Application of INEPT nitrogen-15 and silicon-29 nuclear magnetic resonance spectrometry to derivatized fulvic acids

The Science of the Total Environment, 81/82 (1989) 209-218 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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APPLICATION OF INEPT NITROGEN-15 AND SILICON-29 NUCLEAR MAGNETIC RESONANCE SPECTROMETRY TO DERIVATIZED FULVIC ACIDS

K.A. THORN 1, D.W. FOLAN1, J. B. ARTERBURN2, M.A. MIKITA3, and P. MACCARTHY4 1U.S. Geological Survey, Water Resources Division, Denver Federal Center, Mail Stop 408, Box 25046, Denver, Colorado 80225 2Department of Chemistry, University of Arizona, Tucson, Arizona

85721

3Department of Chemistry, California State University at Bakersfield, Bakersfield, California 93311 4Department of Chemistry, Colorado School of Mines, Golden, Colorado 80401 ABSTRACT Use of the INEPT experiment has been examined in two derivatization studies of the Suwannee River fulvic acid. In the first study, the fulvic acid was derivatized with 15N enriched hydroxylamine. The quantitative 15N NMR spectrum, acquired with a 45° pulse angle, 2.0 second pulse delay and inverse gated decoupling, showed that oxirnes (390-340 ppm) were the major derivatives, followed by nitriles (270-240 ppm), hydroxamic acids (170-160 ppm), secondary amides (150-115 ppm), and lactams (115-90 ppm). The INEPT 15N NMR spectrum was acquired using refocussing delays and polarization transfer times optimized for signal enhancement of singly protonated nitrogens. INEPT greatly enhanced the amide and lactam resonances, and showed that resonances downfield of 180 ppm in the quantitative spectrum represented nonprotonated nitrogens. In the second study, the fulvic acid was first methylated with diazomethane and then silylated with hexamethyldisilazane. The 29Si NMR spectra exhibited two major peaks, from approximately 33 to 22 ppm, representing silyl esters of carboxylic acids, and from 22 to 13 ppm, representing silyl ethers of alcohols and phenols. The INEPT 29Si NMR spectrum was virtually identical to the quantitative 29Si spectrum, acquired with a 90 ° pulse angle, 5.0 second pulse delay, inverse gated decoupling, and relaxation reagent. INEPT therefore can be used for quantitative analysis of trimethylsilyl derivatives of the fulvic acid, saving spectrometer time and eliminating the need for relaxation reagents.

0048-9697/89/$03.50

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1989 Elsevier Science Publishers B.V.

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INTRODUCTION The INEPT (Insensitive nuclei enhancement by polarization transfer (ref. 1)) pulse sequence can serve as both a multiplicity sorting experiment and a signal enhancement technique. For nuclei such as 15N and 29Si, which have low sensitivities, negative magnetogyric ratios, and oftentimes long spin lattice relaxation times, Tl'S, INEPT offers two important advantages. First, an enhancement factor of approximately 7A/YX is obtained (7=magnetogyric ratio; A = proton; X = observe nucleus, 15N or 29Si) and is independent of the relaxation processes that contribute to the relaxation of A and X. The theoretical maximum signal enhancements are approximately 10 and 9, respectively, for 15N and 29Si. Secondly, the pulse sequence repetition rate is a function of T1 for the protons, which typically have much shorter relaxation times than 15N and 29Si. Because of negative magnetogyric ratios, signal degradation can occur in normal pulsed experiments in 15N and 29Si NMR when NOE (nuclear Overhauser enhancement) is retained, for those 15N and 29Si nuclei which have unfavorable NOE factors. In this report, INEPT is examined in two derivatization studies involving 15N and 29Si. Witanowski et alo (ref. 2) have reviewed INEPT in their recent text on 15N NMR, and Blinka et al. (ref. 3) have reviewed polarization transfer techniques in 29Si NMR. In the first study, the fulvic acid was derivatized with 15N enriched hydroxylamine and examined by 15N NMR (ref. 4). The objective was to determine the reaction products of the fulvic acid with hydroxylamine, and thereby learn more about the carbonyl functionality of the fulvic acid. In the past, reaction with hydroxylamine followed by titrimetric determination of excess reagent has been used to measure the carbonyl content of humic substances (refs. 5-6). However, it has never been known to what extent the hydroxylamine reacts with groups other than ketones, aldehydes, and quinones, to form derivatives other than oximes. For example, hydroxylamine may react with naturally occurring esters in fulvic or humic acid molecules to form hydroxamic acids. Direct observation of the labelled nitrogen nuclei by 15N NMR would allow the identification and quantitation of the derivatives formed with hydroxylamine. Thusfar, 15N NMR has received limited application in humic substances related studies. Preston et al. and Benzing-Purdie et al. (refs. 7-8) used 15N NMR to analyze synthetically produced melanoidans, and Benzing-Purdie et al. (ref. 9) used 15N NMR to follow the chemical and biological transformations of 15N enriched glycine in peat. In the second study, the fulvic acid first was methylated with diazomethane, silylated with hexamethyldisilazane, and then analyzed by 29Si NMR. Previously, we have used a permethylation procedure to analyze the hydroxyl group functionality of humic substances, which employed methylation with 13C-enriched diazomethane followed by methylation with sodium hydride and 13C-enriched methyl iodide (refs. 10-11). A shortcoming of this method was that the amount of derivative formed with diazomethane could not be differentiated from the amount formed with CH31/NaH, in the 13C NMR of the permethylated samples. By first methylating with diazomethane and recording the 13C NMR, and then silylating residual hydroxyl groups and recording the 29Si NMR, the

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amounts of derivative formed with each step can be measured. The specific reactivity of the individual reagents can then be exploited in assigning the NMR spectra of the derivatives. Silylation in conjunction with 29Si NMR is being used increasingly for analysis of the hydroxyl group functionality of such diverse geologic materials as lignins (refs. 12-14), asphaltenes, and other polar constituents of petroleum and liquified coals (refs. 15-17). According to Blinka et al. (ref. 3), INEPT is slightly superior to DEPT (Distortionless enhancement by polarization transfer) for measurement of proton decoupled 29Si NMR spectra of trimethylsilyl groups. METHODS IDQrivatization

of fulvic acid with hvdroxvlamine Suwannee River fulvic acid was isolated using the procedure of Leenheer (ref. 18), and obtained from J.A. Leenheer. The derivatization was performed by dissolving 0.19 g of hydroxylamine hydrochloride (99 atom percent 15N) and 0.81 g of the fulvic acid (H-saturated form) in 100 mL of deionized, distilled water, titrating to pH 5 with 1N NaOH, and refluxing for 4 hours at 88°C. The derivatized fulvic acid-was then Hsaturated on a cation exchange column and freeze dried. The amount of product recovered was 682 rag, which was dissolved in 2 g DMSO-d6 for NMR analysis. MQthvlation and Silvlation of Fulvic Acid Approximately 100 mg of fulvic acid was methylated with diazomethane generated from N-methyl, N-nitroso-ptoluenesulphonamide. The diazomethylated fulvic acid was dissolved in 10 mL distilled pyridine (kept over KOH) and charged with 3.2 mL (15.0 mmol) of hexamethyldisilazane and 0.16 mL (2.05mmol) trifluoroacetic acid as catalyst. The reaction was allowed to proceed 12 hours at 25°C. The derivatized sample was dissolved in 2 mL chloroform-d for NMR analysis. NMR SDectrosconv Carbon-13, nitrogen-15, and silicon-29 NMR spectra were recorded on a Varian* XL-300 NMR spectrometer at 75.4, 30.4, and 59.6 MHz, respectively, using a 10 mm broadband probe. Acquisition parameters for the quantitative natural abundance 13C NMR spectrum of the fulvic acid included a 50,000 Hz spectral window, 45 ° pulse angle, 0.2 second acquisition time, 8.0 second pulse delay, inverse gated decoupling, and 20 Hz line broadening; the concentration of the H-saturated sample was 200 mg in 2 g of DMSO-d6, 13C depleted. In this paper, quantitative spectrum refers to normal pulsed spectra which were acquired with pulse delays o f sufficient duration to eliminate differential saturation effects, and inverse gated decoupling to eliminate NOE. The quantitative 15N NMR spectrum was acquired using a 21,276.6 Hz spectral window, 45 ° pulse angle, 0.5 second acquisition time, 2.0 second pulse delay, inverse gated decoupling, and 5.0 Hz line broadening. A spectrum acquired using identical acquisition parameters but with an 8.0 second pulse delay showed no differences in peak areas, indicating that the 2.0 second pulse delay was adequate for quantitative results.

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Additionally, a spectrum was acquired, but not shown here, using the same conditions as the quantitative spectrum except that continuous decoupling was employed to retain the NOE. The INEPT spectrum was acquired using a polarization transfer time and refocussing delay both equal to 1/4J (1JNH -- 90.0 Hz), or 2.78msec, which have been reported to be optimal for signal enhancement of singly protonated nitrogens (ref. 2). The delay for proton relaxation was 2.0 seconds. An INEPT spectrum recorded to obtain enhancement of all protonated nitrogens was found to be identical to the spectrum reported here except that it had lower signal to noise. The polarization transfer times and refocussing delays for the INEPT 29Si NMR spectrum were 74.6 msec and 16.1 msec, respectively. These values have been reported by Schraml et al. to be optimal for signal enhancement of trimethylsilyl derivatives of heteroatoms (ref. 16). The delay for proton relaxation was 2.0 seconds. After the INEPT spectrum was recorded, chromium (111) acetylacetonate (0.04M) was added to the sample, and the quantitative spectrum was recorded. Acquisition parameters included a 3321.1 Hz spectral window, 90 ° pulse angle, 0.5 second acquisition time, 5.0 second pulse delay, inverse gated decoupling, and 10.0 Hz line broadening. RESULTS AND DISCUSSION The quantitative natural abundance 13C NMR spectrum of the fulvic acid is shown in Figure 1. This spectrum has been described in more detail elsewhere (refs. 19-20); a 13C NMR spectrum of the sample methylated with 13C enriched diazomethane has also been reported (ref. 10). In brief, the peak from 0 to 60 ppm represents primarily aliphatic carbons bonded to other carbons, and the peak from 60 to 90 ppm represents primarily aliphatic carbons bonded to oxygens, specifically, ether and alcohol carbons, including carbohydrates. Aromatic carbons occur from 90 to 165 ppm, with phenolic carbons in the range from 135 to 165 ppm. Acetal carbons may overlap with aromatic carbons in the range from 90 to 110 ppm. The peak from 160 to 180 ppm represents primarily carboxyl carbons, with possible overlap of amide, ester, and lactone carbons. Attached proton test (APT) spectra have shown the peak from 180 to 220 ppm to represent nonprotonated carbons, allowing the assignment of these carbons as ketones (ref. 19). Quinones may overlap in the region from approximately 180 to 190 ppm. The 15N NMR spectra of the hydroxylamine derivatized fulvic acid are shown in Figure 2. The quantitative spectrum indicates that oximes (390 to 340 ppm) are the major derivative. Other peaks occur from 270 to 255 ppm, 255 to 240 ppm, 216 to 201 ppm, 201 to 191 ppm, 191 to 160 ppm, 150 to 115 ppm, and 115 to 90 ppm; the latter two peaks have such low signal to noise that it it questionable whether they are real or not. The most likely assignments for the resonances from 270 to 240 ppm, 150 to 115 ppm, and 115 to 90 ppm, are nitriles, secondary amides, and lactams, respectively. These are secondary reaction products arising from Beckman rearrangements of the initial oxime derivatives (ref. 21). The chemical shifts of hydroxamic acids range from 168 to 164 ppm ; these are evident in the upfield portion of the peak from 191 to 160 ppm, and

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DMSO-d6

I 300

I

I 200

I

I 100

I

I 0

I

I -100 ppm

Figure 1. Quantitative natural abundance 13C NMR spectrum of Suwannee River fulvic acid.

Beckman Rearrangements of Oxlmes Reactions of Model Compounds (Acidic Conditions) With Hydroxylemine 615N, ppm 61SN, ppm N/'OH O .HOH

R-- c---R

N''°H

I, R- ~ "--R

Ketone

Oxime

390-340

2° Amide

150-105

~

OH =.....

1,2Quinone O

Monooxime NH20H

R--~-~O --R'

Ester

,

Nitrosophenol

430

O

H ~N ~ J '~ ~ D~

F~---NHOH + HO--R'

Hydroxamic acid

Nitrile

170-160

3

270-240

R2 3

120-110.

Scheme 1. Reactions of model compounds with hydroxylamine, B e c k m a n rearrangements of oximes, and 16N NMR chemical shifts.

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are evidence for the presence of esters in the fulvic acid. The pertinent reactions and chemical shift data are summarized in scheme 1. The peaks in the spectrum from approximately 216 to 180 ppm remain unidentified as of this writing. These peaks have also been observed in other samples which we have analyzed. The INEPT spectrum was recorded to obtain enhancement of singly protonated nitrogens, assuming an average 1JNH of 90.0 Hz. The INEPT experiment indicates that all nitrogens downfield of approximately 180 ppm in the quantitative spectrum are nonprotonated. Alternatively, if nitrogens downfield of 180 ppm are protonated, proton exchange is occurring too fast on the NMR time scale for the nitrogens to be observed. The upfield portion of the peak from 191 to 160 ppm in the quantitative spectrum, assigned as hydroxamic acid, is more clearly resolved in the INEPT spectrum. Additionally, the resonances assigned as secondary amides, centered at 129 ppm, and lactams, centered at 106 ppm, are greatly enhanced in the INEPT spectrum, removing any doubt as to their authenticity in the quantitative spectrum. In fact, polarization transfer is the only way to observe these peaks in any detail. In the NOE retained spectrum, the peak centered at 129 ppm could not be observed at all, and the peak centered at 106 ppm was reduced in intensity compared to the quantitative spectrum, indicating that these nitrogens have unfavorable NOE factors. Thus, INEPT has served the dual functions of spectral editing and signal enhancement in this 15N NMR study. A final observation is that the severe baseline roll in the quantitative spectrum, arising from acoustic ringing (ref. 22), is much alleviated in the INEPT spectrum. The INEPT and quantitative 29Si NMR spectra of the methylated and silylated fulvic acid are presented in Figure 3. The spectra exhibit two major peaks, from approximately 33 to 22 ppm, corresponding to silyl esters of carboxylic acids, and from 22 to 13 ppm, corresponding to silyl ethers of alcohols and phenols. These silyl esters and ethers represent hydroxyl groups which were not methylated with diazomethane. More extensive compilations of 29Si NMR chemical shifts of trimethylsilyl derivatives of model compounds have been reported (refs. 12-17). From integration, silyl esters comprise 61% and silyl ethers 39% of the total peak areas in the INEPT spectrum, and 58% and 42%, respectively, in the quantitative spectrum. Experimentally, therefore, INEPT provides quantitatively reliable results for trimethylsilyl derivatives of the fulvic acid. This results in two practical advantages. INEPT obviates the need for paramagnetic relaxation reagents, so that samples can be recovered for other analyses after the NMR is recorded. The savings in spectrometer time make INEPT a convenient method for studying the silylation reaction i t s e l f - evaluating the effectiveness of the multitude of available silylating reagents, determining reaction conditions, etc. In our laboratory, for example, we have found variability in the ability of different silylating reagents to form silyl esters from the carboxylic acids unreacted with diazomethane. The two derivatization studies are not without consequence for one another. The broad nature of the ketone peak in the 13C NMR of the fulvic acid, extending from 180 to 220 ppm and encompassing diaryl, alkyl-aryl,

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Oxime ~ 3~-340

Quantitative

Reagent

Hydroxamic acid

N tr le 170;160 270~240 ] J~ Secondary • amide

j

i\\~

~ \ ~o,~

Lactam 115-90

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I

I

I

I

I

,ne,

I

I

106,,

I

IJ Reagent

129

I

400

I

I

300

I

I

200

I]

I

I

100

I

I

0 ppm

Figure 2. Quantitative (top) and INEPT (bottom) 15N NMR spectra of hydroxylamine derivatized Suwannee River fulvic acid.

216

Inept

R--O--Si(CH3) 3

/s

S

R--CO2--Si(CH3) 3 S = reagent

\ I

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Quantitative

I

30

20

10

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Figure 3. INEPT (top) and Quantitative (bottom) 29Si NMR spectra of diazomethylated and silylated Suwannee River fulvic acid.

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and dialkyl ketones, suggests that ketones occur in a large number of different structural configurations in the fulvic acid, some of which are likely enolizable. With organic compounds containing enolizable ketones, it is customary to form the methoxime derivatives of the ketones with methoxylamine prior to silylation of hydroxyl groups, in order to prevent enol silylation. The same argument applies to diazomethane, since it may methylate enols as well. However, methoxylamine would react with naturally occurring esters in the fulvic acid in the same manner as hydroxylamine, in effect hydrolyzing the ester, forming the m e t h o x y analog of the hydroxamic acid, and creating free hydroxyl groups in the form of alcohols or phenols. Methyiation or silylation of the alcohols or phenols formed would introduce error into the quantitation of hydroxyl groups in the original fulvic acid. The quantitative 15N NMR spectrum provides an estimate of the degree to which hydroxylamine reacts with esters, and therefore an estimate of any error in quantitation of hydroxyl groups by combined 13C .and 29Si NMR if methoximation is performed prior to methylation and silylation. ACKNOWLEDGMENT The authors thank J. A. Leenheer for the Suwannee River fulvic acid sample. REFERENCES

1. 2. 3. 4. 5. 6. 7.

8.

G. A. Morris and R. Freeman, Enhancement of nuclear magnetic resonance signals by polarization transfer, JACS, 101(3) (1979) 760762. M. Witanowski, L. Stefaniak, and G. A. Webb, Nitrogen NMR Spectroscopy,in: G. A. Webb (Ed.), Annual Reports on NMR Spectroscopy, 18, Academic Press, London, 1986. T . M . Blinka, B. J. Helmer, and R. West, Polarization Transfer NMR Spectroscopy for Silicon-29: The INEPT and DEPT Techniques, in Advances in Organometallic Chemistry, 23, 1984, pp. 193-218. K.A. Thorn, J. B. Arterburn, and M. A. Mikita, 15N NMR investigation of hydroxylamine derivatized humic substances, Book of abstracts, 8th Rocky Mountain Regional ACS Meeting, Denver, Co., June 8-12, 1986. M. Schnitzer and S. U. Khan, Humic Substances in the Environment, Marcel Dekker, New York, 1972, p.43-45. F.J. Stevenson, Humus Chemistry, John Wiley & Sons, New York, 1982, pp. 230-231. C . M . Preston, B. S. Rauthan, C. Rodger, and J. A. Ripmeester, A hydrogen-I, carbon-13, and nitrogen-15 nuclear magnetic resonance study of p-benzoquinone polymers incorporating amino nitrogen compounds, Soil Science, 134(5) (1982) 277-293. L. Benzing-Purdie, J. A. Ripmeester, and C. M. Preston, Elucidation of the nitrogen forms in melanoidans and humic acid by 15N cross polarization-magic angle spinning nuclear magnetic resonance spectroscopy, J. Agric. Food Chem., 31 (1983) 913-915.

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*Use of tradenames in this report is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.