7F3 CFzCHCHzCFz Two calibration plots were made, one of the sum of the relative peak areas for CF3H and HFP produced during pyrolysis against the weight percent of HFP calculated from l9F NMR data (9) and the other of the relative peak area for VF2 produced during pyrolysis against the weight percent VF2 calculated from 19FNMR data (9).A least square fit was calculated for each calibration plot and the slope and intercept were determined.
RESULTS AND DISCUSSION The two calibration plots demonstrate a linear relationship between the pyrolysis products and the NMR data. The slopes of the two calibration plots are close to 1,the slope for weight percent H F P is 0.99 and that for VF2 is 0.97. The monomer composition of the series of H F P N F 2 copolymer was calculated from the pyrolysis data and compared to that calculated from NMR data. These data are summarized in Table I. The difference in weight percent calculated, for the two techniques, averages for H F P to be f0.60% and for VF2 4~0.85%. Pyrolysis-gas chromatography shows promise of being a
very effective tool for both the identification of monomers and the quantitative analysis of monomer composition of fluorocarbon polymers.
ACKNOWLEDGMENT The author is indebted to T. L. Pugh and D. R. Wonchoba for their assistance in obtaining the mass spectra of the pyrolyzate and to E. G. Brame and V. A. Brown for their assistance in obtaining the 19FNMR spectra.
LITERATURE CITED (1) W. Simon and C. L. Buhler, J. Chromatogr. Sci.. 8, 323 (1970). (2) F. Farre-Fius and G. Guiochon, Anal. Chem.. 40, 998 (1968). (3) R. L. Levy and D. L. Fanter, Anal. Chem., 41, 1465 (1969). (4) R. L. Levy J. Gas Chromatogr., 5, 107 (1967). (5) C. E. R. Jones and G. E. J. Reynolds, J. Gas Chromatogr., 5 , 25 (1967). (6) W. Simon, P. Driemler, J. A. Voellmin, and H. Steiner, J. Gas Chromatogr., 5, 53 (1967). (7) W. Simon and H. Gracobbs, Chem. Eng. Techno/.,37, 109 (1965). (8) J. S.Fok and E. A . Abrahamson, Am. Lab., 8 (6), 63 (1965). (9) E. G. Brame and F. W. Yeager, Anal. Chem., in press.
RECEIVEDfor review April 16, 1976. Accepted August 9, 1976.
Theoretical Considerations in Stable Isotope Dilution Mass Spectrometry for Organic Analysis John F. Pickup*
and Kllm McPherson2
Divisions of Clinical Chemistry and Computing and Statistics, Medical Research Council Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ, U.K.
A fundamental theory of stable Isotope dllutlon mass spectrometry in organlc analysis is developed whlch exploits the analogy between the relative abundance of Isotopes and their probability of occurrence. A procedure for model studies of Isotope dilutlon assays is described based on the use of blnomial probability theory to evaluate the effect of changlng isotopic abundance on the mass spectra of individualfragment ions. Examples of the use of such model studles are given. The nature of the calibration graph In isotope dilution Is examlned and it Is found In general to be a curve, with some speclal cases of practical use which are stralght Ilnes.
The use of materials labeled with stable isotopes as internal standards in quantitative assays by gas chromatography-mass spectrometry has grown enormously in the ‘past few years. This growth is probably due equally to the increasing availability of compounds labeled with stable isotopes and to the recent advances in instrumentation for isotope ratio measurement ( I ). Apart from a brief discussion by Holland et al. (2) and a note by Chapman and Bailey (3), there is no guidance available to the analyst on the theory behind stable isotope dilution assays when applied to organic analytes. The lack of such theory is evidenced in the confusion which surrounds the nature of the calibration graph commonly used in isotope dilution studies-a graph of measured isotope ratio Present address, Department of Pharmacy, Royal Cornwall Hospital, Treliske, Truro, Cornwall, U.K. Present address, Department of Social and Community Medicine, 8, Keble Road, Oxford, U.K.
vs. the relative proportion of natural and labeled (“spike”) material. Thus, the calibration graph has been variously described as a straight line with slope equal to unity and intercept zero (4), as a straight line with slope not equal to unity and a nonzero intercept (e.g. ( 5 ) )or as a curve of undefined shape (6). Obviously, accurate results are more likely to be obtained if the theoretical equation for the calibration graph is known and is used for standardization. Accordingly, this paper sets out in detail a theoretical basis for stable isotope dilution assays. Stable isotope dilution analysis can be viewed as a special case of the well-established technique of assay by internal standard-a special case in which the internal standard is as near ideal as is possible, the only difference between the analyte and the internal standard being a small difference in molecular mass. As a consequence, the “internal standard: analyte” ratio is an isotope ratio and must almost invariably be measured by mass spectrometry. The following discussion is based on the assumption that a mass spectrometer is to be used and, accordingly, the terminology of mass spectrometry has been adopted. The theory developed below, however, is intended to be general, and not related in any way to the instrumentation used to realize the theory in practice. The procedure commonly used in quantitative work is to examine the mass spectrum of the analyte and to identify a prominent peak in the spectrum which can be used as a measure of the natural analyte. When a labeled internal standard is introduced, the corresponding peak is found to be displaced to a different position in the spectrum, according to the nature of the label used. Subsequently, the ratio of the two masses in the spectra of mixtures of labeled and natural material is taken as a measure of their relative proportions. In organic chemistry, however, a particular fragment in-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
* 1885
I
0
100
%
200
150
314
/1
I
I .I.I.I.
300
2%
350
mle Flgure 1. The
70-eV mass spectrum of tris(triemthylsi1yl) phosphate
variably displays not one but several peaks in the spectrum, due to the natural occurrence of stable isotopes such as 13C, l5N, 2H, etc. As a preliminary to examining the value of the ratio as an analytical response, it is worthwhile to consider the exact nature of the group of “isotope peaks” which is invariably seen.
NATURE OF ISOTOPE PEAKS AND THEIR PREDICTION Three assumptions are made in the following discussion: i) The relative abundance of an isotope of any element is equivalet to the probability that an atom of the element has any particular corresponding relative atomic mass in the population from which it is drawn. ii) This probability is independent of the chemical environment of a particular atom in a molecule and, in particular, is independent of the isotopes of other elements in the molecule. iii) For clarity, it is assumed that relative atomic and molecular masses are integers; relaxation of this assumption should not invalidate the arguments displayed below. With a simple molecule such as COZ, it is apparent that the molecular mass of 44 can only be achieved by the atomic masses of carbon and oxygen being 12 and 16, respectively. However, a molecular mass of 46 can be achieved by a number of distinct isotopic combinations in the molecule, namely, 13,16,17; 12,18,16; and 12,17,17. If the probabilities of occurrence of each of these different atomic masses are known, then the proportion of molecules with each possible molecular mass can be calculated using the elementary laws of probability. However, in a more complex molecule, it quickly becomes very complicated to compile an exhaustive list of isotope combinations which can constitute a given molecular mass, and it is correspondingly difficult to calculate the relative abundance of any particular molecular mass. For instance, let a given molecule be constituted of n elements without isotopes, with atomic masses a, of which there are s, atoms in the molecule (1 ,< i ,< n).Let there be m elements with isotopes, each with lowest (base) atomic mass = b,, and d, further isotopes, of which there are t, in the molecule (1 6 i 6 m ) . Since it is assumed that the atomic mass of such atoms takes only integer values in the analysis, the atomic mass of each of the d, isotopes is b,, b, 1. . . , b, + d, (b,, d, integer). So in the case of oxygen for example, where b, = 16 and d, = 2, the atomic mass of the three isotopes is 16,17,18. This approximation is sufficient for low resolution mass spectrometry. An element such as chlorine, with atomic masses of 35 and 37 could be considered to have an isotope 36 with associated probability = 0. The lowest (base) molecular mass for such a molecule is evidently:
+
rn
5 a,s, + c b,t,
L=l
and the highest: 1886
a
c=l
(1)
n i=l
+ ~i=lc i(bi + di)ti m
~
i
Let us say that the probability of the j t h isotope of the ith element is pij ( 0 6 j 6 di)and
( p , is~ the probability of the element occurring in the form with the lowest mass). Then the relative abundance of any molecular mass can, in principle, be calculated exactly. The difficulty is that for m larger than 3 or 4 and some di greaterthan 1, there are too many combinations to be practicable for hand calculation. In fact for each element i, there are
(4) unique combinations of the various isotopes. As an example, let us say that there are nine atoms of carbon in a molecule for which d, = 1;then there are 10 unique arrangements of the possible isotopes giving contributions to the molecular mass of 108 through 117. If there are four atoms of oxygen far which di = 2, then there are (6 X 5)/2 = 15 possibilities. Since the base atomic mass of oxygen is 16, the lowest contribution to the molecular mass of four atoms is 64 and the highest is 72, it being apparent that some of these 15 possibilities give rise to the same contribution to the molecular mass. For example, if this analysis is applied to the M - 15fragment (C~H2404Si3P)in the mass spectrum of the tris(trimethylsilyl) phosphate (7) (Figure 1)for which n = 1 (phosphorus) and m = 4 (carbon, hydrogen, oxygen, and silicon), in terms of the above nomenclature we have: Phosphorus al=31 sl=l Carbon i=l bl=12 ti28 dl=l Hydrogen i=2 bz=1 t2=24 dz=1 t3=4 d3=2 Oxygen i=3 b3=16 Silicon i=4 bq=28 t4=3 dq=2 So the number of unique molecular structures which needs to be evaluated in determinations of the relative abundance of all molecular masses is (from Equation 4) 9 X 25 X (6 X 5)/2 X (5 X 4)/2 = 33 750 unique structures. The general expression for this number is
In order to calculate from the relative abundancies of particular isotopes the proportion of molecules which will be expected to have any one of the unique combinations of isotope given by Equation 5, we have to examine in more detail the arrangements of the isotopes in the t, atoms. When di = 1, the situation is relatively straightforward. There are in general ( t i 1) combinations and the relative abundance of each is given by the binomial distribution:
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
+
1.
1
0Calculated Found
27 0
265
269
r l , 300
mle
Flgure 2. Data comparing the mass spectrum of the molecular ion of hexachlorobutadiene predicted by the method described in the text with that obtained by experiment
ti! ( ~ i o ) ~ ( p i l ) ~k i = - ~0 , 1,.. . (ti - l),ti (6) k!(ti - k ) ! (by convention O! = 1). Note that there are always (ti 1)values of k representing the ( t , 1) unique combinations. The factorial part of expression 6 is simply the number of ways of arranging k atoms of minimum atomic mass among t , atoms. Consistent with expression 3, the sum of the results of expression 6 through all values of k will be unity because that represents all possibilities. So, given values of p,j, the probability that any combination of isotopes will occur can be calculated. When d, > 1,the situation is more complicated; thus examining the case d; = 2, then in general there are [(t, l ) ( t i 2)]/2 possibilities.
+
+
+
+
ti!
k ! k ’ ! ( t i - k - k’)!
( PLO. )k(Pi0)k’(Pi2)(ti-h-k’)
k = 0 , . . . , t,; h‘ = 0 , . . . , ( t , - k ) ; each k .
(7)
The simultaneous restrictions on k and k’ are constraints provided by the necessary condition that the sum of the three terms in the denominator without the factorials should be t,, Le., k k‘ t , - k - k’ = t, for 0 < k < t , and 0 < k’ < t,. Evaluation of these restrictions will confirm that they are satisfied in [(t, l)(t, 2)]/2 ways. As before, the factorial part of the expression is the number of ways of ordering k atoms of minimum atomic mass, h’ atoms of (minimum atomic mass 1)and consequently t , - k - k’ atoms of (minimum atomic mass + 2) among t , atoms. The generalization of expression 7 for d, > 2 is straightforward. Obviously for molecules as complex as the one described above, a computer offers the only practical means of calculation. In principle, the program works through each of N , possibilities given by the expressions 6 and 7, calculates the contribution to the molecular mass, which for expression 6 is
+ +
+
+
+
kb,
+ ( t , - k ) ( b , + 1)
(8)
and for expression I where d, = 2, it is kb,
+ k’(b, + 1)+ ( t , - k - k’)(b, + 2)
(9)
For d, > 2, the expression is similar except that it is the sum
305 mle
310
Figure 3. Data comparing the mass spectrum of the M - 15 fragment predicted by the method from tris(trimethylsily1)phosphate (90 % l80) described in the text with that obtained by experiment
+
of d, 1 terms. The “1” in expression 8 and “1”and “2” in expression 9 are consequences of the simplifying assumption made above that all atomic weights of all isotopes are integers. The relaxation of this assumption where appropriate would not greatly increase the complexity. In the example above for C8H2404Si3P)the molecular mass for any one isotope arrangement is evidently 2
4
,=l
1=3
+ C hb, + ( t , - k ) ( b , + 1) + C kb, + k’(b, + 1)+ ( t , - k - k’)(b, + 2)
(10)
for values of k and k’ given by expressions 6 and 7 and for the isotope arrangement of each element. Thus the probabilities given by expressidns 6 and 7 for corresponding values of k and k’ for all m elements would be multiplied together to form the contribution of that arrangement to the relative abundance of the molecular mass given by expression 10. Then the product of the probabilities for each of the m elements for each unique isotopic arrangement will give the contribution of that arrangement to the total molecular mass-given by the sum of all terms like expressions 8 and 9 added to the first part of expression 1 for the n elements with constant atomic mass. Many different arrangements will, in general, contribute to the same molecular mass, so that the relative abundance in the mass spectrum for a given molecular mass will be the sum of all such probabilities. Elements, the isotopes of which do not increase in atomic mass in steps of one, are easily handled by assuming the existence of the missing isotopes and making pV = 0 where appropriate. A computer program was written to perform the calculations as described above (the evaluation of factorials must be performed with care to avoid word under- or over-flow) and was used to test the validity of this theory by applying it to the calculation of the relative abundancies of the isotope peaks in the spectra of natural hexachlorobutadiene (molecular ion) and ls0-enriched tris(trimethylsily1) phosphate (M - 15 ion). Data comparing the calculated spectra with those found by experiment are shown in Figures 2 and 3. It can thus be seen that the factors contributing to the value of the ratio determined in the course of an assay are very complex. In spite of this, it is possible to describe a theory of stable isotope dilution which is of practical value provided that
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
1887
the assumption that “relative abundance” and “probability of occurrence” are equivalent is accepted.
THEORY OF ISOTOPE DILUTION Suppose there are N atoms (or molecules) of natural material, existing in (d l)isotopic forms with minimum atomic (or molecular) mass W , then the different forms will have masses W , W 1, W 2 , . . . (W d ) . Further if each isotopic form has an associated probability of occurrence pj
+
+
+
+
then the number of atoms or molecules having each isotopic form will be given by the series: Npo, Np1, . . . Npd. In the case of molecules, isotopically distinct forms having the same nominal mass are considered to be identical (hence, the masses form an integer series). Similarly, let there be M atoms (or molecules) of labeled material, also possessing ( d 1) isotope forms with masses: W , W 1, . . . W d , each with an associated probability 4;
+
+
+
(15) Here, x is the mass of the natural material and y the mass of labeled material. Webster (9) gives, but does not show the derivation of a similar equation. For values of d greater than 2 or 3, however, determination of the terms pJlpl and q,,/q1 will prove troublesome and small errors in each determination will progressively degrade the overall performance of the method. Several problems arise in biochemical analysis which make direct solution of Equation 15 difficult. These problems stem mainly from the large number of isotope forms possessed by organic molecules; generally a molecule composed of M different elements, each having d, isotopes, of which there are t, atoms in each molecule exhibits
9.
1=1
+
Note that the set of (d 1) isotopes is almost always the same for both natural and labeled material; it could only differ if, for example, an entirely artificial isotope were incorporated in the labeled material. If N atoms (or molecules) of natural material and M atoms (or molecules) of labeled material are mixed, the mixture will also display (d 1) isotopic forms and the amounts of material having each form will be given by the series (Npo + Mqo), ( N P +~ M q i ) , ( N P Z+ M q z ) , . . . (NPd + Mqd). Thus, in general, the ratio of (numbers of atoms or molecules in the kth (j = k ) form) to (the number of atoms or molecules in the Zth (j = I ) form), Rkl, is given by
+
In an isotope dilution assay, M is known and N is to be found. Accordingly Equation 11may be rewritten (12)
But whereas the ratios qk/ql, Pk/P1, and Rkl can be measured experimentally, q1/p1 cannot be determined directly. In the special case when the natural and labeled material exist in only two forms, the relationship po + p1 = qo + q1 = 1 (h = 0 , l = 1) is true and Equation 12 may be rewritten so as to eliminate qJp1 ( = 1 - qo/I - PO)
Thus, in this case, by measuring the three ratios p1/po, 41/40, and Rol and knowing M , N may be found. In the more general case, the ratio ql/pi could be found experimentally by mixing known amounts of natural and labeled material, measuring Rki and using a modification of Equation 12
different molecular weights (cf. Equations 1 and 2 ) . Thus a molecule such as cholesterol (C27H460) will have (1 X 27) (1 X 46) ( 2 X 1) = 7 5 different molecular masses. Each molecular mass is, for the purpose of Equation 15, a different isotopic entity. The proper solutions of Equation 15 will therefore call theoretically for a total of 150 ratio measurements on pure natural and pure labeled materials, in addition to a ratio measurement on each mixture to be analyzed. In practice, many of the pj and qJ values will be vanishingly small and thus the respective pJ/pl ( W j ) and qJ/qi ( W j ) terms will be close to zero and could be disregarded. On the other hand, some values of pJ may be very large compared with p1 and the ratio of the two may be so large as to present intractable problems in measurement. Thus, in general, Equation 15 is not suitable for use in organic systems. As mentioned, most workers who have used this general technique have adopted an empirical approach to the calcuation of results. The procedure most commonly adopted has been to plot a calibration graph of measured isotope ratio (&) vs. x l y (in mass units). For numbers of molecules, the line is given by Equation 11 and clearly, in this case, Rkl is not linearly related to NIM. For mass units, Equation 11 must be rewritten
+
+
+
+
where A = Avogadro’s number and x and y are the masses of natural and labeled material, respectively. E and F are the molecular masses of natural and labeled material, respectively, and are given by E=C d (W+j)P; j= 0
and
Equation 16 may be shortened to Hintenberger (8) gives essentially similar equations although he does not mention the use of a standard material to derive a value for q1/pi (Equation 14). If Equation 12 is rewritten so as to refer to the masses of natural and labeled material rather than the number of atoms or molecules, ql/pi may also be eliminated 1888
X
EPl+
from which the relationship
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
Y
(.ly.$)
Table I. Mass Spectra of the Ion at m/e 538 from the Diheptafluorobutyl Derivative of 5-Hydroxyindole Acetic Acid Derived from the Data Given by Bertilsson et al. (12)
+Q1 F
Peak heights given as probabilities
may be deduced. This relationship is not the general linear relationship R k l = a b ( x / y ) ;the error introduced by assuming that such a linear relationship exists will depend on the actual values of the various parameters in Equation 17. In the special case that q k and p1 tend to zero (there being no interference of natural material at higher mass or labeled material at lower), then
+
The relationship is now linear and a calibration graph would be expected to pass through the origin, although the slope of the graph would not necessarily be unity. In the more general case, when pl only is negligible
which corresponds to a straight line with the same slope, but in this case, the intercept is qhIq1. An alternative form of calibration graph was used by Klein et al. (10)who plotted the logarithm of the percentage increase in R k l vs. the logarithm of the percentage of labeled material in the sample. In the notation used above, this is equivalent to plotting
for numbers of molecules. Again, the relationship will only be truly linear in the special cases mentioned above. A number of workers have prepared calibration graphs using ranges of values of x l y of the order of 0.001 to 0.005, e.g., (11),but this extreme case has no special merit so far as the linearity of the line is concerned. The theory given above, and in particular the validity of Equation 17, was tested by applying it to data given by Bertilsson et al. (12) who described an assay for 5-hydroxyindolacetic acid (5HIAA) using 2H-labeled 5HIAA as internal standard. From their mass spectra of natural and labeled SHIAA after derivatization, mass spectra of the ions
may be tabulated as in Table I, and the molecular masses of the two forms may be approximated by applying Equations 16a and 16b. These data may now be entered into Equation 17 so as to give the expected points for a calibration curve covering the range 5-50 ng natural 5HIAA per 20 ng labeled (the range covered by Bertilsson et al.). The graph thus produced is shown in Figure 4 which also gives the points found experimentally by Bertilsson et al. I t is seen that there is good
/e
Natural
Labeled
0.77 0.04
541 54 2
...
0.09 0.30 0.48
Total
1.00
0.12 0.01 1.00
538 539 540
0.19
2.0
-
1.5
-
cPredicted
--* Found
1. 0
0. 5
5
20
10 (20
30
50
40
nglml (5 HIAA) ng internal standard)
Figure 4. Data comparing the calibration graph for the determination of 5-hydroxyindoleacetic acid given by Bertilsson et al. ( 72)with that predicted by the theory given in the text
agreement between the calculated calibration graph and the experimentally determined graph. The discrepancy between them is probably accounted for by the difficulty of assessing peak heights from a single mass spectrum as published in a journal. It may be concluded that Equation 17 adequately predicts the relationship between measured isotope ratios and the relative proportions of natural and labeled material in an isotope dilution assay.
DISCUSSION With the rapid increase in use of stable isotopes as internal standards in quantitative analysis, it is imperative that there should be a clear understanding of the nature of the analytical process being used. The theory set out above should aid that understanding, and may be applied in practice to the evaluation and assessment of isotope dilution assays. The first stage in developing an isotope dilution assay is the selection of a suitable labeled internal standard. The chemical considerations involved-stability of the label during workup and on storage, etc.-are beyond the scope of this paper but the theory described above concerning this distribution of an ion among its various possible molecular masses can be of value in selection of the label. The nature of the calibration graph eventually obtained is a function inter alia of the values of p , and qj (Equation 17) and these in turn depend on the nature of the stable isotopes incorporated-the increase in molecular mass obtained and the degree of enrichment obtained. Of course, application of the first part of the theory is
ANAL.YTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
0
1889
not likely to lead to exact values for p j and q1 since the isotopic composition of the constituent elements of the compound being studied will never be known exactly. If isotope abundancies were known exactly, such calculations would yield exact results. Thus, in theory, a calibration graph for a particular determination could be drawn without reference to any experimental observation using a standard material. In practice, natural variation in the isotopic composition of elements will be sufficient to make such a procedure unsafe and, in addition, an accurate value for the enrichment achieved in the labeled material is unlikely to be available. On the other hand, the values obtained will be sufficiently close approximations to the truth to act as a guide to the likely success or failure of a potential assay procedure. Such calculations may also be of use in assessing the enrichment achieved vs. the enrichment expected for labeled internal standards. The second part of the theory, summarized in Equation 17 is of particular use in calculating the results of assay procedures. In theory, it will always be better to fit the data corresponding to an experimentally determined calibration graph to Equation 17 since this equation should exactly describe the data. In practice, it may be that as the special cases outlined in Equations 18 and 19 are approached, fitting the points to the equation for a straight line will not give significant error. Whether or not this is so will depend on the circumstances of each individual assay procedure. In any event, an assay procedure is always improved by a
proper understanding of its underlying principles; accordingly, it is to be hoped that the relationships outlined above will be of use in analytical practice.
LITERATURE CITED (1) A. M. Lawson and G. H. Draffan, Prog. Med. Chem., 12, 1 (1975).
(2) H. F. Holland, R. E. Teets, M. A. Bieber, and C. C. Sweeley, "A Proposed Standardisation of the Method used in the Calculation and Presentation of Data from isotope Dilution Experiments," 21st Ann. Conf. Mass Spectrom., San Francisco, Calif., 1973. Am. SOC.Mass Spectrom., N.C.-4, pp 55-9 (1973). (3) J. R. Chapman and E. Bailey, J. Chromafogr., 89, 215 (1974). (4) M. G. Horning, W. G. Stillwell, J. Nowlin, K. Lertratanangkoon, D. Carrol, I. Dzidic, R. N. Stillwell, and E. C. Horning, J. Chromafogr., 91, 413 (1974). (5) W. F.Holmes, W. H. Holland, B. L. Shore, D. M. Bier, and W. R. Sherman, Anal. Chem., 45, 2063 (1973). (6) J. E. Holland, C. C. Sweeley, R. E. Thrush, R. E. Teets, and M. A. Bieber, Anal. Chem., 45, 308 (1973). (7) J. F. Pickup, Ann.,Clin. Blochem., 13, 306 (1976). (8) H. Hintenberger, A Survey of the Use of Stable Isotopes in Dilution Analyses", in "Electromagnetically Enriched Isotopes and Mass Spectrometry", M. L. Smith, Ed., Butterworth, London 1956, p 177. (9) R. K. Webster, "Isotope Dilution Analysis" in "Advances in Mass Spectrometry", I. D. Waldron, Ed., Pergamon, London, 1959, p 1. (IO) P. D. Klein, J. R. Haumann, and W. J. Eisler, Anal. Chem., 44, 490 (1972). (11) B. Samuelson, M.Hamberg, and C. C. Sweeley, Anal. Biochem., 38, 301 (1970). (12) L. Bertilsson, A. J. Atkinson, J. R. Althous, A. Harfast, J-E. Lindgren, and B. Holmstedt, Anal. Cbem., 44, 143 (1972).
RECEIVEDfor review February 18, 1976. Accepted June 29, 1976.
Characterization of Sulfur-Containing Polycyclic Aromatic Compounds in Carbon Blacks M. L. Lee and Ronald A. Hites" Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Mass. 02 139
Computerlzed gas chromatographic mass spectrometry and high resolution mass spectrometry have been used to identify sulfur-containing polycyclics and polycyclic aromatic hydrocarbons in carbon blacks obtained from sulfur-containing petroleum feedstocks. Twenty-eight compounds have been identlfied, seven of whlch are sulfur-containing polycyclics.
Carbon black is a material of considerable commercial importance: More than 1.5 billion pounds per year of domestic carbon black are used in the manufacture of tires alone (1). It is also a material of potentially great environmental concern because of (a) wide environmental distribution of carbon black, primarily in automobile tire dust, and (b) the potent carcinogenicity of a number of compounds adsorbed on carbon black such a,s certain polycyclic aromatic hydrocarbons (PAH). These considerations have led to several studies of the organic compounds associated with carbon black. For example, two recent studies (2, 3 ) reported the identification of cyclopenta[cd]pyrene as a major constituent of carbon black extracts; in addition, 11other PAH ( 2 )and several oxygenated polycyclics ( 3 )were also reported. This paper reports on the analysis of organic extracts of several carbon blacks which were manufactured under varying conditions. Of particular interest is the first reported identification of sulfur-containing polycyclics in carbon black. In addition, the detection of high-boiling PAH has been extended to include compounds of molecular weights up to 376 (C30H16). Capillary column gas chromatography combined with mass 1890
spectrometry (GC/MS) has allowed the positive identification of 21 compounds and the tentative identification of 10 others (see Figure 1).High-resolution mass spectrometry (HRMS) of these same samples has verified the elemental composition of individual compounds, especially for the sulfur polycyclics.
EXPERIMENTAL Samples of four different furnace blacks (see Table I) were obtained from a commercial source (Cabot Corporation, Boston, Mass). The aromatic feedstocks used in the production of three of these furnace blacks were derived from refinery and naphtha-based ethylene type tars. They were over 90% aromatic hydrocarbons, and had a considerable amount of organic sulfur (1.2-3.1%). Appropriate amounts (see Table I) of each furnace black were extracted with methylene chloride for 18 h in a Soxhlet apparatus. Soxhlet thimbles were extracted with Nanograde methylene chloride (Mallinckrodt)for several hours prior to each sample extraction to remove any organic contaminants in the thimble or apparatus. The methylene chloride extracts were then evaporated to minimal volumes (1-10 ml) by rotary evaporation under Figure 1. Compounds identified in carbon blacks by GC/MS and HRMS a Structure is presumed correct but has not been verified by comparison with authentic compounds. Exact position of benzo group is not known. The increase in molecular weight of PAH species also increases the number of possible isomers: the lack of authentic compounds in this molecular weight range prevents the elucidation of the exact structures for these particular GC peaks. The structures given are examples only; many other isomers are possible. Detected only by high resolution mass spectrometry. The structures given are examples only: many other isomers are possible
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
