CHAPTER 10 Using Nuclear Magnetic Resonance Spectroscopy to Determine Structure Physical and Chemical Tests 10-1 Purification: Chromatography Distillation Recrystallization Comparison to known compounds: Melting point Boiling point … many other properties When the properties of an unknown purified substance match those in the literature for a known compound, the identity and structure of the substance are still not known with certainty. Many new substances are newly synthesized for the first time and their properties are not in the literature. Elemental analysis reveals the gross composition of the sample. Chemical tests identify the functional groups present. For larger molecules, knowledge of the composition and functional groups present in a substance are not enough to determine the chemical structure of the substance. For instance, the alcohol C7H16O: Defining Spectroscopy 10-2 Spectroscopy is a technique for analyzing the structure of molecules, usually based on how they absorb electromagnetic radiation. Four types are most often used in organic chemistry: Nuclear Magnetic Resonance spectroscopy (NMR) Infrared spectroscopy (IR) Ultraviolet spectroscopy (UV) Mass spectroscopy (MS) NMR spectroscopy of C and H provides the most detailed information regarding the atomic connectivity of a molecule. Defining Spectroscopy 10-2 Molecules undergo distinctive excitations. Electromagnetic radiation can be described as a wave having a wavelength (λ), a frequency (ν) and a velocity (c). The speed of light in a vacuum is 3 x 1010 cm s-1 or 3 x 108 m s-1. The units of wavelength must match those used for the speed of light. The units of frequency are cycles s-1 (or just s-1) or Hertz (Hz). Molecules absorb energy in discrete packets called “quanta.” A quanta of electromagnetic radiation is referred to as a photon. The energy of a photon is determined by the frequency of the incident radiation: E = hν When a photon of energy is absorbed by a molecule, it causes electronic excitation or mechanical motion to occur. The electronic excitations and motions of a particular molecule are also quantized so only certain frequencies of radiation are able to be absorbed. An analysis of the frequencies of electromagnetic radiation absorbed by a molecule provides information about the arrangement of the atoms in the molecule. The lowest energy state of a molecule is called the ground state. Absorption of electromagnetic radiation causes the molecule to move to an excited state. The difference in energy between the excited state and the ground state must be exactly equal to the energy of the photon absorbed. Absorption of X-rays results in the promotion of electrons from inner atomic shells to outer ones (electronic transitions). This requires X-ray energies greater than 300 kcal mol-1. UV and visible absorption excites valence shell electrons, typically from a filled bonding to an unfilled antibonding orbital. This involves energies between 40 and 300 kcal mol-1. IR absorption causes bond vibration excitation: 2 to 10 kcal mol-1. Microwave radiation excites bond rotations: ~10-4 kcal mol-1. Radiowaves, in the presence of a magnetic field, produces alignment of nuclear magnetism: ~10-6 kcal mol-1. This is the basis of NMR. In this diagram, frequency is specified in units of wavenumbers, defined as 1/λ, which is the number of waves per centimeter. Wavenumbers are used to specify energy in infrared spectroscopy. A spectrometer records the absorption of radiation. Continuous Wave Spectrometry (CW) Radiation of a specific wavelength (UV, IR, NMR, etc.) is generated and passes through a sample. The frequency of the radiation is continuously changed and the intensity of the transmitted beam is detected and recorded. Frequencies that are absorbed by the sample appear as peaks deviating from a baseline value. Fourier Transform Spectroscopy (FT) A much faster technique. A pulse of electromagnetic radiation covering the entire spectrum under scrutiny (NMR, UV, IR) is used to obtain the whole spectrum instantly. The pulse may be applied multiple times and the results accumulated and averaged, which provides for very high sensitivity. The signal measured is actually the decay, with time, of the absorption event. This signal is then mathematically transformed using a Fourier transform, producing the more familiar frequency versus absorption plot. Proton Nuclear Magnetic Resonance 10-3 Nuclear spins can be excited by the absorption of radio waves. Many nuclei can be thought of as spinning on their axes, either clockwise or counterclockwise. One such nucleus is the hydrogen nucleus: 1H. A 1H nucleus is positively charged and its spinning motion generates a magnetic field. In the presence of an external magnetic field, H0, the magnetic field of the hydrogen nucleus can be oriented either with H0 (lower energy) or against H0 (higher energy). These two states are called and spin states, respectively. The difference in energy between the and states depends directly on the external magnetic field strength, H0. 21,150 G 90 MHz 42,300 G 180 MHz 70,500 G 300 MHz The actual energy difference is small. At 300 MHz, the energy difference for a proton is about 3 x 10-5 kcal mol-1. Because the energy difference is so small and the equilibrium between the two states is so fast, the numbers of nuclei in the two states are nearly equal, however, a slight excess will be in the state because of the external magnetic field. When electromagnetic radiation having the same energy as energy difference strikes the nucleus, the electromagnetic radiation is absorbed and the slight excess of nuclei in the state is reduced. Many nuclei undergo magnetic resonance. In general, nuclei composed of an odd number of protons (1H and its isotopes, 14N 19F, and 31P) or an odd number of neutrons (13 C) show magnetic behavior. If both the proton and neutron counts are even (12C or 16O) the nuclei are non-magnetic. In a hypothetical scan of CH2ClF in a 70,500-G magnet, the following spectrum would be observed: High-resolution NMR spectroscopy can differentiate nuclei of the same element. In the NMR spectrum of ClCH2OCH3 at 70,500-G from 0 to 300 MHz, one peak would be observed for each element present. Using high-resolution NMR spectroscopy, the region around each of these peaks can be expanded and additional spectral details can be observed. Using NMR Spectra to Analyze Molecular Structure 10-4 The position of an NMR absorption of a nucleus is called its chemical shift. Chemical shifts depend upon the electron density around a nucleus and are thus controlled by the structural environment of the nucleus. The NMR chemical shifts provide important clues for determining the molecular structure of a chemical compound. The position of an NMR signal depends on the electronic environment of the nucleus. In the high-resolution 1H NMR spectrum of chloro(methoxy)methane above, two separate resonance absorptions of hydrogen are observed. These absorptions reflect the differing electronic environments of the two types of hydrogen nuclei present. Electrons in the bonds connecting the hydrogen atoms to the molecule affect the NMR absorptions. Bound hydrogens are connected to a molecule by orbitals whose electron density varies: Bond polarity Hybridization of the attached atom Presence of electron withdrawing/donating groups The electrons in these orbitals are affected by the external magnetic field, H0, in such a way as to generate a small local magnetic field, hlocal, opposing H0. The total magnetic field seen by the hydrogen nucleus is the sum of these two fields and is thus reduced. The hydrogen nucleus is said to be shielded from H0 by its electron cloud. The degree of shielding of a nucleus depends upon its surrounding electron density. Adding electrons increases shielding. Removing electrons causes deshielding. Shielding causes a displacement of an NMR peak to the right in the spectrum (shifted upfield). Deshielding causes a displacement to the left (shifted downfield). Chemically equivalent hydrogens in a molecule all have identical electronic environments and therefore show NMR peaks at the same position. In the NMR spectrum of 2,2-dimethyl-a-propanol, there are three different peaks due to absorptions by: Nine equivalent methyl hydrogens on the butyl group (most shielded); One hydrogen on the OH; Two equivalent methylene hydrogens. The chemical shift describes the position of an NMR peak. Rather than reporting the exact frequency of each resonance in an NMR spectrum, we measure frequencies relative to an internal standard, tetramethylsilane, (CH3)4Si. To remove the effect of differing applied magnetic fields using different spectrophotomers, the frequencies relative to tetramethylsilane are divided by the frequency of the spectrometer. This yields the chemical shift (δ), a field-independent number measured in ppm. For (CH3)4Si, δ is defined as 0.00. The spectrum above would be reported as: 1H NMR (300 MHz, CDCl3) δ = 0.89, 1.80, 3.26 ppm Functional groups cause characteristic chemical shifts. Each type of hydrogen in a molecule has a chemical shift which depends upon its chemical environment. The absorptions of alkane hydrogens occur at relatively high field. Hydrogens close to an electron withdrawing group (halogen or oxygen) are shifted to relatively lower field (deshielding). The more electronegative the atom, the more the deshielded methyl hydrogens are relative to methane. Multiple substituents exert a cumulative effect. The deshielding influence of electron withdrawing groups diminishes rapidly with distance. Hydroxy, mercapto, and amino hydrogens absorb over a range of frequencies. The absorption peak of the proton attached to the heteroatom may be relatively broad. This variability of chemical shift is due to hydrogen bonding and depends upon: Temperature; Concentration; Presence of H-bonding species such as water (moisture). When line broadening is observed, it usually indicates the presence of OH, SH, or NH2 (NHR) groups. Tests for Chemical Equivalence 10-5 In general, chemically equivalent protons have the same chemical shift. To identify chemically equivalent nuclei, we often have to resort to symmetry operations to decide on the expected NMR spectrum for a compound. Tests for Chemical Equivalence 10-5 Molecular symmetry helps establish chemical equivalence. Rotational symmetry results in equivalent protons when the group of protons is rapidly rotating, as in a methyl group. Conformational interconversion may result in equivalence on the NMR time scale. In the case of the rapid rotation of the methyl group in chloroethane, or the rapid conformation flip in cyclohexane, the observed chemical shifts are the averages of the values that would be observed without the rapid rotation or flip. In the case of cyclohexane, the single line in the NMR spectra at δ = 1.36 ppm at room temperature becomes two lines at a temperature of -90o C, one at δ = 1.12 ppm for the six axial hydrogens and one at δ = 1.60 for the six equatorial hydrogens. At this temperature, the conformational flip of the benzene is slower than the NMR time scale. In general, the lifetime of a molecule in an equilibrium must be on the order of one second to allow its resolution by NMR. Integration 10-6 Integration reveals the number of hydrogens responsible for an NMR peak. The area under an NMR peak is proportional to the number of equivalent nuclei contributing to the peak. By comparing peak areas, it is possible to quantitatively estimate the relative numbers of contributing protons. The areas are obtained by the controlling computer and plotted on top of the regular spectrum by choosing integration mode. Chemical shifts and peak integration can be used to determine structure. Consider the monochlorination of 1-chloropropane: NMR spectroscopy distinguishes all three isomers: 1,1-Dichloropropane: Three NMR signals in the ratio of 3:2:1. Δ = 5.93 ppm (CH), 2.34 pm (CH2), and 1.01 (CH3). 1,2-Dichloropropane: Three NMR signals in the ratio of 3:2:1. δ = 4.17 ppm (CH), 3.68 ppm (CH2), and 1.70 ppm (CH3). 1,3-Dichloropropane: Two NMR signals in the ratio of 2:1. δ = 3.71 ppm (CH2Cl) and 2.25 ppm (CH2). Spin-Spin Coupling: The Effect of Non-Equivalent Neighboring Hydrogens 10-7 When non-equivalent hydrogen atoms are not separated by at least one carbon or oxygen atom, an additional phenomenon called “spin-spin splitting” or “spin-spin coupling” occurs. Instead of single peaks (singlets), more complex patterns occur called multiplets (doublets, triplets or quartets). The number and kind of hydrogen atoms directly adjacent to the absorbing nuclei can be deduced from the multiplicity of the peak. One neighbor splits the signal of a resonating nucleus into a doublet. Consider two protons, Ha and Hb. The population of each of these protons is very close to 50% and 50% in the external magnetic field, H0. This means that in 50% of the molecules, Ha protons will have Hb protons in the state and in 50% of the molecules, Ha protons will have Hb protons in the state. The total field seen by 50% of the Ha protons will therefore be slightly greater than H0 and slightly less than H0 for the other 50 % of the Ha protons. What would have been a singlet NMR peak is now split into a doublet of peaks, symmetrically displaced from the original peak. The chemical shift of the Ha nucleus is reported as the center of the doublet. The amount of mutual splitting is equal. The distance between the individual peaks making up the doublet is called the “coupling constant” (J). Here J is 7 Hz. Coupling constants are independent of the field strength of the NMR spectrometer being used. Spin-spin splitting is usually observed only between hydrogen atoms bound to the same carbon (geminal coupling) or to adjacent carbons (vicinal coupling). Hydrogen nuclei separated by more than two carbon atoms (1,3 coupling) is usually negligible. Finally, equivalent nuclei do not exhibit mutual spin-spin splitting. Ethane exhibits only a single line at δ = 0.85 ppm. Splitting is observed only between nuclei with different chemical shifts. Local-field contributions from more than one hydrogen are additive. Consider the triplet above. It corresponds to the methyl protons being split by the methylene protons. The methylene proton spins will statistically orient in the external magnetic field as , , and . Each methyl proton will see an increased field 25% of the time (), no change 50% of the time ( and ), and a decreased field 25% of the time (). The integrated intensity of the triplet will be 6 since there are a total of 6 equivalent methyl protons. In the case of the methylene protons, the methyl proton spins will statistically distribute as , , , , , , , and . This will result in a 1:3:3:1 quartet of peaks. The integrated intensity of the quartet will be 4, corresponding to the 4 equivalent methylene protons. In many cases, spin-spin splitting is given by the N+1 rule. A simple set of rules: Equivalent nuclei located adjacent to one neighboring hydrogen resonate as a doublet. Equivalent nuclei located adjacent to two hydrogens of a second set of equivalent nuclei resonate as a triplet. Equivalent nuclei located adjacent to a set of three equivalent hydrogens resonate as a quartet. This table illustrates the N+1 rule: Nuclei having N adjacent equivalent neighbors split into N+1 peaks. The heights of the N+1 peaks follow Pascal’s triangle. It is important to note that non-equivalent nuclei split each other. A split in one requires a split in the other. In addition, the coupling constants will be the same for each type of nuclei. Two additional examples: Spin-Spin Splitting: Some Complications 10-8 Complex multiplets sometimes occur when there is a relatively small difference in δ between two absorptions. The N+1 rule may not apply in a direct way if several neighboring hydrogens having fairly different coupling constants are coupled to the resonating nucleus. The hydroxy proton may appear as a single, even if coupled to vicinal hydrogens. Close-lying peak patterns may give rise to non-first-order spectra. The intensity patterns in many NMR spectra do not follow the idealized pattern of Pascal’s triangle but instead are skewed towards each other. The intensities of the lines facing each other is slightly larger than expected. Perfectly symmetrical splittings are observed only when the resonant frequency difference of the two groups of protons is much larger than the coupling constant between them. When ν >> J, the spectra is said to be first-order. Non-first-order spectra assume more complex shapes and can only be analyzed with the help of computers. Since the resonant frequency difference increases with higher field strengths (J remains the same), a complicated spectrum can be made first order by measuring it at higher field strengths. Coupling to non-equivalent neighbors may modify the simple N+1 rule. The spectrum of 1,1,2-trichloropropane illustrates the effects of two sets of non-equivalent neighbors. The Ha proton is split by the Hb proton into a doublet as expected. This doublet is at low field due to the effect of two adjacent chlorine atoms. The methyl protons are also split by the Hb proton into a doublet as expected. This doublet is at high field. The Hb proton is split by both Ha and the methyl protons. In this case eight lines are observed because Ha and the methyl protons have different coupling constants to Hb. The methyl group splits the Hb resonance into a quartet (1,3,3,1). Each line of the quartet is then split into a doublet by the Ha proton (1,1,3,3,3,3,1,1). In the case of 1-bromopropane, the hydrogens on C2 are also coupled to two non-equivalent sets of neighbors. A theoretical analysis of this resonance would predict as many as 12 lines (a quartet of triplets). Because the coupling constants are very similar, however, many of the lines overlap, thus simplifying the pattern. Fast proton exchange decouples hydroxy hydrogens. In the spectra of 2,2-dimethyl-a-propanol, the OH absorption appears as a single peak and is not split by the CH2 protons. In addition, the CH2 protons are not split by the OH. The OH proton is weakly acidic and is both between alcohol molecules and traces of water on the NMR time scale at room temperature. This type of decoupling is called “fast proton exchange.” It may be slowed or removed by removal of traces of water or acid or by cooling. Rapid magnetic exchange self-decouples chlorine, bromine, and iodine nuclei. Fluorine is the only halogen that exhibits spin-spin coupling to 1H in a proton NMR spectra. Chlorine, bromine, and iodine exhibit a fast internal magnetic equilibration on the NMR time scale which precludes an adjacent proton from recognizing them as having different alignments in the external field. This is termed “self-decoupling,” in contrast to exchange-decoupling, as exhibited by the hydroxy protons. Carbon-13 Nuclear Magnetic Resonance 10-9 The NMR spectroscopy of 13C is of greater potential utility than that of 1H NMR. The 13C spectra of an organic compound is much simpler than the 1H spectra because spin-spin coupling between adjacent carbon atoms and between carbon and hydrogen atoms can be avoided. Carbon NMR utilizes an isotope in low natural abundance: 13C. Carbon occurs as a mixture of two principle isotopes, 12C (98.89%) and 13C (1.11%). Of these, only 13C is active in NMR. Because of the low abundance of 13C and its weaker magnetic resonance (1/6000 as strong as 1H), FT NMR is usually used for 13C spectroscopy because multiple pulsing and signal averaging allows the accumulation of strong signals than would otherwise be possible. Carbon-carbon coupling is absent in 13C spectra due to the very low probability of two 13C nuclei being adjacent to each other in a single molecule (.0111 x .0111 ~ .0001). 13C-1H coupling is present, however, the chemical shift range of 13C is much greater than the splittings due to 1H, which precludes the overlapping of adjacent multiplets. The 13C chemical shifts are reported relative to an internal standard, usually (CH3)4Si. The 13C NMR spectra of bromoethane above exhibits 13C splitting by the methyl and methylene protons. The 13C peak due to C1 is first split by its own two hydrogens to produce a triplet. Each of the triplet peaks is then split into a quartet by the adjacent protons on the C2 carbon. The 13C peak due to C2 is first split by its own three hydrogens to produce a quartet. Each of the quartet peaks is then split into a triplet by the adjacent protons on the C1 carbon. Hydrogen decoupling give single lines. 13C-1H coupling can be completely removed by a technique called “broad-band hydrogen (or proton) decoupling.” In this technique, a strong broad radio frequency signal covering the entire resonant frequency range for hydrogen is simultaneously applied at the same time as the 13C signal. The broad-band hydrogen signal causes rapid - flips of the hydrogen nuclei, effectively averaging their local magnetic field contributions. Using this technique simplifies the spectra of bromoethane to two single lines. Proton decoupling is particularly powerful when analyzing complex molecules because every magnetically distinct carbon gives only a single peak. The decoupled 13C spectra of methylcyclohexane shows only five peaks, revealing the twofold symmetry in the structure. A limitation of FT 13C NMR spectroscopy is that, due to difficulties in integration, peak intensities (areas) no longer correspond to the numbers of nuclei present. Carbon, like hydrogen, has chemical shifts which depend upon its chemical environment. Electron withdrawing groups cause deshielding. The chemical shifts go up in order: primary < secondary < tertiary carbon. The numbers of non-equivalent carbons in the isomers of C7H14 are clearly demonstrated by the numbers of 13C peaks in their NMR spectra. Advances in FT NMR are greatly aiding structure elucidation: DEPT 13C and 2D-NMR. Using sophisticated time-dependent pulse sequences (two-dimensional NMR), it is now possible to establish coupling (e.g. bonding) between close-lying hydrogens (homonuclear correlation) or connected carbon and hydrogen atoms (heteronuclear correlation). This, in effect, determines the molecular connectivity of a molecule by measuring the magnetic effect of neighboring atoms on one another along a carbon chain. On such pulse sequence is the distortionless enhanced polarization transfer (DEPT) 13C NMR spectrum. DEPT gives information specifying which type of carbon gives rise to a specific signal in the normal 13C spectrum: CH3, CH2, CH, or Cquaternary. A DEPT experiment consists of three spectra: Normal broad-band decoupled spectra; DEPT-90 pulse sequence spectra, which reveals signals only of carbons bound to one hydrogen; DEPT-135 pulse sequence spectra which produces normal CH3 and CH signals, but negative absorptions for CH2 and no peaks for quaternary carbons. Limonene DEPT spectra: Spectrum A: All 10 lines – 6 alkyl C at high field, 4 alkenyl C at low field Spectrum B: CH carbons Spectrum C: CH carbons, positive signal CH3 carbons, negative signal CH2 carbons Spectrum A lines – Spectrum C lines: quaternary carbon We can apply 13C NMR spectroscopy to the problem of the monochlorination of 1-chloropropane. Both 1,1- and 1,2-dichloropropane should exhibit three carbon signals each. The groups of three signals would be spaced differently due to the different arrangement of chlorine substituents. 1,3-dichloropropane should exhibit only two carbon signals. The two deshielded chlorine bearing carbons assignments in 1,2-dichloropropane can be made using DEPT data. The signal at 49.5 ppm appears inverted in a DEPT-135 spectrum (CH2). The signal at 55.8 ppm is the only absorption in a DEPT-90 spectrum Important Concepts 10 NMR – Most important spectroscopic tool for elucidating organic structures. Spectroscopy – Based on lower energy forms of molecules being converted into higher energy forms by the absorption of electromagnetic radiation. NMR – Based on alignment of the nuclei of certain nuclei (i.e.. 1H and 13C) with () and against () a strong magnetic field. to transition affected by radio-frequency radiation leading to resonance and characteristic absorption spectra. High magnetic fields lead to higher resonant frequencies. High Resolution NMR – Allows differentiation of 1H and 13C nuclei in different environments. Spectral positions are measured as the chemical shift, δ, in ppm from an internal standard (TMS). Important Concepts 10 Chemical Shifts – Highly dependent on presence (shielding) or absence (deshielding) of electron density. Electron donor substituents shield. Electron withdrawing substituents deshield. Hydrogen bonding or proton exchange result in broad peaks. Chemical Equivalency – Equivalent hydrogens or carbons have the same chemical shift. Integration of peak area indicates number of contributing hydrogens. Spin-Spin Splitting – Pattern determined by number of hydrogen neighbors (N+1 Rule). Equivalent hydrogens show no mutual splitting. Important Concepts 10 Non-First-Order Spectra – Complicated patterns created when chemical-shift difference between coupled hydrogens is comparable to their coupling constant. Non-Equivalent Neighboring Hydrogens – (N + 1) rule is applied sequentially. Carbon NMR – Utilizes low abundance 13C nuclei. C-C coupling is not observed. C-H coupling can be removed by proton decoupling. DEPT 13C NMR – Allows peak assignment to CH3, CH2, CH, and quaternary carbons respectively.