alumina, and activated silica gel, having a p H much lower than the iittapulgus fuller's earth, promote only faint reactions. The use of silica gel to which sodium bicarbonate has been added results in an increased hut still small amount of iodination. .i mixture of sodium hicarbonate with celite shorn no activity. The relative ability of different aromatic hydrocarbons to form molecular complexes with Lewis acids has been discussed bjseveral authors (1-3, 12, I S ) and related by Mulliken (13) and Brown ( 3 ) to the ease with which these compounds may be halogenated. Brown found a simple linear relationship between the logarithms of the relative rates of halogenation of several methylbenzenes and the logarithms of the relative stabilities of their deeply colored onium-ion complexes with hydrogen fluorideboron trifluoride. I n dealing with mixtures of polycyclic aromatic hydrocarbons the diffei,enc>ebetween the basicity of the
MATERIALS
Iso-octane, Phillips Petroleum Co., pure grade, redistilled. Benzene, C.P. thiophene-free, redidlled. Solution of benzo [alpyrene, Edcan Laboratories, c.P., 5.00 mg. in 100.0 ml. of benzene. Developing solvent, mixture of 200 ml. of iso-octane (2,2,1-trimethylpentane) with 200 ml. of benzene. Solution of iodine (reagent grade), 1 gram in 10 ml. of benzene, freshly prepared. ilctivated alumina, Alcoa, Grade F-20, 80 to 200 mesh. Solution of sodium thiosulfate, c.P., approximately 5 gram-, with auuroximatelr 50 my. of Dotassum iodide, c.P.. in 200 ml. of distille7 water. ' Sand, nonporoup, white si1ic:t. l i
~
PROCEDURE
Volume of Developing Solvent Required for Satisfactory Chromatographic Fractionation. A chromatographic column of alumina is prepared as shown in Figure 4. Because the quantit\ of adsorbent used is measured volumetrically in the column itself, it is important that the procedure for packing the columns in thih and the next step of the procedure be as uniform as possible. h satisfactory routine consists of tapping the loosely filled column six times with a pencil. Ten milliliters of the developing solvent are poured on to the column followed by 10 i 0.05 ml. of the solution of benzopyrene and then by an additional 100 ml. of the solvent. Each portion of liquid should be allowed to enter thr top layer of sand before the next is added. Additional solvent is then added, 10 ml. at a time, until the top of the benzopyrene band, a s indicated b y blue-violet fluorescence under ultraviolet light (the General Electric Co. Purple-X lamp bulb is a satisfactory source of light for this purpose), is approximately 0.25 inch below the top of the lop-er section of alumina. At this point, the fluoresrent compound will usually be spread over about one third of the lower section of alumina.
V O L U M E 2 7 , NO. 2, F E B R U A R Y 1 9 5 5 JVith adsorbent of normal activity, this development will require a total volume of about 120 to 150 ml. of the solvent (not including the two 10-ml. portions used to prewet the column and as solvent for the benzopyrene). T h e total volume of solvent thus det,ermined is used a s the "standard volume" for the separay r y chromatography (below)). T h e use of alumina requiring a 'standard volume" of solvent less than 100 ml. is to be avoided if possible, a s the effectiveness of the separation of benzopyrene from interfering components is thereby reduced. T h e only kno\vn disadvantage in the use of alumina requiring a standard voliime greater than 170 ml. is the additional cost in time and material. Alumina of unusually high or low activity could prob:il)ly I)e used satisfactorily by modification of the composition of the developing solvent, but should in that case be checked furt,her w i t h pure benzopyrene to determine whether it has a satisfactory c*:it>xlyticaction in the iodination reaction described belox. -42 a rule, the activat,ed adsorbent needs to be calibrated only oriw foi, a given series of analyses, so long as all of the alumina is takcn from the same can and adequate care is taken to minimize the periods during which the cxn is open and t o keep it tight]!. sexled a t other times. Preparation of Fraction for Iodination. Approximately 100 mg. (weighed to the nearest 0.1 mg.) of the material to be analyzed a r r dissolved in 5 nil. of benzene. After the material is completely in solution, 5 nil. of iso-octane are added. (If part of t'he inaterial is insoluble in benzene, or if precipit,ation occurs on adtiition of iso-octane, the sample should be subjected to a sliecial procedure. See modification below-.) The material is t1it.n fractionated on a freshly packed chromatographic coluniri iri the exact manner described above, substituting this solutiori of thr unknown for the solution of benzopyrene used to calibrate t h r :ilamina. After the standard volume of the developing solvent h:te IUII tliivugh the column, the sections are separated and the material :iclsorbed on the lower section is eluted by 50 ml. of a solution of 20yoethyl alcohol and 80% benzene (by volume). Most of the jolverit is removed from the eluate by evaporation on a steam bath. The remainder is removed by blox-ing under a stream of nitrogen with care to avoid loss by spattering. T h e residue, wpresenting a concentrate of a n y benzopyrene present in the s:mple, is then ready for the iodination step. T h e suspension of 100 mg. of the sample in lIoDIFrc.\TIoN. c.ither 5 ml. of benzene or 10 nil. of the 50 to 50 mixture of benzene :ind iso-octane, as the case may he, is poured onto a column of alumina 1 inch in diameter anti 1 inch deep (comparable to the up1)er section of the column in Figure 4). T h e flask in which the suspension was prepa1,ed is n-ashed thoroughly with 10-ml. portiona of benzene, and the washings are added to the column. .\ftrr this material has passed into the adsorbent', the column is eluted with 100 ml. of benzene. IIost of the solvent is removed f r o m the eluate by evaporation on a steam bath, the remainder, by c:irei"ul blowing under a stream of nitrogen. The residue nl)t:iiiicd is dissolved in 5 ml. of benzene, 5 ml. of iso-octane a w :idtled, and the analysis resumed a t the point of interruption. Iodination. The residue from the step above is dissolved iri 13 + 0.05 ml. of benzene, and two 5-ml. portions, labeled I A n t i IT, of the resulting solution, are transferred to two clean 125-3111. flasks, using the same pipet for each. Five milliliters of the solution of iodine are added t o I, and 5 ml. of benzene added to 11. Two parallel columns of alumina are prepared, each 1 inch deep and 1 inch in diameter, covered with 0.5 inch of sand. Each ttoluinn of adsorbent is wet by 15 nil. of benzene and allowed to dr:iiii until the frequency of drops is less than one in 10 seconds. Clean, tared, 125-ml. flasks are then placed under t,he columns. Five milliliters of the solution of iodine, diluted with 5 ml. of l)enzene, are poured onto t,he first column, and 10 ml. of benzene alone are poured onto the second. Solutions I and I1 are then atided to the respective columns, the flasks \+-hichhad contained them are rinsed by 10 ml. of benzene, and the rinsings added t'o the proper columns. Each is then eluted b y 70 ml. of benzene a n d allowed to drain a s before. T h e eluate from column I, containing the iodine, is then shaken with 200 ml. of the aqueous sodium thiosulfate in a 500-ml. separatory funnel, after which i t is washed with two 100-ml. portions of distilled water. T h e eluate obtained from column I1 is used directly in the final step without further handling. T h e prxessed fractions, I and 11, should be subjected to the ..l)~,ct,rophotomet,ricanalysis without delay, since the iodinated sample occasionally proves to be unstable on standing overnight. Determination and Analysis of Difference Spectrum. T h e algebraic differences bet.ween t,he absorbances of the final fractions, I and 11, in the 370 to 420 mp range are now measured directly by use of a Beckman DC spectrophotometer. Sample I is placed in the 1-cm. cell normally used for solutions, and sample I1 in the 1-cm. cell used for blanks. Obviously, the cells used must be well matched as to length. T h e instrument is operated
25 1 i n the normal manner except that, when I 1 has the higher absorbance, the machine is balanced on I, and the absorbance is recorded a s a negative value (see Figure 2). T h e slit width must be kept, moderately lo~v,below 0.1 mm. being desirable. The sensitivity knob should therefore be turned to a point near its extreme counterclockwise position (minimum sensitivity), arid the t,ungsten lamp should be used. T h e reduced slit widths arts necessary because the iodinated solution I usually has a markedly lower !eve1 of fluorescence than the solution 11. This differenc assumes critical importance when compared with the differen in absorption rather than with the usual t,otalabsorption. Hence, if the slit .\\-idthrequired a t 380 mk exceeds 0.15 mm., 10.00 ml. of he diluted n i t h 50.00 ml. of benzene prior t(J 'ses may be obtained from difference spectra in which the measurcd alisorbance a t 389 or 405 mp is 0.02, 01' greater, so long as each point, is determined carefully and the absorbance is plottkd on a suitable scale. If the absorbance is less than 0.02, longer cells (5- or 10-cm.) should be used. Alternat,ively, solutions I and I1 may be concentrated by the following procedui,e: T h e \\-eights of the solutions are determined. Then they are evaporated on a steam bath sufficiently t o obt:Lin the desircd level of absorhancc,. Their reduced weights are adjusted to equal percent3ges of their respective original weights vith benzene (this percent,agcis then used as the value of the facto],,T', in the equation below). The difference in absorl1:ince is determined at 380, 389, arid -405 nip s i i r l a t every interval of 4 mp in the region 420 to 370 nip. The following base-line technique (15, 20) is applied to the differenw spcctrum obtaincd: A line is drawn intersecting the curve a t t,he points corresponding to 40t5 and 380 mp. The d i s t m w ( i n absorliance units) along the vertical coordinate a t 389 nip from P Figure 2). the curve to this line is designated as L ~ S(see Geometrical analysis of the system provides thf, folloi\.ing rc.lntionship: &IBP
=
+ 0.:36.4$(5 - '4
o.(~~.'~.;8(8
i,u
Direct use of this equxtion \\-ittiout plottiiig is \\.ai,rantecl only when expei,ience has provided satisfactory qualit:it ire rvideiice of the presence of henzop!-rriic~ nnd :ihence of Pigriificant i n t ~ r f e r ence f w m perylene. T h e percentage of benzopyrene in thts oi~igirial s:iinple is ralculated from the equation:
hew TI7$
1- =
oi,iginal weight of sample in milligrams 01' concentration of t,he f i r i a l solutions was required 600 if dilution was required
1- =
T172
=
1. = 100 if no dilution
TI-:
rr, x
100 if concentration was required
the \\-eight of the iodinated fraction aft,er concentration Tl-, = the weight of the iodinated fraction before conwntration i = length of spectrophotometer cells in centimeters T h e constant, 0.23, is the slope of the essentially linea1,relationship hrtween AABP and the content of beneopyrrne over the range- of concentrations studied, 0.01 t o I .5yo. =
SENSITI\-ITY AND REPRODUCIBILITY
Consideration of various factors inherent in the procedure indicates that the limit of sensitivity of the method under ideal ronditions is about 0.0001 mg. of benzopyrene. In actual anal>.Pes,the presence of other conipounds, which modify the shape of the final difference spectra, limits the practicaal sensitivity to about 0.001 mg. Quantities of pure benzopyrene sufficient to provide from 0.01 to 0.5% were dissolved in several different oils of widely varying type, to which this analytical procedure had been applied previously. I n each case in which the amount of added benzopyrene represented a significant increase in the total present, the standard analypis yielded a reasonably precise measure of the quantity added, as shown by the examples in Table I. Data from a number of analyses involving duplication by four different operators indicate :i reproducibility within the limits of
ANALYTICAL CHEMISTRY
252 Table I.
Oil Number 1 2 3 4
5
A Benzo[ alpyrene Found in Original Sample,
used to calculate the content of benzopyrene, should then be corrected by subtracting the quantity 0.1 A A p where:
Precision of Method E
%
A and B Indicated Total, %
by Analyses,
0.015 0.428 0.0012 (0.0002) 0.086
0.101 0.490 0.0100 0.0099 0.198
0.116 0.918 0.0112 0.0101
0.114 0,909 0.0111 0.0098 0,283
0.284
A A p = 0.5.4ilzz
Tot+ Determined,
Concentration of Benzo[ alpyrene .4dded, %
%
about &20% for samples with benzopyrene concentrations around O.Ol%, 2~10%for concentrations of Q.lO%, and is% for concentrations of 0.50% or greater. Seven other research laboratories are now cooperating in a further evaluation of the analvtical method.
11. In cases of samples 1 and 2, percentages are based on the total benxopxrene indicated by summation of the weight of synthetic benzopyrene added and that determined by independent ordinary analysis of fraction A. The table shows that the apparent benzopyrene from the oil does not differ chromatographically from synthetic benzopyrene. There is some reason, therefore, to believe that alkylbenzopyrenes are creating no serious interference in the analysis of the oils. Although perylene and some of its alkyl derivatives remain in the benzopyrene fraction obtained from chromatography, their concentration in cracked products is usually so low, relative to that of the benzopyrene, that it does not interfere significantly. I n the analysis of certain straight-run distillates, however, the perylene family does create a positive error. A high, positive slope of the final difference spectrum in the 410 to 420 mp range is an indication of such an interference, which may be confirmed by finding maxima due to iodoperylenes at 450 to 458 mw and 422 to 425 mp, and corresponding minima due to the hydrocarbons at approximately 438 and 410 mp. The value of A A B P ,
O.5A:ji
I n the analysis of certain types of materials, such as crude residua, difference spectra are occasionally obtained which lack sharp maxima and minima or ne11 defined inflections and do not furnish satisfactory qualitative evidence of the presence of benzopyrene. Hence, the significance of results obtained by application of the base-line technique to such spectra is uncertain. Various methods of eliminating the obscuring interference are being examined.
Tahle 11. Chromatographic Comparison of Apparent Benzopyrene from a Complex Oil with Pure Compound
INTERFEREKCES
Many compounds other than benzopyrene react Tvith iodine under the conditions of the iodination step of the analysis. Among these are anthracene, p j rene, 3-methylcholanthrene, and perylene. The majority of such compounds are separated from benzopyrene by the chromatographic fractionation step. Of the compounds which accompany benzopyrene through this separation and the subsequent iodination, the alkyl derivatives of benzopyrene are perhaps the most important potential sources of interference, if present in any significant concentration. It is not known how their presence would effect the final analysis. The following experiments were carried out in an effort to obtain some estimate of the extent of such interference, if any. Analysis of a 20% residuum from distillation of the oil from which benzopyrene was isolated ( S o . 2 in Table I ) indicated the presence of 1.8% of this hydrocarbon. A second sample of this residuum was fractionated according to the second step of the procedure. One fifth (termed fraction A in Table 11)of the chromatographic fraction containing the benzopyrene was then subdivided by a more refined chromatography into three fractions containing approximately equal portions of benzopyrene, and these were analyzed by iodination and difference spectra. A measured quantity of synthetic benzopyrene was then added to a similar one-fifth portion of the standard chromatographic fraction from the oil. This sample was then subdivided by a careful reproduction of the previous chromatography, and the three resulting fractions were analyzed. Duplications were then made for both experiments. Finally, pure benzopyrene was processed chromatographically in duplicate expel iments similar to the preceding ones, and the resulting fractions nere analyzed. The results of these experiments are shown in Table
- -4438 f
1. 2
3
Sample Fraction -4 of cracked residuum Fraction . I added benzopyrene Pure benzopyrene
+
Total Benzo[a] pyren’e Present, % Frartion Fraction Fraction Fraction 1 2 3 1 + 2 + 3 31
34 31
39 42 42 41 47
28
25 22 20
46
26
29 31
31 27
44
100 98 47 98 100
.4 technique has been devised which, applied to such materials, provides evidence that the actual concentration of benzopyrene is, at most, no higher than that indicated by the standard procedure. The values in question have usually been less than 0.003%. It has been found that a significant proportion of the compounds responsible for the obscuration of the difference spectra may be modified in composition (possibly polymerized) by subjection of a Tveighed quantity (approximately 100 nig.) of the sample to what amounts to a flash distillation a t atmospheric pressure without reflux. Application of the standard method of analysis to the distillate then yields a much improved difference spectrum. I n our experience, the value of the concentration of benzopyrene, thus determined, has not proved to be higher than that obtained by the routine method. Indeed, agreement between the values obtained by the t\ro procedures has been rather striking. 4PPLICABILITY
The method was developed primarily for application to vai ious petroleum products resulting from refining operations. It has been applied to more than 35 such materials and the analytical data thus far obtained have been plausible in the light of the history and the physical properties of the samples. The use of the method has been extended, in a limited manner, to substances derived from other sources, including coal tars and fractions thereof. No apparent difficulties were encountered n ith these materials. It seems likely that the analysis may be applied to mixtures extracted by benzene from a variety of additional materials such as soot, carbon black, condensates from atmospheric samples, and other materials which are derived from fossil fuels or the incomplete combustion of organic mixtures. I t should be recognized that the results of analyses of solid materials of limited solubility in benzene would not necessarily indicate the total amount of benzopyrene in the original sample, since the complete extraction of benzopyrene from such materials is frequently very difficult to achieve (16). LITERATURE CITED (1) Benesi, H. A . ,
and Hildebrand, J. H., J. Am. Chem. Soc., 70,
2832 (1948). (2) Ibid., 71, 2703 (1949). (3) Brown, H. C., and Brady, J. D., Ibid., 74, 3570 (1952). (4) Clar, E., “Aromatische Kohlenwasserstoffe,” Berlin, SpringerVerlag, 1941. (5) Cook, J., and Hewett, C., J . Chern. Soc., 1933, 398.
V O L U M E 2 7 , NO. 2, F E B R U A R Y 1 9 5 5 Falk, H. L., Steiner, P. E., Goldfein, S..Rreslow, A., and Hykcs, R., Cancer HesParch, 11, 318 (1951). Fieser, L. F., and Campbell, W.P., J . Am. Chem. SOC.,60, 1142 (1938). Goulden, E . and Tipler, ll.,Brit.J . Cancer, 3, 157 (1949). Hartwell, J. I,., “Survey of Compounds Which Have Been Tested for Carcinogenic Activity,” Public Health Service, Pub. 149, 2nd ed., pp. 197-232, 1951. Hieger, I., J. Chent. Soc., 1933, 398. Jones, I{. S . . J . .4m.Chem. Soc., 6 7 , 2127 (194.5). llulliken, R. S., Ibid.. 72, 600 (1950)
253 (13) Ihid., 74, 511 (1952). (14) Pestemer, AI., and Freiber, E., Ber., 74, 964 (1941). ASAL. C m x , 19, 29s (15) Seyfried, W. D., and Hastings, S. H., (1947). (16) Steiner, P. E., Cancer Research, 14, 103 (1954). (17) Waller, R. E., Brit. J . Cancer, 6 , 8 (1952). (18) Wedgwood, P., and Cooper, R., Analyst, 78, 170 (1953). (19) Windaus, A., and Raichle, K., Ann., 537, 157 (1939). (20) Fright, AI., IND.ESG. CHEM.,ASAL.ED.,13, 1 (1941). for review May 10, 1964. Accepted October 23, 1964. T h e majority of this work was carried out as part of t h e American Petroleum Institute Research Project MC-1. RECEIVED
Copper( I)-2,2’-biquinoline~ Complex in Aqueous Dimethylformamide RONALD T. PFLAUM, ALEXANDER I. POPOV, and NEIL C. GOODSPEED Department of Chemistry, State University of /owe, /owa City, lowa
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The copper(I)-2,2’-biquinoline complex was investigated in order to determine the nature of the absorbing species in a water-miscible solvent and the feasibility of utilizing this solvent in determining copper. The bis2,2’-biquinoline-~opper(I)ion was found to be responsible for the characteristic purple color developed in the reaction of the reagent with cuprous copper. A mixture of equal parts by volume of dimethylformamide and water w-asa satisfactory medium for this chelation reaction. Such a solvent mixture functions in the role of a reducing agent in the reduction of copper(II) to copper( I ) ion. 4 s a consequence, it was unnecessary to add a reductant to the s>stem. Reducible ions and those that enter into precipitation reactions interfere in the detelopnlent and measurement of color. The results obtained on the determination of copper in selected samples indicate the feasibility of employing the described s>stem for such determinations. An unreported maximum in the absorption curve of the complex was found and described. The ultraviolet absorption curves for the reagent in dimethylformamide and in isoamyl alcohol were obtained.
T
HE color reaction between the organic base, 2,2’-biquinoline, and copper(1) ion was first observed by Breckenridge, Lewis, antl Quick in 1039 (1). Since that time, several investig:rtors have *tiitlied the colored system from the standpoint of the selectivity of the reaction (3,5, 6). Breckenridge and coworkers (1)utilizetl g1:tciitl acetic acid as a solvent, in their studies whereas Hoste antl coworkers (3, 6, 6) have restricted themselves to the use of .rvater-ininiiscil,le alcohols. These latter investigators have tlctermined that the reagent is specific for copper and have proposed that a bis-2,2‘-hiquinoline-copper( I ) species existed in these nonaqueous media. The specificity of this reaction has led to some interesting proposals for. the steric requirements of the reagent in the chelation process ( 2 ) und lends great analytical significance t o its use. Applications of the colored complex to the determination of copper in a variety of samples have been described (4,7 , 8). All the determinations have been carried out in amyl or isoamyl alcohol with the use of hydroxylamine hydrochloride for the reduction of copper(I1) t o copper(1) ion. The standard analytical procedure requires a change in the oxidation st,ate of the metal ion and an extraction of the colored species out of aqueous solution into a nonaqueous phase. I n view of the specificity of this cuproirie reagent,, it seemed desirable to carry out a rigorous determination of the formula of t h e colored species involved in the reaction. This investigation
is concerned with such a study, together with the application of new conditions in the determination of copper. These permit the ube of a simplified procedure in the analysis of certain selected samples. APPhRATUS AND REhGEYTS
All spectrophotometric measurements \sere made with a Cary recording spectrophotometer. One-centimeter matched silica cells %ereused in every measurement. A Beckman Model C, pH
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~ ~ e ~ from the ~ Rohm and ~ Haas Co. Purification was effected by treatment with barium oxide for a period of 24 hours with subsequent rectification in an all-glass system. The fraction boiling a t 151” =t 1” C. was utilized a s the purified solvent. T h e 2,2’-biquinoline, obtained from the G. Frederick Smith Chemical Co., was used as received. A standard solution of copper(I1) ion was prepared by dissolution of pure copper nire in nitric acid and conductivity water. T h e p H of the final solution was adjusted to 5 with sodium hydroside’ All other chemicals used were of reagent grade quality. EXPERIlIENTA L
Investigation of Reagent. An investigation of the reagent was undertaken initially in order to ascertain its solubility and absorption characteristics. It was found that 2,2’-biquinoline is sparingly soluble in ethyl alcohol, dioxane, acetonit,rile, and isoamyl alcohol, ‘but very soluble in diniethylformamicle. Dissolution in dimethylformamide is not accompanied by any color change nor is color developed on standing for a t least 2 months. The reagent easily retains its chelative powers for this period of time. Thus, dimethylforninmide appears to be an ideal solvent for 2,2’-biquinoline. Ahsorption curves for 2,2’-biquinoline in the ultraviolet region of the spectrum are shown in Figure 1. Curves for the reagent in isoamyl alcohol and in dimethylformamide are presented. The same absorption characteristic? were ohserved in both solvents. S o variations were apparent for fresh and aged solutions. Recr:-stallization of the reagent from aqueous alcoholic mixtures did not result in changes in the absorption curve. Effect of Solvent on Color Reaction. The addition of solid 2,2’-biquinoline to a dimethylformamide solution containing 1 X 10-3JI copper(I1) ion praduced a faint purple color. The subsequent addition of 0.05 gram of hydroxylamine hydrochloride completely removed an>- purple color formed, and resulted in a colorless solution. The addition of water to a solution containing the reagent and copper(I1) ion in dimethylformamide or t o a dimethylformamide solution containing the reagent, copper ion, and hydroxylamine hydrochloride resulted in the formation of an intense purple color. The maximum color in the reartion was developed in the absence of hydroxylamine hydro-
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