Quantitation of partial structures of aquatic humic substances by one

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Anal. Chem. 1989, 61,628-631

Quantitation of Partial Structures of Aquatic Humic Substances by One- and Two-Dimensional Solution I3C Nuclear Magnetic Resonance Spectroscopy Joachim Buddrus,* Peter Burba, Helmut Herzog, and Jorg Lambert I n s t i t u t f u r Spektrochemie u n d angeccandte Spektroskopie, Postfach 10 1352, 0-4600 D o r t m u n d 1 , Federal Republic of G e r m a n y

One- and two-dimensional solution 13C NMR spectroscopy has been applied to elucidate partial structures of aquatic humic substances. Conventional "C NMR spectra have been taken by the spin echo technlque rather than by the one-pulse technique because phase corrections and thus quantitative measurements are more accurate to perform. The pulse sequences DEPT (distortionless enhancement by polarization transfer) and QUAT (quaternary-only carbon spectra) provide the ratio of the fragments CH, CH,, CH,, and Cque,,the proportion of C, being unusually high (78 % ). Comparing these results with elementary analysis gives the fraction of hydrogen connected to carbon and oxygen. I n addition, the DEPT spectra give the ratio of aromatic (olefinic) and aliphatic protons, which is in good agreement with the ratio derived from WEFT (water elimination by Fourier transformation) 'H NMR spectra. Two dimensional J-resolved 13C NMR spectroscopy partly conflrms these results and provides some evidence of CH coupling. Spin-spin relaxation rates of 13C nuclei are determined and reveal complexation of paramagnetic metal ions wRh carboxylate groups in humic substances.

Humic substances (HUS) are important components of the organic matter in the biosphere ( 1 , 2). Transport, storage, and reactivity of numerous organic and inorganic trace substances largely depend on the natural buffer and exchange capacity of HUS. The presence of oxygen-containing ligands stemming from aliphatic, aromatic, and carbohydrate moieties in HUS is responsible for the formation of strong complexes (3,4 ) with many heavy-metal ions. Humic substances are subdivided into humus, humic acids (HA), and fulvic acids (FA) according t o their solubility in acid and base (2). Many chemical and physical methods provide information on the structure of HUS, the most valuable physical methods being pyrolysis/MS ( 5 ) ,IR spectroscopy (2),proton NMR (6),and particularly carbon NMR (7). Carbon NMR spectra of fulvic and humic acids display four broad signal groups that can be assigned t o aliphatic, hydroxylic, aromatic, and carbonyl carbons. Modern NMR multipulse techniques have only scarcely been applied for structural analysis of HUS: distortionless enhancement by polarization transfer (DEPT) in the 135" version to analyze fulvic acids (a),spin echo Fourier transform (SEFT)to analyze HA and FA (9),lignins, and some humic substances (10). All these techniques usually result in highly complex spectra. Two-dimensional NMR spectra of HUS have not been reported so far. In this paper, for the first time, a complete set of edited CH, subspectra obtained by the D E P T and quaternary-only carbon spectra (QUAT) techniques and, in addition, the first two-dimensional J-resolved carbon NMR spectrum of a HUS (fulvic and humic acid together) is

presented. From the CH, subspectra the ratio of the fragments CH3, CH2, CH, and Cquatis derived, and with the elementary analysis taken into account, the amount of oxygen (-OH, -C02H) is determined. EXPERIMENTAL SECTION Isolation of Aquatic HUS. Fulvic and humic acids dissolved in groundwater were separated on technical scale (drinking water workup facilities at Hannover-Fuhrberg, FRG) on a strongly basic anionic exchanger (Lewatite MP 500 A, Bayer AG, Leverkusen, FRG) (11). After elution using a basic NaCl solution (10% NaC1, 2% NaOH), inorganic salts were removed from the dissolved humic and fulvic acids by dialysis at pH 6.0. Their subsequent fractionation was performed by ultrafiltration (upper mass limit 5000). Fulvic and humic acids with masses below 5000 (Fuhrberg-FHA) were isolated as sodium salts by employing freezedrying . Elementary Analysis. The combustion analysis yielded the following (in percent) C, 37.3, 37.2; H, 3.6, 3.5; N, 1.1, 1.1;Na, 7.2. Assuming that the remainder is oxygen, the empirical formula is C3,1H3,,No,303,2. Neglecting nitrogen and exchanging sodium by hydrogen gives C3,1H3,&2 (error within 5%). This formal composition will be the basis for later discussion. NMR Measurements. The NMR spectrometer was a JEOL GX 400. The pulse width of a 90" 13Cand 'H pulse (20 and 100 ps, respectively) was roughly determined in a solution of 2-butanol in D20 and then refined in a solution of HUS in D,O. 6 values refer to sodium 2,2-dimethyl-2-silapentanesulfonate, the largest signal of which being set to zero; this scaling was applied to the subsequent HUS measurement performed under identical spectrometer conditions. The frequency range was 48000 Hz and there were 16k data points for all one-dimensional spectra. Free induction decays (FIDs) were multiplied with an exponential filter function equivalent to 70 Hz. Complete Spectrum. These spectra were taken by either the conventional 13CNMR technique or by the spin echo technique. The conventional technique was performed by the procedure of pulse-acquisition without proton decoupling: pulse length, 45'; acquisition time, 0.17 s; relaxation delay, 0.4 s. The spin echo technique (see ref 9), yielding spectra free from base-line roll normally associated (12) with very broad signals (line width, several kilohertz), was executed according to the scheme 9 0 O - 7 180°-r-acquisition-relaxation: T , 20 ps; acquisition time, 0.17 s (after 0.1 s the FID is on noise level); relaxation delay, 1.83 s. Enlargement of the delay to 10 s was found to have no influence on relative intensities of the signals of the spectrum. In order to avoid sample heating, the decoupler was switched off during the relaxation delay (Figure 1). T 2 Determination. A series of spin echo spectra with delay times of 0.1, 0.3, 0.5, 1.0, and 1.5 ms was recorded. The height h of the carbonyl signal decreased markedly with increasing 7 delay, whereas the heights of the other signals were not influenced (65% at T = 1.5 ms). The decay of the carbonyl signal intensit>h was found to be exponential [ h = h, exp(-(2~)/T,)]. Leastsquares fit of In h and 2r to a straight line yielded TJcarbonvl) = 5 ms (correlation coefficient 0.98). DEPT Spectra. DEPT spectra were recorded by using the technique of Pegg et al. (13)under the following conditions: T = (ZJcH)-l = 3.45 ms (corresponding to J = 145 Hz); relaxation

0003-2700/89/0361-0628$01.50/0 E 1989 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989

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Flgure 1. 100-MHz 13C NMR spectra of 1 g of the sodium salt of Fuhrberg-FHA, completely dissolved in 4 mL of D,O, p D = 6.0. (a) Pulse-acquisition spectrum: pulse width 45', 22 800 transients. (b) Spin echo spectrum: T = 20 gs, 48 000 transients, pulse repetition rate of 2 s. Note the smooth base line.

delay, 0.22 s. 6' values of 45", 90°, and 135" yield the subspectra S45,S,, and SIs5,respectively. An equal number of transients was collected to get the S45,S,, and S i 3 5 subspectra. This procedure is contrary to that in ref 13, which takes twice the number of transients for the S , spectrum compared to the other types of CH, spectra and thus needs more measurement time. The CH, subspectra (Figure 2b-d) were received according to eq 1-3. CH spectrum = 2lI2Sw

(1)

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(2)

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(3)

QUAT Spectrum. The pulse technique suggested by Bendall et al. ( 2 4 ) was used under the following conditions: T = 3.45 ms; relaxation delay, 1.83 s; no decoupling during relaxation (Figure 2e). Two-DimensionalJ-Resolved Spectrum. The gated decoupling variant of the experiment was used with decoupling in the second half of the pulse sequence (15). Experimental conditions were as follows: 128 t l increments of 4096 transients each; acquisition time, 0.089 s; relaxation delay, 0.45 s; frequency range in f 2 dimension 23000 Hz (4k points), in fl 500 Hz (128 points). Exponential filtering with a line broadening of 3.9 (fl) and 70 Hz (fi) was used for sensitivity reasons. Power spectra were calculated (Figure 2f,g).

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RESULTS AND D I S C U S S I O N Qualitative Evaluation. The conventional I3C NMR and the spin echo NMR spectra of Fuhrberg-FHA are shown in Figure 1. In both spectra typical signal groups for purely aliphatic carbons (0-60 ppm), carbonhydrate carbons (70-85 ppm), aromatic carbons (115-160 ppm), and carbonyl (165-190 ppm) carbons can be observed. Figure 2b-d shows the spectra for CH3, CH2, and CH carbons and Figure 2e that of quaternary carbons. In Figure 2f a contour plot of the J-resolved carbon spectrum is displayed. This plot reveals some deviations from ideality in fl (see for instance the distorted triplet of the CH2 fragments a t 40 ppm) and f 2 (tailing of the signals along f l = 0 Hz) caused by heavy exponential weighting in both dimensions (3.9 and 70 Hz, respectively). Figure 2g shows the symmetrized J-resolved spectrum. Range 0-60 ppm. This signal group arises from CH, fragments bonded to carbon atoms. A surprisingly small fraction of methyl carbons is found. Figure 2f shows the methyl carbons as doublets; the missing outer signals of the expected quadruplet are lost in noise. CH, groups appear as strongly asymmetric triplets a t 40 ppm.

Figure 2. 100-MHz 13C spectra of the same solution as described in Figure 1. (a) Same spectrum as in Figure l b . (b-d) DEPT spectra: T = 3.45 ms (corresponding to JCH = 145 Hz), 276000 transients, pulse repetition rate of 0.39 s. (e) QUAT spectrum: T = 3.45 ms, 500 000 transients, pulse repetition time of 2 s. The gain is such that 'the addition of the spectrum to the sum of spectra b-d gives the envelope of spectrum a in Figure 3. The carbonyl signal intensity is attenuated (see text). (f) Two-dimensional J-resolved spectrum: 128 t , increments of 4096 transients each, pulse repetition time of 0.54 s. (9) Same as b, but symmetrized.

Range 60-100 ppm. These signals arise from 0-CH, fragments (n = 0-2). The signal a t 62 ppm (Figure 2c) can

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ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989

The integrals ID^^ and IQ"AT of the D E P T and QUAT spectra do not give quantitative information on the CH, ( n = 0-3) fragments, because the sensitivities of the two techniques differ. Therefore, an empirical correction is proposed for the different sensitivities of D E P T and QUAT involving the addition of the QUAT spectrum, weighted by a factor a , to the three DEPT subspectra. A variation of a is carried out until coincidence of the envelopes of the sum spectrum and the spin echo spectrum is achieved. This proposal follows mainly a suggestion of Netzel (18), who faced the same problems in an investigation of fossil-fuel-derived oils. xIDEPT(CHn)

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?' Flgure 3. (a) Sum of the 13C subspectra 2b-e. The dashed signal results after correction of carbonyl signal intensity for losses due to T , relaxation (see text). (b) Spin echo spectrum for comparison (identical with Figure lb).

be assigned to the C-6 carbons of carbohydrates. Integration of this signal will give the carbohydrate content of Fuhrberg-FHA. Range 100-165 ppm. The signals arise mainly from aromatic (olefinic)CH and C, carbons. The well resolved signal at 117 ppm with J(C-2,2-H) = 170 Hz is confirmed (6) to come from CH fragments in position 2 of phenols of type A; the strong signal a t 130 ppm (Figure 2d,f) is assigned to CH fragments in the 2-position of fragments of type B. The signal OH

C

+ aIQUAT = bIecho

(4)

(The factor b represents different gain settings of the two types of spectra and is of no special importance). With a = 1.4, sufficient coincidence is achieved (Figure 3) except for the carbonyl signal, the intensity of which is too low in the sum spectrum. This deficiency is caused by rapid spin-spin relaxation ( T z = 5 ms) of the carbonyl magnetization during the preparation phase (27 = 6.90 ms) of the QUAT experiment. Multiplication of the carbonyl signal height in the sum spectrum by a correction factor, c = exp(27/Tz) = 4, should eliminate this discrepancy. This procedure, however, makes that signal larger (Figure 3a) than it is in the spin echo spectrum, probably because off-resonance effects are more severe in the spin echo experiment. Integration of the subspectra (Figure 2b-d) gives 21121DEpT(CH)= 18.5

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The fractions x of quaternary, methine, methylene, and methyl carbons are calculated from eq 5 and 6: A

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at 140 ppm and the shoulder at 150 ppm indicate quaternary aromatic carbons (C-1 in B and C-1 in A, respectively). Range 165-220 pprn. The signals arise from quaternary carbons as demonstrated by Figure 2e. The broadening of fl of the carboxyl signals a t 180 ppm (Figure 2f) is due to the rapid relaxation ( T 2 = 5 ms) caused by complexation of carboxylate groups with paramagnetic ions (e.g. Fe3+). The iron content of the Fuhrberg-FHA was determined by atomic absorption spectroscopy (AAS) to be 760 pg/g of FHA (flame AAS; 10 mg/mL FHA, dissolved in water). Quantitative Evaluation. Quantitative determination of various fragments (CH,; hydrogen bonded to oxygen or carbon) was achieved by comparing D E P T and QUAT spectra with the complete spectrum and the elementary analysis. D E P T was preferred over INEPT editing (16). D E P T spectra result from pulse sequences in which the overall evolution time 37 is the same for all B values. I N E P T spectra are derived from pulse sequences of different evolution times, thus yielding subspectra of different sensitivities. Furthermore, the DEPT sequence employs less pulses than insensitive nuclei enhanced by polarization transfer (INEPT) and is therefore much less sensitive to pulse imperfections. QUAT was preferred over SEFT ( 1 7)because incorrectly set T delay times yielding crosstalks are less crucial in QUAT than in SEFT. The spin echo technique (Figure l b ) was preferred over the conventional technique (Figure la), because the spin echo spectrum has no base-line roll (12).

0.78:0.16:0.05:0.01

(7)

The content of quaternary carbon is 78% and thus unexpectedly high. The ratio of carbon versus carbon-bonded hydrogen is

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Comparison with the element analysis of Fuhrberg-FHA (C3,1H3,603,2) reveals that only 25% of the hydrogen content is connected to carbon, but 75% to oxygen. The D E P T spectra, in addition, allow the determination of the content of aromatic (olefinic) and aliphatic protons. Comparing the 13C NMR signals of methine carbons in the aromatic (olefinic) region 110-160 ppm (Figure 2a) with the sum of the signals in Figure 2b-d yields 18% aromatic (olefinic) and 82% aliphatic protons. This result is in good agreement with the water elimination by Fourier transformation (WEFT) (29) 'H NMR spectra yielding 16% and 84%, respectively. Possible Sources of Error. Measurements were carried out with a 10-mm multinuclear probe head; the 90° pulse lengths were rather long: 20 (lac)and 100 ps (lH). Accordingly, considerable off-resonance effects are anticipated for carbon signals a t the outer range of the spectrum. This is substantiated by spin echo measurements under on-resonance conditions, yielding up to 20% larger (in height) signals for CO and CHB. Other sources of error are estimated to be of minor importance.

Anal. Chem. 1989, 6 1 , 631-632

CONCLUSIONS The spin echo technique is well suited for the measurement of conventional carbon NMR spectra of HUS. Spectra with smooth base lines are readily obtained, and quantitative evaluations of the spectra become feasible. The combined use of the D E P T and QUAT pulse techniques allows a quantitation of the carbon type distribution in aquatic FA and HA. A surprisingly large fraction (78%) of quaternary carbons is found. Moreover, comparing NMR results and elementary analysis reveals that about 75% of the hydrogen of the investigated HUS is bound to oxygen. Short spin-spin relaxation times of the carboxylate carbons in HUS confirm the latter as preferred ligands for paramagnetic ions. Considerable improvement of the accuracy of carbonyl and methyl carbon determination is anticipated (a) from the implementation of composite pulses (20) into the pulse sequences used and (b) from the use of probe heads with shorter pulse widths.

ACKNOWLEDGMENT The stimulating discussions with Professor G. Tolg, Dortmund, are gratefully acknowledged.

631

(3) Florence, T. M.; Batley, G. E. Chemical Speciation in Natural Waters. CRC Crit. Rev. Anal. Chem. 1980, 9 ,219-296. (4) Ryan, D. K.; Weber, J. H. Anal. Chem. 1982, 5 4 , 986-990. (5) De Haan, H. In Aquatic and Terrestrial Humic Materials; Christman, R. F., Gjessing, E. T., Eds.; Ann Arbor Science: Stoneham, MA, 1983; pp 165-182. (6) Wershaw, R. L. In Humic Substances in Soil, Sediment and Water; Aiken, G. R., McKnight, D. M., Wershaw, R. L., Maccarthy, P., Eds.; Wiley: New York, 1985; pp 561-582. (7) Wilson, M. A. NMR Techniques and Applications in Geochemistry and Soil Chemistry; Pergamon Press: 1987; pp 182-203. (8) Hanninen, K. I. Sci. Environ. 1987, 62, 193-200. (9) Preston, C. M.; Blackwell, B. A. Soil Scl. 1985, 739,88-96. (10) Steelink, C.; Petson, A. Sci. Environ. 1987, 62, 165-174. (1 1) Kolle, H. Erfahrungen bei der Aufarbeitung eines reduzierten huminstoffhaltigen Grundwassers im Wasserwerk Fuhrberg der Stadtwerke Hannover; Abschlussbericht zum Forschungsvorhaben 02 WT 606 des BMFT; Hannover, FRG, 1981. (12) Turner, C. J. Prog. Nucl. Magn. Reson. Spectrosc. 1984, 16, 311-370, especially p 314. (13) Pegg, D. T.; Doddrell, D. M. J . Chem. Phys. 1982, 7 7 , 2745-2752. (14) Bendall, M. L.; Pegg, D. T. J . Magn. Reson. 1983, 53,272-296. (15) Freeman, R.; Kempsell, S. P.; Levitt, M. H. J . Magn. Reson. 1979, 3 4 , 863-667. (16) Morris, G. A.: Freeman, R. J . Am. Chem. SOC. 1979, 707, 760-762. (17) Brown, D. W.; Nahashima, T. T.; Rabenstein, D. L. J . Magn. Reson. 1981, 4 5 , 302-314. (18) Netzel, D. A. Anal. Chem. 1987, 59, 1775-1779. (19) Pan, S.L.; Sykes, B. D.J . Chem. Phys. 1972, 56,3182-3184. (20) Levin. M. H. Prog. Nucl. Magn. Reson. Spectrosc. 1988, 78, 61-122.

LITERATURE CITED (1) Bohn, H. L. SoilSci. SOC.Am. J . 1978, 4 0 , 468-469. (2) Stevenson, F. J.. Humus Chemistry, Wiley: New York, 1982.

RECEIVED for review July 11, 1988. Accepted December 1, 1988.

CORRESPONDENCE Model for Conductometric Detection of Carbohydrates and Alcohols as Complexes with Boric Acid and Borate Ion in High-Performance Liquid Chromatography Sir: In recent articles ( I d ) , Okada has demonstrated the utility of indirect conductometric detection of electrically neutral sugars and alcohols through their complexes in boric acid solution. The use of a boric acid eluent provides a highly sensitive means of detection for monosaccharides, lactose, and sugar alcohols but not for polysaccharides (other than lactose) and simple alcohols. Addition of sorbitol, mannitol, or fructose to the boric acid eluent allows detection of the polysaccharides and simple alcohols, as well as lactose, glucose, fructose, and presumably other monosaccharides and sugar alcohols. These results were interpreted ( I ) in terms of the ability of an analyte to form either dissociated or undissociated complexes with boric acid. This interpretation was quantified with a mathematical description of the complexation equilibria and the conductivity due to ionic species. Unfortunately, the mathematical model contains some incorrect assumptions that severely limit the utility of the derived equations and may prevent optimization of this potentially important technique. We present here a more general mathematical model that does not suffer from these limitations. A basic flaw in the earlier interpretation was the assumption that the complex between boric acid and an analyte either dissociates or remains nonionic. Actually, most polyols complex to some degree either with boric acid (undissociated) or with borate ion (dissociated). Addition of any poly01 to a boric acid solution generally lowers the pH (6, 7), indicating that

the complex with borate ion is stronger than that with the acid. Mannitol, sorbitol, and fructose show very strong effects, while the drop in pH is considerably less for comparable concentrations of glucose, maltose, glycerine, or ethylene glycol. In describing the eluent containing mannitol, sorbitol, or fructose and the analyte, Okada focused on the conductivity due to ionic borate complexes. However, since no other cations are present, the change in concentration of hydrogen ion must be equivalent to the sum of the changes in concentrations of all anions. Therefore, any changes in conductivity are primarily due to changes in concentration of the highly mobile proton. For a generalized model, the eluent is assumed to contain boric acid (HB) at formal concentration CBand a complexing agent (R) a t formal concentration CR. These are a t equilibrium with protons (H'), borate ions (B-), and a series of complexes that may be dissociated (BRk-) or undissociated (HBR,). Normally, j and k are no greater than 2. The eluent may also contain an analyte (S) and its complexes which also may be dissociated (BS-) or undissociated (HBS). Because of its low concentration, only 1:l complexes of the analyte are considered. There is also the possibility or borate complexes of higher order, but consideration of these species contributes more confusion than illumination a t this point. The equilibria may be described

0003-2700/89/0361-0631$01.50/0 0 1989 American Chemical Society