Properties of High Boiling Petroleum Products Nonbiological Laboratory Methods for Predicting Carcinogenicity L. T. EBY, WILLIAM PRIESTLEY, JR., AND JOHN REHNER, JR.
Esso Laboratories, Standard Oil Development Go., Linden, N . J . AND
.MATNARD E. HALL' Humble Oil and Rejining Go., Raytown, Tex.
The only methods heretofore described for predicting carcinogenic activity have been limited to series of pure aromatic compounds or to high]>-specialized types of petroleum products. After examination of a number of different reactions and properties of polycyclic aromatic compounds, five nonbiological methods have been developed for the prediction of tumor potency of high boiling petroleum products. Each method gave about the same degree of correlation with biological data. These physical-chemical methods are all based upon measurements of certain types of polycyclic aromatic hydrocarbons. The results support the premise t h a t the carcinogenicity is a function qf the concentration of these types of compounds in oils. The methods, therefore, obviate the necessity of isolating or analyzing the actual carcinogenic compounds. The caffeine number method is preferred for routine analytical work. It is based upon a n ultraviolet examination of a n aqueous caffeine extract of the aromatic hydrocarbons. A standard deviation of 8.4 was obtained for the difference between the predicted and the biological tumor potency values of 123 different products. Reproducibility of the caffeine number was better than t h a t of the biological data. Comprehensive analytical data for all five methods are presented for a variety of high boiling petroleum products. These demonstrate t h a t a choice of methods is available, depending upon the particular types of products to be analyzed and the facilities a t hand.
S
EVERAL physical and chemical properties of polycyclic
aromatic hydrocarbons have shown limited correlation with carcinogenic activity. These include reactions with perbenzoic acid ( 7 ) ,lead tetraacetate (g), osmium tetraoxide ( 2 , 3 , 1 8 ) ,p-nitrobenzenediazonium chloride ( 8 ) , and maleic anhydride ( I d , 18). However, none of these reactions has proved to be particularly useful as a method for predicting the carcinogenic properties of high boiling petroleum fractions. [The term "carcinogenic" as used in this paper applies to those materials which produced a significant tumor and/or cancer response when repeatedly a p plied to the skin of certain experimental mice under specified conditions ( 1 4 ) .] I t is well known that Kennaway, Hieger, and others in England have investigated fluorescence spectroscopy as a means of identifying certain carcinogens, such as benzopyrene. The authors have investigated fluorescence spectra of chromatographic fractions of a variety of high boiling petroleum products. Although some correlation in limited groups of samples has been found, the method was not found to be generally applicable to a large variety of such products. Where the products are of a limited type-e.g., oils from certain virgin crudes, there appears t o have been some success in using refractive index and density measurements to predict tumor forming properties (1, 16, 17). After examining a large number of petroleum fractions from different types of refinery processes and sources (6),the following conclusions have been reached concerning the carcinogenic activity of petroleum products. 1. The major part of the carcinogenic activity resides in the portion boiling between 700" and 1000" F. Very little if any is associated with that portion boiling below 700" F. 2. None of the:carcinogenic activity is associated with the 1
Present address, C h e m t r a n d , h e . , Decatur, Ala.
nonaromatic fraction (paraffins, olefins, and naphthenes) of petroleum oils (14). 3. Most if not all of the activity is due to polycyclic aromatic hvdrocarbons. Some may be due to derivatives thereof, containing oxygen, sulfur, and nitrogen. 4. There is suggestive evidence that some of the oxidized material in high boiling petroleum samples may act to reduce the carcinogenic potency which the sample would have in the absence of the oxidized material. These four basic facts have led to the development of five methods for predicting the tumor forming potency of petroleum fractions. Two of the methods have been briefly described (6, 18). These and the other methods will be treated in detail in this paper. Also, the validity of each method for predicting tumor potency will be evaluated. BIOLOGICAL TUMOR POTENCY
It is neceseary to have a biological evaluation of carcinogenicity, or tumor forming potency, of a large number of different types of high boiling petroleum fractions before any correlations of chemical or physical tests can be reliably used to predict the biological activity. Biological researchers have not yet reached common agreement on which particular animal test may best measure the tumor forming activity of given material. In the present study, a large number of petroleum samples were available which had been painted on a single strain of mice in a standard manner (14) by the same observers over a relatively short span of time. It would have been difficult to obtain a more consistent set of results on such a large group of samples. Since many samples were not very active, the best expression of activity was in terms of total tumor formation, regardless of whether the growths were benign or malignant. Early tumor formation invariably resulted in a high incidence of cancer in later stages, but in cases where tumor formation was late in starting, the mice usually died before rancers appeared. In order to express the results of these
1500
,
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 teats in a single number for correlation purposes, an inverse function of the time for 50% of the mice to obtain tumors was employed (4). Thus, the biological tumor potency (BTP) is expressed as BTP 1O,OOO/~jo where t 5 0 = the number of days to 50% tumors, and % tumors = T T / ( S D T ) , where Ti" = total number of mice dead and alive with tumors, S = total number of survivors with and without tumors, and DT = number of mice dead with tumors. This method of evaluation omits those dead without tumors from both the numerator and denominator, which is equivalent to omitting those dead from nontumor causes from the calculation. The determination of the time of the most significant point of tumor incidence (50%) is made by a probability plot of percentage of tumors versus the logarithm of time. Here also, the numerical expression of the biological tests may be made in other ways ( 4 ) . However, the values of biological tumor potency presented here are self-consistent, and this is important for a comparison of predicted tumor potencies (PTP) obtained by nonbiological methods. By means of extrapolation, some' consistent values of biological tumor potency in the range of 15 to 20 can sometimes be measured. HoIvever, the average life span of the mouse is insufficient to measure a biological tumor potency of 10 or lower. This being the case, a biological tumor potency of zero is a fictitious number; therefore, 10 is the minimum value used for any inactive sample. Thus, an inactive sample is defined as having a biological tumor potency of 10 in this paper, even though it may have been assigned a value of zero in earlier publications ( 4 , 6, I O ) . Likewise, the minimum value in this paper for predicted tumor potency is 10. [A more complete description of the biological tests would, of course, also include a biological cancer potency (BCP) calculated in a similar manner, but considering only malignant growths. Biological cancer potency would always have lower numerical values than biological tumor potency since benign tumors usually precede malignant ones.]
+
METHODS OF PREDICTING TUMOR POTENCY
,411 of the methods described in this paper appear to be useful for predicting the tumor potencies of high boiling petroleum products. These methods share the common feature of being based on the measurement of certain types of polycyclic hydrocarbons. The premise underlying all of these methods is that the concentration of carcinogenic compounds in the high boiling petroleum fractions occurs in a nearly fixed ratio to the concentration of certain types of polycyclic hydrocarbons that are present. 90tests have been devised which are specific for carcinogena present in these types of mixtures. Consequently, exceptions may be found in the correlation of any of these tests with biological data, especially when synthetic blends of carcinogenic hydrocarbons are employed. It was found that the different methods do not always predict the same tumor potency value for a given sample. Hoir-ever, it will 1~ shown later that the degree of correlation with the biological potency values is nearly the same for all of these methods. Even though the over-all correlations of the methods appear more or less equivalent, discrepancies in individual samples will vary with the particular method chosen because each method is subject to its own type of interference from noncarcinogenic components in the oil or tar mixtures. More evidence is accumulating (11, 13, 1 6 ) to show that the carcinogenic effect of a mixture of polycyclic hydrocarbons is not simply additive and is not always predictable. It is possible that the carcinogenic activity of an oil is not necessarily dependent upon the presence of a minute concentration of a particular carcinogenic compound, but it may depend upon the resultant effects of many compounds of certain polycyclic types which, when
1501 taken alone, are not regarded as carcinogenic. Therefore, it is possible that, as in the present study, the proper method for predicting tumor potencies of mixtures of a multitude of compounds, like a petroleum product, can be derived from empirical correlations based on properties of the types of compounds present. Tests for specific compounds, for this reason, may even fail to correlate with biological tests on a wide variety of products. For example, unsuccessful attempts were made to correlate the biological data with coupling reactions of the diazonium compounds and with chromatographic-fluorescence identification of benzopyrene for a group of petroleum samples in this laboratory. Ultraviolet Method. This was one of the first methods applied generally to high boiling petroleum fractions ( I O ) . It was based on the segregation of a 650' to 1000O F. distillation fraction and an ultraviolet absorption measurement a t 360 mp thereon. Since the alkylated benzenes, naphthalenes, and phenanthrenes boiling in this range do not absorb a t this wave length, the absorption is due to certain polycyclic hydrocarbons with which the carcinogenic hydrocarbons are associated. A viscosity correction factor was also applied because it was found that some very viscous samples had a high ultraviolet absorption but were not biologically active. SimplifiedUltravioletMethod. I t is somewhat difficult t o obtain a reproducible 650' to 1000° F. distillation fraction for a routine test. After a close examination of a large number of spectra of different undistilled samples, it became apparent that a correlation could be made using the absorptivities a t three wave lengths, 314, 364, and 470 mp, which would be as good as the correlation obtained with the absorptivity of distilled sample a t 360 mp. The viscosity correction factor was retained. N o other combination of wave lengths seemed to be as adequate for general use. The same absorptivities may be measured in isc-octane if the wave lengths of 340 and 360 mp are used in place of 344 and 364 mp with benzene. The first studies were made with iso-octane so that more complete spectra could be examined. A bathochromic shift of about 4 mp occurs in this region for some polycyclic hydrocarbons, like pyrene, in going from iso-octane to benzene as a solvent. Caffeine Number. This method is based upon the ultraviolet absorption of an extract of the sample, using an aqueous caffeine solution as the extractant ( I O , 18). The very high molecular weight polycyclic hydrocarbons, which are not carcinogenic, are insoluble in the caffeine solution, and the lower aromatics (benzenes, naphthalenes, and phenanthrenes) do not absorb a t 340 mp. Oxygenated materials are quite soluble in the aqueous caffeine solution; some of those which interfere are corrected for in the 460 m p measurement since the caffeine number (CY) is the difference in absorbance of the extract a t 340 and 460 mp. This test is easily performed and is quite sensitive. Care is taken to avoid undue exposure of the sample to oxygen before testing, since the oxygenated polycyclics are the largest source of error. The correlation of this test was improved for general use when a correction was made for the amount of material boiling above 700" F. The first three methods of predicting tumor potency are all based in part on ultraviolet absorption. Ultraviolet measurement is particularly useful since there is no interference from the nonaromatic hydrocarbons as with other physical tests like refractive index and density. KOadvantage vas found in using the ultraviolet spectra of the aromatic fraction separated from the nonaromatic and oxy (6) fraction*. High Aromatics Characterization. .klthough the refractive indev of a high boiling petroleum sample could not be used to predict tumor potenry. the refractive indev of its aromatic fraction w-a* quite useful for this purpose. -4chromatographic procedure employing elution through dicta gel was used to isolate the aromatic fraction (6). as well as the nonaromatic and the oxy fractionq.
ANALYTICAL CHEMISTRY
1502 Correlations of this test with the biological data gave values which predicted low tumor potencies for samples with eonsiderable oxy content. A correction was therefore applied for concentrations of oxy fractions up to 5%. Higher contents of this fraction showed no further influence on tumor potency. The correlations of this test were also improved for general use when a correction was made for the amount of material boiling above 700" F. This high aromatics characterization (HAC) was found to be of broader applicability than the caffeine number, since it could be used for samples of coal tar and shale oil, the tumor potencies of which could not be predicted by the caffeine number because of the interference from the oxy and nitrogen compounds. Polarographic Method. Several different polarographic procedures were applied with some success. In each case they were based upon the fact that the reduction potentials for the anthracenic derivatives are lower than for other aromatic compounds. Even though the large number of polycyclics in the high boiling petroleum fractions result in a lack of definition in the polarograms, the diffusion current corresponding to the potential range for anthracenic derivatives has proved to be useful for predicting tumor potency. The procedure for the polarographic reduction was adapted from Wawzonek and Laitinen (19) who used tetra-n-
_-
NO.6
101 104 105 106 107 108 109 110 111 114 145 146 151 159 160 161 168 169 171 173 175 186 188 189 191 193 195 208 208 211 212 214 215 216 217 218 222 226 227 245 246 247 252 344 374 378
CORREL4TIOV EQUATIONS
In order to establish valid correlations between the biological tumor potencies and the various physical and chemical measurements, it was necessary to employ a large number of samples in view of the uncertainties already mentioned. The procedural details of each method will be given. In each case, the physical or chemical measurement was carried out on a sample which y a s tested biologically. Plots were made of the biological tumor potency values (BTP) versus the respective analytical values. Lines of best fit were drawn for establishing the best obtainable correlations, after empirical trials with various functions of the measurements and correction factors. In cases where a specific type of sample is employed, it is possible that a modified correlation may be used to advantage. However, the best correlation equation9 found for general use with high boiling petroleum wnples nre given for each method.
.. . .
._
Table I. Sample
Lutylammonium iodide as the electrolyte and dioxane as the qolvent. Since it was difficult to obtain dioxane in sufficiently high purity, it was replaced with a mixture of benzene and methanol. The mercury pool anode was used in place of the calomel electrode and was found to be about 0.47 volt more positive. ilgain, the inclusion of a viscosity correction factor gave improved correlation.
~
.... .
Heavy catalytic as oil Raffinate from S& extraction of wax from 101 Raffinate from Son extraction of dewaxed 101 Acid-treated Columbian distillate Acid-treated Columbian distillate U.S.P. white oil T a r from steam cracking Thermal reformer t a r Coal tarb Virgin residuum (West Texas) Slack wax fSan Joaouin) oil 6 5 0 + O F. from 101 treated with 3 lb. 66' BB. HzSOl/bbl
650+" F. from 101 treated with 3 lb. 98% HzSOdbbl. 6 5 0 C " F. from 101 treated with 8 lb. 98% H&Od/bbl
Log T'L.30
1.83 1.71 1.83 2.71 2.03 2.00 3.73 2.50 2.56 5.23 1.88 1.88 1.90 1.74 2.22 2.38 1.85 1.84 1.83 2.11 3.60 1.72 1.86 2.48 1.84 2.06 2.02 1.81 3.34 2.40 2.21 3.22 3.34 1.82 1.63 2.85 2.13 2.91 3.19 2.06 5.15 3.88 1.83 2.00 3.48
Distillation D a t a Wt. % of 650Vol. % of 1000° F. 700+'F. 42.3 89.7 99 98.9 42 87.1 100 99.7 42.2 89.3 30.3 84.3 54.4 52.5 48.7 38.8 63.9 39.6 98 0 9.2 91.7 81.9 97.3 89.6 84.0 95.2 45.0 81.0 66.1 64 3 98.2 100 50.3 96.1 46.2 96.6 47.3 97.5 69.1 86.1 81.4 61.6 1.0 91.3 95.8 99.7 98.4 91.6 43.8 90.0 100 96.3 99.3 96.7 59.5 82.8 53.3 87.2 94.1 92.3 81.3 80.2 92.8 45.5 27.0 49.2 23.6 73.7 1.0 10.4 50.3 88.6 77 81.4 85 54.5 35.6 81.7 49 9 47.8 99.1 28.9 72.5 60.9 95.0 40.0 97.0 97.5 92.5 51.5
_
Ultraviolet Absorptivities Kaso of K of total sample a t 6501000° F. 344 ma 36$ ma 470 m p 0.908 2.50 1.15 0.000 0.06 0.00 .. 1.05 0.41 .. 0.390 .. 0.23 0.250 0.83 0.11 0.144 0.28 .. 0.00 0.000 0.00 0 : r; 4.55 4.8 7.6 1.0 2.43 4.4 6.7 on 7.9 14.1 11.3 1 . r, 5,2 1.00 7.6 0.07 0.17 0.100 .. 0.02 0.05 0.047 0.08 0.15 0.066 1.2 1.07 4.1 3.8 3.24 10.7 2.0 1.71 5.3 1.15 2.70 0.880 0.96 2.50 0.83 0.78 2.14 0.662 o'!, 3.5 2 24 5.7 0 3 2.00 2.93 0.370 0.42 0.333 0.67 1.0 0.78 4.0 0 3 4.0 2.76 7.4 1.25 .. 0.930 2.66 2 20 1.54 4.76 .. 2.65 5.80 1.56 2.79 2.11 5 70 1:ir 4.44 7.10 1.31 0 . r, 3.05 5.25 1.80 0 . .i 5.00 12,70 4.62 0.8 4.20 2.07 7.50 .. 1.96 3.10 0.74 2.62 2.33 6.80 0.12 0.22 0.24 1.1 3.40 5.25 1.09 0 6 4.50 3.36 9.00 2.36 3.80 1 .03 1 0 3.40 B .30 1.12 3.22 5,90 2 53 3.20 6.05 1.94 1:05 2.91 12.2 6.6 .. 2.28 0.75 0.75 1.80 1.39 4.08 0 : 75 3.77 1 61 6.65
Filtered 101 700+O F. residue of 191 193 free from heptane insoliil~lre Catalvtic clarified oil Bunkkr fuel oil 6f30f°F. residue from catalytic cycle stock Catalytic clarified oil Bunker fuel oil Bunker fuel oil Catalytic clarified oil Heavy catalytic cycle stock Bunker fuel oil Catalvtic clarified oil Bunkkr fuel oil Bunker fuel oil T a r from suspensoid cracking Phenol extract from T i a Juana distillate T a r from suspensoid cracking 6 O O f O F. residue b y atmospheric distillation of 101 40-100% residue of 191 (7OO+ y F.) 25% of 693+O F. catalytic clarified oil in 114 s n d 108 20% of 6 9 3 f 0 F. thermally cracked catalytic clarified oil in 114 2 46 91.7 4.40 42.7 a n d 108 430+O F. tar from visbreaking 25% catalytic clarified oil and 381 2 .46 7 9 . 5 3.70 44.3 75% virgin residuum 2.85 86.0 93.5 2.69 39 1 Aromatics from 344 on silica gel 3 .61 2 . 0 1 . 7 3 9 7 . 1 405 650-700° F. distillate of 191 0 65 99.0 99.6 1.83 700-750° F. distillate of 191 406 1 28 9 9 . 5 100 1 . 9 7 750-800° F. distillate of 191 407 2.01 100 99,7 2.18 409 900-930" F. distillate of 191 2.06 100 2.20 99.3 410 930-950" F. distillate of 191 2.43 100 2.26 99.7 411 950-965' F. distjllate of 191 2.52 100 2.34 99.5 965-980° F. distillate of 191 412 3.40 99.8 100 2.49 4 13 980-1O1Oo F. distillate of 191 6.35 57.2 2.04 64.6 Residuum from fluid catalytic cracking 478 a T h e sample numbers employed in this paper correspond t o similar sample numbers in previous publications (4-6, 14). b Coal t a r is the only nonpetroleum sample listed. ~
_
Data for 57 Samples Used in Establishing Correlations of Physical and
Petroleum Product
-~
~
-.
~
-
..
9.00
5.05
0 9
10.0 10.0 0.70 2.40 5.20 5.10 5.25 5.10
5.5 3.6 0.39 0.68 1.30 2.09 2.19 2.45 2.61 3.42 5.0
0.8
5.70
6.65 9.5
.. .. .. .. .. , .
.. .. 0:i
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3
1503
These are later compared with biological data for a large set of eamples. Ultraviolet Method.
where W = u t . % of the 650" to 1000" F. distillation fraction KSsn= absorptivity at 360 mpof the 650" to 1000" F. fraction in benzene Vloo= viscosity of total sample in S.S.U. a t 100" F. Simplified Ultraviolet Method. When K470is less than or equal to 0.5, then
PTPsuv
=
4
+ 53[(Kwt - k'ju)/lOg
PTPsuv = 4
+ 50[0.5
(K341
- K364)/K470 log V100I':-
where Kjl4,K3s4,and K4;oare absorptivities of the whole sample in benzene a t 344, 364, and 470 mp, respectively, in liters prr grain per centimeter. Caffeine Number.
PTPcr = 56.1
PTP1Ti.c
=z
I55 C I1 log A log B
V1001'/9
is greater than 0.5, then
When
n-here CN = caffeine nuniber B = volume per cent boiling above 700" F., except that B is assigned a minimum value of 5 for all eamples containing less than 5% of 700+ O F. material It is noted that, because of the logarithmic terms, PTPch- is not sensitive to the value of B at higher values of the latter. Hence, if B is known to exceed 50% (as will often be known from the nature of the sample in question-e.g., a bunker fuel), then B need not be determined, and a mean value corresponding to the class of materials of interest may be used in the equation. High Aromatics Characterization.
+ 46.4 log (CN log B )
where A = weight per cent aromatics B = volume per cent boiling above 700+" F., except that B is awigned a minimum value of 5 for all samples containing less than 5% of 700+ O F. material C = 1-0.10 (per cent oxy), except that C is assigned a minimum value of 0.5 for all samples containing over 5% of oxy fraction
D =
fig aromat,ra - 1.5000
Chemical Propertiefi with Biological T u m o r - F o r m i n g Properties 1IhC
cs
A
0.510 0.013 0.141 0.071 0.073 0.003 0.583 0.163 15.5 0.025 0.105 0.063 0.077 0.780 1 078
34.4 0.6 21.8 39.2 36.0
0.560
0.415 0.392 0.196 0 405 0 068 0.234 0.460 0.510
0.568 0 712 0.585 0.399 0 246 n 441 1.078 0.352 0.208 0.799 0.159 0.237 0 918
0.6
87.8 79.8 55.4 53.8 10.4 4.4 9.2 36.6 75.2 35.8 33.8 33.6 32.8 42.4 39.4 33.8 31.0 89.8 82.2 34 2 33.4 39.6 55.0
41.9 59.8 57.2 50.0
D 0.1421 0.0345 0.0943 0.0599 0.0603 0.0010
~
~- Polarographic__ PTPUV aa
C
0.86 0.90 0.96 0.80 0.88 1.00
0.1721 0.1121 0.2347 0.0953 0.0668 0.0485 0,0746 0.1622 0.1755 0.1539 0.1348 0.1362 0.1334 0.1750 0.0774 0.1182 0.1361
0.64
0.1654 0.1316
0.60
0.1493 0.1323 0.1668 0.1227 0.1479 0.1982 0.1153 0.0422 0.1710 0.1610 0.1013 0.1779 0.0981
0.80 0.50 0.50
0.80 1.00 0.60
0.60 0.78 11.76 0.94 0.94 0.96 0.90 0.62 0.94 0.94 0.50 0.70 0.74 0.94
.. .. 0:91
.. .
I
2:70 2:99 0 : i2
.. ..
4:Ol
.. .
I
5:i9 1.27
..
s:59 2.80
48 10 34 25 19 10
56 45 101 14 19 15
;;
69 60 49 48 44 67 21 32 47 71
..
40
0.50
4:97 3.10 4.17 8.78 4.14
0.84 0.90
5'i5
69 35 60 89 40 21 68 13 :3 4 78 34 28
0.50
0.84 0.82
60 62
Predicted Tumor Potency PTPsrv PTPcN PTPRAC PTP 47 52 47 14 10 10 34 27 31 .. 26 17 24 18 19 13 21 .. 10 10 10 .. 48 58 56 .. 38 30 35 45 47 123 .. 57 24 23 10 25 16 .. 17 24 12 14 11 10 14 13 .. 18 68 39 61 .. 92 72 69 .. 63 58 50 56 50 51 49 .. 30 .. 51 48 34 -ii .. 51 50 55 73 71 15 30 24 18 23 20 .. 19 67 54 .. 69 53 56 53 63 48 55 46 26 59 63 50 59 47 66 .. 49 67 69 75 41 32 36 32 52 53 51 61 ii; 76 104 86 49 11 31 40 33 35 35 79 58 56 86 12 17 26 .. 29 40 31 29 67 70 .. 82 39 27 32 27 31 33 43 25 58 61 31 .. 41 .. 29 27 44 46 61 n0 40 46 47 58 60 52 49 32 37 41 40
..
..
n 227
51.4 29.4 49.0 55.6 43.4 48.0 62.2 70.6 89.4 30.4 33.4 43.2
n 289
51.2
0.1469
0.50
..
37
40
45
39
0 398
56.4 93.9 32.8 32.0 32.0 32.0 32.6 34.6 37.0 41.4 57.8
0,1668 0.1431 0.1298 0.1371 0.1451
0.50 0.88 0.92 0.86 0.82 0.74 0.76
4.95
41 70 34 44 58 67 67 72 72 80 97
48 81
50
43 75 20
0 .I55
0.277 0 654 0.130 0.730 0.399 0 610
0
752
0 275 0 500 0 637
0 703 0 678 0 675 0 685 0 815 1 57
0.1003
I . 00
0.56 0.90
0.56 0.50
2:24 2:i3 3.19
..
56
0.129P 0,0928 0.1510 0.1340 0.1424 0.1210
0.50 0.50 0.54 0 94 0.72 0.52
2:32 3.51 4.01
26 53 45 58 37
0.1595
0.1645 0.1585 0.1647 0.1772 0.1790
0.64
0.64 0.62 0 70
..
..
.. .. .. .. .. ..
25
52 74 63 63 58 62 61 60
63 23 56 61 63 62 62 62 66 76
255 59 49 52 55 60
.. 42
.. .. .. .. .. .. .. .. .. I
.
PTP-BTP ~~
BTI'
6rV
BSUV
55
-7
10 45 31 28
0 -11
-8 4
10
63 32 60 10
-6 -4 0
-7
13 41 4
31
-12
I .5 10
0
53 58
66
7 -0
ii -1
-6
63
- 1; - 2.,
65 10
27 r, 7 AA
__ __
.I.)
e
'$ - 10
6 -fi ,j
.,.I
77
- 15
61
8
x?,
43 1no
2 17 -11
39
10
7i
1 11
--o
8
31 68
in
27
1 11
.4.5 10
46 45
67
0
- 15 6 - 13 14 - 15 -4
4 15
34 7
x
n
16 7 0
- !?I 1
36
0
0 -a
- 14 - 15 -7
-2
-3 53 7 0
2 5
4
16 31 0
5 -9 5
-8
15 4
k .i
20 1
10 0 -3 13 - 29
-7 -7
67
0
15
-7 -1 8 8 11
2 -6
-9 0
4 -11 6 3 9
0
13 -3
-7
- 12
14 7
.. .. ..
10
-2
14
- 14
-7 -3
20
-9 -19
32 8
0
-4
-5
-4
- 18
- 1.5 - 35 - 15
-8
21
6~
-8
- 13 -22 - 10
2
10 34
-9
-5
0J
64
-11 -5
6HhC
-3
~ C N
5
8 - 18
- 12
8
10
- 24 10
25
- 19
2 9 -1 -2 16 13 19 15 2
-7 4 0 0
--l?
0
3
- 14 14 0
-4 - 12 - 18 8 14 -8 2 - 12 - 29 -5
--RO
8 -1
18
- 14
-8 2c5 -21 16 -1
14 -7 -2
17
-2 1
-- 418
4
-3
.. ..
.. .. ..
-6
'G 8
..
..
-2 -9
.. , .
14
-1 8 4 1
..9 .. ..
-2
....6 ..
-5
- 15
1
-,
-8
-7 6
18 - 13
'3
..
13
..
1
-3
..
-6
- 14
9
..
-7
- 1.1
- 26
13 8 7
- 13
10 7 2
-9
- 13
- 297
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 The comments made above concerning the use of a mean value of B for certain classes of samples are applicable to this equation also. Polarographic Methods. PTPP = 10
+ 23.6 U , / I O ~ Vim
where a, = diffusion current difference in microamperes per gram per liter between -1.2 and -2.0 volts as described ANALYTICAL PROCEDURES
Ultraviolet Method. Thi. procedure is a slight modification of one previously described (11). The elements of the analysis are: ( a ) vacuum pot distillation (ASTM D 1160-51T) to obtain a 351 to 628" F./5 mm. fraction, which corresponds to a 650' to 1000' F. boiling range a t atmospheric pressure, ( b ) measurement of the absorptivity, in liters per gram per centimeter, of this fraction in benzene a t 360 mp with a Beckman DU spectrophotometer, and ( c ) determination of the Saybolt universal viscosity in seconds of the total sample a t 100' F. This may be an extrapolated value using an ASTM viscosity-temperature chart (D 341-43) with viscosities measured a t higher temperatures (150' and 210' F.). The correlation equation was based on 81 samples with BTP ranging from 10 to 100. Simplified Ultraviolet Method. The viscosity is determined as described above. The absorptivities, in liters per gram per rentimeter, of the total sample in benzene are determined a t 344, 364, and 470 mp. The correlation equation was based on 57 samples with B T P ranging from 10 to 100. C d e i n e Number. The caffeine number determination h m been described previously (18). I n brief, CN is the difference in the absorbances a t 340 and 460 mp of an aqueous caffeine extract of a solution of 2.00 grams of sample in 10.0 ml. of n-heptane and 10.0 ml. of toluene. The solution employed in the reference cell of the spectrophotometer is the 0.1 M caffeine solution used for the extraction. It is sometimes found with samples such as bunker fuels that, on centrifugation of the caffeine extract, the emulsion is not completely broken. In such cases, one can clarify the extract by inserting a stirring rod into the 15 X 125 mm. test tuhe and rotating it slowly against the side to collect suspended tar. This will often clear up the extract immediately and eliminate the need for repeated centrifuging. The correlation equation was based on 127 samples with B T P ranging from 10 to 100. The present correlation was found to be more generally applicable than a preliminary linear correlation for which PTP = 111 (CN). The volume per cent boiling above 700' F. mag be obtained by a vacuum pot distillation (ASThl 5-mm. distillation D 116051T, residue a t 390' F./5 mni., or a 10-mm. Engler distillation, residue a t 421' F./10 mm.). High Aromatics Characterization. The experimental details of the HAC procedure are given elsewhere (6). This involves a chromatographic separation of the sample on silica gel by elution with n-heptane, benzene, and pyridine. The data employed for predicting tumor potency from this procedure are as follows: yo aromatics (fraction eluted with benzene) 9 oxy (fraction eluted with pyridine) nz~aromst,cs (refractive index of aromatics a t 20" C.) The volume per cent boiling above 700' F. is obtained by a vacuum pot distillation as described above under caffeine number. Polarographic Method. The viscosity is determined as dem i b e d above. The polarographic diffusion current for the sample is determined a t -1.2 and -2.0 volts with a dropping-mercury cathode and a mercury-pool anode in 0.1 J1 tetra-n-butylammonium iodide solution, using equal volumes of benzene and methanol as solvent. Since the polarographic determination is very sensitive and this procedure for analysis of high boiling petroleum fractions has not been previously described, some detail Kill be given. A water-jacketed electrolysis cell (Sargent 529307) with nitrogen inlet tip 10 mm. from the bottom was employed a t 25' C . The 6- to 12-second capillary (Sargent S29350) was used for the cathode with mercury level adjusted to give 5 seconds per drop at - 1.2 volts and 3 seconds per drop a t -2.0 volts for the solventelectrolyte system employed. The Sargent Model XXI rerording polarograph was employed, but an accurate indicating instrument may be used. The reagents employed were: Mercury, purified, distilled Methanol, absolute, Baker's reagent grade Benzene, Baker's C.P.
1505 Tetra-n-butylammonium iodide (Southwest Analytical Chemicals, 1107 West Gibson St., Austin 4, Tex.) Anthracene, Eastman Kodak No. 480-X Nitrogen, from cylinder, but if not completely free from oxygen, it must be purified A stock electrolyte solution of 0.2 -11tetra-n-butylammonium iodide (7.39 grams per 100 ml.) in absolute methanol is mixed with an equal volume of benzene for a blank, or with a benzene solution of the sample for a determination. A benzene solution of anthracene (0.4 gram per liter) is used for calibration of both the potential and diffusion current. A fine slit in the stopper holding the capillary cathode serves as the sole exit for nitrogen from the electrolysis cell. Fresh mercury is added to a clean cell for each determination. Exactly 5 ml. of benzene or benzene solution is added, and nitrogen bubbling is started at 50 ml. per minute, then exactly 5 ml. of the stock electrolyte solution is added, and the cell is closed. Bubbling is continued a t this rate for exactly 7 minutes and is then changed to nitrogen blanketing at the same rate of 50 ml. per minute. The diffusion current is then recorded for a t least 1 minute a t -1.2 and a t -2.Ovolts The difference in diffusion current between these two voltages must be below 1.0 microampere; if not, the reagents must be purified. This diffusion current difference for the blank is designated UR. .4 similar diffusion current difference for the anthracene Polution (0.2 gram per liter in the cell) is designated U A . A check on the half-wave potential of anthracene should be made to see that it is -1.51 zk 0.02 volts; if it is not, instrument adjustment should be made to get the true potentials a t -1.2 and -2 0 volts. [The half-wave potential of anthracene may readily be chosen where the diffusion current is 0.5(m o - U I 2).1 equal to A , 2 About 0.2 gram of sample ( 1 0 05 gram) is weighed to four places in a 50-ml. beaker. About 25-m1. of benzene (same as used in the hlank) is added to dissolve the Sam le The solution is rinsed into a 50-ml. volumetric flask with EenTen,, filled to the mark, and mixed thoroughly The diffusion current of thin solution mixed with the stock electrolyte, as described above, should be determined the same day. The diffusion currents a t -1.2 and -2.0 volts. a, 2 volts and a2 0 volts, respectively, are used to calculate the diffusion current difference of the sample, a,.
+
a, =
6.50
(~2.0
-
2
- ~e'i/
10 (grams of sampleha pa./gram/liter
The grams of sample for this equation is the total weight which was dissolved in the 50 ml. of benzene. The value of 6.50 is a standard value chosen for CIA so that all instrumental and procedural variations (such as variation in capillary size and drop rate of mercury) may be corrected to a single value for anthracene. The value of a, is used in the correlation equation. I t is often difficult t o obtain steady values of diffusion currents a t the more negative voltage for some high boiling petroleum products. In these instances, it is best to average replicate results. Favorable correlations were first established with 57 samples using a polarographic procedure similar to that described. For the present procedure, the correlation equation was derived with results from 22 samples having B T P values ranging from 10 t o 100. These samples were aelected from those of the original 57 samples to typify different kinds of products which gave good correlation in the earlier polarographic procedure DISCUSSIOli
The analytical results required for predicting the tumor potencies of 57 samples are shown in Table I. This particular group of samples was chosen for correlation studies by various methods because it was a heterogeneous group of high boiling petroleum samples for which biological tumor potencies were available. Included in the group were samples which had given difficulty in attempted correlations n i t h biological data. A randoni group of samples would be expected to show better correlation than this group. Furthermore, if one of the methods is used for a particular class of samples, it may be modified to produce an even higher degree of correlation with biological data. I t is preferable that a comparison of these methods be made with the same group of samples. The complete data used for this purpose are shown in Tables I and 11. Three different statistics are used to express the degree of correlation between predicted and biological potencies. They are the mean deviation of PTP-BTP, the standard deviation of PTP-BTP, and the percentage of samples having values of PTP-BTP greater than f15. Comparisons are shorvn below for four groups of samples.
ANALYTICAL CHEMISTRY
1506 Group I. Method
All Samples Tested
No. of Samples
___
Mean
PTP-BTP S.D. % >f15
Ouiitting samples 111, 265, and 392 where Cpi is not applicable.
On the basis of the above data, the polarographic method appears to he superior to the other methods. Also, the high aromatics characterization method appears better than the ultraviolet. However, probably neither of these comparisons is valid because ( a ) different group sizes were employed above, and ( b ) data given below show that, for a common group of samples, these differences practically vanish. This is seen in the following statistics, which are based on given groups of samples. All the samples tested by the ultraviolet method are included in Group 11, below. Samples Tested by Ultraviolet PTP-BTP No. of Samgles Mean S.D. % > =t15 8.3 11.0 11 82 8.3 10.8 12 81 8.3 10.9 12 82
Group 11. Method
uv
(2x0
HAC
a
Omitting sample 111 where CN is not applicable.
These data show that the three methods indicated predict the tumor potency with about the same confidence. The data available for the simplified ultraviolet method are derived from the 57 samples of Table I, and their statistics are given below in Group 111. Again, there is little or no difference in tmhepredicting capacities of the four methods. Group 111. Method
SUV UV CNO
HAC a
Samples Tested b y SUV
No. of Samples 57 57 56 57
Mean 9 2 8 9 9 2 10 0
PTP-BTP S.D. % >*15 12.1 119 12.1 12 5
12 14 18 18
Omitting sample 111 where C S is not available.
Only 22 samples were utilized for establishing the polarographic method. The corresponding statistics and comparisons with other methods are shown below for Group IV. Group IV. Method
Mean
22 22 22 22 22
6 5 3 6 7 5 9.i
suv uv
CN HAC
6 d
0 5 5 14 18
8.0 8.0 7.5 10.5 12.4
Instrument Required I'olarograph Spectrophotometer SI,ectrnr)hotoineter >pecrrophotometcr Refractometer
Table 111. Repeatability of Predicted Tumor Potency by Different Methods i n Different Laboratories
Method
suv P
cs HAC
Laboratory A
Average of PTP
of All 40 Samples
B C
35 2 34 6 35.5 33 !I
B
33 0 31 1 35.2 36 2 37 0 34.4 33.0 31 7 31.6 34.1 35.2 34.6 29.5
D A C D A B C A C
Elapsed Time '/n hour 1/2 hour '/2 hour '/t day
day More training and careful control w e required for the polarograph than for the other methods. The time does not include control work.
"
Five different physical-chemical methods have been developed for predicting carcinogenic properties of high boiling petroleum products. These methods, in general, correlate with biological data to about the same degree; however, some may be more applicable to certain types of products and to certain laboratories than others. The predicted tumor potencies are a t least as precise as the biological values with which the methods were correlated. Only further use of these methods will determine the superiority of a particular method in a given field of application. While the carcinogenic activity of a given product has been expressed in terms of a single tumor potency value, in the interests of simplicity and for purposes of correlation, it is not meant to imply that one need not examine more fully the complete animal
PTP-BTP S.D. %>il5
The polarographic method appears to be comparable to the other methods in its precision for predicting tumor potencies of high boiling petroleum fractions. Since all five methods were similar in their ability to predict tumor potency, the choice of one of them as a preferred method also devolves upon other criteria. Among these were reproducibility and simplicity, or ease of adaptation to routine analysis. The latter may be evaluated as follom: Method
CONCLUSIONS
Sainples Te5ted h y Polarograph
KO.of Samplr-
P
In older to evaluate reproducibility, a cross-check program involving four different laboratories was carried out on 20 different high boiling petroleum samples in duplicate. The 40 samples were coded, bottled by one laboratory, and distributed to all four laboratories simultaneously. The repeatability within a given laboratory, as calculated from paired duplicates, is given ill Table 111. As a result of an analysis of variance, the standard deviation associated with the difference between the laboratories for the 40 samples is shown in Table IV The latter represents the reproducibility of the methods for a random sample determined in any one of the laboratories. >lost of these values could possibly be improved with better training of personnel and more experience with the methods. The samples were not easy to handle; these were all bunker fuels and ingredients of such fuels. Despite the good repeatability of the polarographic method, its poor reproducibility would be expected from the variation in the average predicted tumor potency values between laboratories, as shown in Table 111. This method is, therefore, too difficult to control for routine use, in its present state of development. The most reproducible has been the simplified ultraviolet method. It is also the easieqt to carry out in the laboratory. However, current data which are not yet complete indicate that it is inferior to the caffeine number in its correlation ryith biological data for certain samples. The caffeine number method is the present choice for routine analysis because its reproducibility is good, it is simple and short, and it predicts tumor potency with as much reliability as any of the other methods. However, all methods may be used to advantage in research. This is pnrticularly true of high aromatics characterization, which provide< considerably more data concerning chemical composition
1/z
D
UV
A"
B"
c
D
" Both laboratories A and B
39 1
From Paired Duplicates -Mean Standard deviation deviation 1 1 1 4 0.9 1 2 1 8 2 7 2 4 4 4 2.9 3.7 3.7 5.8 1.4 1.7 3.2 4.2 2.4 3.3 3.1 5.5 3.9 5.9 3.8 5.9 4.4 7.5 1.6 2.2 2 5 3 2 3.1 5.3 1 8 2.2 5 0 7.8
used the same distillate for their measurements. Laboratory B carried out the distillations. Laboratory A should be omitted for reproducibility of UV method.
V O L U M E 2 5 , N O . 10, O C T O B E R 1 9 5 3
1507 LITERATURE CITED
Table IV. Reproducibility of Single Determination of Random Sample in Any One of the Laboratories (From analysis of variance of 40 samples in 3 or 4 laboratories) Method
Degrees of Freedom
Reproducibility of P T P , Standard Deviation 4.5 26.6 5.6 6.7 18.8
test data. This point has been referred to in a previous publication ( 5 ) . ACKNOWLEDGhIENT
The authors are pleased to acknowledge the contributions of U'. A. Dietz, B. F. Dudenbostel, Jr., W.H. King, Jr., and G. G. Wariless in helping to establish the various assay methods. Thanks are also due to H. G. 31. Fischer, R. E. Eckardt, and t,heir associates for helpful advice and discussion. The animal test data were obtained and supplied by W.E. Smith and associates of the Sexv Tork University Bellevue Medical Center. Valuable support to the program was given by C. L. Brown, R.M.Shephardson, M. W. Swaney, and the managements of the Standard Oil Co. (N. J.) and the Standard Oil Development Co. Part of the work reported here was carried out in the laboratories of the Humble Oil and Refining Co. under a research contract with the Standard Oil Development Co.
(1) Auld, 9. J. M.,,J. I n s t . Petroleum Technol., 24,577 (1938). (2) Badger, G. II.,J . Chem. SOC.,1950, 1809. (3) Badger, G. Il.,and Lynn, K . R., I b i d . , 1950,1726. (4) Blanding, F. H., King, R. H., Jr., Priestley, R., Jr., and Rehner, J., Jr., A r c h . I n d . Hug. Occupational M e d . , 4, 335 (1951). (5) Diets, W.h..King, IT. H., Jr., Priestley, W., Jr., and Rehner, J., Jr., I n d . E n g . Chem., 44, 1818 (1952). (6) Eby, L. T., AXAL.CHEX.,25, 1057 (1953). (7) Eckhard, H. J., Ber., 73, 13 (1940). (8) Fieser, L. F., and Campbell, W.P., J . Am. Chenz. SOC.,6 0 , 1142 (1935). (9) Fieser, L. F., and Putnam, S. T., Ibid., 69, 1041 (1947). (10) Fischer, H. G. ll.,Priestley, W., Jr., Eby, L. T., Wanless, G . G., and Rehner, J., Jr., A r c h . I n d . Hug. Occupational M e d . , 4, 315 (1951). (11) Hill, W.T., Stanger, D. W., Pizao, 4.,Riegel, B., Ghubik. P., and Wartman, W.B., Cancer R e s e a w h , 11, 892 (1951). (12) Jones, R. X., Gogek, C. J., and Sharpe, R. W., Con. J . R e search, B26, 719 (1948). (13) Riegel, R . , Wartman, W.B., Hill, W.T., Reeb, B. B., Shuhik, P., and Ptanger, D. W,, Cancer Research, 11, 301 (1951). (14) Smith, IT. E., Sunderland, D. .4.. and Sugiura, K., A r c h . I n d . Hug. Occzcpational M e d . , 4, 299 (1951). (15) Steiner, P.E., and Falk, H. E., Cancer Research, 11, 56 (1951). (16) Twort. C . C., and Twort, J. AI., J . H y g . , 33, 464 (1933). (17) Twort, C. C., and T w o r t , J. Il.,Oil Colour T r a d e s .J., 94, 215, 315 (1938). (18) TTTanless, G. G., Eby, L. T., and Rehner, J . , Jr., ANAL.C m x , 23, 563 (1951). (19) Wawsonek, S.,and Laitinen, H. d.,J . A m . Chem. SOC.,64, 2365 (1942). RECEIVED for review June 20, 1953. Accepted July 27, 1953. Presented before the Division of Petroleum Chemistry a t the 124th Meeting of t h e .kNERIC.AS C H E \ I I C I L SOCIETY, Chicago, 111.
Fractionation of Organic Acids in Sugar Beets by Ion Exchange Resins H 1 R I i I S. OWER'S, ALAN E. GOODBAN, ~ N J. D BENJAMIN STARK Western Regional Research Laboratory, Bureau of Agricultural and Industrial C h e m i s t r y , .4gricrcltctral Research ..idministration, L-. S. D e p a r t m e n t of .Igriculture, .4lbany 6 , Calif. Development of improved processing methods in the beet sugar industry and of new varieties of beets with better processing characteristics requires basic knowledge of the composition of beet juice and industrial processing liquors. This knowledge will be acquired only as rapid methods become available for the separation, identification, and analysis of the various constituents. The background and details of a chromatographic method based upon the use of ion exchange resins are given for the fractionation of organic acids in sugar beet diffusion juice. The method has proved satisfactory and may be useful in other fields. The naturally occurring organic acids in beets appear in the following order: citric > oxalic > malic > glycolic. Pyrrolidone carboxylic and lactic acid also appear in diffusion juices, b u t the former probably arises from glutamine, the latter from a lactic fermentation. I t will now be possible to follow the effect of processing, variety, and other agronomic factors on the organic acid content of sugar beets.
K
NOiI-LEDGE of the composition of sugar beets is important for StudieE leading t o improved processing niet,hods, for development of vitrieties of sugar beets with better processing characteristics, :ind t o make optimal use of by-products and waste liquors from the sugar beet industry. One of the major groups of nonsugar constituents is the organic :icids, which account for about 8 0 7 of the anionic impurities in beet juice. Approximately GOC; of the organic acids are betaine and amino acids, reported 011 elsrn-here (3j.mid t,he remainder arc nonamino
a d s , the subject under discussion. rilthough gross analysis of acidic constituents is relatively easy, analysis for each of the acids is a task which would have been tedious and inconclusive before the recent advances in chromatography. Chromatograph)- offers a t least three methods for the analysis of organic acids. Cellulose columns based upon the present knowledge of paper chromatography (18, 62, 24) might be used. Columns of silica gel are quite satisfactory for separation of a wide variety of acids (3, 7 , 19, 20). The third possibility is ion
