ultra-high resolution nmr - American Chemical Society

Mar 15, 1987 - laboratory (1-6) increases the power of ... 0.1 Hz. We have demonstrated the fea ... 0003-2700/87/0359-441 A/$01.50/0 ..... A state-of-...
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RESOLUTION ULTRA-HIGHRESOLUTIONNMR This new technique makes it possible to use NMR for the nonseparative structural and quantitative analysis of complex mixtures Adam Allerhand Steven R. Maple Department of Chemistry Indiana University Bloomington, Ind. 47405

Chromatography is nearly always the method of choice for the analysis of complex mixtures. Chromatography alone, however, is often inadequate; it is frequently necessary to use a "hy­ phenated" procedure in which chroma­ tography is used for separation of the mixture into pure substances and then mass spectrometry, infrared spectros­ copy, or some other spectroscopic method is used for structural identifi­ cation of each component. Although nuclear magnetic resonance (NMR) spectroscopy has been very successful in structural determinations of pure compounds, it is seldom used for iden­ tification of specific molecules in com­ plex mixtures. Here we show that methodology recently developed in our laboratory (1-6) increases the power of NMR for analysis of complex mixtures. We wish to emphasize that, because of the low sensitivity of NMR spectros­ copy, the large amounts of material re­ quired for an analysis will restrict the applicability of the methods presented below. Nevertheless, the examples in this report illustrate the many impor­ tant analytical problems that can be solved by means of ultra-high resolu­ tion NMR methods. We have used two terms, complex mixtures and ultra-high resolution NMR, that deserve further elabora­ tion. In this article, we use two arbi­ trary but convenient definitions of complex mixtures: A complex mixture of Class A contains many compounds 0003-2700/87/0359-441 A/$01.50/0 © 1987 American Chemical Society

of very similar structures in any pro­ portions; a complex mixture of Class Β contains at least two similar com­ pounds in lopsided concentration ra­ tios, such as 104:1. In this report we present one Class A application, the composition of gasoline, and two Class Β cases, a nonequilibrium trace amount of the aldol condensation di­ rtier of acetone contained in reagentgrade acetone and the equilibrium con­ centration of a linear isomer of D-glucose.

proton-decoupled 13C NMR spectra on standard commercial NMR instru­ ments with the use of standard large (10-mm diameter) sample tubes, large (3 mL) sample volumes, and standard single-pulse excitation (5). Proton-decoupled 13C NMR spectra were recorded at 50.3 MHz on a slightly modified (see below) Nicolet NT-200 NMR spectrometer equipped with a 10-mm probe from Cryomagnet Sys­ tems (5). Temperature drift was dimin­ ished by prestabilizing the tempera-

INSTRUMENTATION We use the term ultra-high resolu­ tion NMR to describe operating condi­ tions under which the NMR instru­ ment makes a negligible contribution to the observed line widths of the NMR peaks (1). Ultra-high resolution meth­ odology can be achieved on typical commercial high-resolution NMR in­ struments as a result of minor and in­ expensive modifications (2-5). Our studies suggest that the resolution per­ formance of typical commercially available high-resolution supercon­ ducting magnets is much better than has generally been believed, and that the ultimate available resolution has been masked by broadening from chemical shift gradients caused by temperature gradients in the sample (4) and temperature drift during signal accumulation. Manufacturers of NMR instruments typically specify an instrumental con­ tribution to the line width of at least 0.1 Hz. We have demonstrated the fea­ sibility of achieving an instrumental contribution of as little as 0.003 Hz in

ture of the compressed air used for sample temperature control within the probe. The compressed air was first run through a long metal tube coil im­ mersed in a large constant-tempera­ ture (±0.02 °C) water reservoir (2). Temperature gradients within the sample were diminished by using effi­ cient low-power proton decoupling (3, 4) and by increasing the flow of com­ pressed air from the "normal" 5-10 L/min to about 20 L/min (2, 4). These simple and inexpensive changes were enough to yield our reported ultra-high resolution performance (1-6). Fourier transform NMR, however, requires a digitization of the signal and computer storage of the data. Therefore, a resolu­ tion improvement increases the data memory requirements. Specific infor­ mation about computer requirements appears later in this article, following the three examples of analytical appli­ cations. From the standpoint of minor com­ ponent detection, it is noteworthy that ultra-high resolution NMR not only di-

ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987 · 441 A

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13

Recorded at 50.3 MHz and 26.5 °C with an acquisition time of 20.45 s, a spectral width of ±1602.56 Hz (quadrature detection), 128K time-domain points, and 2100 scans. Spectrum a is a 25-fold horizontal and 2-fold vertical expansion of the 32.0-34.0 ppm region of spectrum b. Fifty-four time-domain batches of 60 scans each were acquired. Homogeneity adjustment (Z, gradient only) was automatically implemented prior to each 60-scan batch. The 35 batches

minishes the instrumental contribu­ tion to the line width at half height, but it also greatly diminishes the instru­ mental contribution to the line shape at the skirts of the peaks, such as the width at 0.1% of full peak height (5). It is also desirable to reduce the size of spinning sidebands. High-resolution NMR spectroscopy of liquids requires sample spinning to minimize the broadening effect of magnetic field gra­ dients perpendicular to the spinning axis; the field gradient along the spin­ ning axis is minimized with field-gra­ dient shim corrections to high order (7). Spinning of the sample causes spinning sidebands at frequencies ±nvs relative to the centerband, where η = 1, 2, 3 . . . , and cs is the rate of rotation (7). Even relatively weak "high-order" spinning sidebands (at large multiples of ±e s ) of strong peaks can interfere severely with the study of minor com­ ponents. We use a twofold strategy to eliminate spinning sidebands. First, we have developed field-gradient adjust­

C NMR spectrum of Phillips 66 unleaded gasoline (with with the narrowest line widths were consolidated into 7 time-domain spectra and processed with 0.01 Hz exponential broadening. After Fourier transformation, the resulting 7 frequency-domain spectra were added in the double-precision (40-bit word) mode. Horizontal scale is in parts per million relative to internal Me4Si. The vertical scale was adjusted to truncate the tallest peak at 5% of its full peak height (see text).

ment procedures that minimize the amplitude of spinning sidebands. Sec­ ond, we use a modified version of a re­ ported method (8) of vs modulation to "smear out" the residual spinning side­ bands. The three structural studies present­ ed below were carried out with the use of 13C NMR spectroscopy; however, our conclusions can be extrapolated to the more sensitive 1 H, 19 F, and 31 P NMR techniques and to low-sensitivity spin-1/2 nuclei such as 15N and 29Si. Mixture of numerous similar substances: gasoline

A typical gasoline consists of more than 200 compounds, many with very simi­ lar structures, and is a complex mixture of Class A. Chromatography has been the best method for the analysis of pe­ troleum fractions (9). Liquid chroma­ tography was used in the monumental Research Project 6 of the American Pe­ troleum Institute (10, 11). Today, gas chromatography (GC) is used for re­

442 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987

solving individual molecules (12, 13), whereas high-performance liquid chro­ matography (HPLC) is used for hydro­ carbon group-type analysis (14, 15). NMR has been used for group-type analysis of crude oil, petroleum frac­ tions, shale oil fractions, and liquefied coal fractions (16-18). As far as we know, only one 13C NMR spectrum of a gasoline fraction has been reported (16); there is also a published 13C NMR spectrum of Petsol, a commercial aro­ matic petroleum solvent that boils in the gasoline range (17). In any case, the reported 13C NMR spectra of fossil fuel fractions did not have enough resolu­ tion to yield resolved peaks of individ­ ual compounds, and therefore no at­ tempts have been made in the past to use 13C NMR for the identification and quantitative analysis of individual molecules in fossil fuel fractions. Spectra. The regions of saturated and unsaturated carbons of the 13C NMR spectrum of a sample of unlead­ ed gasoline are shown in Figures l b and

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ppm Figure 2. Region of unsaturated carbons in the proton-decoupled Spectrum a is a 22-fold horizontal and 2-fold vertical expansion of the 126.0128.0 ppm region of spectrum b. Sample and procedures were the same as for Figure 1, except that the acquisition time was 28.64 s, the sweep width

2b, respectively. The vertical scale has been set to show the numerous small peaks. Therefore, many strong peaks are truncated at a small fraction of their full peak height. The tallest peaks in Figures lb and 2b, the resonance of C-l of 2-methylbutane at 22.464 ppm and that of C-3 of toluene at 128.559 ppm, respectively, are truncated at only five percent of their full peak heights. 2-Methylbutane and toluene constitute about 6.5 and 3 vol %, re­ spectively, of our gasoline sample (see below). We observe about 1200 and 600 peaks in the saturated and unsaturated carbon regions, respectively. The spec­ tra of Figures lb and 2b are too com­ pressed horizontally for a detailed analysis. Figures la and 2a show hori­ zontal (and also twofold vertical) ex­ pansions of small portions of the satu­ rated and unsaturated carbon regions, respectively. In each case, the expand­ ed region, which covers only 2 ppm, yields about 90 peaks. Furthermore, some segments of the expanded spectra are still very crowded. In particular, consider the region from 32.70 to 32.80 ppm of Figure la (shown more expand­ ed in Figure 3), which covers a total

13

C NMR spectrum of gasoline. was ±1144.16 Hz, the best 35 out of 61 batches were used, and the chemical shift scale was established from a separate 240-ppm spectrum that contained the resonance of Me4Si.

32.72

32.70

Figure 3. The crowded region from 32.70 to 32.80 ppm of the spectrum of Figure 1a. ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987 · 443 A

chemical shift range of only 0.10 ppm. Figure 3 can be contrasted with the fact that the most extensive published tab­ ulation of |:!C chemical shifts of paraf­ fins, published in 1971 (19), lists a pre­ cision of ±0.1 ppm. More recent re­ ports often cite i;iC chemical shifts with a precision of ±0.01 ppm (20). Peaks 4 and 5 of Figure 3 have a chemical shift difference of 0.0016 ppm (0.080 Hz). Peak 8. shown truncated in Figure 3, has a width at half height of 0.083 Hz, which includes a 0.010 Hz contribution from digital Lorentzian broadening. This value is typical throughout the spectra of Figures 1 and 2. Identification of individual mole­ cules. A detailed examination of Fig­ ures 1 and 2 reveals that most of the observed 1800 peaks are resolved reso­ nances of individual compounds. This observation has major significance only if the resonances can be assigned to specific molecules. Although assign­ ments to types of structural groups can be made on the basis of published l:iC chemical shifts, the close proximity of many resonances in our gasoline spec­ tra prohibits assignments based on chemical shift values alone. To make specific assignments, we took advan­ tage of the high resolution in our spec­ tra. We compared the spectrum of the original gasoline sample with another spectrum that had been doped with a specific compound. If this compound was originally present in the gasoline, no new peaks appear, but some peaks of the original spectrum grow upon ad­ dition of the known compound. There may be minor difficulties when com­ paring the two spectra because of the extreme sensitivity of 13C chemical shifts to solvent variations. Details will be given elsewhere. So far, we have identified the resonances of 32 com­ pounds. The procedure is fast, and it is particularly suitable for gasoline be­ cause so many gasoline hydrocarbons are available in pure form. A few of the assigned resonances fall within the expanded windows of Fig­ ures la, 2a, and 3; they are shown as numbered peaks in these figures. Peaks 1, 2, 3, and 4 of Figure 2a arise from C-2 of 1.3,5-trimethylbenzene, C-4 of mxylene, C-4 of o-xylene, and C-4 of ethylbenzene, respectively. Peaks 1 and 2 of Figure la arise from C-4 of 2,3,3-trimethylpentane and C-5 of 2methylheptane, respectively. Peaks 9, 10, and 11 of Figure la are assigned to C-3 of fz-heptane, n-hexane, and 2methylbutane, respectively. The re­ maining assignments presented here refer to Figure 3. Peak 3 is assigned to C-6 of 2-methyloctane; peaks 8, 7, and 6 are the C-3 resonances of rc-octane, nnonane, and n-decane, respectively; peak 5 contains contributions from C-3 of n-undecane, C-7 of 2-methylnonane, and C-8 of 2-methyldecane; C-3 of n-

dodecane, n-tridecane, and 3,3-dimethylpentane contribute to peak 4. We have deliberately described here a "worst-case" region (Figure 3) of car­ bons with very similar chemical shifts. Most resonances of Figures lb and 2b are well resolved. The observation of separate C-3 resonances for n-octane, M-nonane, n-decane, and n-undecane in Figure 3 is an illustration of the structural sensitivity of 13C chemical shifts of hydrocarbons. The C-3 reso­ nances of the smaller n-alkanes are ac­ tually far outside the range of Figure 3 and far apart from each other. Most importantly, branching creates large chemical shift variations (19). For closely spaced peaks, some cau­ tion must be exercised in the applica­ tion of our assignments to 13C NMR spectra of other gasoline samples be­ cause 1;iC chemical shifts of hydrocar­ bons are subject to large solvent ef­ fects. For groups of closely spaced peaks, even small changes in composi­ tion may produce ambiguities in rela­ tive chemical shifts if the solvent ef­ fects vary from peak to peak. We plan to measure these effects systematically and develop methodology for direct identification of molecular compo­ nents from a single 13C NMR spectrum of an undoped gasoline. Diastereomers. So far, we have only attempted to identify one gasoline component containing two or more asymmetric centers. Specifically, we have determined the separate propor­ tions of the meso and d,l-diastereomers of 3,4-dimethylhexane. In the past, GC has only yielded the combined propor­ tion (12,13). Knowledge about relative proportions of diastereomers may yield information about the chemical history of the compounds (21). Diastereoisomerism induces large nonequivalence of l;lC chemical shifts when the two asymmetric centers are near each other (19, 20). We have identified the reso­ nances of the meso and ci,/-diastereo­ mers of 3,4-dimethylhexane in the spectrum of gasoline (Figure lb) by us­ ing the reported 13C chemical shifts of the pure compounds (22) and our method of comparing the spectra be­ fore and after doping with compounds of interest. We find a total of 0.09% (by volume) 3,4-dimethylhexane, similar to the reported 0.08% measured by GC for a different sample (13). Relative in­ tegrated intensities of the 13C reso­ nances indicate that there is more d,lthan meso- isomer in our sample. Quantitative analysis. The use of integrated intensities of 13C resonances for determination of the proportions of gasoline components is subject to pos­ sible errors caused by differences in spin-lattice relaxation times (7Ί) and nuclear Overhauser enhancement (NOE) factors (23). On the basis of available data (23) and our own recent

444 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987

observations, we believe that T\ and NOE differences will not significantly degrade the accuracy of quantitative determinations of hydrocarbon mix­ ture components by 13C NMR, provid­ ed that nonprotonated carbons such as quaternary aliphatic and substituted aromatic ones are excluded from the analysis. Nevertheless, additional sys­ tematic measurements of T\ and NOE values of hydrocarbon mixture compo­ nents are desirable to firmly establish the required conditions for high-accu­ racy quantitative analysis. Additives. Alcohols in gasoline have been determined by GC (24) and Ή NMR (25). After we doped our gasoline sample with a small amount of meth­ anol, the 13C NMR spectrum yielded a peak at 49.448 ppm, which was absent from the spectrum of the undoped sample. On the basis of Figure lb, we place an upper limit of 0.05 vol % on the amount of methanol in our gasoline sample. The reported lower limits of detection are 0.08 and 0.065% for the GC (24) and l H NMR (25) methods, respectively. Other oxygen-bearing ad­ ditives should also be easy to determine by 13C NMR. Carbons bonded to oxy­ gen are expected to yield 13C chemical shifts in an uncluttered region outside the range shown in Figure lb. Comparison of GC and 13C NMR. High-resolution NMR instrumenta­ tion is much more expensive than GC. The comparison of technical perfor­ mance presented below is not intended to imply that NMR can replace GC for petroleum fraction analysis when cost considerations are critical. However, because of the complexity and econom­ ic importance of petroleum fractions, and the relatively low cost of NMR spectra on multiple-use time-shared NMR equipment, we anticipate that ultra-high resolution NMR can become an important additional technique in petroleum analysis. At present, GC can detect gasoline components down to the 0.0005% level (13), whereas Figures l b and 2b have a detection limit of about 0.01%. Better signal-to-noise ratios can be achieved, however, with more scans per spectrum (at the expense of more time) or with the use of spectrometers equipped with stronger magnetic fields than that of our instrument. The GC method is very good at resolving hydrocarbons of dif­ ferent molecular weight, but it has low­ er resolving power for structural iso­ mers (12, 13). Just the opposite is true in the case of 13C NMR. Therefore, from the standpoint of resolution, the two methods are complementary. For all but the smallest alkanes, the number of possible isomers is large, and some of them may have indistin­ guishable GC retention times. In such a case, a single chromatographic peak is weak evidence of a pure component.

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There is much less danger of such "de­ generacy" in an ultra-high resolution 13 C NMR spectrum, for two reasons. First, except for very small symmetric hydrocarbons, each compound yields multiple peaks, the equivalent of sever­ al retention times per compound. Sec­ ond, under ultra-high resolution condi­ tions, chemical shift differences as small as 0.002 ppm are resolvable. To­ tal accidental degeneracy for two com­ pounds would imply degeneracy of all their 13C chemical shifts to within 0.001 ppm, an occurrence of very low proba­ bility. Analysis of minor components

The classic example of application of NMR to the study of minor compo­ nents in a liquid sample is the observa­ tion of Ή NMR spectra of a dilute sol­ ute, say 10 mM of some compound in H 2 0. Here the dynamic range D, de­ fined as the ratio of the peak heights of the major and minor components, is about 10,000. Historically, NMR spec-

13

C NMR spectrum of acetone (with 2 0 % v/v

domain spectra were added in the double-precision (40-bit word) mode. For the upper scale, in Hertz, the main peak has been set to zero frequency. The lower scale, in parts per million relative to Me4Si, was obtained from a sepa­ rate spectrum of a sample that contained Me4Si. The 1JCc satellites of 13CH313 CO-12CH3, labeled c, and c2, are truncated at about 10% of their full peak height in spectrum a.

troscopists have avoided this dynamic range problem by means of various wa­ ter-peak suppression schemes (26). We believe that careful attention to dy­ namic range and line shape quality en­ ables the NMR spectroscopist to study minor components without suppres­ sion of the major resonances, even when each minor peak has only 10~4 the intensity of a major one and the separation between the two peaks is only 100 Hz or even less. Our method­ ology is particularly helpful when there are many large peaks, when one or more of the small peaks is very close to a large one, and when the relative in­ tensities of small and large peaks are being studied. Our studies involve the use of l:,C NMR to observe minor com­ ponents whose resonances are very close to those of major ones. We present two examples. In the first one, the line width (after introduction of some digital Lorentzian broadening) of the large resonance is about 0.08 Hz. In the second case, the line width of the

446 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987

major resonances is about 1 Hz. We feel that these two examples cover the range from very narrow to typical line widths in 13C NMR spectroscopy of simple and complex organic molecules. Minor components in "pure" ace­ tone. Figure 4 shows the methyl carbon resonance of acetone of natural isotopic composition after 6000 scans. The main peak, that of l;i CH ;i - 12 CO12 CH.·,, is truncated at 0.6% (Figure 4b) and at 0.06% (Figure 4a) of its full peak height. The other two relatively large peaks, at about ±20 Hz from the main peak (labeled cj and c2), are the in C- 13 C satellites that arise from the 1.1% (rela­ tive to 1:,CH;i-12CO-12CH,) of l3 CH 3 l;i CO-l2CH;i molecules. Firm evidence to be presented elsewhere indicates that peaks dj, d2, and d3 of Figure 4a arise from the 0.045% (relative to 13 CH 3 - 12 CO- 12 CH 3 ) of l:i CH 2 D- 12 COl2 CH :) molecules. Within experimental error, each of these "deuterium satelli­ tes" has an integrated intensity of 0.015% of that of the main resonance,

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as expected on the basis of the natural isotopic H/D ratio. The deuterium sat­ ellites constitute an ideal system for testing dynamic range and resolution of minor resonances very close to large peaks. Peaks di, d->, and d3 are at +7.033 Hz, -12.435 Hz, and -31.909 Hz, respectively, relative to the main resonance. The dynamic range factor D is 10,000, which is greater than the ratio of integrated intensities, because of a contribution from 13C-2H scalar relax­ ation to the line widths of the deuteri­ um satellites. On the basis of evidence presented elsewhere, we assign peaks ai and a2 of Figure 4a to C-l and C-5, respectively, of CH ;f -CO-CH 2 -(OH)C(CH 3 ) 2 , com­ monly called "diacetone alcohol," which is the aldol condensation dimer of acetone. Integrated intensities, cor­ rected for differences in NOE values, indicate that our sample of reagent grade acetone contained about 0.01% diacetone alcohol. This amount is 3 orders of magnitude less than the equilibrium proportion. Figure 4 suggests that minor compo­ nents with D values much lower than those of peaks ai, a2, and di-d3 can be observed if their separation from the major resonance is 40 Hz or more. We believe that D values of 105 are accessi­ ble if the signal-to-noise ratio is high enough. The example of Figure 4, how­ ever, involves a line width (of the strong resonance) of less than 0.1 Hz. For larger line widths, D values of 104 or more will require separations of much more than 40 Hz, as demonstrat­ ed below for line widths of about 1 Hz. Aldehyde and aldehydrol a n omers of glucose. The equilibrium composition of aqueous reducing sug­ ars has been investigated by carbohy­ drate chemists for the last 140 years. It is well known that each aldopentose or aldohexose in aqueous solution exists as an interconverting mixture of at least six isomeric species (anomers): the two pyranoses, the two furanoses, the acyclic aldehyde form, and the acy­ clic hydrated aldehyde (gem-diol or al­ dehydrol) form (27). The equilibrium proportions and rate constants of in­ terconversion are of considerable inter­ est because they are major determi­ nants of chemical and biochemical be­ havior. The equilibrium proportions of five of the six anomers mentioned above are known with varying degrees of accuracy for most of the aldopentoses and aldohexoses. The exception is the hydrated aldehyde form (27), which has only been reported for aque­ ous solutions of the two aldotetroses erythrose (28) and threose (28, 29), which contain about 10% aldehydrol at room temperature. The anomeric carbon resonance of the aldehydrol of one aldohexose, D-[l- 13 C]idose, has been detected recently (30), but the

CIRCLE 126 ON READER SERVICE CARD 448 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987

proportion of this anomer was not re­ ported. It is generally taken for granted that NMR is not suitable for detecting an anomer whose proportion is much less than one percent (27), even though there are isolated examples of NMR detection of much lower proportions, notably the recent report of 0.03% alde­ hyde in aqueous D-[l- 13 C]ribose (30). The detailed anomeric composition of aqueous D-glucose is undoubtedly the greatest challenge of the remaining gaps of knowledge about anomeric equilibria, not only because of the bio­ logical importance of glucose, but also because of the overwhelming domi­ nance of the two pyranose anomers (27). The reported proportions of the /3-furanose and aldehyde anomers are 0.14% (at 43 °C) and 0.002% (at 20 °C), respectively (27). The α-furanose and aldehydrol forms have never been de­ tected (27). We expect the anomeric carbon resonance of the aldehydrol to be in close proximity to the corre­ sponding resonances of the pyranose forms. In contrast, the anomeric carbon resonance of the aldehyde an­ omer is expected about 100 ppm downfield of the saturated anomeric carbon resonances (29, 30). With the use of our ultra-high resolu­ tion methodology, we have observed all six anomers of a sample of aqueous D-[l-13C]glucose, and we have obtained equilibrium proportions as a function of temperature. Figure 5 shows the an­ omeric carbon region in the 13C NMR spectrum of 1.4 M D-[l-13C]glucose in H 2 0. It is important at this point to comment about the peaks labeled X in Figure 5, which are instrumental arti­ facts. It is well known that very large signal-to-noise ratios (for the large res­ onances), obviously required for the study of minor components without "solvent" peak suppression, reveal spurious peaks caused by imperfect data accumulation and processing (31, 32). Although these artifacts are very small relative to the major resonances, they can be mistakenly identified as peaks of minor components. We are confident that these artifacts can be reduced to levels even lower than those in Figure 5 and that systematic proce­ dures can be developed for distinguish­ ing residual artifacts from real peaks. In the meantime, we have distin­ guished the resonances of minor an­ omers of glucose from artifacts by ob­ serving the temperature dependence of the ,3 C NMR spectra; the intensities of the peaks assigned to minor anomers changed with temperature, all by more than a factor of 4 when going from 27 to 82 °C. We assigned peaks 1, 2, 3, 4, and 5 of Figure 5 to the /î-furanose, α-furanose, /3-pyranose, α-pyranose, and linear gem-diol anomers, respectively, and we obtained the proportions of the an-

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ppm Figure 5. Region of the anomeric carbon resonances in the proton-decoupled 13C NMR spectrum of 1.4 M D-[1- 13 C]glucose (99.6% 13 C, obtained from MSD Isotopes) in H 2 0, with 10% v/v dioxane-d 8 and 1 % v/v dioxane. Recorded at 50.3 MHz and 37 °C with an acquisition time of 3.28 s, a spectral width of ±10,000 Hz (quadrature detection), 128K time-domain points, and 28,000 scans, which were the final result of 140 batches of 200 scans each. Z, gradient homogeneity was automatically adjusted prior to each batch. Im­ mediately after completion of accumulation of a batch, it was automatically added to a double-precision (40-bit word) data file that contained the sum of all prior batches. The final 28,000-scan double-precision time-domain spectrum

omers. The carbonyl region of our spectra yielded the proportion of the aldehyde anomer. Here we shall focus on the proportions of the aldehydrol (0.0062% at 37 °C) and tt-pyranose (39.1% at 37 °C). The aldehydrol resonance (peak 5) is only 121.7 Hz away from that of the «-pyranose anomer (peak 4). The a-pyranose/aldehydrol peak-height ratio (D) is 7200, which differs from the anomeric concentration ratio of 6300 because the «-pyranose and aldehydrol resonances have different line widths: 0.99 and 1.14 Hz, respectively. These values include a 0.5 Hz contribution from digital Lorentzian broadening. Computer requirements

Ultra-high resolution NMR strains the computer capabilities of existing com-

was processed in the floating-point mode with 0.5 Hz digital broadening and Fourier transformation. The peaks designated with C are the 1 J œ satellites that arise from the pyranose anomers of the 1.1 % of molecule that have 13C nuclei at C-1 and C-2. Chemical shifts are expressed in parts per million from Me4Si, but they were measured relative to internal dioxane, taken to have a chemical shift of 67.47 ppm.

mercial NMR instruments in both the horizontal direction (number of data points or "words") and the vertical direction (number of bits per word). Our instrument is equipped with the standard Nicolet-1280 computer provided by General Electric (previously Nicolet) NMR. This computer can contain a maximum of 256K (20-bit) time-domain data points, which results in a maximum of 128K (20-bit) data points after Fourier transformation. This level is inadequate for covering the full range of 13C chemical shifts, even at our relatively low resonance frequency of 50 MHz. Therefore, we normally record partial spectra, such as the separate ones for the saturated and unsaturated carbon regions of gasoline in Figures 1 and 2. Our computer memory limitation becomes even more severe when

450 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987

the 20-bit word is inadequate for the vertical dynamic range required to observe small peaks in the presence of big ones, as in all three examples discussed in this report. In such cases, we use double-precision (40-bit words), which diminishes the maximum number of frequency-domain data points to 64K. We are attempting to convince users and manufacturers of NMR equipment that a minimum of one million 32-bit data words should be available for NMR, together with disk storage space of at least 400 Mbytes. Such a requirement would have been prohibitively expensive a few years ago, but now computer memory and disk storage cost (not including markups by NMR manufacturers) is an insignificant portion of the price of an NMR instrument. Furthermore, the newest micro-

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(1) Allerhand, Α.; Addleman, R. E.; Osman, D. J. Am. Chem. Soc. 1985,107, 5809-10. (2) Allerhand, Α.; Dohrenwend, M. J, Am. Chem. Soc. 1985,107, 6684-88. (3) Allerhand, Α.; Addleman, R. E.; Osman, D.; Dohrenwend, M. J. Magn. Reson. 1985, 65, 361-63. (4) Maple, S. R.; Allerhand. A. J. Magn. Reson. 1986, 66, 168-71. (5) Allerhand, Α.; Bradley, C. H. J. Magn. Reson. 1986,67,173-76. (6) Maple, S. R.; Allerhand, A. J. Am. Chem. Soc, 1987,109, 56-61. (7) Conover, W. W. In Topics in Carbon-13 NMR Spectroscopy; Levy, G. C , Ed.; Wi­ ley: New York, 1984; Vol. 4, Chapter 2, and references cited therein. (8) Bammel, B.; Evilia, R. F. Anal. Chem. 1980,52,1999-2000. (9) Altgelt, K. H.; Gouw, T. H., Eds. Chro­ matography in Petroleum Analysis; Marcel Dekker: New York, 1979. (10) Rossini, F. D.; Mair, B. J.; Streiff, A. J.

Hydrocarbons from Petroleum; Reinhold: New York, 1953. (11) Camin, D. L. In Chromatography in Petroleum Analysis; Altgelt, Κ. Η.; Gouw, T. H., Eds.; Marcel Dekker: New York, 1979; Chapter 1. (12) Whittemore, I. M. In Chromatography in Petroleum Analysis; Altgelt, Κ. Η.; Gouw, T. H., Eds.; Marcel Dekker: New York, 1979; Chapter 3. (13) Johansen, N. G.; Ettre, L. S.; Miller, R. L. J. Chromatogr. 1983, 256, 393-417. (14) O'Brien, A. P.; Ray, J. E. Analyst 1985, 110, 593-97, and references cited therein. (15) Hayes, P. C , Jr.; Anderson, S. D. Anal. Chem. 1986, 58, 2384-88, and references cited therein. (16) Gray, G. A. Anal. Chem. 1975, 47, 546A-564A. (17) Cookson, D. J.; Smith, Β. Ε. Fuel, 1982, 61, 1007-13. (18) Petrakis, L.; Fraissard, J. P., Eds. Magnetic Resonance-Introduction, Ad­ vanced Topics, and Applications to Fossil Energy; Reidel: Dordrecht, 1984. (19) Lindeman, L. P.; Adams, J. Q. Anal. Chem. 1971, 43, 1245-52. (20) Dalling, D. K.; Pugmire, R. J.; Grant, D. M.; Hull, W. E. Magn. Reson. Chem. 1986, 24, 191-98. (21) Patience, R. L.; Yon, D. Α.; Ryhack, G.; Maxwell, J. R. Phys. Chem. Earth 1979, 12, 287-93. (22) Moller, M.; Ritter, W.; Cantow, H.-J. Polym. Bull. 1981, 4, 609-16. (23) Cookson, D. J.; Smith, B. E. J. Magn. Reson. 1984, 57, 355-68. (24) Shofstahl, J. H.; Hardy, J. K. Anal. Chem. 1986, 58, 2412-14, and references cited therein. (25) Renzoni, G. E.; Shankland, E. G.; Gaines, J. Α.; Callis, J. B. Anal. Chem. 1985,57,2864-67. (26) Hore, P . J . J. Magn. Reson. 1983, •55, 283-300, and references cited there­ in. (27) Angyal, S. J. Adv. Carbohydr. Chem. Biochem. 1984, 42, 15-68, and references cited therein. (28) Angyal, S. J.; Wheen, R. G. Aust. J. Chem. 1980, 33, 1001-11. (29) Serianni, A. S.; Pierce, J.; Huang, S.-G; Barker, R. J. Am. Chem. Soc. 1982, 104, 4037-44. (30) Synder, J. R.; Serianni, A. S. J. Org. Chem. 1986,57, 2694-2702. (31) Cooper, J. W.; Mackay, I. S.; Pawle, G. B. J. Magn. Reson. 1977, 28, 405-15. (32) Shaka, A. J.; Barker, P. B.; Bauer, C. J.; Freeman, R. J. Magn. Reson. 1986, 67, 396-401.

Adam Allerhand {left) is professor of chemistry at Indiana University, Bloomington. His research interests include development of NMR methods and their application to problems of chemical and biochemical interest.

Steven R. Maple {right) is a graduate student at Indiana University, where he is working on development and ap­ plications of ultra-high resolution NMR. He received his B.S. and M.S. degrees from West Virginia University.

processor chips can address e n o r m o u s a m o u n t s of memory. For example, t h e new Intel 80386 chip can a d d r e s s 4 bil­ lion bytes (one billion 32-bit words) of m a i n m e m o r y a n d 64 trillion bytes of virtual memory. In t h e near future, in­ expensive mass-produced personal c o m p u t e r s using t h e 80386 chip will be m u c h more powerful t h a n t h e c o m p u t ­ ers t h a t are built i n t o c u r r e n t l y avail­ able commercial i n s t r u m e n t s .

Conclusion Ultra-high resolution methodology ex­ p a n d s t h e range of applications of N M R s p e c t r o s c o p y to q u a n t i t a t i v e analysis of complex mixtures. We can t h i n k of m a n y examples beyond those p r e s e n t e d above. R e a d e r s who have possible applications are encouraged to contact us.

Acknowledgment T h i s work was s u p p o r t e d by t h e N a ­ tional Science F o u n d a t i o n (Grant P C M 83-04699) a n d t h e N a t i o n a l Insti­ t u t e s of H e a l t h (Grant G M 22620). We t h a n k Mr. R o b e r t E. A d d l e m a n and Mr. Deon O s m a n for their help.

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452 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987