Since its discovery in the 1940’s, nuclear magnetic resonance (NMR) spectroscopy has had a spectacular growth and acceptance in chemistry. Until recently, sensitivity considerations had limited its application to high abundance magnetically active nuclei such as ‘H, 19F,and 31Pfor which a conventional field or rf frequency sweep can elicit strong resonances, even on very small samples. Progress in instrumentation has centered on: increasing the applied magnetic field to increase spectral dispersion and simplify resonance patterns; improving the homogeneity of the applied magnetic field for greater resolution of spectral lines; stabilization of frequency field ratios for drift-free operation; sensitivity improvement; multiple-resonance capability (decoupling); and “other nuclei” observation. These developments in conventional swept (continuous wave or CW) NMR have resulted in the availability of low-cost routine proton NMR spectrometers and their consequent widespread use both in research and practical endeavors. The low sensitivity (Table I) and often small natural abundance of nuclei “other” than ‘H, 19F,and 31Phave retarded their use until recently. Given a choice, a chemist would certainly consider the NMR spectrum of carbon to be of fundamental and desirable importance, while recognizing that the practical development of the field has been guided primarily by what has been permissible technologically. And while it is now possible to obtain I3C NMR spectra, the very fac-
Table I. NMR Nuclei Isotope
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Spin
e
FreRe1 quency sensi- (MHz) dance, % tivity a t 2 3 kG Abun-
tors that have retarded its development are actually now recognized to be advantageous. This seeming contradiction is easily explained. The low natural abundance of the stable magnetically active nuclide of carbon (13C, 1.1%natural abundance, spin = Ih) makes the chances of two 13C’sbeing in one molecule very small, obviating any complications of homonuclear spin-spin (J)coupling. (If desired, extended time-averaging or isotopic enrichment does make this observation possible.) Upon complete proton decoupling (see below), therefore, the 13C NMR spectrum consists of single lines, one for each chemically shifted carbon. The routine application of this technique has depended on several technological advances such as broadbanded proton decoupling, time-averaging, and pulsed Fourier transform NMR.
saves a factor of nine in time-averaging in attaining a desired signal-tonoise ratio. The above decoupling experiment can be termed “coherent single-frequency” decoupling since only one type of proton in the proton spectrum was irradiated. For a complicated molecule this approach is impractical, and one resorts to application of broadbanded decoupling in which the coherent proton rf is modulated with “white” noise. If the frequency spread of this noise is greater than the range of proton frequencies and high enough power is employed, the various l3C-IH multiplets are collapsed, giving single lines for all carbons formerly coupled to protons. Further advantage is realized since no detailed consideration is necessary for selection of the ‘H rf frequency.
Proton Decoupling in 13C NMR Since the proton is a s p i n - s nucleus, it can spin couple to 13C and produce a “coupled” spectrum. For example, a methyl carbon would appear as a quartet with intensities of 1:3:3:1. If an intense ‘H rf decoupling field is used to irradiate the protons of the methyl group, collapse will occur, giving a narrow line centered at the chemical shift of the methyl carbon. In comparing the areas of the coupled and decoupled spectra, it is usually noticed that one obtains up to almost three times as much area as expected from simple multiplet collapse. This “extra” intensity is the nuclear Overhauser enhancement. I t results in considerable sensitivity enhancement since a factor of three greater signal
Single-scan NMR spectra have provided and still provide the vast majority of proton NMR spectra. Weak nuclei such as I3C and low-level proton spectra require time-averaging (multiscanning) for extraction of the signals from within the noise. In the CW mode this requires very long periods of time since each scan usually takes -500 sec. This large expenditure of time has discouraged its routine use for signal-to-noise enhancement in the CW mode. One principal advantage of the pulsed Fourier transform method is that each scan requires only on the order of 1 sec. Hence, 100 scans can give up to a factor of 10 in signal-tonoise improvement and lo4 scans up to a corresponding 100-fold improvement (the signal-to-noise ratio im-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
Time-Averaging
Report
Nuclear Magnetic Resonance Spectroscopy George A. Gray Varian Instrument Division 25 Route 22 Springfield. NJ 07081
proves as the square root of the numher of scans). The latter improvement alone can overcome the unfavorable natural abundance factor for 13C. Pulsed Fourier Transform NMR In a nuclear spin system a t equilihrium the steady state and impulse response form a Fourier transform (FT) pair. That is, the time response of the spins can he calculated from their frequency domain spectrum and vice versa. This is extremely useful for obtaining spectra quickly so that multiscan averaging can he performed. The basic FT experiment is illustrated in Figure 1. Rather than a continuous, weak (mW) rf field, we employ a powerful (kW) field which is pulsed on for times as short as a few microseconds. The response of the entire spin system is picked up in the normal manner, amplified, and detected in the spectrometer system, The resultant signal is digitized in an analog-to-digital converter (ADC) and stored in a computer. Subsequent “transients” are taken in a similar manner and are coherently added to the previously stored data for signal-to-noise improvement. After completion of the data accumulation and the Fourier transformation, the frequency domain data may he plotted in the normal manner. There are a few simple relationships which are fundamental to an understanding of FT spectroscopy. First, to unambiguously assign a frequency of “f’Hz, the ADC must perform conversions a t a rate of a t least “2f” conversionslsec. Hence, a spectral width of 1000 H z (the transmitter frequency is defined as 0 Hz) requires an ADC
rate of 2000 conversionslsec. Each conversion will result in a time-indexed sampling of the transient response which must be added to the appropriately stored samplings in the computer memory. Therefore, the length of time spent acquiring data will determine the number of conversions and hence the number of memory channels used for data storage. Of course, the normal case is that of a limited memory capacity, typically 8192 channels or “words”. Hence, for a full data table, 8192 = (ADC rate) X (acquisition time); 8192 = 2 X SW X AT. Another fundamental relationship is that the attainable resolution is determined by the length of time recording the response of the spin system to the pulse (the free induction decay or
-
FID), Le., resolution 1/AT (Figure 2). Of course, the limiting resolution attainable will be that corresponding to the natural linewidth (liTd-lor magnet homogeneity-dictated linewidth. Characteristics of 13C NMR Spectrum In many respects, the chemical shift scale (Figure 3) closely follows that of the proton, even including the commonly accepted internal reference tetramethylsilane (TMS). Taking TMS as the zero of the scale, resonances to higher frequency (at constant field) are considered to have positive chemical shifts in ppm-the same as the proton “6” scale. Aliphatic carbons are a t higher shielding (lower frequencies) and therefore have the
Figure 1. Schematic of FT spectrometer ANALYTICAL CHEMISTRY, VOL. 47. NO. 6 , MAY 1975
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CFT.20 SPECTRUM NO, OPERATOR DATE SAMPLE
LOCK SIGNAL SPIN RATE
rps
TEMP
'C
ACQUISITION SPECTRAL WIDTH (SW] NO OF TRANSIENTS I N T I ACQUISITION TIME (AT) PULSE WIDTH (PW1 PULSE DELAY (PDi DATA POINTS (DPI
Hz SK
" sec SK
TRANSMITTER OFFSET (TO1 HIGH FIELD LOW FIELD RECEIVER GAIN IRG) DECOUPLER MODE I D M ) DECOUPLER OFFSET IWi NOISE BANDWIDTH I N B i
kHz
DISPLAY SENS ENHANCEMENT (SEI WIDTH OF PLOT I W P l I N D OF PLOT IEPI WIDTH OF CHART IWCI END OF CHART IECI VERTICAL SCALE (VSI 7EFERENCE LINE (RLI
SK HZ HZ
HZ HZ
Figure 2. Signal-to-noise improvement obtained (at expense of some resolution) by multiplying raw FID by decaying exponential function e"(-SE),thus favoring high SIN portion of FID over low S/N portion
smaller chemical shifts. With increasing substitution, and particularly heteroatom substitution, the resonances appear a t greater chemical shifts. Olefinic and aromatic carbons occur at 90-170 ppm, whereas carbonyl carbons have chemical shifts at 160-220 ppm. Note that in ppm the carbon scale is greater than 20 times as wide
as that of the proton. Since the linewidths are similar, this represents a true increase in dispersion. Considerable success has been realized in empirical prediction of 13C chemical shifts based on substituent parameters. Usually, this approach relies on model compounds and a minimal number of substituted com-
pounds. For example, Grant and Paul (I) and later Lindeman and Adams (2) have systematically developed regressional analyses for linear and branched hydrocarbons which give excellent comparisons between calculated and experimental shifts. Thus, the chemical shift of a paraffinic carbon k can be expressed as: 6'
= A
+
XBp,, i
Figure 3. I3C chemical shift correlations 548A
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
where A represents an appropriate reference compound, Bi the ith substituent effect, and nik the number of substituents of type i relative to carbon k. Typical substituent effects (appropriate for the reference methane) are 9.1 ppm for replacement of a proton by a carbon ( a effect), 9.4 ppm for replacement of a /3 hydrogen, and -2.5 for replacement of a y hydrogen. Further parameters are necessary for branching situations, but they are straightforward, and calculating shifts for an arbitrary branched.or linear hydrocarbon is simply an arithmetic exercise. Similar parameter sets have been developed for cyclic hydrocarbons ( 3 ) . Replacement of a substituent carbon with a polar substituent leads to extremely large effects on the 13C shifts.
ir=-----
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Introducing something NEW under the sun for liquid chromatography-the constaMetric II-optimized for HPLC to provide pulsationless solvent delivery against pressures to 5000 psig. This newest member of the M i l t o n Roy/LDC family of pumps for the laboratory combines the proven performance of the Instrument minipump with the pulsationless delivery achieved by the Solvent Delivery System. In addition to constant flow, the constaMetric II adds constant pressure control capability plus digital display of five different operating parameters of your LC set-up. Designed to be compatible with all chromatographs, the constaMetric SI is the ultimate choice for upgrading the performance of any existing HPLC system. All of LDC's controlled volume pumps meter extremely precise volumes of solvent with repetitive accuracy of better than f0.3%. So now you can choose from an assortment of LDC pumps to maintain flow rates of 6 t o 1000 ml/hr, in addition to the
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CIRCLE 1 5 0 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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CFI.20 SPECTRUM NO OPERATOR DATE
Gray
20
J3J4.J-5
5
*I1
1
'I
c
i &
r,-
0
cbc/,
LOCK SIGNAL SPIN RATE 3 Q r p r
TEMP3r'C
ACQUISITION
Yqk?r Hr
SPECTRAL WIDTH iSW1 NO OF TRANSIENTS !NT) ACQUISITION TIME (AT) PULSE WIDTH I P W I PULSE D E L A Y i P D i Q DATA POINTS iDPl 8 1
0.92bsec
/a
,sec rec
92
96
TRANSMITTER OFFSET ~TOI HIGH FIELD LOW FIELD J RECEIVER GAIN IRG)
y
DECOUPLER MODE IDM) D E C O W L E R OFFSET (DO) NOISE BANDWIDTH ( N B )
DISPLAY
f /0
kHz
is
SENS ENHANCEMENT WIDTH OF PLOT i W P i END OF PLOT IEP) WIDTH OF CHART I W C I E N D OF C H A R T i E C I VERTICAL SCALE i V S i REFERENCE LINE I R L )
4?f$
z: '
8 2 9
HZ
HZ Ht
0
-
YO
Figure 4. Closely related steroids showing sensitivity of 13C shifts to chemical structure. Note only a few resonances may be assigned in any proton spectrum even at 100 MHz, and proton shift range covers 6 ppm whereas carbon shift ranges over 200 ppm
CFT.20 SPECTRUM NO. >PERATOR DATE fa/&
GPflr
I
-C
-
OCK SIGNAL ,PIN RATE
!
I
ips
TEMP
'C
KQUISITION P E C T R A L WIDTH !SWl 10 OF TRANSIENTS (NT) \CQUlSlTION TIME !AT1 'ULSE WIDTH i P W i 'ULSE DELAY IPD) IATA POINTS I D P I
HZ
rec
" Sec rec
RANSMITTER OFFSET (TO) IIGH FIELD LOW FIELD IECEIVER GAIN IRGI IECOUPLER MODE IDM) IECOUPLER OFFSET 100) 1OlSE BANDWIDTH I N 8 1
)ISPLAY
f 9
a
&HZ
-#*b 16 do5:t / b 80 li
,ENS E N H A N C E M E N T i S E VIDTH OF P L O T I W P I N D OF PLOT i E P ) VIDTH OF CHART (WCI N D OF CHART (ECI lERTlCAL SCALE iVS1 IEFERENCE LINE iRL1
$0
firs
Figure 5. Off-resonance spectra allow determination of degree of protonation of carbon. Only changes necessary are moving decoupler frequency out of proton region and removal of noise bandwidth 550A
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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CIRCLE 221
ON
READER SERVICE CARD
Changes of I3C Chemical Shifts upon Replacement of Methyl Group by Polar Substituent Substituent
-OR -OH -0COR -NH2
-c1 -F -cox
-COOR -COOH -CN
C-1 +45 +40 +43 +20 $23 +61 +l5 +10
+12 -2
C-2 -3 +1 -2 +2 +2 -1 -5 -1 -3 -1
C-3 -1 -1 -1 -1 -1 -2 0 -1 -1 -1
R = alkyl, X = C1 or NH2 Depending on substituents, carbonyl shifts range from 152.0 ppm for (CsH50)&=0 to 215.8 ppm for di-tbutylketone. Aromatic and olefinic carbons exhibit similar large ranges of shifts and have also been systematically cataloged (4). This inherent sensitivity to substituents is responsible for the power of 13C NMR in structural studies (Figure 4). The shifts alone give significant information in this regard but are usually insufficient, except in the simplest cases, to completely assign the resonances and subsequently formulate a structure for an unknown com-
pound. T o attain these objectives, the chemist has several powerful diagnostic techniques: Coherent single-frequency offresonance decoupling Gated decoupling Suppressed Overhauser spectra Selective proton decoupling I3C-lH shift cross-correlation Deuteration Specific labeling Spin-lattice relaxation time measurements. Coherent Single-Frequency Off-Resonance Decoupling When the IH decoupling power is insufficient and/or the IH frequency not exactly set a t the resonance frequency of the proton(s) coupled to the carbon of interest, there will be some residual coupling, manifested as a line broadening or actual breaking up of the singlet into the t y p e of multiplet characteristic for the number of protons on the carbon. Thus, a methyl carbon will appear as a compressed quartet, a methylene as a triplet, and a methine as a doublet. The advantages of this are the characterization of the carbon as to degree of protonation, retention of the full nuclear Overhauser effect, and reduced splittings, making interpretation convenient. In most cases, useful “off-resonance” spectra
can be obtained in about the same time as the noise-decoupled spectra (Figure 5). Gated Decoupling
A coupled spectrum may be obtained simply by turning off the proton decoupler, although a significant sensitivity penalty is often paid since no decoupling is permitted. It is possible to gain back some of the Overhauser enhancement by turning on the decoupler for a period just prior to the monitoring pulse and acquisition (Figure 6). Since the pulse samples the state of magnetization existing immediately prior to the pulse, the coupled spectrum and Overhauser enhancement may be obtained by automatically turning off the decoupler before the pulse and on again after completion of acquisition. Couplings appear instantaneously so that a fully coupled spectrum is obtained. The reverse “gating” sequence accomplishes the “suppressed Overhauser spectrum”. Suppressed Overhauser Spectrum In this mode the decoupler is turned on just prior to the pulse and off after acquisition. Any Overhauser enhancement built up during the acquisition does not affect the signal being recorded since it only contributes to the magnetization along the magnetic
TRANSMITTER OFFSET (TO) 75HIGH FIELD J LOW FIELD RECEIVER GAIN IRGI ‘f
2 10
DECOUPLER MODE (DM) DECOUPLER OFFSET 100) r 6 NOISE BANDWIDTH INBi /‘o
kHz
Figure 6. Greater signal-to-noise ratios obtained in given amount of time through use of gated decoupling Insertion of pulse delay and setting DM = 2 automatically sets proper conditions. Note about a factor of two in S/N gained through use of gated decoupling. or same S/N as in lower trace could have been obtained in one-fourth of time. Obviously, even in very simple molecules, a very substantial SIN price paid to obtain coupled spectrum 552A
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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O N READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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LOCK SIGNAL
ACQUISITION
2000 /oL?
SPECTRAL WIDTH (SW! Hz NO OF TRANSIENTS I N T I ACQUISITION TIME IAT! 2804'7 sec PULSE WIDTH IPWl /g "set PULSE DELAY IPD! 000 rec DATA POINTS (DP! r/q
#
TRANSMITTER OFFSET (TO! 5-0 HIGH FIELD / LOW FIELD RECEIVER GAIN IRG!
3(4,
DECOUPLER MODE IDMI DECOUPLER OFFSET (DO] 5 NOISE BANDWIDTH INB!
n
/(d
/
kHz
DISPLAY SEiS-ENHANCEMENT ISEl WIDTH OF P L O T I W P I / 2 # Q 3/2. END OF PLOT (EPI WIDTH OF CHART (WCI END OF CHART (EC! VERTICAL SCALE (VSI REFERENCE LINE IRLI
-/
sec
/aQO
Hz Hz Hz
3/
4Z=/
-h d 2
lnswr ~ a m noise c
/**rl,* A re. Figure 7. Only difference in two spectra is presence or absence of decoupling prior to pulse and concomitant nuclear Overhauser enhancement. Ratio of intensities or, better, areas, gives NOE "factor" directly. Decoupler gating automatic as specified by selecting DM parameter 1 or 3
cdc/,
LOCK SIGNAL SPIN RATE 3 V rps
TEMP
71' 'C
ACQUISITION
SPECTRAL WIDTH ISWI 2703 NO OF TRANSIENTS (NT! /&X ACQUISITION TIME (AT! /f/>PULSE WIDTH IPW! /5 PULSE DELAYIPDI 0 DATA POINTS ( D P l &"'
HZ
sec "5%
sec
TRANSMITTER OFFSET ITO! 5 ' HIGH FIELD W ' LOW FIELD RECEIVER GAIN (RGI 'f DECOUPLER MODE I D M I 1 DECDUPLER OFFSET (DO! s d / $ ' O NOISE BANDWIDTH INB! D kHz
DISPLAY
SENS ENHANCEMENT (SEI -/o WIDTH OF PLOT (WP! /SJ-O END OF PLOT (EPI /4Wf-2WIDTH OF CHART (WC! /530 END OF CHART (ECI / f l O / - Z M VERTICAL SCALE 1VS) fd REFERENCE LINE IRL! TM
k h s
mpanr?%se
sec Hz Hz HZ HZ
observe&
fori Si,CfJ
Figure 8. Method uses at least two off-resonance spectra (containing TMS) to establish proton shifts For case of two as shown, baselinesare positionedto provide convenient vertical proton frequency scale which can be converted subsequently to ppm. Two proton offsets bracket proton spectrum. Proton shift numbers taken from more accurate plots of residual couplings for spectra where proton offsets were 5800 (DO = 58),5600, 5100, 4200, 4000, and 3800 Hz (DO = 38). Proton spectra of compound would only permit methyl, CHOH, and olefinic proton assignment
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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field (z) axis, while the receiver coil only picks up signals along one of the other orthogonal axes. Any subsequent monitoring pulse would measure signals with Overhauser enhancement (Figure 7). This may be avoided by making the interval between the end of acquisition and the next pulse a t least 3-5 times as long as the acquisition time. If this delay is also 13-5 T l m a r (see below), each carbon will have completely returned to its equilibrium magnetization. The spectral integrations, therefore, are reflective of carbon concentrations and useful for quantitative analysis. Further, when this measurement is compared with data obtained under identical conditions except that the decoupler is left on continuously, the ratio of line intensities gives directly the nuclear Overhauser enhancement factors (minimum of 1.00 to maximum of 2.988). These factors are valuable in analyzing and using spin-lattice relaxation time data.
Deuteration Replacement of hydrogen by deuterium is a standard and well-used technique in chemistry, and it has dramatic effects in the I3C NMR spectrum. Deuterium has a spin of 1, is not affected by the proton decoupling, and produces a 13C-2H multiplet in the I3C spectrum. If a carbon is completely deuterated, its spin-lattice relaxation time is greatly increased, so that its resonance is easily saturated and consequently its intensity in the normal rapidly pulsed mode is greatly diminished, perhaps to within the noise level. The absence of a resonance therefore indicates the deuterated carbon. Neighboring protonated carbons, however, still spin couple to the deuterium atoms, marking those carbons within two bonds of the originally labeled carbon. Sequence information is therefore an additional benefit of this technique.
Further information may be obtained by relating one particular carbon to a proton (or protons) to which it is coupled, particularly if the proton spectrum gives structurally unambiguous information. Specific decoupling (single-frequency on-resonance) will result in a sharp singlet for any carbon directly bonded to protons resonating a t that frequency. Certain complications can occur in those cases with overlapping l3C-lH satellite spectra (recall that it is the satellite peaks which must be irradiated, not those in the normal proton spectrum) or when the number of signals is too large for practicability. Both of these objections are circumvented by a simple graphical technique.
Specific Labeling Deuterium is just one example of specific labeling. This may alternately be done with both magnetically active or inactiue nuclei. For example, labeling with 13C depleted material would also give rise to one or more missing resonances when compared to the natural abundance 13C spectrum. 15N enrichments of -90% are available commercially. Use of the 15N will produce 13C-15N splittings of those resonances corresponding to carbons in proximity to nitrogen. Of course, 13C enrichment is a classic avenue toward better signal-to-noise, but it can also be used to mark one type of carbon and follow its resonance unambiguously. This is extremely important for low concentration biochemical samples or in following metabolic or process development.
13C, ’H Chemical Shift Cross-Correlation Selective decoupling is a time-consuming and tedious operation and therefore of limited use. A far faster and more informative technique utilizes two or more “off-resonance” spectra in a graphical manner to correlate individual carbons and protons (Figure 8). The residual 13C-IH couplings are plotted as a function of decoupler frequency, and points of intersection give exact decoupling frequencies. When an internal reference such as T M S is used, the decoupling frequencies can be placed on the chemical shift ppm scale, thus connecting the observed proton shifts with the observed carbon shifts. This is done simultaneously for all protonated carbons and hence is productive and useful as a routine diagnostic tool.
Spin-Lattice Relaxation ( Tl) The essentially instantaneous sampling of the magnetization state of the sample via a pulse allows following of time-dependent phenomena which were very difficult or impossible by traditional CW spectroscopy. One time-dependent process is the actual magnetization of the sample following placing the sample into the probe. In CW spectroscopy the relative slowness of the spectral determination made this time inconsequential. Our interest in this “relaxation” process (recall that the equilibrium populations in the spin levels are equal prior to placing the sample in the magnetic field; therefore, the sample is in an “excited” state) arises from the observation that difficult spectral lines in the NMR spectrum can and do have different relaxation times. differences
Selective Proton Decoupling
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
that can be interpreted in terms of chemical and physical concepts. Spin-lattice relaxation times can be determined experimentally using a two-pulse sequence as diagrammed in Figure 9. The 180’ pulse inverts the magnetization to along the -z axis. At an arbitrary time later, the state of magnetization is sampled by a 90’ pulse. For very short times between the 180’ and 90’ pulses, the resulting spectrum will appear inverted. Of course, as the interval time becomes very long, it is just equivalent to a single 90’ sampling pulse, producing upright peaks. When a set of different experiments is performed a t different interval times, a sequence of spectra is obtained in which the peaks start out negative and systematically go positive. The value of this process is the variability of rates of return to equilibrium for the various lines in the spectrum, a variability which can be tied to the chemistry of the molecule. Although more specialized theory is necessary to explain 2’1’s of macromolecules tumbling very slowly in solution, there is a very simple form for the T1 of a carbon in intermediate to small molecules (MW
