Effect of Residual Vanadyl on the Spectroscopic Analysis of Humic Acids

1. Effect of Residual Vanadyl on the Spectroscopic Analysis of Humic Acids Etelvino H. Novotny University of Limerick, Ireland Embrapa Solos Post-Doctoral fellowship holder of IRCSET

2. Introduction • Humic Substance characterization • Spectroscopic methods such as: • Fluorescence; • Nuclear magnetic resonance (NMR); and • Electron paramagnetic resonance (EPR). • Fluorescence structures  minor HS constituents • However intrinsic fluorescence can provide information about structure, conformation, heterogeneity and interaction with metallic ions.

3. Introduction

4. Introduction

5. Introduction • Evaluation of structural alterations due to different managements systems by NMR: • Labile and recalcitrant proportions; • Identification of compounds such as: lignins, tannins, carbohydrates, peptides, aliphatic biopolymers…

6. Methoxyl, N-alkyl Alkyl COO/Amide O-alkyl Aryl di-O-alkyl O-aryl Introduction

7. Introduction • EPR  structural information, without artifacts or restrictive conditions, about humic substances paramagnetic ions complex. • Detection and quantification of stables organic free radicals.

8. Introduction OFR signal Semiquinones, metoxybenzene and/or N associated free radicals

9. OFR Introduction However, a typical spectrum show others paramagnetic centers

10. Cu 2+ (I = 3/2) Axial symmetry Fe3+ Rhombic symmetry Introduction 100 200 300 400 500 Magnetic Field (mT)

11. VO2+ (I = 7/2) axial symmetry Introduction 100 200 300 400 500 Magnetic Field (mT)

12. OFR + Cu2+ + Fe3+ + VO2+ HA Introduction 100 200 300 400 500 Magnetic Field (mT)

13. Introduction But, do these paramagnetic species interfere in spectroscopic analysis?

14. Experimental • Typic Haplorthox soils • Management systems experiment (NT, CT and MT) installed 12 years ago at Instituto Agronômico de Campinas - Brazil • Depths: 0-5; 5-10; 10-20 and 20-30 cm • The HAs were extracted according to the method recommended by the IHSS • Utilized spectroscopic techniques: NMR, EPR and Fluorescence

15. MI = -3/2 Experimental VOL4 axial  square pyramid

16. Results and Discussion Suppression of OFR signal by VO2+ Probably dipolar interaction

17. Results and Discussion Fluorescence suppression Proximity of fluorophores and binding sites or diffusion

18. Results and Discussion Selective suppression of some resonance signals

19. Results and Discussion Spectral Region (NMR) R O-Alk. (59-91 ppm) -0.92 di-O-Alk. (91-109 ppm) -0.94 COO, Amide (156-186 ppm) -0.90 • Complexation of VO2+ by oxygenated groups from carbohydrates and carboxylic (uronic acids)

20. Results and Discussion • The direct interpretation of spectroscopic data to determine intrinsic properties of HS samples, such as humification degree, can be affected. • Is it possible to isolate this effect of paramagnetic ions in such a way that would be possible allow use of these data to obtain information about intrinsic characteristics of HS?

21. Results and Discussion • A technique that can be used for this is the multivariate statistic, specifically Principal Components Analysis. • New set of variables (factors or PC) • Each factor is a linear combination of the original variables. • The factors are orthogonal and with maximal variance • In this way is possible “separate the components in a mixture” and isolate different variation sources

22. Results and Discussion PC Spectroscopy Variance (%) R Fluor: Em. (ex=243) 99.98 -0.92 Em. (ex=455) 99.81 -0.93 Ex. (em=500) 97.36 -0.95 13C-NMR 24.73 -0.95 OFR (EPR) 64.61 -0.88

23. Results and Discussion Loadings with red shift

24. Long chain Alkyl Lignin Results and Discussion

25. O-Alk. COO di-O-Alk. Results and Discussion

26. Results and Discussion • The effect of the tillage is restricted to upper layers • The contribution of lignin associated structures is low in the 20-30 cm layer More intense tillage  Higher contents of lignins residues and long chain alkyl

27. Results and Discussion

28. Results and Discussion The signal of OFR is due at least two paramagnetic centers

29. Results and Discussion The 1.a PC, that was correlated negatively with [VO2+], have a higher g-value  VO2+ suppressed preferentially OFR with electronic density is delocalized over O atoms

30. Results and Discussion On the other hand, the 2.a PC, associated to the humification degree, have a lower g-value

31. Conclusions • The ion VO2+ drastically affected the results of spectroscopic analysis, causing the suppression of: • NMR signal of hydrophilic groups; • Intensity of fluorescence emission; • EPR signal of OFR

32. Conclusions • Due to the selective suppression of signals from COO and those associated with carbohydrates it is possible to conclude that these structures are possibly directly involved in the VO2+ complex formation • The OFR concentrations and the suppression of the fluorescence signal indicate that either the fluorophores and OFR were relatively close to the binding site or the paramagnetic effect has an efficient diffusion in the structure of the HA

33. Conclusions • The multivariate analysis facilitated the isolation of this effect of the VO2+ and indicated that the OFR is due to at least two paramagnetic centers and that the VO2+ suppress preferentially the signal with lower g-value (O) • The new obtained variables (PC) indicated that the more intense tillage caused a relative accumulation of: • Recalcitrant structures, lignin and long chain alkyl; • Structures whose fluorescence spectra were presented red shifted; • Paramagnetic centers with lower g-value.

34. Collaborators • Prof. Heike Knicker (Technische Universität München) • Dr. Ladislau Martin-Neto (Embrapa) • Dr. Luiz A. Colnago (Embrapa) • Prof. Rodrigo B.V. Azeredo (Universidade Federal Fluminense) • Prof. Antônio Riul Jr (Universidade Estadual Paulista) • Prof. Michael H.B. Hayes (University of Limerick)