Robert W. SiJverstein and G. Clayton Bossier
Downloaded via UNIV OF SOUTH DAKOTA on September 14, 2018 at 07:36:00 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Stanford Research Institute Menlo Park, California
Spectrometric Identification
of Organic Compounds
Consider the plight of the chemist confronted with a few milligrams of a completely unknown organic liquid which Ire has isolated by means of gas chromatography from a complex mixture. Gas chromatography, though fantastically successful as a tool for isolation, affords practically no help in identification, and has the further characteristic of being most effective with small samples. Let us look at the classical procedures in which the analyst has been trained. First, he smells the liquid. Then he carries out a fusion and qualitative tests for the elements. This is followed by a series of chemical reactions designed to establish a class based on a functional group. Solubility tests, a boiling point, and a refractive index narrow down the possibilities to several in one of the standard tables. A crystalline derivative, whose melting point agrees with the literature and is not depressed by an authentic sample, usually serves for “conclusive” identification. If no satisfactory fit can be obtained, recourse is then had to combustion analyses, to a molecular weight determination, and to degradations. It takes a skilled and determined analyst to get by on much less than 20 milligrams of a compound that has been described in the literature. A good deal more may be necessary for a compound that has not been described. Infrared spectrometry can probably now be considered as a classical tool, and it, of course, saves much chemical probing to establish functional groups. Actually the classical methodology is an extremely useful device for teaching laboratory skills and organic chemistry per se, and students develop their first “feel” for organic chemistry in the qualitative organic analysis course. But, in contrast with methods now available and used in research but not systematically taught in universities, the techniques just described are pretty feeble. Over the past few years, wc have been engaged in isolating small amounts of organic compounds from complex mixtures, and identifying these compounds spectrometrically. At the suggestion of Dr. A. J. Castro of San Jose State College, we developed a one unit course entitled "Spectrometric Identification of Organic Compounds,” and presented it to a class of first year graduate students and industrial chemists during the 1962 spring semester. The invitation to teach at San Jose State College was extended by Dr. Bert M. Morris, Head, Department of Chemistry, who kindly arranged the administrative details. A book1 bearing the same title as this paper has evolved from the materia] gathered for the course. ! Silvekstein, R. M., and Rassler, G. C., “Spectrometric Identification of Organic Compounds,” John Wiley and Sons, Inc., New York, in press.
546
j
Journal of Chemical Education
Our presentation was based on our experience in identifying organic molecules by their response to four energy probes; the responses of the molecules are recorded as spectra. We are concerned with the following kinds of spectra: mass, infrared, nuclear magnetic resonance, and to a lesser extent, ultraviolet. Only a rather modest level of sophistication and expertise in each of these areas of spectrometry is required to solve a gratifying number of identification problems. Extension of the methodology from identification of rather simple compounds, about which little or no information is available, to elucidation of structural details of complex molecules, about which quite a bit is known, should be obvious. A higher level of competence in any of the areas of spectrometry is probably best achieved in connection with specific research problems. In a large number of cases, identification of a completely unknown compound can be made from mass, infrared and ultraviolet spectra obtained on a tenth of a milligram or less; and all except the mass spectrometer sample can be recovered. If a milligram sample is available, the unique data afforded by a nuclear magnetic resonance spectrum can be obtained. These data extend the range of identifications manyfold; again, all save the mass spectrometer sample can be recovered. The orientation throughout the course was on the rationalizations involved in translating spectra into chemical structures. We dealt only with identification of pure organic compounds. "Pure,” in this context, is a relative term, and all we can say is: the purer, the better. Probably the ultimate practical criterion of purity (for a sufficiently volatile compound) is chromatographic homogeneity on two capillary columns (several hundred feet in length), one containing a nonpolar substrate, the other, a polar. Another test is effusion through the micro-leak of a mass spectrometer. Various forms of liquid phase chromatography (adsorption and liquid-liquid columns, paper, thinlayer) are applicable to relatively nonvolatile compounds. All of the spectra presented in the course were obtained on samples that were purified by recrystallization to constant melting point or by gas c hromat ography. There is one limitation to the methodology we espouse. A mass spectrum is dependent on a degree of volatility and of thermal stability. And since mass spectrometry is our primary tool, this limitation can be serious. However, mass spectra have been obtained on a large number of high molecular weight compound, e.g., steroids, terpenoids, and alkaloids. Techniques for inserting a sample directly into the ionizing beam of the mass spectrometer promise to overcome the limitations of volatility and stability.
The problem of cost of necessary instrumentation will be raised, and answered by pointing to the amazing evolution of commercial instruments. The time saved, the smaller sample required, and the information made available far overbalance the cost. Infrared and ultraviolet spectrometers have been developed beyond the stage of reliable instruments in the hands of a trained technician. They are now cheap, rugged, and simple enough to be used as a bench tool by the organic chemist. A nuclear magnetic resonance spectrometer is still a fairly expensive, complicated instrument that requires the service of a trained technician. Even here,
the recent reduction in cost (by about one-half) and complexity indicates the trend toward use by relatively unskilled personnel, backed by a network of factorytrained servicemen. Almost from its inception, the utility of nuclear magnetic resonance spectrometry to the organic chemist has been evident, though its utility in identification in combination with mass, infrared and ultraviolet spectrometry has not been stressed. Mass spectrometry has had a somewhat different history. Developed by the physicist and utilized extensively by the petroleum chemist, it has been ignored almost completely by the organic chemist concerned with identification and structure determination. Even today there is only a handful of laboratories in which the application of mass spectrometry to these problems is appreciated. It is still an expensive, very complex instrument which requires considerable skill in its use and maintenance. And yet, as we shall show it is undoubtedly the most powerful tool of the four we use. The sequence of spectra chosen would depend on circumstances. If less than a tenth of a milligram is available, infrared and ultraviolet spectra can be run in solution in microeells, the solvent removed, and a mass spectrum obtained. A volatile sample from which solvent removal is difficult could be handled in an infrared gas cell and transferred directly to the mass
spectrometer. The course given at San Jose State College consisted of 11 hours of lectures and 3 hours of discussion. No laboratory work was involved. Three hours of lecture were devoted to mass-, IR-, and NMR-spectrometry. Two hours were devoted to ultraviolet, spectrometry. The discussions were concerned with translation of sets of spectra into organic structures; ten sets of spectra were covered. The final examination (open book) consisted of the translation of a single set of spectra. Our lectures on IR-, UV-, and NMR-spectrometry require no elaboration here. A brief summary of our presentation of mass spectrometry to the organic chemist may bo of interest, followed by discussions of two sets of spectra. A bibliography of selected general references is appended. Mass Spectrometry
A mass spectrometer bombards the substance under investigation (in the gas phase) with an electron beam, and records the damage as a spectrum of positive ion fragments and their relative abundance. The positive ions are separated on the basis of mass. The usual sample size ranges from several milligrams to less than 0.1 milligram. At an electron beam energy of about 9 to 15 ev or so, depending on the molecule involved, a
molecular radical ion (parent ion) is formed by interaction with the beam electrons. In effect, a single electron has been removed from the molecule. It is important to be able to recognize the parent ion because it gives us the molecular weight of the compound. This is an exact numerical molecular weight, not merely the approximation obtained by all other molecular weight procedures familiar to the organic chemist. The parent peak is usually the peak of highest mass except for isotope contributions. The mass spectrometer is usually operated at an electron beam energy of 70 ev, and under these conditions, numerous fragment ions are formed. A presentation of the masses of the fragment ions (including the parent ion) versus their relative concentrations constitutes the mass spectrum of the sample. The largest peak in the spectrum, called the “base” peak, is assigned a value of 100%, and the other peaks are reported as percentages of the base peak. The mass spectrum is presented as a table of two columns: one headed m/e; and the other, % of base peak. In a separate table, the parent peak is set at 100%, and the isotope peaks are reported relative to the parent peak. In most eases, these will be the parent + 1 and the parent -f 2 peaks. Parent + 4, parent + 6, parent + 8, etc., peaks are given for compounds which contain several chlorine or bromine atoms. These isotope peaks arise, of course, because a certain number of molecules contain heavier isotopes than the common isotopes. The mass spectrometer can distinguish between the molecule containing, for example, the I2C isotope and the molecule containing the 13C. We shall limit ourselves to compounds containing C, H, O, N, S, Cl, and Br. Suppose a compound contains a single carbon atom. Then the parent + 1 peak will be about 1.1% of the intensity of the parent peak. If a compound contains one sulfur atom, the parent -f- 2 peak will be about 4.4% of the parent peak. The first step in the identification of a compound is to attempt to establish an empirical formula from the parent mass and the isotope contributions. We shall not always succeed. In some cases, the parent peak is so small that the isotope contributions cannot be accurately measured. We settle for the molecular weight. In some cases, the parent peak may be missing; we then try to establish the molecular weight from the fragmentation pattern and from the other spectra at hand. In some cases, we may resort to preparation of appropriate derivatives, or to other methods of obtaining molecular weights. Selection of likely empirical formulas is greatly facilitated by a table constructed by Beynon. The table is limited to compounds containing C, H, 0, and N. The presence of S, Cl, or Br is usually readily apparent Principal Stable Isotopes and Relative Abundances Isotopes i-'C !1I iso 15
N
34g
mCl 81Br
Percent of isotope of lowest mass 1.1 0.015
0.2 0.37 0.78 4 4
32.5 98.0
Volume 39, Number 11, November 1962
/
547
Infrared Spectrum FREQUENCY (CM'1)
5000
lOOOO
3000 2500
2000
1600
1400
1200
1000
900
800
750
700
ABSORBANC
m/e 27 38 39 41
42 43
50 51
52 02 03 04 05 77
% of base peak 6. 5. 20. 4. 3. 73. 11. 23.
0. 3. 9. 3.
Mass Spectral Data (Relative Intensities) m/e % of base peak 77 22. 78 6. 79 26. 89 90 91 92 105 100 107 108
18.
109 150
22
151
151
47. 71. 6. 6. 3. 20.
152
8.
28.70 2.84 0.26
NMR Spectrum
PPM Figure 1.
548
/
Data for benzyl acetate.
Journal of Chemical Education
m/e 150
13.
100.
152
Isotope Abundances
( cT )
(P) {P + 1) (P + 2)
% of P 100.
9.9 0.9
Ultraviolet Data 208 204 202
158 147
257 252 248 (shoulder) 243 (shoulder)
194 153 109 78
101
650
Infrared Spectrum FREQUENCY
m/e 20 27
% of base peak
28
(3
29
28 04 9
31
32 33 34 3,5
42 43 44 45
11, 3,3.
12 8 7 7
Mass Spectral Data (Relative Intensities) m/e % of base peak 25. 46
Isotope Abundances m/e % of P
47
100.
48 49
65.
78
10.
.50
3. 6.
(P + 1) 80 (P + 2)
57 58 59 60 61
21
62 78
58
79
24
(CM"'l
80
100.
(P)
3.48 5.0
79
11.
Ultraviolet Data
36. 98. 7. 5. 34. 1.18 1.7
232
infl.
(infl.) =
136
inflection
NMR Spectrum
Volume 39, Number
1
1, November
7
962
/
549
from a large parent +2 peak. The number and combinations of chlorine and bromine atoms can be determined by means of another table.2 The procedure for obtaining an empirical formula will be demonstrated as we work through the sets of spectra presented below. We found it advisable to remind students of the “nitrogen rule”: an odd-numbered molecular weight permits only an odd number of nitrogen atoms, and an even-numbered molecular weight permits only an even number of nitrogen atoms (including zero). Now let us consider the fragmentation pattern. A number of general rules for predicting prominent peaks can be written and rationalized using concepts of statistics, resonance, hyperconjugation, polarizability, and inductive and steric effects. For example: 1.
Cleavage is favored at branched carbon atoms
Aromatic compounds generally give a larger parent peak than do aliphatic compounds 3. Double bonds favor allylic cleavage 4. Saturated rings lose side chains at the a-carbon; special case of branching 5. In alkyl substituted aromatic compounds, cleavage is most probable at the bond beta to the ring 6. A heteroatom will induce cleavage at the bond beta to it. A feeling for these modes of cleavage, plus a reference library, form the basis for use of mass spectrometry for identification purposes. Identification is complicated, however, by rearrangements. These, too, can usually be rationalized on the basis of low energy transition pathways, and increased product stability. 2.
Examples We are now in a position to interpret the first set of spectra (Fig. 1). The first step in translating the set of four spectra in Figure I into a molecular structure is to establish an empirical formula. The parent peak in the mass spectrum is 150; thus we have the molecular weight. The parent peak is an even number. We are therefore permitted either no nitrogen atoms or an even number of them. The P + 2 peak obviously does not allow for the presence of sulfur or halogen atoms. We now look in Beynon’s table under molecular weight 150. We are faced with twenty nine empirical formulas of molecular weight 150 containing only C, H, N and 0. Our P + 1 peak is 9.9% of the parent peak. We list the empirical formulas whose calculated isotopic contribution to the P + 1 peak falls—to be arbitrary—between 9.0 and 10.7 (our isotope contribution peaks are often somewhat higher than the calculated values): we also list their P + 2 values. We immediately eliminate three of these formulas because they contain an odd number of nitrogen atoms. The P + 2 peak is 0.9% of the parent; this best jilts C9H10O2 which we shall tentatively designate as our empirical formula. We make a mental note that both the intensity of the parent peak and the C to H ratio of the empirical formula indicate aromaticity. We turn now to the infrared spectrum and note the C=C band at about 1730 cm.-1 (5.76 /u). This, together with the presence of two O atoms in the empirical formula, suggests an ester. We look for conBeynon, J. H., “Mass Spectrometry and its Applications to Organic Chemistry,” Elsevier Publishing Co., Amsterdam, 2
1960.
550 / Journal of Chemical Education
firmation in the C—O—-C stretching region and note the large broad band at about 1225 cm-1 (8.15 ju) characteristic of an acetate. Two large bands at about 740 cm-1 (13.5 fi) and 700 cm-1 (14.3 yu) suggest a singly substituted benzene ring. Further evidence may be adduced from the aromatic C—H stretching peak at 3065 cm-1 (3.28 n) and from the ring-stretching peak at 1502 cm-1 (6.65 m)We have tentatively established the presence of a benzene ring, and are quite confident about the acetate group. Furthermore, we note from the position of the carbonyl band that the C=0 moiety is not conj ugated with the ring. This is confirmed by the wavelengths and intensities of the ultraviolet absorption peaks, which also eliminate a ketone from consideration. If we subtract a singly substituted benzene ring and an acetate group from the empirical formula we obtain Empirical formula:
C9H,0O2
O
C6H5
+ CH3C—O:
C8H302
CH2
Remaining:
It takes no great imagination to insert the CH2 between the ring and acetate group, and write benzyl acetate o II
CH20CCH3
The NMR spectrum provides almost conclusive confirmation for the above structure. We see three sharp unsplil peaks in the following positions and with the following integrated intensities. t
5
Intensity
2 75
7.25 5.00 2.02
5 2
5.00 7.08
3
The five protons at r 2.75, 8 7.25 are the five benzene ring protons. The singlet of two protons at, r 5.00, 8 5.00 represents the methylene group substituted by a phenyl and an ester group. The singlet of three protons at t 7.98, 8 2.02 represents the methyl group. We can obtain additional confirmation by returning to the mass spectrum and considering the fragmentation pattern in view of the information at hand. The base peak at 108 is a rearrangement peak representing cleavage of an acetyl group (43) and rearrangement of a single hydrogen atom. The large peak at mass 91 is the benzyl (or tropylium) ion formed by cleavage beta to the ring. The large peak at mass 43, of course, represents the acetyl fragment. The peaks at 77, 78, and 79 are additional evidence for the benzene ring. We can state with a high degree of confidence that the compound is benzyl acetate. Formula
P +
C,H10Nh
9.25 9.23 9.61 9.98 9.96 10.34
C8H8N02 C8H10N2O C8H12N3 C9H10O2
C9H12NO
C„H„N,
1
10.71
P
+
2
0.38 0.78 0.61 0.45 0.84 0.68 0.52
Infrared Spectrum FREQUENCY (CM'1) IOOOO
5000
3000
2000
1600
(400
(000
1200
900
600
700
750
650
ABSORBANCE
m/e 26 27 29 50 51 74 76 77 78 105 106 122 150 151 152
Mass Spectral Data (Relative Intensities) % of base peak Isotope Abundances 3. m/e 13. 150 (P) 10. 151 (P + 1) 12. 152 (P + 2) 29. 3. 4. Ultraviolet Data 52. 6. 100. 229 11.
272 280
30. 16.
%ofP 100.
10.2 0.88
)
OH
4.08 2.90 2.85
1.72 0.14
NMR Spectrum
PPM Figure 3.
(
