sy I I I I I I I I I1 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 “hyphenated” procedure in which chromatography is used for separation of the mixture into pure substances and then mass spectrometry, infrared spectroscopy, or some other spectroscopic method is used for structural identification of each component. Although nuclear magnetic resonance (NMR) spectroscopy has been very successful in structural determinations of pure compounds, it is seldom used for identification of specific molecules in complex mixtures. Here we show t h a t methodology recently developed in our laboratory (2-6)increases the power of NMR for analysis of complex mixtures. We wish to emphasize that, because ofthe low sensitivity of NMR spectroscopy, the large amounts of material required for an analysis will restrict the applicability of the methods presented below. Nevertheless, the examples in this report illustrate the many important analytical problems that can he solved by means of ultra-high resolution NMR methods. We have used two terms, complex mixtures and ultra-high resolution NMR, that deserve further elaboration. In this article, we use two arbitrary hut convenient definitions of complex mixtures: A complex mixture of Class A contains many compounds 0003-2700/87/0359-441A/501.50/0
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American Chemical Society
of very similar structures in any proportions; a complex mixture of Class B contains a t least two similar compounds in lopsided concentration ratios, such as 104:1. In this report we present one Class A application, the composition of gasoline, and two Class B cases, a nonequilibrium trace amount of the aldol condensation dimer of acetone contained in reagentgrade acetone and the equilibrium concentration of a linear isomer of D-glucose.
proton-decoupled ’:IC NMR spectra on standard commercial NMR instruments with the use of standard large (IO-mm diameter) sample tubes, large (3 mL) sample volumes, and standard single-pulse excitation (5). Proton-decoupled I3C NMR spectra were recorded at 50.3 MHz on a slightly modified (see below) Nicolet NT-200 NMR spectrometer equipped with a IO-mm probe from Cryomagnet Systems ( 5 ) .Temperature drift was diminished by prestabilizing the tempera-
1 We use the term ultra-high resolution NMR to describe operating conditions under which the NMR instrument makes a negligible contribution to the observed line widths ofthe NMR peaks (1). Ultra-high resolution methodology can be achieved on typical commercial high-resolution NMR instruments as a result of minor and inexpensive modifications ( 1 - 5 ) . Our studies suggest that the resolution performance of typical commercially available high-resolution superconducting 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 contribution to the line width of at least 0.1 Hz. We have demonstrated the feasibility 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 immersed in a large constant-temperature (*0.02 “C) water reservoir (2). Temperature gradients within the sample were diminished by using efficient low-power proton decoupling (3, 4 ) and by increasing the flow of compressed air from the “normal” 5-10 L/min to about 20 Llmin ( 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 resolution improvement increases the data memory requirements. Specific information about computer requirements appears later in this article, following the three examples of analytical applications. From the standpoint of minor component detection, it is noteworthy that ultra-high resolution NMR not only di-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15. 1987 * 4 4 1 A
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. . Recorded a1 50.3 MHz and 28.5 O C wkh an acquIsillQIU r n 01 20.45 6. a 8Ira1 w i d h of 1602.58 Hz (qusdnmrs demclwnl. 128K l i r n - j o m i n poim. and 2100 s m . Sp&um a Is a 25-told h o r i m l and 2 M d vertical expanohm of he 32.0-34.0 ppm re~lm01 apenum b. FlW-four Urndomain b a t 6 (15 of 80 scans ea& m e acquired. Homooeneliy a d l M M ( 2 , gradlent only1 was aU(omal1~llylmpkmmed pior 10 each 8 0 a m M c h . The 35 batches
minishes the instrumental contribution to the line width a t half height, but it also greatly diminishes the instrumental contribution to the line shape at the skirts of the peaks, such as the width a t 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 gradients perpendicular to the spinning axis; the field gradient along the spinning axis is minimized with field-gradient shim corrections to high order (7). Spinning of the samde causes spinning sidebandsat frequencies in”, relative to the centerband, where n = 1, 2, 3 . . . , and u. is the rate of rotation (7). Even relatively weak “high-order” spinning sidebands (at large multiples of fu,) of strong peaks can interfere severely with the study of minor components. We use a twofold strategy to eliminatespinningsidehands.First, we have developed field-gradient adjust-
11
w l h the MROwesl line wdths were consolldaled inm 7 11-dmin spma and proanaed I& 0.01 HI exponmllal broadening. Ana Fouriff lran8tgrme lion. the resulting 7 hequency-domaln s p n r a wwe adad in he doublegrecision 140-bl word1 mode. kbrlromal scab is in par16 pff million relative lo ~r. leml MerSi. The wrtlull scale was adlusted 10 rmrale the tallm peak at 5% 01 I(s lull peak hsigm Isw ma).
ment procedures that minimize the amplitude of spinning sidebands. Second, we use a modified version of a reported method (8)of v. modulation to “smear out” the residual spinning sidebands. The three structural studies presented below were carried out with the use of I3C NMR spectroscopy: however, our conclusions can he extrapolated to the more sensitive ‘H, 19F, and 3lP NMR techniques and to low-sensitivity spin-1R nuclei such as 15N and 29%.
‘Mure lzlmoTous substances: gasollne A typical gasolineconsists of more than 200 compounds, many with very similarstrunures,andisacomplexmixture of Class A. Chromatography has been the best method for the analysis of petroleum fractions (9). Liquid chromatography was used in the monumental Research Project 6 of the American Petroleum Institute (10. 11). Today, gas chromatography (GC) is used for re-
442A * ANALYTICAL CHEMISTRY. VOL. 59. NO. 6. MARCH 15. 1987
solving individual molecules (12, 13), whereas high-performance liquid chromatography (HPLC) is used for hydrocarbon group-type analysis (14, 15). NMR has been used for group-type analysis of crude oil, petroleum fractions, shale oil fractions, and liquefied coal fractions (16-18). As far as we know, only one I3C NMR spectrum of a gasoline fraction has been reported (16);there is also a published 13C NMR spectrum of Petsol, a commercial aromatic petroleum solvent that boils in the gasoline range (17). In any case, the reported I3C NMR spectra of fossil fuel fractions did not have enough resolution to yield resolved peaks of individual compounds, and therefore no attempts have been made in the past to use I3C 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 unleaded gasoline are shown in Figures l b and
Flgure 2. Region of unsaturated carbons in the protondecoupled 13C NMR spectrum of gasoline. Spctrum a IS a 22fald horizonla1 and ?-fold venical expansion 01 the 126 0128 0 ppm reglo" Of spechum b Sample and procedures were the same 85 ecqulsltlon tlmS war 28 64 5 , the Sweep wldlh for Figure 1, except mat
2b, respectively. The vertical scale has been set to show the numerous small peaks. Therefore, many strong peaks are truncated a t a small fraction of their full peak height. The tallest peaks in Figures Ib and 2b, the resonance of C-1 of 2-methylbutane at 22.464 ppm and that of C-3 of toluene a t 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 %, respectively, of our gasoline sample (see below). We observe about 1200 and 600 peaks in the saturated and unsaturated carbon regions, respectively. The spectra of Figures l h and 2b are too compressed horizontally for a detailed analysis. Figures l a and 2a show horizontal (and also twofold vertical) expansions of small portions of the saturated and unsaturated carbon regions, respectively. In each case, the expanded 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 expanded in Figure 3), which covers a total
32.80
was i 1144 16 Hz. the best 35 out of 61 batches were used. and the chemical *hilt scale was established from a separate 240-ppm spectrum mat contained the resonance 01 Me,%
32.7,
32.78
Ppm Flgure 3. The crowded region from 32.70 to 32.80ppm of the spectrum of Figure la. ANALYTICAL CHEMISTRY, VOL. 59, NO. 6. MARCH 15. 1987
443A
chemical shift range of only 0.10 ppm. Figure 3 can be contrasted with the fact that the most extensive published tabulation of l'C chemical shifts of paraffins. published in 1971 (19),lists a precision of f O . l ppm. More recent reports often cite chemical shifts with a precision of fO.O1ppm (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 molecules. A detailed examination of Figures 1 and 2 reveals that most of the observed 1800 peaks are resolved resonances of individual compounds. This observation has major significance only if the resonances can be assigned to specific molecules. Although assignments to types of structural groups can be made on the basis of published '"C chemical shifts, the close proximity of many- resonances in our gasoline spectra prohibits assignments based on chemical shift values alone. To make specific assignments. we took advantage of the high resolution in our spectra. 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 addition of the known compound. There may he minor difficulties when comparing the two spectra because of the extreme sensitivity of chemical shifts t o solvent variations. Details will be given elsewhere. So far, we have identified the resonances of 32 compounds. The procedure is fast, and it is particularly suitable for gasoline because so many gasoline hydrocarbons are available in pure form. A few of the assigned resonances fall within the expanded windows of Figures 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 l,S,5-trimethylbenzene, C-4 of mxylene, C-4 of o-xylene, and C-4 of ethylbenzene, respectively. Peaks 1 and 2 of Figure l a arise from C-4 of 2,3,3-trimethylpentane and C-5 of 2methylheptane, respectively. Peaks 9, 10, and 11 of Figure l a are assigned t o C-3 of n-heptane, n-hexane, and 2methylbutane. respectively. The remaining assignments presented here refer to Figure 3. Peak 3 is assigned t o C-6 of' Zmethyloctane; peaks 8, 5 , and 6 are the C-3 resonances of n-octane, n nonane, 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 444A
dodecane, n-tridecane, and 3,3-dimethylpentane contribute to peak 4. We have deliberately described here a "worst-case" region (Figure 3) of carbons with very similar chemical shifts. Most resonances of Figures I b and 2b are well resolved. The observation of ieparate C-3 resonances for n-octane, ri-nonane, n-decane, and n-undecane in Figure 3 is an illustration of the structural sensitivity of I T chemical rhifts of hydrocarbons. The C-3 resonances of the smaller n-alkanes are actually 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 caution must be exercised in the application of our assignments to NMR spectra of other gasoline samples because l 'C chemical shifts of hydrocarbons are subject to large solvent eftects. For groups of closely spaced peaks, even small changes in composition mag produce ambiguities in relative chemical shifts if the solvent effects vary from peak to peak. We plan to measure these effects systematically and develop methodology for direct identification of molecular components from a single *?CNMR 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 proportions of the meso and d,l-diastereomers of 3.4-dimethylhexane. In the past, GC ha5 only yielded the combined proportion (12,13).Knowledge about relative proportions of diastereomers may yield information about the chemical history of the compounds (21). Diastereoisomerism induces large nonequivalence of "C chemical shifts when the two asymmetric centers are near each other (19, 20). We have identified the resonances of the meso and d,l-diastereomers of 3,4-dimethylhexane in the spectrum of gasoline (Figure Ib) by using the reported I3C chemical shifts of the pure compounds ( 2 2 ) and our method of comparing the spectra before 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 integrated intensities of the 13C resonances indicate that there is more d,lthan meso- isomer in our sample. Quantitative analysis. The use of integrated intensities of l3C resonances for determination of the proportions of gasoline components is subject to possible errors caused by differences in spin-lattice relaxation times ( T I )and nuclear Overhauser enhancement (NOE) factors (23). On the basis of available data (23)and our own recent
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
observations, we believe that 2'1 and NOE differences will not significantly degrade the accuracy of quantitative determinations of hydrocarbon mixture components by NMR, provided that nonprotonated carbons such as quaternary aliphatic and substituted aromatic ones are excluded from the analysis. Nevertheless, additional systematic measurements of TI and NOE values of hydrocarbon mixture components are desirable to firmly establish the required conditions for high-accuracy quantitative analysis. Additives. Alcohols in gasoline have been determined by GC (24) and IH NMR (25).After we doped our gasoline sample with a small amount of methanol, 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 l b , 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 'H NMR (25) methods, respectively. Other oxygen-bearing additives should also be easy to determine by 13C NMR. Carbons bonded to oxygen are expected to yield % chemical shifts in an uncluttered region outside the range shown in Figure lb. Comparison of GC and 13C NMR. High-resolution NMR instrumentation is much more expensive than GC. The comparison of technical performance 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 economic 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 petroieum 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 different molecular weight, but it has lower resolving power for structural isomers (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 indistinguishable GC retention times. In such a case, a single chromatographic peak is weak evidence of a pure component.
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rqurr 4. Region of the methyl resonance in the protondecoupied 13C NMR spectrum of acetone (with 20% viv cyclohexane-dln). Recwdedat 50.3 M k and 26.5 ‘C. with an acquisition time of 54.59 5. a spectral widlh of +300.12 Hz (quadraturedetection). 64K timedomain points. and 6000 scans. Specifically. 380 time-domain batches 01 20 scans each were acquired 2 , gradient homogeneity was automaticBlly adjusted prior to each batch. The 300 batches with the narrowest line widlhs were conrolidated into 30 timedomain spectra, each 01 Which was Subjected to 30 mHz e x p ~ nemial broadening and Fourier transformation. The resulting 30 hequency-
There is much less danger of such “degeneracy” in au ultra-high resolution 13C NMR spectrum, for two reasons. First, except for very small symmetric hydrocarbons, each compound yields multiple peaks, the equivalent of several retention times per compound. Second, under ultra-high resolution conditions, chemical shift differences as small as 0.002 ppm are resolvable. Total accidental degeneracy for two compounds would imply degeneracy of all t h e i r W chemical shifts to within 0.001 ppm, an occurrence of very low probability. Analysis of minor components The classic example of application of NMR to the study of minor components in a liquid sample is the observation of ‘H NMR spectra of a dilute solute, say 10 mM of some compound in H?O. Here the dynamic range D, defined as the ratio of the peak heights of the major and minor components, is about 10,000. Historically, NMR spec-
domain Spectra were aMed in the double-precision (40-bit word) d e . FM the upper scale, in Hertz. the main peak has been set to zero frequency. The lower scale, in parts per million relative to MeLi. war obtained from a separate spectrum 01 a sample that contained MeLi. The ‘Jcc satellites 01 ”CHr ‘3CO-‘zCH3,labeled c1 and c2. are truncated at about 10% of their lull peak height in spectrum a.
troscopists have avoided this dynamic range problem by means of various water-peak suppression schemes (26).We believe that careful attention to dynamic range and line shape quality enables the NMR spectroscopist to study minor components without suppression of the major resonances, even when each minor peak has only the intensity of a major one and the separation between the two peaks is only 100 Hz or even less. Our methodology 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 intensities of small and large peaks are being studied. Our studies involve the use of NMR to observe minor components 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
446A * ANALYTICAL CHEMISTRY. VOL. 59. NO. 6, MARCH 15, 1987
major resonances is about 1Hz. 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” acetone. Figure 4 shows the methyl carbon resonance of acetone of natural isotopic composition after 6000 scans. The main peak, that of 13CH3-12CO“CH:,, is truncated at 0.6% (Figure 4b) and a t 0.06%(Figure 4a) of its full peak height. The other two relatively large peaks, a t about f 2 0 Hz from the main peak (laheled el and 4 , are the 13C-13C satellites that arise from the 1.1%(relative to ’3CH3-12CO-12CH3)of W H 3 l:’CO-”CH3 molecules. Firm evidence to he presented elsewhere indicates that peaks d,, dp, and d3 of Figure 4a arise from the 0.045% (relative to 13CH3-’2CO-12CHs) of 13CH2D-12COI2CH3molecules. Within experimental error, each of these “deuterium satellites” 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 satellites constitute an ideal system for testing dynamic range and resolution of minor resonances very close to large peaks. Peaks di, dz, 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 13G2Hscalar relaxation to the line widths of the deuterium satellites. On the basis of evidence presented elsewhere, we assign peaks a1 and a2 of Figure 4a to C-1 and (2-5, respectively, of CH,I-CO-CH2-(OH)C(CH3)2,commonly called “diacetone alcohol,” which is the aldol condensation dimer of acetone. Integrated intensities, corrected 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 components with D values much lower than those of peaks al, a2, and dl-dS can be observed if their separation from the major resonance is 40 Hz or more. We believe that D values of lo5 are accessible if the signal-to-noise ratio is high enough. The example of Figure 4, however, involves a line width (of the strong resonance) of less than 0.1 Hz. For larger line widths, D values of lo4 or more will require separations of much more than 40 Hz, as demonstrated below for line widths of about 1 Hz. Aldehyde and aldehydrol anomers of glucose. The equilibrium composition of aqueous reducing sugars has been investigated by carbohydrate 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 acyclic hydrated aldehyde (gem-diol or aldehydrol) form (27). The equilibrium proportions and rate constants of interconversion are of considerable interest because they are major determinants of chemical and biochemical behavior. 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 aqueous 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-13C]idose, has been detected recently (301,but the
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ANALYTICAL CHEMISTRY, VOL. 59. NO. 6, MARCH 15, 1987
proportion of this anomer was not reported. 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% aldehyde in aqueous ~ - [ l - ’ ~ C ] r i b o(30). se 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 biological importance of glucose, but also because of the overwhelming dominance of the two pyranose anomers (27). The reported proportions of the ?-furanose and aldehyde anomers are 0.14% (at 43 “C) and 0.002% (at 20 “C), respectively (27). The a-furanose and aldehydrol forms have never been detected (27). We expect the anomeric carbon resonance of the aldehydrol to be in close proximity to the corresponding resonances of the pyranose forms. In contrast, the anomeric carbon resonance of the aldehyde anomer is expected about 100 ppm downfield of the saturated anomeric carbon resonances (29,30). With the use of our ultra-high resolution 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 anomeric carbon region in the 13C NMR spectrum of 1.4 M D-[l-13c]glucose in HzO. It is important at this point to comment about the peaks labeled X in Figure 5, which are instrumental artifacts. It is well known that very large signal-to-noise ratios (for the large resonances), 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 procedures can be developed for distinguishing residual artifacts from real peaks. In the meantime, we have distinguished the resonances of minor anomers of glucose from artifacts by observing the temperature dependence of the I3C 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 p-furanose, a-furanose, ?-pyranose, a-pyranose, and linear gem-diol anomers, respectively, and we obtained the proportions of the an-
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Whether your requirement is for a single or dual detector gas chromatograph, only an 8000 Series instrument can offer you: Screen Graphics to display your chromatogram in real time andat the end of a run to replot and examine all or part of it. Combine lis unique feature with ............. Integral Data Handling and you lave the ultimate in self-contained las chromatographs, presenting lath chromatogram with overlaid :omponent names and all the equired integration data. The iptional reintegration facility ,aves valuable time in method levelopment and, when the day Jst isn't long enough ................ Automation Control can integrate he AS-8300 autosampler with the 1000 Series instrument control to iandle the analysis of up to 100 ;amples in a completely inattended mode of operation. 'inally. via RS232-C, ................. CompLrer ComrnJn cat ons [ne 1000 Ser cs sysiem can oc n6co o a UI oe range of compuer ;ysicms nc i.dlng Per6 n-E mer's :nrornarograpny -aborarory Uomat on Sysiem (C-ASj 10 )rov ae me mosi povverk isir-menrai on lor iooay's IJIy iuiornalco aooraiory The 8000 Series - the peak of ,=flection! For further information please contact Perkin Elmer Corp Analytical Instruments Main Ave Norwalk CT 06859-0012 U S A Tel 12031 . . 762-1000 Perkin-Elmer Ltd , Post Office Lane, Beaconsiield, Bucks HP9 1QA. England Tel. Beaconsiield (049 46) 6161 Bodenseewerk Perkin~Elmer& Co., GmbH, Postiach 1120, 7770 Ueberlingen. Federal Republic of Germany. Tel: (07551)81 0
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Figure 5. Region of the anomeric carbon resonances in the protondecoupled I3CNMR spectrum of 1.4 M D-[1-'3C]glucose (99.6% '%, obtained from MSD Isotopes) in H20, with 10% vlv dioxaneda and 1% vlv dioxane. Recorded at 50.3 MHr and 37 "C with an acquisition time 01 3.28s, a spectral width of &10,000 Hz (quadrature detection). 128K time-domain points. and 28.000 scans, which were the final result 01 140 batches Of 200 scans each 2 , gradient homogeneity was automatically adlusted prim to each batch. Im mediately aner completion of accumuiation Of a batch, it was automatically added to a dOublegrecision (40-bit word) data tile that contained the sum of all prior batches. The final 28.000-scandouble-precision time-darnain spectrum
umers. Thecarhonyl region of our spectra vielded the proportion of the aldehyde anomer. Here we shall focus on the proportions of the aldehydrol (0.0062% at 37 "C) and &-pyranose (39.1% a t 37 "C). The aldehydrol resonance (peak 5) is only 121.7 Hz away from that of the a-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 a-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 requlremenfs Ultra-hieh " resolution NMR strains the computer capabilities of existing com-
was processed in the Ilmting-pain! mode with 0.5 Hz digital broadening and Fourier transformation.The peaks designated with Care me 'Ja: saiellites that wise horn the pyranose anomerr of the 1.1 % 01 molecule that have '3c nuclei at C-land C-2.Chemical shin5 are expressed in parts per million f r m Me&. but they were measured relative lo internal dioxane, taken to have a chemical Shin of 67.47 ppm.
mercial N M R instruments in both the horimntal 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-hit) time-domain data points, which results in a maximum of 128K (20-hit) data points after Fourier transformation. This level is inadequate for covering the full range'of 13Cchemical shifts, even a t 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 comnuter memorv limitstion becomes even more severe when
450A * ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
the 20-hit word is inadequate for the vertical dynamic range required to ohserve 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-hit 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 he available for NMR, together with disk storage space of at least 400 Mhytes. Such a requirement would have been prohibitively expensive a few years ago, hut 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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Hydrocarbons from Petroleum; Reinhold New York, 1953. (11) Camin, D. L. In Chromatography in Petroleum Analysis; Altgelt, K. H.; Gouw, T.H., Eds.; Marcel Dekker: New York, 1979: Chapter 1. (12) Whittemore, I. M.In Chromatography Ln Petroleum Analysis; Altgelt, K. H.; 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) OBrien, 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. And. Chem. 1975, 47,
processor chips can address enormous amounts of memory. For example, the new Intel 80386 chip can address 4 billion bytes (one billion 32-bit words) of main memory and 64 trillion bytes of virtual memory. In the near future, inexpensive mass-produced personal computers using the 80386 chip will be much more powerful than the computers that are built into currently available commercial instruments.
conclusion Ultra-high resolution methodology expands the range of applications of NMR spectroscopy to quantitative analysis of complex mixtures. We can think of many examples beyond those presented above. Readers who have possible applications are encouraged to contact us.
546A-564A. (17)Cookson,D. J.;Smith,B. E. Fuel, 1982, 61,1007-13. (18) Petrakis, L.; Fraissard, J. P., Eds.
Magnetic Resonance-lntroduetion, Adoaneed Topics, and Applications to Fossil Energy; Reidel Dordrecht, 1984. (19) Lindeman, L. P.; Adams, J. Q. Anal. Chem. 1971,43,124%52. (20) Dalling, D. K.;Pugmire, R. J.; Grant, D. M.; Hull, W. E. Mogn. Reson. Chem.
Acknowledgment This work was supported by the National Science Foundation (Grant PCM 83-04699) and the National Institutes of Health (Grant GM 22620). We thank Mr. Robert E. Addleman and Mr. Deon Osman for their help.
1986,24,191-98. (21) Patienee,R.L.;Yon,D.A.;Ryhack,G.; Maxwell, J. R. Phys. Chem. Earth 1979, 12,287-93. (22)MBller, 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) Shafstahl, J. H.; Hardy, J. K. Anal. Chem. 1986.58, 2412-14. and references
References (1) Allerhand,A.;Addleman, R. E.; Osman, D. J. Am. Chem. Soc. 1985,107,5809-10. (2) Allerhand, A,; Dohrenwend, M. J . Am. Chem. Soe. 1985,107,6684-88. (3) Allerhand, A.; Addleman, R. E.; Osman,
cited therein.
(25) Renzoni. G. E.; Shankland, E. G.;
Gaines, J. A,; Callis, J. B. Anal. Chem.
1985.57.2864-67, (26) Hore, P.J. J. Magn. Reson. 1983, 55, 283300, and references cited there-
D.; Dohrenwend, M. J. Mogn. Reson.
1985,65,361-63. (4) Maple, S.R.; Allerhand, A. J . Magn. Reson. 1986.66.168-71, (5) Allerhand, A,; Bradley, C. H. J. Magn. Reson. 1986,67,173-76. (6) Maple, 8. R.; Allerhand, A. J. Am. Chem. Soc., 1987,109,5641. (7) Conover, W. W. In Topics in Carbon-I3 NMR Spectroscopy; Levy,G. C., Ed.; Wiley: New York, 1984; Vol. 4, Chapter 2,
in.
(27) Angyal, S. J. Ad". Corbohydr. Chem. Biochem. 1984.42, 15-68. and references
cited therein.
(28)Angyal, S. J.; Wheen, R. G. Aut. J. Chem. l980,33,1001-11. (29)Serianni, A. S.;Pierce,J.; Huang, S.-G; Barker, R. J. Am. Chem. SOC.1982.104, 403744. (30) Synder, J. R.; Serianni, A. S. J . Oig. Chem. 1986,51,2694-2702. (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,
and references cited therein.
(8) Bamrnel, B.; Evilia, R. F. Anal. Chem. 1980,52,1999-2OOO. (9) Altgelt, K.H:; Gouw, T. H., Eds. Chromatography ~n Petroleum Analysis; Marcel Dekker: New York, 1979. (10) Rossini, F.D.; Mair, B. J.;Streiff, A. J.
C. J.: Freeman. R. J. Maen. Reson. 1986.
67,396-401.
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Adam Allerhand (left) is professor of chemistry a t Indiana Uniuersity, Bloomington. His research interests include development ofNMRmethods and their application to problems of chemical and biochemical interest.
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Steven R. Maple (right) is a graduate student a t Indiana Uniuersity, where he is working on development and applications of ultra-high resolution NMR. H e received his B.S. and M.S. degrees from West Virginia Uniuersity.
