Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue4.2000:

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Note

Solid-state 2-D double cross polarization magic

angle spinning

15

N

13

C NMR spectroscopy on degraded

algal residues

Heike Knicker *

Lehrstuhl fuÈr Bodenkunde, Technische UniversitaÈt MuÈnchen, 85350 Freising Weihenstephan, Germany

Received 6 December 1999; accepted 27 January 2000 (returned to author for revision 29 December 1999)

Abstract

Solid-state 2-D15_N13_{C NMR spectroscopy was applied for the ®rst time to reveal the chemical nature of nitrogen in}

degraded algae. Cross peaks indicating coupling between13_{C and}15_{N were detected only for carboxyl/amide-C and}

N-substituted-alkyl-C. Cross peaks correlating15_{N intensity to sp}2_{-C and supporting the presence of heteroaromatic-N}

were not observed. The Bloch decay15_{N NMR spectrum demonstrated that the lack of pyridine-type-N and/or}

imine-N signals in the CPMAS15_{N NMR spectrum is not caused by inecient magnetization transfer. The results give}

fur-ther evidence that amide-N in peptide-like structures comprises the major form of nitrogen in degraded algal residues.

Keywords:Solid-state 2-D15_N13_{C NMR; Bloch decay; Refractory organic nitrogen; Degraded algae}

1. Introduction

Solid-state 15_{N NMR spectroscopic studies of the}

algal-derived sapropel from Mangrove Lake, Bermuda, indicated that a considerable part of humi®ed organic nitrogen (HON) is bound in amides (Knicker et al., 1996). Evidence for the occurrence of higher amounts of heteroaromatic-N was not obtained for this sapropel. This ®nding, however, contradicts former studies indi-cating that the formation of heteroaromatic-N already occurs at an early stage of sediment diagenesis (Patience et al.,1992). On the other hand, one has to keep in mind that spectra of the sapropel from Mangrove Lake were obtained with the cross-polarization-magic-angle-spin-ning (CPMAS) technique, where the sensitivity of the

15_{N is increased by magnetization transfer from the}1_H

spin system to the15_{N spin system. The eciency of this}

transfer is dependent upon the distance between the two

coupling nuclei. Consequently, the concentration of15_N

that is not directly bound1_{H (e.g. pyridine-type-N or}

imine-N) may suer a relative intensity loss and may be underestimated in the respective spectrum.

The reliability of solid-state CPMAS15_{N NMR}

spec-tra can be tested by comparison with solid-state Bloch decay (BD) MAS 15_{N NMR spectra, acquired after}

direct excitation of the15_{N spin system. Because of the}

the low sensitivity of15_{N, this technique was introduced}

only recently as a possible tool for characterization of HON (Knicker et al., 1999).

Another problem that may lead to underestimation of heteroaromatic-N in solid-state 15_{N NMR spectra of}

humi®ed organic material is the overlapping of the chemical shift region of amide-N with that of pyrrole-type-N or indole-pyrrole-type-N (Witanowski et al., 1993). An unambiguous assignment of signals to speci®c chemical compounds is dicult. On the other hand, modern NMR spectroscopy provides several techniques that can overcome this problem. One of those techniques is a solid-state double cross polarization (DCP) MAS NMR experiment, resulting in spectra that show only signals

Organic Geochemistry 31 (2000) 337±340

www.elsevier.nl/locate/orggeochem

* Corresponding author. Tel.: 8161-71-4423; fax: +49-8161-71-4466.

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of13_{C or}15_{N which interact intra- and intermolecularlly}

(Schaefer et al., 1979). Performed as a 2-dimensional (2-D) experiment, such interactions are revealed in the form of cross peaks correlating the signals of the 13_C

NMR spectrum with those of the15_{N NMR spectrum.}

Although a promising technique, no published report of its application for the characterization of HON is known to the author.

In the present study, this experiment and the Bloch decay technique are introduced as potential tools to improve characterization of HON. The main goal was to elucidate the extent to which heteroaromatic-N is formed during degradation of algal-derived residues.

2. Experimental

2.1. Sample material

A mixed algal culture was prepared from equally mixed strains of Chlamydomonas, Chlorella, Closterium and Scenedesmusas previously described (Zelibor et al., 1988). The mixture was grown with13_{C-enriched CO}

2in

a liquid medium containing 15_{N-enriched potassium}

nitrate. The freeze-dried algal material (13_{C: 10 at%;}15_N:

90 at%) was mixed with quartz sand (6:100), inoculated with 1 cm3_{of an aqueous extract from a natural}

com-post, and incubated under aerobic conditions at 25_C

(Knicker and LuÈdemann, 1995). After two months the sample was harvested. The residual organic material was mechanically separated from the quartz sand and lyophilized.

2.2. NMR

A solid-state 1-D CPMAS NMR spectrum was obtained on a Bruker MSL 300 instrument. The stan-dard cross-polarization-magic-angle-spinning-technique was applied with a spinning speed of 5.5 kHz, a contact time of 0.7 ms and a pulse delay of 300 ms. After accu-mulation of 5000 single scans, a line broadening of 25 Hz was applied before Fourier transformation.

For the solid-state 2-D DCP MAS 15_N 13_{C NMR}

spectrum a Bruker DMX 400 instrument with a triple resonance 7 mm probe was used. The 15_{N resonance}

frequency was 40.54 MHz; that of the 13_{C was 100.61}

MHz. A consecutive matched spin-lock transfer was used, ®rst from1_{H to}5_{N (contact time}_t

1=0.7 ms) and

then from 15_{N to} 13_{C (contact time} _t

2=8 ms). During

the latter, protons were decoupled using a frequency-shifted Lee-Goldburg sequence. Duringt1a ramped1

H-pulse was used, and during t2a ramped15N-pulse was

used. Both pulses were shaped from 100% down to 50% (Peersen et al., 1993).

Sixty-four spectra were obtained with mixing times increasing at an increment of 0.062 ms and at a spinning

speed of 5.5 KHz. For one spectrum, 2048 scans with a pulse delay of 300 ms were accumulated. The15_N

che-mical shifts are referenced to the nitromethane scale (=0 ppm) and were calibrated with glycine (ÿ347.6

ppm). The13_{C chemical shifts are referenced to the}

tet-ramethysilane (=0 ppm) and were also calibrated with glycine (carboxylic carbon=176.04 ppm).

The Bloch decay (BD) MAS15_{N NMR spectrum was}

obtained on the Bruker DMX 400 instrument, with15_N

pulse width of 4ms and a pulse delay of 10 s. A

ring-down-elimination (RIDE) sequence with a 36-step phase cycle was used (H. FoÈrster, Bruker, Karlsruhe, personal communication). 1031 scans were accumulated. A line broadening of 100 Hz was applied.

3. Results

More than 80% of the intensity in the CPMAS 15_N

NMR spectrum of the degraded algae (Fig. 1a) is found betweenÿ220 andÿ285 ppm, in the region tentatively

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assigned to amide-N. The signals denoted by asterisks are spinning side bands of the amide signal, arising due to incomplete removal of chemical anisotropy. A further signal at ÿ347 ppm is often attributed to amino-N of

terminal amino acids or amino sugars. Less intense sig-nals are observable betweenÿ285 andÿ325 ppm. These

resonances can be assigned to NH2or NR2groups of

guanidines or additional amino groups of some amino acids, but could also derive from aromatic amines. The signal at ÿ358 ppm is best explained by ammonium,

formed during microbial degradation.

Intensity between ÿ145 and ÿ220 ppm may derive

from N in purines, indoles, imidazoles, pyrrole-like compounds, carbazoles or proline-N in peptides. The chemical shift region of some of these heterocyclic compounds, however, can expand to ÿ270 ppm.

Con-sequently, the determination of their contribution may be augmented by the broad amide signal. A distinction between the contribution of amide-N and hetero-aromatic-N to the total signal intensity of the peak at

ÿ260 ppm can be made by solid-state 2-dimensional

double-cross-polarization-magic angle-spinning (2-D DCP MAS) NMR. During this experiment, magnetiza-tion is transferred ®rst from1_{H to}15_{N and then to}13_C.

The resulting spectrum shows cross peaks correlating

15_{N signal intensity to} 13_{C chemical shifts of carbons}

that have strong dipolar interactions with neighboring

15_N.

In the solid-state 2-D DCP MAS 15_N 13_{C NMR}

spectrum of the degraded algae (Fig. 2), cross peaks are

observed only for carboxyl/amide-C (185±160 ppm) and C giving rise to signals between 60 and 45 ppm (N-sub-stituted alkyl-C). The cross peaks around 230 ppm and 120 ppm of the13_{C dimension are spinning side bands}

of the signal at 173 ppm. No cross peaks are detected for C in the sp2 13_{C chemical shift region that could give}

direct evidence that pyrrole-type-N is present in higher

concentration. This CPMAS 15_{N NMR spectrum}

clearly shows that the intensity betweenÿ220 andÿ285

ppm is entirely assignable to amide-N.

15_{N intensity of the}15_{N chemical shift region between}

ÿ220 andÿ285 ppm also correlates to the13C chemical

shift region between 60 and 45 ppm. The color code indicates that this peak and the cross peak between 185 and 160 ppm of the 13_{C dimension have comparable}

intensity. Consequently almost all of the nitrogen in amide bonds is additionally bound to alkyl carbon as would be expected for peptides.

The signal at ÿ306 ppm in the15N dimension also

correlates with the alkyl region between 60 and 45 ppm, but shows no cross peak with the 13_{C chemical shift}

region of sp2_{-carbons (160±110 ppm). This ®nding}

strongly supports an assignment of this peak to alipha-tic rather than to aromaalipha-tic amines.

In Fig. 1b the solid-state Bloch Decay (BD) MAS15_N

NMR spectrum of the degraded algae is shown.

Comparable to the corresponding CPMAS 15_{N NMR}

spectrum, it reveals its main intensity in the amide-N region. Signals of pyridine-type- or imine-N between

ÿ25 and ÿ150 ppm cannot be distinguished from the

noise, demonstrating that the lack of such resonance lines in the CPMAS15_{N NMR spectrum is not}

explain-able by insucient eciency of cross polarization. This result supports the conclusion that those structures were not formed to an appreciable extent during algal degradation.

Further comparison of the BDMAS and CPMAS15_N

NMR spectra suggests an underestimation of the inten-sity of the ammonium signal (ÿ358 ppm) in the CPMAS 15_{N NMR spectra. This can be explained by the high}

mobility of ammonium and resultant weak dipolar interaction of its nitrogen with the1_{H spin system. This}

experiment supports earlier assumptions that inorganic N cannot be quantitatively determined (Knicker and LuÈdemann, 1995) using the common NMR parameter set-up for organic nitrogen in humi®ed material.

4. Summary and conclusions

In the present study, solid-state 2-D DCP MAS 15_N

13_{C NMR and BDMAS}15_{N NMR spectroscopy were}

applied for the ®rst time to15_{N- and}13_{C-enriched algal}

material that was microbiologically degraded. No evi-dence was obtained that could support the formation of heteroaromatic-N during microbial degradation of the

Fig. 2. Solid-state 2-D DCP MAS15_N13_{C NMR spectrum of a} highly15_{N- (90 at%) and}13_{C- (10 at%) enriched algal mixture,} aerobically degraded for 2 months. Asterisks indicate spinning side bands.

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algal remains. The assignment of15_{N signal intensity in}

the region between ÿ220 andÿ285 ppm of the

solid-state15_{N NMR spectrum of the degraded algal remains}

to amide structures is con®rmed. This result strongly supports former conclusions that some biogenic pep-tide-like material in algae can survive microbial

degra-dation rather then being involved in abiotic

recondensation reactions (Knicker and LuÈdemann, 1995; Knicker et al., 1996). The respective solid-state

2-D 2-DCP13_C15_{N MAS NMR and BDMAS NMR}

spec-tra were obtained within feasible measurement times of 12±24 h. It is clearly demonstrated that in spite of the low sensitivity of 15_{N, solid-state 2-D DCP MAS} 13_C 15_{N NMR spectroscopy has the potential to contribute}

to a better understanding of the chemical nature of HON.

Acknowledgements

The author thanks the German Federal Ministery of Education and Science (BMBF) for ®nancial support. Dr. H. FoÈrster (Bruker, Karlsruhe) is gratefully acknowledged for his help in setting up the 2-D NMR experiments. I also thank Dr. P.G. Hatcher (Ohio State University, Columbus) for providing the15_{N- and}13

C-enriched algal material.

Associate EditorÐJ. Curiale

References

Knicker, H., Bruns-Nagel, D., Drzyga, O., von LoÈw, E., Stein-bach, K., 1999. Characterization of15_{N-TNT residues after} an anaerobic/aerobic treatment of soil/molasses mixtures by solid-state 15_{N NMR spectroscopy. I. Determination and} optimization of relevant NMR spectroscopic parameters. Environmental Science and Technology 33, 343±349. Knicker, H., LuÈdemann, H.-D., 1995. N-15 and C-13 CPMAS

and solution NMR studies of N-15 enriched plant material during 600 days of microbial degradation. Organic Geo-chemistry 23, 329±341.

Knicker, H., Scaroni, A.W., Hatcher, P.G., 1996.13_{C and}15_N NMR spectroscopic investigation on the formation of fossil algal residues. Organic Geochemistry 24, 661±669.

Patience, R.L., Baxby, M., Bartle, K.D., Perry, D.L., Rees, A.G.W., Rowland, S.J., 1992. The functionality of organic nitrogen in some recent sediments from the Peru upwelling region. Organic Geochemistry 18, 161±169.

Peersen, O.B., Wu, X., Kustanovich, I., Smith, S.O., 1993. Variable-amplitude cross-polarization MAS NMR. Journal of Magnetic Resonance 104, 334±339.

Schaefer, J., McKay, R.A., Stejskal, E.O., 1979. Double-cross-polarization NMR of solids. Journal of Magnetic Resonance 34, 443±447.

Witanowski, M., Stefaniak, L., Webb, G.A., 1993. Nitrogen NMR Spectroscopy. Academic Press, London.