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Anal, Chem. 1982, 54, 2380-2382
1 ,
Following the appearance of the water/dioxane peaks, there is base line separation of xylose (k’ = 1.2) from ribose (k’ = 2.9) and fructose (k’ = 3.5) which are clearly separable. The use of borate complex formation to produce anionic sugar species results in a dramatic improvement in selectivity. The formation constant for the glucose-borate complex is far smaller ( 10’) than the correspondingfructose complex ( lo4) (7). As a result, glucose does not give any detectable response under the experimental conditions employed here, making the determination of fructose entirely free from glucose interference. Changing these conditions to accommodate the lower stability of the glucose complex, e.g., by increasing borate ion concentration,would very likely permit determination of glucose also. Although further work is required to clearly establish the optimum conditions necessary to separate all sugars, it would appear that reactions occur on the column to give suitable products for effective separation and detection. Further work along these lines is under way in this laboratory. N
k +
i
15
T I W (Pins.)
N
LITERATURE CITED
Flgure 1. Chromatographic behavior of monosaccharides: xylose (I), ribose (2),fructose (3); mobile phase, 0.1 M borate buffer pH 7.5, methylene blue M, 2% MeOH/H,O (v/v); flow rate, 1.0 mL/min; sample, 10 pL 1.0% (wlv) solutions.
column saturation and subsequent chromatography was 1.0 mL/min. Solutions (1%w/v) of the fructose, xylose, and ribose in the mobile phase were added through a 10-pL loop sampling valve (rotary valve injector SP-419-0410), As may be seen from the data, reasonable separation of the three sugars tested was obtained at submicrogram detection limits ( g).
(1) Ladish, M. R.; Tsao, Q. T. J. Chromatogr. 1978, 166,18-100. (2) Palla, G. Anal. Chem. 1881, 53, 1966-1967. (3) Linden, J. L.; Lawhead, C. L. J. Chromatogr. 1975, 705, 125-233. (4) Conrad, E. C., Palmer, J. K. Food Technol. (Chicago) 1976, 84-92. (5) Gnanasambandan T.; Freiser, H. Anal. Chem. 1981, 53, 909-911. (6) Gnanasambandan, T.; Freiser, H.Anal. Chem. 1982, 5 4 , 1282-1285. (7) Roy, G. L.; Laferriere; Edwards, J. 0. J. Inorg. Nucl. Chem. 1957, 4 , 106.
T.Gnanasambandan H.Freiser* Department of Chemistry University of Arizona Tucson, Arizona 85721 RECEIVED for review April 26,1982. Accepted August 16,1982. This work was supported by the Office of Naval Research.
Identification of the Origin of Natural Alcohols by Natural Abundance Hydrogen-2 Nuclear Magnetic Resonance Sir: It is well-known that the overall content of stable isotopes in natural compounds depends on a number of biochemical, physical, and environmental factors. Deuterium, in particular, is an atom whose distribution is largely widespread over the surface of the earth since the range of 2H contents of natural molecules, represented by the (D/H)ppm ratios, undergoes a %fold variation on the SLAP-SMOW scale (1, 2). However, most of the methods applicable to the determination of the deuterium content give only the overall 2H percent of the molecule and the use of mass spectrometry, which is certainly the most accurate technique, requires a prior chemical transformation of the sample to be studied. We have shown recently that very important variations may be found in the internal distribution of deuterium within a molecule ( 3 , 4 ) . In fact, quantitative 2H NMR can provide invaluable information on the selectivity of the 2H repartition and we shall demonstrate here that we have, a t our disposal, a very powerful tool for the identification of the origin of natural alcohols. Indeed, a proton decoupled deuterium spectrum represents a quite different situation from that normally encountered in proton spectroscopy since each line in the 2H spectrum corresponds to a given deuterated species. If we consider the case of 95% ethanol, the four lines observed correspond to the molecules CH2DCH20H(I), CH3CHDOH (II), CH3CH20D (111),and HOD (IV). Quantitative results may be obtained by using carefully selected experimental parameters and an
appropriate treatment of the spectra (5). The 2H spectra were recorded a t the natural abundance level at 298 K and 38.41 MHz with a Bruker WM 250 spectrometer in the lH broadband decoupling mode. The acquisition parameters were selected after a careful analysis of the relaxation processes. Due to the quadrupolar properties of 2H,the dipolar relaxation is relatively ineffective and no noticeable Nuclear Overhauser effect was detected. Although we have observed that values of the longitudinal relaxation time ranging from 4.5 s to 7.7 s may be observed for molecules such as CH3N02,(CH3)&0, CH30H (6), and CH3CN, the TI values of ethyl derivatives are substantially shorter (1-2.5 s) for C2H5Br, C2H61,and (C2H5)3Nand, for four azeotropic samples of ethanol containing 4.4% of water, we have determined, at 303 K, T,(I) = 0.95 f 0.05 s, T,(II) = 1.10 A 0.05 s, and T1(III) E 0.2 s. This behavior enabled the following acquisition parameters to be selected: spectral width, 1200 Hz; pulse width, 100 to 115 X lo4 s (90’ pulse angle); acquisition time, 6.8 s (memory size 16K). The relative isotopic composition can be readily obtained and expressed either in terms of molar fractions, f ( i ) = S(i)/CS(i)(where S(i) is the signal area associated with molecule i = I to 111) or in terms of internal ratios R(i) = 3S(i)/S(I). When i = 11, the parameter R represents the relative enrichment of the methylene site with respect to the methyl group. It should be noted that a value R(I1) = 2 would be expected on the basis of a statistical distribution of deu-
0003-2700/82/0354-2380$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
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Table I. Variations in the Deuterium Content of Ethanols Obtained from Fermentation of Various Plants and from Chemical Synthesis (Ethiylene) ethylene ethylene corn sugar cane wheat potato sugar beet apple contact (HISO,)
a
(D/H)a 122.1 i- 2 123.2 2.5 (D/H)I 111.6 i- 2 111.2 i 2.5 (D/H)II 123.9 i- 2 127.8 i: 2.5 Average overall deuterium content (9-1 1 ).
20
25
30
119.5 103.5 128.3
117.6 i: 4 97.3 i: 4 131.8 i 4
9
Figure 1. Internal distribution of 2H at the natural abundance level in varlous ethanols obtained from C3 or C4 plants (by fermentation)and from ethylene (by chemical synthesis). terium within the ethyl fragment. We have investigated a series of ethanols obtained from different plants or synthesized from ethylene. Figure 1shows that very large differences exist in the values of the R parameter which has been determined 10 times for a given sample. The standard deviation sR which is equal to 0.01 represents the repeatability of the NMR measurement in its present state of development. For each kind of plant, several ethanols were investigated, and a natural dispersion can be determined with the confidence interval ofR at the 90% level, measured for various alcohols. The natural dispersion seems to be higher for certain plants. It reaches f0.05 for the various samples of ethanol extracted from sugar beet for example. Secondary effects, which will be discussed later, are responsible for the observed scattering in the R values, but there is no doubt that the nature of the plant is the prime factor governing the internal distribution of deuterium. Thus, an ethanol sample extracted from wheat (R = 2.47-2.50), for example, is unambiguously distinguished from a sample extracted from corn (R = 2.18-2.'25). Interestingly we note that the plants in which the sugar is synthesized via a Hatch-Slack C4 cycle (7) (corn, sugar cane) have a relatively low R value (I? < 2.35) whereas the plants characterized lby a higher R value are produced via a Calvin C3 cycle (8)which needs a different quantity of water with respect to the C4 cycle. The phosphosynthesis cycle and the origin of the sugar therefore seem to have a major influence on the 2H relative abundance within the ethyl fragment. In order to compare the internal deuterium proportions in a given molecular site, we have introduced selective (D/H), pstrameters which are d e f i e d according to the relation @ / H I , = (f,/F,)(D/W where f, and F, represent, respectively, the experimental and statistical molar fractions of species i and (D/H) is the average overall deuterium content of the chemical molecule considered. It is easily shown that ( D / H ) = 1/22(D/H)1+ %(D/H)II -I- 1/6(D/H)III (D/H) can be measured by NMR using a working standard contained in a coaxial tube fitted in the 15 mm 0.d. cell or
114.7 i 2 94.1 i- 2 128.0 i- 2
118.7 100.9 129.7
133.9 * 1 123.3 i- 1 141.8 i: 1
136 120.9 151.7
by mass spectrometry (9-11). The experimental results are given in Table I. If we compare, for example, the ethanols obtained from corn and sugar beet, which are characterized by very different values of R and total (D/H), we observe that the natural abundance of deuterium in the methylene site, (D/H)=, does not change significantly ( e 5 % ) whereas the 'H content of the methyl site (D/H)I undergoes a greater variation (-20%). These results support the fact that the glycolysis cycle has a leveling effect on the natural abundance in the methylene site but preserves the relative abundances in the methyl site which are therefore primarily determined by the (D/H) values of the starting sugars. In addition, by a joint examination of the 2H and lH spectra of a given ethanol 288 K) we have sample in the slow exchange region (2' checked that the deuterium contents of the hydroxyl site and of the remaining water are very close to each other (D/H)III N (D/H)H,D The (D/H) value of water contained in ethanol can therefore be determined from the 2H spectrum recorded with an external reference (C6H6)(even in the fast exchange limit) and calibrated in the SLAP-SMOW scale. The (D/H)m values are close to the average value of 151 ppm which has been used in the calculations. The dispersion of the values given in Table I corresponds to the range of the various samples investigated for a given species. Moreover, we notice that the smaller variations of R(i) detected for a given vegetal may be related to differences in geographical origin and climatological conditions. This second-order effect of climate is clearly revealed for the C3 sugar beet plant grown under different conditions. Thus, for eight samples of ethanol extracted from sugar beet grown in the north of France between 1972 and 1976, we obtain a good correlation between the isotopic ratio R(II), expressed as the reduced centered value Rc, and the average temperature, 8 ("C), and rainfall, h (mm), in the appropriate period of time Rc = 4.68 - 0.628 0.00264h r = 0.99 sRC= 0.1
+
(the Rc values were computed from the relation Rc = (R, R,)/s where R, is the mean value of R over a series n of experiments performed with different plants and s is the standard deviation on R,J. It appears that when the rainfall increases and the temperature decreases, the isotopic abundance ratio R(I1) increases significantly. This behavior probably reflects the influence of the climatological variations on the isotopic fractionation via vaporespiration of the plant. Quantitative 2H NMR at the natural abundance level therefore provides a new method for investigating biomechanisms. This technique enables, more easily than mass spectrometry although less accurately, the overall deuterium contents to be measured. More interestingly we have shown that very spectacular variations in the internal 2H distribution can be directly determined and the access to selective enrichments, even in molecules with many sites, provides an important source of mechanistic information and sample recognition. Work is in progress to improve our knowledge of the deuterium fractionation occurring in various fermentation processes.
LITERATURE CITED (1) Craig, H. Sclence 1961, 133, 1833. (2) Hagemann, R.; Nlef, G.;Roth, E. Tellus 1970, 22,712.
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(3) Martin, G. J.; Martin, M. L. Tefrabedron Lett. 1981,3525. (4) Martln, G. J.; Martin, M. L. C.R. Hebd. Seances Acad. Sci. 1981, 293, 31. 151 Martin. M. L.: Delouech. J. J.: Martin. G. J. “Practical NMR Spectroscopy”t Heyden: London, Philadelphia, 1980; Chapter 9. Mantsch, H. H.; Salto, H.; Smlth, I. C. P. I n “Progress in NMR Spectroscopy”; Emsley, J. W., Feeney, J., Sutcliffe, L. H., Eds.; Pergamon Press: London, 1977; Vol. 1I, p 237. Hatch M. D.;Slack C. R. B/ocbem. J . 1986, 101, 103. Calvin, M.; Bassham, J. A. “Photosynthesis of Carbon Compounds”; W. A. Benjamin: New York, 1962. Bricout, J. Rev. CyW. Biol. Veg. 1978, 1 , 133. Bricout, J.; Fontes, J. C.; Merlivat, L.; Pusset, M. Ind. Agric. Aliment. 1975,375.
(11) Rauschenbach, P.; Slmon, H.; Stichler, W.; Moser, H. Z.Naturforscb ., C: Biosci. 1979, 34C, 1.
Gerard J. Martin* Maryvonne L. Martin Franpoise Mabon Marie-Jo Michon Laboratoire de Chimie Organique Physique, ERA 315 Facult6 des Sciences (F), 44072 Nantes Cedex, France RECEIVED for review April 19, 1982. Accepted July 21, 1982.
AIDS FOR ANALYTICAL CHEMISTS Long Optical Path Electrochemical Cell for Absorption or Fluorescence Spectrometers Michael J. Slmone, Wililam R. Heineman, and George P. Kreishman* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 1
Since their introduction, optically transparent electrodes have been extensively exploited to make a variety of electrochemical measurements. Presently, their use in the determination of Eo’ (formal redox potential) and n (electron stoichiometry) is commonplace. Of particular interest to the bioanalytical chemist has been the use of spectroelectrochemistry in the study of biological redox components ( I ) . The heterogeneous electron transfer rate can be very slow for biological materials whose redox centers are shielded from interaction with the electrode surface. The use of mediator titrants, together with optically transparent electrodes, has provided effective coupling of the biomolecule to the electrode and has allowed for the subsequent characterization of these molecules with optical techniques (2). Yildiz et al., capitalizing on the inherently greater sensitivity of fluorescence over absorbance, used an optically transparent thin-layer electrode (OTTLE) in conjunction with a spectrofluorometer to study the highly fluorescent molecule perylene (3). In a similar manner, previous work in our laboratory attempted to make use of the inherent fluorescence of tryptophan-59 of horse heart cytochrome c to study possible conformational changes upon oxidation and reduction ( 4 ) . A difference in the fluorescence intensity between the oxidized and reduced forms of cytochrome c was detected and the observed change was quantitatively consistent with previously postulated conformational changes (5) and with that observed for tuna cytochrome c in the solid state (6). A number of technical difficulties associated with the use of the conventional OTTLE cell complicated the preliminary studies. In order to compensate for the relatively short path length (0.02 cm) of the OTTLE, high concentrations of cytochrome c coupled with high instrumental gain settings were used. These conditions resulted in unusually high Rayleigh light scattering relative to fluorescence intensity. In addition, because of the right angle geometry of the fluorometer, difficulties were encountered in reproducibly positioning and taking spectra while thermostating the OTTLE. The present paper is a report on the construction and evaluation of a new long optical path electrochemical cell which can be used for both absorbance and fluorescence studies. Several investigators have reported novel long optical path cell designs optimized for particular uses (7-12). These cells, although elegant in design, generally require large outlays of time and/or expense due t o their complexity. The cell
described in this report has as its attributes, (1) ease and quickness of construction from inexpensive, commercially available materials, (2) the ability to be used with relatively inexpensive instrumentation, and (3) small sample volume (0.5 mL) requirement.
EXPERIMENTAL SECTION The gold resinate solution (GOLD #8300,28% Au content) was purchased from Englehard Corp., East Newark, NJ. K,Fe(CN), and o-tolidine were purchased from Fisher Scientific; NaC1, NaH2P04,and Na2HP04(reagent grade) were purchased from MC/B chemicals. All chemicals were used without further purification. Cyclic voltammograms were obtained with a Bioanalytical Systems (BAS) Model CV IB potentiostat coupled to a Keithly Model 178 digital multimeter and a Hewlett-Packard Model 7015A X-Y recorder. Potential step experiments were performed with a Princeton Applied Research (PAR) Model 173 potentiostatgalvanostat. All potentials are reported relative to the standard calomel electrode (SCE). Absorbance measurements were made with a Gilford Model 250 absorbance spectrophotometer and fluorescence spectra were obtained with a Perkin-Elmer Model 650-10s fluorescence spectrophotometer equipped with an Hewlett-Packard Model 7015A X-Y recorder. In a typical experiment, the absorbance or fluorescence spectrum was taken after equilibrium had become established at the applied potential. Applied potentials were selected randomly between the oxidizing and reducing potential limits. The equilibration time was from 30 to 40 min and was indicated by a cessation in spectral response change. This equilibration time is relatively long when compared to the few minutes necessary for complete electrolysis in the OTTLE cell and illustrates the pseudo-thin-layernature of the long optical path cell. The increase in equilibration time due to the leakage from the bulk solution is minimal due t o the fact that the working electrode surface extends from the bottom of the insert to the four sides separating the active zone solution and the bulk solution. This electrode area is similar to the reactant-getter electrode element utilized previously (3). Eo’and n values were obtained from the y intercepts and slopes of Nernst plots with lines drawn using linear least squares analysis of the data points. A diagram of the long optical path cell is shown in Figure 1. A Teflon block (Cincinnati Plastics) was cut and milled so that the base fit snugly into a standard 1 X 1cm quartz cuvette (Fisher Scientific). A set of four “feet” were milled into the bottom of the base providing a space of 0.5 mm in depth (active zone volume -25 wL. A small hole was drilled into the center of the base t o hold the auxiliary (Pt wire) and reference (SCE) electrodes. A
0003-2700/82/0354-2382$0 1.2510 0 1982 American Chemical Society
