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Determination of Sucrose in Sugar Beet Juices by Nuclear Magnetic Resonance Spectrometry Douglas W. Lowman and Gary E. Maciel" Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523
A rapid, accurate proton Fourier transform (FT) nuclear magnetic resonance (NMR) spectrometry technique has been developed for the quantitation of sucrose in aqueous media, and has been applied to raw, pressed sugar beet juices. The technique is based upon a time-resolution approach-Time Resolution Water Eliminated FT (TRWEFT) NMR---for removal of the water proton resonance. I n TRWEFT NMR, an added paramagnetic relaxation reagent preferentially relaxes the water protons faster than the sucrose protons. Pre-truncating the free induction decay prior to NMR power spectrum calculation removes the water resonance. With an internal standard, TRWEFT NMR gives a linear response over the examined sucrose concentration range 0.0 to 0.810 M (i.e., 0.0 to 26.0 w/w YO in water). Accuracy is generally better than 0.5 YO (absolute) relative to gas-liquid chromatography results. Single solution precision is better than f0.65 % (absolute) at the 15.00 w/w YO sucrose concentration level.
Sucrose concentration of sugar beet juices has been determined by several techniques, including isotope dilution analysis ( I ) , gas-liquid chromatography (GLC) (2, 3 ) , polarimetry ( 3 , 4 ) , colorimetry ( 5 ) , enzymatic analysis (6), titrimetry ( 7 ) ,and fluorometry ( 8 ) . Isotope dilution analysis is the ultimate method for sucrose analysis, but is expensive and time consuming. T h e GLC method is as accurate as the isotope dilution method, easy enough to be used routinely, and requires less time, although the time required is still considerable. In general, sucrose contents obtained from polarimetric analysis exceed the values from GLC analysis because of the presence of optically active species besides sucrose in solution. Accuracy and precision of the other sucrose analysis methods are generally not as good as the GLC and isotope dilution methods. Because of time constraints, costs, and ease of analysis, polarimetric analysis is employed in sugar beet tare laboratories for routine sucrose determination. Although the GLC method, polarimetric analysis with enzymatic inversion, and isotope dilution method give reliable results, a need exists for a new, routine sucrose determination method with greater accuracy and precision and fewer interferences than the polarimetric method and a more rapid analysis time than the currently available accurate methods. One technique not yet applied t o the determination of sucrose (structure I)
is Fourier transform (FT) nuclear magnetic resonance (NMR) spectrometry. Considering the NMK-active isotopes in sucrose 'H NMR appears to be the best for (e.g., 'H, 13C, and l70), a rapid analysis, based upon its high relative NMR sensitivity and natural abundance. T h e proton NMR spectrum of sucrose (Figure 1) shows the anomeric proton (H,) doublet resonance less shielded than the general proton resonance region of the other ring protons and the primary methylene protons of sugars ( 9 ) . Using t h e anomeric proton as a quantitation probe, interference problems associated with other sucrose determination methods might be minimized because the anomeric proton chemical shift is substantially different from the chemical shifts of potential interfering species, such as other sugars and nitrogenous compounds. In sugar beet juices, water protons are approximately 150 times more abundant than the anomeric proton a t typical sucrose concentrations. The water proton and anomeric proton chemical shifts are close enough to make quantitation of sucrose difficult, as seen in a standard 100-MHz 'H NMR spectrum of a 0.63 M solution of sucrose in water (Figure 2). Several instrumentally oriented techniques exist for the elimination or reduction in intensity of a large solvent resonance. These techniques include the power spectrum null method (10); multiple pulse sequences-WEFT (11, 12), SWEFT (13),and VASE (13);steady-state pulse method (14); the use of a notched filter in the analog data channel ( 1 5 ) ; selective saturation (16-18); Fourier synthesized excitation (19);and rapid scan FT, or correlation, N M R spectroscopy (20-22). These methods, designed for optimum resolution, often increase the experiment time over t h a t required in routine F T NMR. Some of the methods require prior knowledge of relaxation times, as well as major hardware modification and software design. Our approach is based upon recognizing t h a t the time constraints were more important than resolution considerations in this particular application. Paramagnetic species in solution (e.g., paramagnetic metal ions) reduce the relaxation times of all protons in solution. Since this reduction in relaxation time is dependent on concentration and distance of closest approach, it seemed possible to find a concentration of a paramagnetic species that would relax the water protons much faster than the sucrose protons. The initial portion of the free induction decay (FID) could then be zeroed, removing the rapidly relaxing water resonance, leaving much of the FID due to the sucrose resonances still remaining. In this way, a time-resolution approach to water resonance removal would be feasible. For routine quantitative NMR determination of sucrose, a method is needed that is simple, reproducible, and does not increase the data collection time. This paper deals with the development of such a method, employing the time resolution approach to solvent resonance elimination.
EXPERIMENTAL All chemicals except the deuterated sucrose used in this research were obtained as reagent grade chemicals from commercial distributors and used without further purification. Paramagnetic relaxation reagents (PARR) were used as their hydrated chloride or nitrate salts. In the preparation of sucrose in which OH protons were replaced by deuterium, all exchangeable sucrose OH protons were 0003-2700/79/0351-0085$01 OO/O
(lZ 1978 American
Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
. 0
Figure 1. Proton NMR spectrum of sucrose (0.632 M)in D,O. Computer resolution = 0.48 Hz. Magnetic shielding ( u ) increases in the direction of the arrow
z
13
Pi'
Figure 2. Proton NMR spectrum of sucrose (0.632 M) in H,O. The region of the anomeric proton is enlarged. Spinning side bands are denoted by
exchanged with deuterium by twice dissolving sucrose in D20. After each dissolution, the deuterated sucrose was taken to dryness
under vacuum. The hydroxyl groups were found to be 98% deuterated by proton NMR analysis, using the method described by Kasler (23). Henceforth, the deuterated sucrose is designated "sucrose-d". Proton NMR spectra were obtained at 29 f 1 "C with a JEOL MH-100 NMR spectrometer operating at 100 MHz in either the continuous wave or FT mode in spinning 5-mm 0.d. NMR tubes. Fourier transform NMR spectra were taken on a FT-modified MH-100 NMR spectrometer (Figure 3) using either a Digilab FTS-NMR/3 data system (for relaxation-time measurements only) or a JEOL EC-100 data system. For measurements of the spin-lattice relaxation time, TI, samples were not degassed. The inversion-recovery pulse sequence ( 2 4 ) was used. Sucrose anomeric proton and tert-butyl alcohol methyl proton T I measurements were performed in D20 solutions. Estimated relative error in T I values was less than f 5 % . A computer program, entitled SUCROS, was written for sucrose quantitation and used on the EC-100 data system. SUCROS computed the ratio of either peak intensities or empirical peak areas for the anomeric proton resonance relative to the internal standard proton resonance. Either ratio could be used to calculate the molar sucrose concentration, using data from the appropriate calibration curve. Procedure. For maximizing signal-to-noise ratio in the 'H FT spectra, audio gain was adjusted to prevent overflow of the analog-to-digital converter by the pre-truncated FID of the higher-concentration calibration sample. Typical data collection parameters were 40 accumulations using 90' pulses, 2000-Hz bandwidth, 2048 data points, 550-ps delay between the transmitter pulse termination and data collection initiation, -10 exponential decay constant, and pre-truncation of 3.5?%of the FID. The NMR power spectrum was calculated from the pre-truncated and exponentially apodized FID prior to quantitation by SCCROS. Pressed sugar beet juices were stored at either -20 or -78 OC between pressing and GLC or NMR analysis. Beet juice pH was Each adjusted to 3.0 by addition of a few drops of 6 M "OB. sucrose-containing sample and two corresponding calibration-curve samples were prepared in 5.00-mL volumetric flasks. Sucrose concentrations for the calibration-curve solutions were chosen to bracket the expected unknown sucrose concentrations. Calibration-curve solutions were prepared daily. Sample preparation and calculation of sucrose concentration were performed by two methods. Method A. To each 5.00-mL volumetric flask was pipetted 0.50 (henceforth referred to as Cr3+(aq)) mL of 0.150 M Cr(N03)B.9H20 solution, 0.50 mL of 0.440 M tert-butyl alcohol solution, and 2.00
I
L-________-____-___--------------
A
Figure 3. Block diagram of the FT-modified JEOL MH-100 NMR spectrometer, labeled for use with the EC-100 data system. Radio frequency and audio signals (+):
control pulse signals (+)
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
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“1 A
8 I
!!
, I
-;:
Lsdx:%e?’
’
2ij
ca
Figure 4. TRWEFT NMR spectrum of sucrose (0.632 M) in H,O wlth 20 mM Cr3+(aq). Conditions: 40 pulses (90’) in 20.5 s, 2000-Hz bandwtdth, and 1024 data points. (A) Unapodized FID; (B) pre-truncated FID; (C) power spectrum plotted in a 660.2-HZ spectral window
Figure 5.
mL sucrose-containing solution, then made t o volume with distilled H20. The juice and additional H 2 0 were carefully run down the side of the flask to avoid formation of suds-like bubbles. The final solution contained sucrose, 13 mM Cr3+(aq)and 0.044 M tert-butyl alcohol. From the SccRos-calculated molar sucrose concentration, CONCN, the weight/weight % sucrose was calculated from the following equation, based on a tabulation correlating percent sucrose and sucrose molarity (25):
spectrum of sucrose in water (Figure 4C). Anomeric proton resonance (H,) broadening is not a problem in this analysis. The broad resonance more shielded than HIis due to the other ring protons and the primary methylene protons of sucrose. T h e region between the two sucrose resonances shows t h a t the water resonance has been effectively eliminated. Application of TRWEFT NMR. T o quantitate the T R W E F T N M R sucrose analysis, a n internal standard was needed. Conditions for a good internal standard have been presented previously (23). Chemical shifts of several possible internal standards were examined. Chemical shifts of the CH protons of malonic acid, methanol, tetramethylammonium bromide, and maleic acid overlap the sucrose proton shifts. Acetate complexes Cr3+ so tightly as t o make it no longer available as a relaxation reagent. Since the methyl proton resonances of dimethylsulfoxide, acetone, acetonitrile, and tert-butyl alcohol did not overlap the sucrose resonances, their Tl values were measured for comparison with t h e anomeric proton T1. On the basis of T , similarity and chemical shift relative to those properties of the anomeric proton, tert-butyl alcohol was deemed the internal standard of choice. T h e optimum PARR concentration was selected by a n examination of the T1values of all pertinent protons in solution. Results are plotted for t h e protons of water (Figures 5 and 6) and the methyl protons of twt-butyl alcohol (Figures 5 and 7), as well as the sucrose-d anomeric proton (Figures 5 and 8), as a function of both sucrose and Cr3+(aq) concentrations. T h e solid lines in these figures represent a second-order polynomial curve fit of the data. Examination of the ratios of Ti's for the tert-butyl alcohol methyl protons and the anomeric proton (Figure 9) indicated t h a t the smallest ratio occurred in the presence of 15..0m M Cr3+(aq). This is the Cr3+(aq)concentration a t which T I of the anomeric proton most nearly equals the T I value of the internal standard proton over t h e entire sucrose concentration range. Also, t h e anomeric proton T1 is about 7 times larger than t h e water proton T1 a t this Cr3+(aq)concentration. Using this “optimized” Cr3+(aq) concentration, various quantitation methods were examined. The methods included intensity, integration, and empirical area ratios for the anomeric proton resonance relative to the tert-butyl alcohol
% = 0.008
+ 34.10(CONcIi)
~
3.87(COhCS)2
(This equation is valid for sucrose concentrations from 0.0 t o 26.0 weight/weight 70.) Method B. Solution preparation and instrumental setup was similar to that in Method A except the beet juice was weighed, not pipetted. To each 5.00-mL volumetric flask was weighed an accurately known amount of sucrose-containing sample (approximately 2 g). Weight/weight % sucrose was calculated as follows: ( C O N C N ) (342.30 g/mo1)(0.005 L)(100) 7c = (weight of sucrose-containing solution in grams)
RESULTS AND DISCUSSION Time Resolution Water Eliminated Fourier Transform (TRWEFT) NMR Spectrometry. Of the PARR investigated in this study, Cr3+(aq) is t h e PARR of choice t o preferentially relax the water protons over the sucrose protons. T h e decision was based on a n analysis of the relative effects of Cu2+,Ni2+,Co2+ Fe3+, Cr3+,and Mn2+ on the linewidths of the water proton iesonance and the sucrose anomeric proton resonance. In the presence of Cr3+(aq),the FID of an aqueous sucrose solution shows two time constants of decay-a fast decay rate for the water protons a n d a slower rate for the sucrose protons. This F I D is shown in Figure 4A. T h e T1 value for water protons in this case is measured to be 4 ms, while that of the sucrose anomeric proton is 39 ms. The initial 18-ms period of the FID covers the time period corresponding to 4.5 Tl’s for the water protons and only 0.5 TI for the anomeric proton. Zeroing, or pre-truncating, this initial portion of the F I D removes 99% of t h e water proton F I D leaving 63% of the sucrose FID. T h e resulting pre-truncated FID is shown in Figure 4B. Computing t h e N M R power spectrum of the pre-truncated FID gives the T R W E F T NMR
Spin-lattice relaxation times, T,, of water protons (O), tert-butyl alcohol methyl protons (O), and sucrose-danomeric proton (A)without cr3+(aq)present as a function of sucrose Concentration. [ tee-Butyl alcohol] = 0.044 M
88
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979 ac,
300 -
I
oo 02
0‘
.
02
04
06
oe
Figure 6. Spin-lattice relaxation times, T,, of water protons as a function of Cr3+(aq)and sucrose concentrations. [Cr3+(aq)]:5.0 mM (0),10.0 mM (O), 15.0 mM ( A ) , 20.0 mM (V), and 25.0 mM (0); [teff-butyl alcohol] = 0.044 M
08
06
04
[Sucrose- d
~Sdcrole!(hll
]
(MI
Figure 8. Spin-lattice relaxation times, TI, of the sucrose-danomeric proton as a function of Cr3+(aq)and sucrose-d concentrations. Legend same as Figure 6. [tert-Butyl alcohol] = 0.044 M Table I. Effect of Variation of tert-Butyl Alcohol and Cr3+(aq)Concentrations and p H on Sucrose Analysis
zoor
( a ) tert-Butyl Alcohol
[ tert- butyl 150
t
alcohol], M
calcd [sucrose]
0.0440a 0.0444 0.0449 0.0462
0.400
i i
0.381 0.378 i 0.357 t
b,cad
M
0.009 0.007 0.012 0.012
(b) Cr3+(as)
[Cr’+(aq)],mM 13.0 14.0 15.0a 16.0 17.0
calcd [sucrose] b , c , d M 0.394 0.386 0.395 0.372 0.371
t
0.018 0.014 0.013
t
0.011
i
t
0.014
I
( c ) PH 0
L
c4
32
1
sdcrare
36 d
0
08
IM
Figure 7. Spin-lattice relaxation times, T I , of the methyl protons of tert-butyl alcohol (0.44 M) as a function of Cr3+(aq)and sucrose-d concentrations. Legend same as Figure 6 methyl proton resonance. T h e “empirical area” was the product of the resonance linewidth and signal intensity. Problems with poor quantitation by computer integration have been reported previously (26). Use of the intensity ratio was found to give the best quantitation. Peak intensities were measured from a base line t h a t was extrapolated from flat spectral regions well outside the sucrose and internal standard spectral regions. Using the intensity ratio method, the actual and computed sucrose concentrations are linearly related with a slope of 1.0 and x- and y-intercepts of 0.0 within a 95% confidence interval over t h e examined sucrose concentration range, 0.0 to 0.810 M (Le., 0.0 to 26.0 weight/weight 70).T h e chemical shift
PH 1.38 2.00 3.20 4.25 5.30
intensity ratioC 0.665 i 0.674 t 0.663 + 0.693 t
0.034 0.013
0.022 0.017
. _ e_ _
a Reference solution contains 0.400 M sucrose, 15.0 mM Cr3+(aq),and 0.044 M tert-butyl alcohol at a p H in the range 2.7-3.0. Sucrose concentration calculated by S U C R O S using the reference solutiona for the calibraEach number is the average of 6 determination curve. tions t the standard deviation. Actual sucrose concentration in each solution is 0.400 M. e See text discussion,
difference between the anomeric proton and the internal standard proton is constant a t 4.18 ppm over t h e entire concentration range. Table I shows how variation of tert-butyl alcohol or Cr3+(aq) concentrations or p H affect the quality of the sucrose analysis. Deviation of the tert-butyl alcohol concentration by as much as 1% in test solutions relative to t h e tert-butyl alcohol concentration in the calibration-curve solution cannot be tolerated. Relative to the calibration-curve solution con-
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
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Table 11. Raffinose Interference with Sucrose Concentration Determination' [raffinose]
[sucrose], M
w/w% molarb 0 0.015 0.036 0.053 0.125 0.165
0 0.74 1.71 2.51 5.86 7.66
calcdC
errord
ratio,e %
0.402 + 0.007 0.403 i 0.014 0.449 i 0.010 0.462 i 0.012 0.531 i 0.010 0.564 i 0.017
0.002 0.003 0.049 0.062 0.131 0.164
0 3.75 9.00 13.25 31.25 41.25
i
'Each solution contains 0.400 M sucrose, 1 5 . 0 mM
Cr3+(aq),and 0.044 M tert-butyl alcohol in H,O with the concentration of raffinose shown, The molar raffinose concentration equivalent t o the stated w/w% raffinose concentration. SucROS-calculated sucrose concentration for a sucrose solution prepared as described in footnote a . Each result shows the average of six sucrose concentration determinations by SUCROS with the standard deviation. Error in the calculated sucrose concentration relative to the sucrose concentration in the reference solution (0.400 M ) with n o raffinose present. e Ratio = [raffinose] /[sucrose], expressed as a percentage. centrations, decrease in the Cr3+(aq)concentration in the test solutions by as much as 2 m M does not affect the calculated sucrose concentration. Too large a Cr3+(aq)concentration reduces the calculated sucrose concentration. Natural Cr { + levels in sugar beets (about 21 pM) (27) will not affect the Cr:'+ concentration in the test solutions to an extent t h a t would alter t h e results. T h e intensity ratio calculated as described above is independent of p H over the p H range of 1.38 to 3.20 (Table I). At p H 4.25, the computed ratio increases, due to Cr3+ precipitation. T h e Cr3+(aq)concentration a t p H 5.30 is so reduced by precipitation that the large water resonance obscures t h e anomeric proton resonance. Possible interferences in this analysis include any compound whose proton N M R resonance has a chemical shift corresponding to t h a t of the sucrose anomeric proton. These compounds include glucose (the anomeric proton) and raffinose (the anomeric proton of the glucose moiety). T h e raffinose anomeric proton of the galactose moiety is 0.39 ppm more shielded t h a n the sucrose anomeric proton. In a T R W E F T NMR spectrum of an aqueous sucrose solution containing raffinose (Figure lo), the anomeric proton resonance of the galactose moiety is a shoulder on the sucrose anomeric proton resonance. Increasing the computer resolution from 1.95 to 0.98 Hertz per data point does not separate the two resonances. Raffinose interference was examined for a constant 0.400 M sucrose concentration (Table 11). It was found t h a t raffinose a t a concentration 3.75% of the sucrose concentration interferes with sucrose quantitation. Similarly,
1 _ C P
Figure 9. T , (tert-butyl alcohol)-to- T , (sucrose-d) ratio as a function of Cr3'(aq) and sucrose-d concentrations [Cr3+(aq)] 0 0 mM (0), 5.0 mM (a),10 0 mM (A),15 0 mM (V), 20 0 mM (0),and 25 0 mM (0)[ tert-Butyl alcohol] = 0 044 M
-
.~
Figure 10. TRWEFT NMR spectrum of 0.400 M sucrose (13.04 w/w%) and 0.165 M raffinose (7.66 w/w%) with 0.044 M fert-butyl alcohol and 15.0 mM Cr3+(aq). Raffinose resonance is denoted by "R". The most shielded resonance is the internal standard
glucose should interfere at about the same concentration ratio. T o date, a means of correcting for the glucose or raffinose interference has not been developed. Possible avenues of research to remove these interferences include the application of shift reagents to increase the chemical shift difference between the sucrose anomeric proton and the interfering protons or the use of multicomponent analysis techniques to separate the overlapping resonances.
Table 111. Summary of Sucrose Analysis Results' in Pressed Sugar Beet Juices by TRWEFT NMR sample methodb 1
A B
2 3 4
A A B A
B
NMR result' solution no. lC solution no. 2c solution no. 3c 15.09 i 0.38 1 5 . 4 4 + 0.57 1 4 . 6 2 5 0.52 1 3 . 5 7 i 0.55 14.89 t 0.50 14.79 i 0 . 6 2 15.11 i 0.34
14.43 I 15.31 I 15.44 i 13.89 i 14.52 2 14.30 r 14.55 2
0.55 0.54 0.55 0.35 0.41 0.69 0.50
14.10 2 15.49 i 14.99 t 14.50 i 14.89 i 15.56 i 14.93 i
0.45 0.25 0.45 0.62 0.25 0.79 0.41
~~
~
averaged 14.54 -i 0.50 1 5 . 4 1 z 0.09 15.02 5 0 . 4 1 13.99 t 0.47 14.77 t 0.21 14.88 i 0.63 14.86 t 0.28
GLC r e s ~ l t ~ ~ ~ 14.38 14.69 14.47 14.36
Expressed as weightiweight % sucrose in H,O. Refers to method of solution preparation and sucrose concentration calculation discussed in the Experimental section. Average of six determinations i the standard deviation. Average of the three averages for the solutions 2 the standard deviation of the three solution values. e Precision: r 0 . 0 5 weight/weight % (absolute).
90
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1 9 7 9 I
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___, 0
Flgure 11. TRWEFT NMR spectrum of a typical raw sugar beet juice. "B" designates the betaine methyl proton resonance
A spectrum typical of the beet juices (Figure 11) has only one resonance due to neither sucrose nor the internal standard. This resonance is due to the methyl protons of betaine. Results from t h e sucrose concentration analysis of four raw sugar beet juices by T R W E F T NMR are given in Table 111. Concentration data from GLC analysis of the same juices are also given. By comparison with the GLC results, accuracy of the T R W E F T NMR analysis is within 0.570 (absolute),except for Sample 1 analyzed by Method B. Precision of replicate analyses on the same solution is generally within &0.650/, (absolute). Similar results are obtained for the T R W E F T NMR analysis of pure aqueous sucrose solutions. At the 95% confidence level, the sensitivity of this method is 0.60 w/w70. Compared t o GLC analysis, the relative accuracy of Method B is less than 3.570,while t h a t by Method A is between -3.3 and 3.5%. This is considerably better than the less than 5% relative accuracy reported for polarimetry compared to GLC analysis ( 3 ) . T h e precision of replicate sample preparations is better when the juice aliquots are weighed (Method B) compared to pipetting (Method A) as determined by a F ratio analysis of variance. The major error source in the analysis by Method B is the instrumental measurement of the sucrose spectrum, not sample preparation. For Method A, the major error source is sample preparation. I t can be expected that further studies could improve t h e precision and accuracy of the T R W E F T approach, mainly by improving sample preparation and computer data analysis. Typical analysis time for one solution by T R W E F T NMR, excluding sample preparation, is on the order of 45 s--20.5 s for data accumulation and 25 s for data treatment and sucrose concentration calculation. These times could be shortened by automated sample preparation, software improvements, and use of a larger NMR sample volume or a different receiver coil design to enhance sensitivity. Implementation of these changes may decrease the total analysis time, including sample preparation, to less than 5 s per sample. CONCLUSIONS This research demonstrates the applicability of proton FT N M R to the rapid, quantitative determination of sucrose in aqueous sucrose juices by employing a time-resolution approach to water resonance elimination. Even at the present
state of the methodology, T R W E F T NMR is more reliable than polarimetric analysis for the sucrose concentration in sugar beet juices. Proton TRWEFT NMR analysis of analytes a t concentrations 0.05 M (1.70 w / w % ) and above in aqueous solutions shows promise as an analysis method for those systems in which relaxation times of the species present can be made to meet the conditions of T R W E F T NMR. Analysis by T R W E F T NMR is not limited to proton NMR. T h e technique is applicable to any nuclear spin system with a large interfering resonance to be eliminated, if the T R W E F T NMR relaxation-time conditions can be met and if a loss of resolution can be tolerated.
ACKNOWLEDGMENT The authors gratefully acknowledge V. J. Bartuska (C.S.U.) for assistance with the design and construction of the MH-100 FT-modification, and S. Martin, USDA Crops Research Laboratory (C.S.U.) for making pressed sugar beet juices available and performing the GLC analyses. The authors also thank James Fischer and t h e Beet Sugar Development Foundation, whose advice during this project and support of earlier related work are gratefully acknowledged.
LITERATURE CITED W. Mauch, Z . Zuckerind., 20. 76 (1970). J. Karr and L. W . Norman, J . Am. SOC.Sugar Beet Technol., 18, 53 (1974). G. W. Maaa and G. H. Sisler. J . Am. SOC.Suaar Beet Technol.. 18. 257 (1975)"Polarimetry, Saccharimetry and the Sugars", Nafl. Bur. Stand. ( U . S ), Cfrc . . C440 - . ., 1942 . E. Van Handel, Anal. Biochem., 22, 280 (1968). I . Satoh, I. Karube, and S. Suzuki, Biofecknol. Bioeng., 18, 269 (1976). S. Belal and R . Soiiman, Pharmazi, 29, 205 (1974). G. G. Guilbautt, P. J. Brignac, Jr., and M. Juneau, Anal. Chem., 40, 1256 (1968). R . U. Lemieux and J. D. Stevens, Can. J . Chem., 44, 249 (1966). A. G. Redfieid. S. D. Kunz, and E. K. Ralph, J . Magn. Reson., 19, 114 (1975). S. L. Patt and B. D. Sykes, J . Chem. Phys., 56, 3182 (1972). F. W. Benz, J. Feeney, and G. C. K. Roberts, J . Magn. Reson., 8 , 114 (1972). T. R. Krugh and W. C. Schaefer, J . Magn. Reson., 19, 99 (1975). E. S. Mooberry and T. R. Krugh, J . Magn. Reson., 17, 128 (1975). R. C. Ferguson, C. C. McDonald, and W. D. Phillips. 13th Experimental NMR Conference, Asilomar, Calif., 1972. I . D. Campbell, C. M. Dobson, G. Jeminet, and R . J. P. Williams, F€BS Letf., 49, 115 (1974). H.E. Bieich and J. A. Glasel, J . Magn. Reson., 18, 401 (1975). N. R. Krishna, J . Magn. Reson., 22, 555 (1976). B. L. Tomlinson and H. D. W. Hili, J . Chem. Phys., 59, 1775 (1973). J. Dadok and R. F. Sprecher, J . Magn. Reson., 13, 243 (1974). R. K . Gupta, J. A. Ferretti, and E. D.Becker, J . Magn. Reson., 13, 275 (1974). Y . Arata and H. Ozawa, Chem. Letf.. 1974, 1257. F. Kasler, "Quantitative Analysis by NMR Spectroscopy", Academic Press, New York, 1973. R. L. Vow. J. S. Wauah. M. P. Klein. and D.E. PhelDS. Jr.. J . Chem. Phvs.. 48, 3831 (1968). "Handbook of Chemistry and Physics", 56 ed.. Chemical Rubber Co.. Cleveland, Ohio, 1975, p D-261. M. L. Jozefowicz, I . K. O'Neiil, and H. J. Prosser, Anal. Chem., 49, 1140 (1977). J. J. Christensen, P. A. Heartz, and R. M. Izatt, J . Agric. FoodChem., 24, 811 (1976).
RECEILXD for review June 13, 1978. Accepted October 2, 1978. Presented in part a t the Rocky Mountain Regional American Chemical Society Meeting, Laramie, Wyo., June 1 7 , 1976.
