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

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

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

10. Cu 2+ (I = 3/2)  Axial symmetryFe 3+  Rhombic symmetry Introduction 100200300400500 Magnetic Field (mT)

11. VO 2+  (I = 7/2) axial symmetry Introduction 100200300400500 Magnetic Field (mT)

12. Cu 2+ + Fe 3+ + VO 2+ + HA Introduction OFR 100200300400500 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. Experimental M I = -3/2 VOL 4 axial  square pyramid

16. Suppression of OFR signal by VO 2+ Probably dipolar interaction Results and Discussion

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

18. Results and Discussion Selective suppression of some resonance signals

19. Results and Discussion O-Alk. (59-91 ppm)-0.92 di-O-Alk. (91-109 ppm) -0.94 COO, Amide (156-186 ppm)-0.90 Spectral Region (NMR) R Complexation of VO 2+ 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 Fluor: Em. ( ex =243) 99.98 -0.92 Em. ( ex =455) 99.81 -0.93 Ex. ( em =500) 97.36 -0.95 13 C-NMR 24.73 -0.95 OFR (EPR) 64.61 -0.88 PC SpectroscopyVariance (%)R

23. Results and Discussion Loadings with red shift

24. Results and Discussion Long chain Alkyl Lignin

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

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. The signal of OFR is due at least two paramagnetic centers

29. Results and Discussion The 1. a PC, that was correlated negatively with [VO 2+ ], have a higher g-value  VO 2+ 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 VO 2+ 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 VO 2+ 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 VO 2+ and indicated that the OFR is due to at least two paramagnetic centers and that the VO 2+ 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)