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The Use of NMR in Structure Elucidation
Chapter 6
The Use of NMR in Structure Elucidation
Of the many applications of high resolution NMR in all branches of chemistry, the most widespread is its use in the elucidation of the struc ture of organic and inorganic compounds. With the background that we have presented in the preceding chapters it is now profitable to consider the way in which the NMR spectrum of a new compound might best be approached. We shall find some features of spectra that can be under stood only in terms of the additional background developed in the suc ceeding chapters, but the examples given in this chapter and in the problems at the end demonstrate that in many cases we can obtain valu able structural information with what we have learned thus far. 6.1 A Systematic Approach to the Interpretation of NMR Spectra In problems of structure elucidation an NMR spectrum may provide useful, even vital data, but it is seldom the sole piece of information avail able. A knowledge of the source of the compound or its method of synthe sis is frequently the single most important fact. In addition, the interpreta tion of the NMR spectrum is carried out with concurrent knowledge of other physical properties, such as elemental analysis from combustion or mass spectral studies; molecular weight; and the presence or absence of structural features, as indicated by infrared or ultraviolet spectra or by chemical tests. Obviously the procedure used for analyzing the NMR spectrum is highly dependent on such ancillary knowledge. The following procedure is suggested, however, as a systematic method for extracting the information from most NMR spectra of new compounds. This proce108
6.1 A Systematic Approach to the Interpretation of N M R Spectra
109
dure is equally applicable to the assignment of NMR features to given nu clei in the spectrum of a compound of known structure. Since proton res onance has thus far accounted for the vast majority of NMR studies, the procedure is aimed principally at proton NMR spectra, and the examples are drawn from such spectra. Some aspects of the interpretation of 13C spectra, which are of increasing importance in organic applications, are given in Section 6.2. The use of NMR data for other nuclei in structure elucidation is also increasing, but must be regarded as sufficiently special ized to preclude our providing a general summary of the best way to ap proach such data. 1. Before attempting to interpret an NMR spectrum, it is wise to as certain whether the spectrum has been obtained under suitable experi mental conditions so that it is a meaningful spectrum. The appearance of the line due to TMS (or other reference) should be examined for sym metry and sharpness (as indicated by adequate ringing in spectra obtained with cw spectrometers). Any erratic behavior in the base line or appear ance of very broad resonance lines should be noted as possibly indicating the presence of ferromagnetic particles (see Fig. 3.11). The trace of the integral should be consistent with proper adjustments of phasing and drift controls (see Fig. 3.9). If these criteria are not met, it is usually desirable to rerun the spectrum under better experimental conditions. It is important to check the calibration of the spectrometer, as indi cated by the spectrum. If the TMS line does not appear exactly at zero, a simple additive correction to all observed lines is sufficient, provided that the overall calibration of the sweep width is correct. Often an indication of gross errors can be obtained from the observed frequency of solvent lines compared with those in Table 3.1 or other sources. It should be noted, however, that small changes (a few hertz) are often found with dif ferent samples due to molecular interactions. Usually the largest value of H0 consistent with adequate resolution is used to maximize chemical shifts. Sometimes spectra at two field strengths provide additional information (see Chapter 7). 2. The presence of any known "impurity" lines should be noted. This includes lines due to the solvent itself or to a small amount of undeuterated solvent. If a proton-containing solvent is used, 13C satellites* and spinning sidebands of the solvent peaks may be prominent. Water is often present in solvents, its resonance frequency depending on the extent of hydrogen bonding to the solvent or the sample and on the concentration of water. * Proton resonance lines from molecules containing 13C (natural abundance, 1.1%); see Section 7.25 for further details, and Table 5.3 for values of 1 3 C - H coupling constants.
110
6. The Use of NMR in Structure Elucidation T
2.0
,,,ι
3.0
4.0
5.0
(ppm)
6.0
00
400
300
■"
■ !
7.0
ι,,., Q uJ
100
i ■ ■ "■
10
9.0
ι.,.,.;.,.,.,..ι: ,Ϊ,1,:
J
-I Θ.0
8.0
200
■
■
7.0
i . . , . ; : . . . . . i . . . . . : . ■ , . , ί i:.'.'.-;::■·■'.·■ i :.·■'.:?:.-.'., i , , . ■ , ;
:
'
.
■
i ' 60J
:'■■
-I
5.0
■■
■ -.
■ '
1 i ■■■'.■■ I . ' . M .
4.0
3.0
,
I
■ ! ■ ■ . , ■ ■ ; ■ ■ ■ .,
2.0
1.0
,
|
0
8 (ppm)
Fig. 6.1 Proton magnetic resonance spectrum (60 MHz) of phenylacetone, CeH5CH2C(0)CH3 in CDC13.
3. An examination of the relative areas of the NMR lines or multiplets (resolved or unresolved) is usually the best starting point for the in terpretation of the spectrum. One should remember that the accuracy of a single integral trace is seldom better than 2% of the full scale value, so that the measured area of a small peak in the presence of several larger ones may be appreciably in error. If the total number of protons in the molecule is known, the total area can be equated to it, and the numbers of hydrogen atoms in each portion of the spectrum established. The opposite procedure of assigning the smallest area to one or two protons and com paring other areas with this one is sometimes helpful, but should be used with caution since appreciable error can be introduced in this way. Occa sionally lines so broad that they are unobservable in the spectrum itself can be detected in the integral trace. 4. The positions of strong, relatively sharp lines should be noted and correlated with expected chemical shifts. This correlation, together with the area measurements, frequently permits the establishment of a number of methyl and methylene groups, aromatic protons (especially in some types of monosubstituted benzene), and in some instances exchangeable protons such as OH and COOH. For example, in Fig. 6.1 the line due to three protons at δ = 2.10 is readily identified as a CH 3 C=0, while that at δ = 3.67 is a methylene group deshielded by nearby substituents. Although the aromatic protons are not precisely equivalent, the dif-
6.1 A Systematic Approach to the Interpretation of NMR Spectra
Fig. 6.2
111
Proton magnetic resonance spectrum (60 MHz) of cholesterol in CDC13.
ferences in their chemical shifts are so small that they give rise to a rela tively sharp single line. Figure 6.2 shows a spectrum typical of a steroid. The protons of the many CH and CH2 groups in the condensed ring system are so nearly chemically equivalent that they give rise to a broad, almost featureless 4 'hump." The angular and side-chain methyl groups, however, show very pronounced sharp lines, the positions of which can provide valuable infor mation on molecular structure (cf. Chapter 4). 5. The approximate centers of all multiplets, broad peaks, and unre solved multiplets should be noted and correlated with functional groups. For example, in Fig. 6.2 the presence of the vinyl proton (~8 = 5.4 ppm) and the proton adjacent to the 3-hydroxyl group (δ = 3.5 ppm) can be identified. At this stage it is unnecessary to worry about exact chemical shifts for complex and unresolved multiplets. The absence of lines in characteristic regions often furnishes important data. For example, the molecule whose spectrum is given in Fig. 6.2 clearly has no aromatic pro tons. 6. First-order splitting in multiplets should be identified, and values of J deduced directly from the splittings. As noted in Chapter 5, the firstorder criterion (vA - vB) > JAB often is not strictly obeyed, resulting in a distortion or "slanting" of the expected first-order intensity distribution. This effect can be seen in Fig. 6.3; note that the "slanting" of the inten sities in one group always increases toward the other group with which it
112
6. The Use of NMR in Structure Elucidation τ (ppm) 3.0
6.0
40 8 (ppm)
Fig. 6.3 Proton magnetic resonance spectrum (60 MHz) of ethyl chloride, showing the almost first-order splitting of the CH3 and CH2 resonances.
is coupled, as we shall see in Chapter 7. The number of components, their relative intensities, the value of J, and the area of the multiplet together provide much valuable information on molecular structure. Commonly occurring, nearly first-order patterns, such as that in Fig. 6.3 due to CH3CH2X, where X is an electronegative substituent, should be recog nized immediately with a little practice. Other patterns that are actually not first order, such as that due to the magnetically nonequivalent protons in p-chloronitrobenzene (Fig. 6.4) are also characteristic and should be easily identified. Para-substituted benzene rings usually display a pattern characterized by four lines symmetrically placed with a weak, strong, strong, weak intensity relation, and a separation between the outer com ponents of about 8 Hz [approximately JHH(ortho)]. There are, however, many less intense lines, as shown in the expanded trace of Fig. 6.4. (Spectra of this type are considered in Section 7.22.) The magnitudes of coupling constants are often definitive in estab lishing the relative positions of substituents. For example, Fig. 6.5 shows that the three aromatic protons of 2,4-dinitrophenol give rise to a spec trum that is almost first order in appearance. The magnitudes of the split tings suggest that two protons ortho to each other give rise to the peaks in the regions of 450 and 510 Hz, and that the latter proton is meta to the one resulting in the lines near 530 Hz. From the known effects of electron-
6.1 A Systematic Approach to the Interpretation of NMR Spectra
113
Fig. 6.4 Proton magnetic resonance spectrum (60 MHz) of l-chloro-4-nitrobenzene in CDC13. Inset shows spectrum with abscissa scale expanded fivefold.
withdrawing and electron-donating substituents (Chapter 4) it is clear that the lowest field protons must be adjacent to the N 0 2 groups. (Note that the slanting of intensities in this spectrum is in accord with the rule men tioned previously.) When magnetic nuclei other than protons are present, it should be re called that some values of J might be as large as many proton chemical shifts. For example, in Fig. 6.6, 2 J H F = 48 Hz, accounting for the widely spaced 1:3:3:1 quartets due to the CH that is coupled to both the fluorine
0"· OH
N0 ?
Fig. 6.5 Proton magnetic resonance spectrum (60 MHz) of 2,4-dinitrophenol in CDCI3, with TMS as internal reference.
114
6. The Use of NMR in Structure Elucidation
and the adjacent methyl group. Since 3 J H F = 21 HZ and 3JHH = 7 Hz, the CH3 resonance is a doublet of doublets. 7. Exchangeable protons (OH, NH, or activated CH) can often be identified by addition of a drop of D 2 0 to the sample (see Section 3.13), and resultant disappearance of peaks. 8. Rerunning the spectrum with the sample dissolved in another sol vent is often good practice in order to resolve ambiguities arising from ac cidental coincidence or overlapping of peaks (see Fig. 6.7). In addition, specific information on configuration or conformation can sometimes be obtained, as we shall see in Chapter 12. 9. Some complex multiplets (nonfirst-order patterns) can be analyzed by simple procedures that we shall develop in Chapter 7. Frequently, coupling constants (or less often, chemical shifts) derived from such anal yses can provide key pieces of information in the elucidation of structure. 10. If there are still ambiguities to be resolved, the technique of spin decoupling is often helpful. By selectively collapsing the splittings of mul tiplets to single lines, one can often determine unambiguously the origins of certain spin couplings. This important technique will be covered in Chapter 9. No amount of discussion of the procedure for analyzing spectra can substitute for practice. The collections of spectra in the Varian catalog95 and in the Sadtler compilation96 provide some excellent readily available examples that should be studied in detail. In addition, there are a number of spectra of "unknowns" assignedattheendof this chapter. Other NMR
Fig. 6.6 Proton magnetic resonance spectrum (100 MHz) of CH3CHFCOOCH2CH3.
6.2 Some Features of Carbon-13 Spectra
200
100
0 Hz
(o)
200
115
>-H->
100
0 Hz
(b)
1
il
*j rk ..
Λ .
■■■.:;■'■ : \ \ v r I , ... i ... , I , ■ . 4.0 3.0
j,
1
/
I 2.0 S(ppm)
1 1.0
0
■ w Ί
—, — ' 1 ■_ - _■'' . ΊI l ^ ^ Lr— 3.0
'
1 r
H— " Λ. Γ■. T.■■■■■■■ 2.0 S(ppm)
I J. 1.0
1 '■ ■ . . . I . T" ' 0
Fig. 6.7 Proton magnetic resonance spectrum (60 MHz) of (CH 3 ) 2 NCH 2 CH 2 C==N (a) as neat liquid and (b) in CDC13. Note accidental coincidence of chemical shifts of protons in the two CH 2 groups in (a) and their separation into a complex nonfirst-order multiplet in (b).
books directed at structure elucidation furnish additional suggestions for procedures and many sample spectra. 97-100
6.2 S o m e Features of Carbon-13 Spectra Carbon-13 NMR spectra are becoming increasingly valuable in the structure elucidation of organic compounds. Since proton NMR generally requires smaller amounts of sample and less complex instrumentation, a proton NMR spectrum is almost always obtained prior to a 13C spectrum, and the 13C data are interpreted in the light of the proton spectrum, as well as non-NMR information. Most of the general comments in Section 6.1 apply, mutatis mutandis, to the interpretation of 13C spectra. Regarding the 10 specific steps recommended there, items 1, 2, and 4 are equally applicable to 13C spectra. As pointed out in Chapter 5, most 13C spectra are obtained ini tially with complete proton decoupling, so that (in the absence of 19 F, 31 P, or other nuclei that might couple to carbon) the spectrum consists of a single line for each chemically different carbon atom in the molecule. Thus, the recommendations regarding spin multiplets are not directly ap plicable. However, as we point out in more detail in Chapter 9, offresonance decoupling leads to simple multiplets in a 13C spectrum and provides information of great interpretive value.
116
6. The Use of NMR in Structure Elucidation
For reasons that we shall discuss in Chapters 9 and 10, 13C spectra are usually obtained under conditions where the areas of chemically shifted lines are not proportional to the numbers of carbon nuclei contributing to the lines. There is no fundamental reason why the theoretically predicted proportionality cannot be obtained, but additional experimental time is re quired, and in many instances it is simply not efficient to spend the time in this way. Since most 13C spectra are obtained by pulse Fourier transform methods, the means exist for determination of spin-lattice relaxation times, Tx (see Chapters 8 and 10), which are occasionally valuable in as signing 13C lines to specific carbon atoms. However, the additional experi mental time required to obtain 7\ data detracts from the utility of this ap proach as a routine matter. Some 13C spectra of "unknowns" are included in Appendix C. Col lections of 13C spectra have been published, and several compilations of 13 C chemical shifts and coupling constants have been mentioned in Sec tions 4.12 and 5.8. 6.3 Structure Elucidation of Polymers Even when the structures of the individual monomer units in a polymer are known, the determination of their sequence and of the geo metrical arrangement, configuration and conformation of the entire polymer presents challenging problems. We can comment on only a few aspects here. The NMR spectrum of a homopolymer may be very simple if the monomeric unit repeats regularly. On the other hand, irregularities, such as head-to-head junctions mixed with head-to-tail junctions, in such cases as vinyl polymers, for example, introduce additional lines that can often be valuable in structure elucidation. When the repeating unit possesses a center of asymmetry, further complexity is introduced into the spectrum. This feature will be taken up in Section 7.27. A synthetic copolymer provides additional degrees of freedom in the arrangement of the repeating units. For example, the spectrum of a copolymer of vinylidine chloride and isobutylene, shown in Fig. 6.8, indi cates that various tetrad sequences (sequences of four monomer units) display significantly different spectra. Copolymers composed of more than two monomer types, including biopolymers, may have much more complex spectra. Bovey has provided excellent discussions of the use of NMR in studies of polymers.83,101
117
6.3 Structure Elucidation of Polymers
(b)
(a)
7L (c)
J
L
J
I
L
\L·
J
I
I
L
Fig. 6.8 Proton NMR spectrum (60 MHz) of (a) polyvinylidene chloride, (b) polyisobutylene, and (c) a copolymer of 70 mole % vinylidene chloride (A) and 30 mole % isobutylene (B). Peaks in (c) can be assigned to various tetrad sequences: (1) AAAA, (2) A A AB, (3) BAAB, (4) AABA, (5) BABA, (6) AABB, (7) BABB (Bovey101).
With the advent of NMR spectrometers operating at higher fre quencies (>200 MHz) and with recent improvements in sensitivity (see Chapter 3), it has become possible to study biopolymers, such as small proteins, nucleic acids, carbohydrates, and lipids. Usually NMR is not a method of choice for analysis of the nature or sequence of monomer units, but it is of great value in providing detailed information on polymer con formation. For example, the four histidine residues in the enzyme Snuclease have substantially different environments in the native protein and have readily distinguishable chemical shifts, as illustrated in Fig. 6.9. On acid denaturation, however, the secondary structure of the protein is lost, and all four histidines shows the same chemical shift in the random coil form of the protein. There are a number of excellent reviews of the use of NMR in studies of biopolymer structure. 103
118
6. The Use of NMR in Structure Elucidation pH 5.04
4.41
8 6
8.5
8.4
8.3
8.2 ppm8l
8.0
Fig. 6.9 Proton NMR spectra (220 MHz) of the C2 proton of histidines in staphylococcal nuclease. The native protein at pH 5.07 was denatured by addition of DCl and renatured by addition of NaOD (Epstein et al.102).
Problems
119
Problems 1. Determine the structural formulas of the molecules giving Spectra 13-20, Appendix C. 2. The proton NMR spectrum of a complex organic molecule has an inte gral with steps of 6.1, 14.0, 22.0, 28.6, 31.9, 43.0, and 61.7 units on the chart paper, (a) Assume that the smallest peak corresponds to one pro ton. Compute the number of protons giving rise to each peak and the total number in the molecule, (b) Suppose, alternatively, that evidence is available from mass spectroscopy that there are 44 protons in the molecule. Compute the number of protons causing each peak. How large an experimental error is required for the discrepancy between the results of parts (a) and (b)?
The Use of NMR in Structure Elucidation
Of the many applications of high resolution NMR in all branches of chemistry, the most widespread is its use in the elucidation of the struc ture of organic and inorganic compounds. With the background that we have presented in the preceding chapters it is now profitable to consider the way in which the NMR spectrum of a new compound might best be approached. We shall find some features of spectra that can be under stood only in terms of the additional background developed in the suc ceeding chapters, but the examples given in this chapter and in the problems at the end demonstrate that in many cases we can obtain valu able structural information with what we have learned thus far. 6.1 A Systematic Approach to the Interpretation of NMR Spectra In problems of structure elucidation an NMR spectrum may provide useful, even vital data, but it is seldom the sole piece of information avail able. A knowledge of the source of the compound or its method of synthe sis is frequently the single most important fact. In addition, the interpreta tion of the NMR spectrum is carried out with concurrent knowledge of other physical properties, such as elemental analysis from combustion or mass spectral studies; molecular weight; and the presence or absence of structural features, as indicated by infrared or ultraviolet spectra or by chemical tests. Obviously the procedure used for analyzing the NMR spectrum is highly dependent on such ancillary knowledge. The following procedure is suggested, however, as a systematic method for extracting the information from most NMR spectra of new compounds. This proce108
6.1 A Systematic Approach to the Interpretation of N M R Spectra
109
dure is equally applicable to the assignment of NMR features to given nu clei in the spectrum of a compound of known structure. Since proton res onance has thus far accounted for the vast majority of NMR studies, the procedure is aimed principally at proton NMR spectra, and the examples are drawn from such spectra. Some aspects of the interpretation of 13C spectra, which are of increasing importance in organic applications, are given in Section 6.2. The use of NMR data for other nuclei in structure elucidation is also increasing, but must be regarded as sufficiently special ized to preclude our providing a general summary of the best way to ap proach such data. 1. Before attempting to interpret an NMR spectrum, it is wise to as certain whether the spectrum has been obtained under suitable experi mental conditions so that it is a meaningful spectrum. The appearance of the line due to TMS (or other reference) should be examined for sym metry and sharpness (as indicated by adequate ringing in spectra obtained with cw spectrometers). Any erratic behavior in the base line or appear ance of very broad resonance lines should be noted as possibly indicating the presence of ferromagnetic particles (see Fig. 3.11). The trace of the integral should be consistent with proper adjustments of phasing and drift controls (see Fig. 3.9). If these criteria are not met, it is usually desirable to rerun the spectrum under better experimental conditions. It is important to check the calibration of the spectrometer, as indi cated by the spectrum. If the TMS line does not appear exactly at zero, a simple additive correction to all observed lines is sufficient, provided that the overall calibration of the sweep width is correct. Often an indication of gross errors can be obtained from the observed frequency of solvent lines compared with those in Table 3.1 or other sources. It should be noted, however, that small changes (a few hertz) are often found with dif ferent samples due to molecular interactions. Usually the largest value of H0 consistent with adequate resolution is used to maximize chemical shifts. Sometimes spectra at two field strengths provide additional information (see Chapter 7). 2. The presence of any known "impurity" lines should be noted. This includes lines due to the solvent itself or to a small amount of undeuterated solvent. If a proton-containing solvent is used, 13C satellites* and spinning sidebands of the solvent peaks may be prominent. Water is often present in solvents, its resonance frequency depending on the extent of hydrogen bonding to the solvent or the sample and on the concentration of water. * Proton resonance lines from molecules containing 13C (natural abundance, 1.1%); see Section 7.25 for further details, and Table 5.3 for values of 1 3 C - H coupling constants.
110
6. The Use of NMR in Structure Elucidation T
2.0
,,,ι
3.0
4.0
5.0
(ppm)
6.0
00
400
300
■"
■ !
7.0
ι,,., Q uJ
100
i ■ ■ "■
10
9.0
ι.,.,.;.,.,.,..ι: ,Ϊ,1,:
J
-I Θ.0
8.0
200
■
■
7.0
i . . , . ; : . . . . . i . . . . . : . ■ , . , ί i:.'.'.-;::■·■'.·■ i :.·■'.:?:.-.'., i , , . ■ , ;
:
'
.
■
i ' 60J
:'■■
-I
5.0
■■
■ -.
■ '
1 i ■■■'.■■ I . ' . M .
4.0
3.0
,
I
■ ! ■ ■ . , ■ ■ ; ■ ■ ■ .,
2.0
1.0
,
|
0
8 (ppm)
Fig. 6.1 Proton magnetic resonance spectrum (60 MHz) of phenylacetone, CeH5CH2C(0)CH3 in CDC13.
3. An examination of the relative areas of the NMR lines or multiplets (resolved or unresolved) is usually the best starting point for the in terpretation of the spectrum. One should remember that the accuracy of a single integral trace is seldom better than 2% of the full scale value, so that the measured area of a small peak in the presence of several larger ones may be appreciably in error. If the total number of protons in the molecule is known, the total area can be equated to it, and the numbers of hydrogen atoms in each portion of the spectrum established. The opposite procedure of assigning the smallest area to one or two protons and com paring other areas with this one is sometimes helpful, but should be used with caution since appreciable error can be introduced in this way. Occa sionally lines so broad that they are unobservable in the spectrum itself can be detected in the integral trace. 4. The positions of strong, relatively sharp lines should be noted and correlated with expected chemical shifts. This correlation, together with the area measurements, frequently permits the establishment of a number of methyl and methylene groups, aromatic protons (especially in some types of monosubstituted benzene), and in some instances exchangeable protons such as OH and COOH. For example, in Fig. 6.1 the line due to three protons at δ = 2.10 is readily identified as a CH 3 C=0, while that at δ = 3.67 is a methylene group deshielded by nearby substituents. Although the aromatic protons are not precisely equivalent, the dif-
6.1 A Systematic Approach to the Interpretation of NMR Spectra
Fig. 6.2
111
Proton magnetic resonance spectrum (60 MHz) of cholesterol in CDC13.
ferences in their chemical shifts are so small that they give rise to a rela tively sharp single line. Figure 6.2 shows a spectrum typical of a steroid. The protons of the many CH and CH2 groups in the condensed ring system are so nearly chemically equivalent that they give rise to a broad, almost featureless 4 'hump." The angular and side-chain methyl groups, however, show very pronounced sharp lines, the positions of which can provide valuable infor mation on molecular structure (cf. Chapter 4). 5. The approximate centers of all multiplets, broad peaks, and unre solved multiplets should be noted and correlated with functional groups. For example, in Fig. 6.2 the presence of the vinyl proton (~8 = 5.4 ppm) and the proton adjacent to the 3-hydroxyl group (δ = 3.5 ppm) can be identified. At this stage it is unnecessary to worry about exact chemical shifts for complex and unresolved multiplets. The absence of lines in characteristic regions often furnishes important data. For example, the molecule whose spectrum is given in Fig. 6.2 clearly has no aromatic pro tons. 6. First-order splitting in multiplets should be identified, and values of J deduced directly from the splittings. As noted in Chapter 5, the firstorder criterion (vA - vB) > JAB often is not strictly obeyed, resulting in a distortion or "slanting" of the expected first-order intensity distribution. This effect can be seen in Fig. 6.3; note that the "slanting" of the inten sities in one group always increases toward the other group with which it
112
6. The Use of NMR in Structure Elucidation τ (ppm) 3.0
6.0
40 8 (ppm)
Fig. 6.3 Proton magnetic resonance spectrum (60 MHz) of ethyl chloride, showing the almost first-order splitting of the CH3 and CH2 resonances.
is coupled, as we shall see in Chapter 7. The number of components, their relative intensities, the value of J, and the area of the multiplet together provide much valuable information on molecular structure. Commonly occurring, nearly first-order patterns, such as that in Fig. 6.3 due to CH3CH2X, where X is an electronegative substituent, should be recog nized immediately with a little practice. Other patterns that are actually not first order, such as that due to the magnetically nonequivalent protons in p-chloronitrobenzene (Fig. 6.4) are also characteristic and should be easily identified. Para-substituted benzene rings usually display a pattern characterized by four lines symmetrically placed with a weak, strong, strong, weak intensity relation, and a separation between the outer com ponents of about 8 Hz [approximately JHH(ortho)]. There are, however, many less intense lines, as shown in the expanded trace of Fig. 6.4. (Spectra of this type are considered in Section 7.22.) The magnitudes of coupling constants are often definitive in estab lishing the relative positions of substituents. For example, Fig. 6.5 shows that the three aromatic protons of 2,4-dinitrophenol give rise to a spec trum that is almost first order in appearance. The magnitudes of the split tings suggest that two protons ortho to each other give rise to the peaks in the regions of 450 and 510 Hz, and that the latter proton is meta to the one resulting in the lines near 530 Hz. From the known effects of electron-
6.1 A Systematic Approach to the Interpretation of NMR Spectra
113
Fig. 6.4 Proton magnetic resonance spectrum (60 MHz) of l-chloro-4-nitrobenzene in CDC13. Inset shows spectrum with abscissa scale expanded fivefold.
withdrawing and electron-donating substituents (Chapter 4) it is clear that the lowest field protons must be adjacent to the N 0 2 groups. (Note that the slanting of intensities in this spectrum is in accord with the rule men tioned previously.) When magnetic nuclei other than protons are present, it should be re called that some values of J might be as large as many proton chemical shifts. For example, in Fig. 6.6, 2 J H F = 48 Hz, accounting for the widely spaced 1:3:3:1 quartets due to the CH that is coupled to both the fluorine
0"· OH
N0 ?
Fig. 6.5 Proton magnetic resonance spectrum (60 MHz) of 2,4-dinitrophenol in CDCI3, with TMS as internal reference.
114
6. The Use of NMR in Structure Elucidation
and the adjacent methyl group. Since 3 J H F = 21 HZ and 3JHH = 7 Hz, the CH3 resonance is a doublet of doublets. 7. Exchangeable protons (OH, NH, or activated CH) can often be identified by addition of a drop of D 2 0 to the sample (see Section 3.13), and resultant disappearance of peaks. 8. Rerunning the spectrum with the sample dissolved in another sol vent is often good practice in order to resolve ambiguities arising from ac cidental coincidence or overlapping of peaks (see Fig. 6.7). In addition, specific information on configuration or conformation can sometimes be obtained, as we shall see in Chapter 12. 9. Some complex multiplets (nonfirst-order patterns) can be analyzed by simple procedures that we shall develop in Chapter 7. Frequently, coupling constants (or less often, chemical shifts) derived from such anal yses can provide key pieces of information in the elucidation of structure. 10. If there are still ambiguities to be resolved, the technique of spin decoupling is often helpful. By selectively collapsing the splittings of mul tiplets to single lines, one can often determine unambiguously the origins of certain spin couplings. This important technique will be covered in Chapter 9. No amount of discussion of the procedure for analyzing spectra can substitute for practice. The collections of spectra in the Varian catalog95 and in the Sadtler compilation96 provide some excellent readily available examples that should be studied in detail. In addition, there are a number of spectra of "unknowns" assignedattheendof this chapter. Other NMR
Fig. 6.6 Proton magnetic resonance spectrum (100 MHz) of CH3CHFCOOCH2CH3.
6.2 Some Features of Carbon-13 Spectra
200
100
0 Hz
(o)
200
115
>-H->
100
0 Hz
(b)
1
il
*j rk ..
Λ .
■■■.:;■'■ : \ \ v r I , ... i ... , I , ■ . 4.0 3.0
j,
1
/
I 2.0 S(ppm)
1 1.0
0
■ w Ί
—, — ' 1 ■_ - _■'' . ΊI l ^ ^ Lr— 3.0
'
1 r
H— " Λ. Γ■. T.■■■■■■■ 2.0 S(ppm)
I J. 1.0
1 '■ ■ . . . I . T" ' 0
Fig. 6.7 Proton magnetic resonance spectrum (60 MHz) of (CH 3 ) 2 NCH 2 CH 2 C==N (a) as neat liquid and (b) in CDC13. Note accidental coincidence of chemical shifts of protons in the two CH 2 groups in (a) and their separation into a complex nonfirst-order multiplet in (b).
books directed at structure elucidation furnish additional suggestions for procedures and many sample spectra. 97-100
6.2 S o m e Features of Carbon-13 Spectra Carbon-13 NMR spectra are becoming increasingly valuable in the structure elucidation of organic compounds. Since proton NMR generally requires smaller amounts of sample and less complex instrumentation, a proton NMR spectrum is almost always obtained prior to a 13C spectrum, and the 13C data are interpreted in the light of the proton spectrum, as well as non-NMR information. Most of the general comments in Section 6.1 apply, mutatis mutandis, to the interpretation of 13C spectra. Regarding the 10 specific steps recommended there, items 1, 2, and 4 are equally applicable to 13C spectra. As pointed out in Chapter 5, most 13C spectra are obtained ini tially with complete proton decoupling, so that (in the absence of 19 F, 31 P, or other nuclei that might couple to carbon) the spectrum consists of a single line for each chemically different carbon atom in the molecule. Thus, the recommendations regarding spin multiplets are not directly ap plicable. However, as we point out in more detail in Chapter 9, offresonance decoupling leads to simple multiplets in a 13C spectrum and provides information of great interpretive value.
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6. The Use of NMR in Structure Elucidation
For reasons that we shall discuss in Chapters 9 and 10, 13C spectra are usually obtained under conditions where the areas of chemically shifted lines are not proportional to the numbers of carbon nuclei contributing to the lines. There is no fundamental reason why the theoretically predicted proportionality cannot be obtained, but additional experimental time is re quired, and in many instances it is simply not efficient to spend the time in this way. Since most 13C spectra are obtained by pulse Fourier transform methods, the means exist for determination of spin-lattice relaxation times, Tx (see Chapters 8 and 10), which are occasionally valuable in as signing 13C lines to specific carbon atoms. However, the additional experi mental time required to obtain 7\ data detracts from the utility of this ap proach as a routine matter. Some 13C spectra of "unknowns" are included in Appendix C. Col lections of 13C spectra have been published, and several compilations of 13 C chemical shifts and coupling constants have been mentioned in Sec tions 4.12 and 5.8. 6.3 Structure Elucidation of Polymers Even when the structures of the individual monomer units in a polymer are known, the determination of their sequence and of the geo metrical arrangement, configuration and conformation of the entire polymer presents challenging problems. We can comment on only a few aspects here. The NMR spectrum of a homopolymer may be very simple if the monomeric unit repeats regularly. On the other hand, irregularities, such as head-to-head junctions mixed with head-to-tail junctions, in such cases as vinyl polymers, for example, introduce additional lines that can often be valuable in structure elucidation. When the repeating unit possesses a center of asymmetry, further complexity is introduced into the spectrum. This feature will be taken up in Section 7.27. A synthetic copolymer provides additional degrees of freedom in the arrangement of the repeating units. For example, the spectrum of a copolymer of vinylidine chloride and isobutylene, shown in Fig. 6.8, indi cates that various tetrad sequences (sequences of four monomer units) display significantly different spectra. Copolymers composed of more than two monomer types, including biopolymers, may have much more complex spectra. Bovey has provided excellent discussions of the use of NMR in studies of polymers.83,101
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6.3 Structure Elucidation of Polymers
(b)
(a)
7L (c)
J
L
J
I
L
\L·
J
I
I
L
Fig. 6.8 Proton NMR spectrum (60 MHz) of (a) polyvinylidene chloride, (b) polyisobutylene, and (c) a copolymer of 70 mole % vinylidene chloride (A) and 30 mole % isobutylene (B). Peaks in (c) can be assigned to various tetrad sequences: (1) AAAA, (2) A A AB, (3) BAAB, (4) AABA, (5) BABA, (6) AABB, (7) BABB (Bovey101).
With the advent of NMR spectrometers operating at higher fre quencies (>200 MHz) and with recent improvements in sensitivity (see Chapter 3), it has become possible to study biopolymers, such as small proteins, nucleic acids, carbohydrates, and lipids. Usually NMR is not a method of choice for analysis of the nature or sequence of monomer units, but it is of great value in providing detailed information on polymer con formation. For example, the four histidine residues in the enzyme Snuclease have substantially different environments in the native protein and have readily distinguishable chemical shifts, as illustrated in Fig. 6.9. On acid denaturation, however, the secondary structure of the protein is lost, and all four histidines shows the same chemical shift in the random coil form of the protein. There are a number of excellent reviews of the use of NMR in studies of biopolymer structure. 103
118
6. The Use of NMR in Structure Elucidation pH 5.04
4.41
8 6
8.5
8.4
8.3
8.2 ppm8l
8.0
Fig. 6.9 Proton NMR spectra (220 MHz) of the C2 proton of histidines in staphylococcal nuclease. The native protein at pH 5.07 was denatured by addition of DCl and renatured by addition of NaOD (Epstein et al.102).
Problems
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Problems 1. Determine the structural formulas of the molecules giving Spectra 13-20, Appendix C. 2. The proton NMR spectrum of a complex organic molecule has an inte gral with steps of 6.1, 14.0, 22.0, 28.6, 31.9, 43.0, and 61.7 units on the chart paper, (a) Assume that the smallest peak corresponds to one pro ton. Compute the number of protons giving rise to each peak and the total number in the molecule, (b) Suppose, alternatively, that evidence is available from mass spectroscopy that there are 44 protons in the molecule. Compute the number of protons causing each peak. How large an experimental error is required for the discrepancy between the results of parts (a) and (b)?