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

Introduction• Humic Substance characterization

• Spectroscopic methods such as:Fluorescence;Nuclear magnetic resonance (NMR); andElectron paramagnetic resonance (EPR).

• Fluorescence structures minor HS constituents

• However intrinsic fluorescence can provide information about structure, conformation, heterogeneity and interaction with metallic ions.

Introduction

Introduction

300 350 400 450 500 550

0

50

100

150

200

250

250 300 350 400 450 500 550

0

200

400

600

800

1000

Synchronous ( = 20 nm)Sample depth: 0-5 cm

Conventional tillage Minimum tillage No-tillage

ex ( nm)

Fluo

resc

ence

Inte

nsity

(a.

u.)

No-tillage Sample ( 20-30 cm) Excitation

em at 500 nm

Synchronous ( = 80 nm) synchronous ( = 20 nm)

ex ( nm)

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…

300 250 200 150 100 50 0 -50

13C Chemical Shift ( ppm)

Introduction Methoxyl,

N-alkylAlkyl

di-O-alkyl

O-alkyl

Aryl

O-aryl

COO/Amide

Introduction

• EPR structural information, without artifacts or restrictive conditions, about humic substances paramagnetic ions complex.

• Detection and quantification of stables organic free radicals.

100 200 300 400 500

Magnetic Field ( mT)

Introduction

OFR signalSemiquinones, metoxybenzene and/or N associated free radicals

100 200 300 400 500

Magnetic Field ( mT)

Introduction

However, a typical spectrum show others paramagnetic centers

OFR

Cu 2+

(I = 3/2) Axial symmetryFe

3+ Rhombic symmetry

Introduction

100 200 300 400 500Magnetic Field (mT)

VO2+

(I = 7/2) axial symmetry

Introduction

100 200 300 400 500Magnetic Field (mT)

Cu2++

Fe3++

VO2++

HA

Introduction

OFR

100 200 300 400 500Magnetic Field (mT)

100 200 300 400 500

Magnetic Field ( mT)

Introduction

But, do these paramagnetic species interfere in spectroscopic analysis?

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

300 320 340 360 380

Magnetic Field ( mT)

Experimental

MI = -3/2

VOL4 axial square pyramid

2 3 4 5 6 7 8

2.4

2.6

2.8

3.0

R = -0.89

Spin

s g-1

det

erm

ined

using

EPR

(10

17)

VO2+ determined using EPR ( a.u.)

Suppression of OFR signal by VO2+

Probably dipolar interaction

Results and Discussion

2 3 4 5 6 7 8

6.0x104

8.0x104

1.0x105

1.2x105

Linear ( R = -0.92)

y = axb ( R = -0.96)

Fluo

resc

ence

(a.

u.)

exc. =

243

nm

VO2+ determined using RPE ( a.u)

Results and Discussion

Fluorescence suppression

Proximity of fluorophores and binding sites or diffusion

2 3 4 5 6 7 8

4.5

5.0

5.5

6.0

Linear ( R = -0.94)

y = axb ( R = -0.98)

di-O-Alk. d

eter

min

ed u

sing

NM

R (

%)

VO2+ determined using EPR ( a.u.)

Results and Discussion

Selective suppression of some resonance signals

Results and Discussion

O-Alk. (59-91 ppm) -0.92di-O-Alk. (91-109 ppm) -0.94COO, Amide (156-186 ppm) -0.90

Spectral Region (NMR) R

•Complexation of VO2+ by oxygenated groups from carbohydrates and carboxylic (uronic acids)

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?

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

Results and Discussion

Fluor: Em. (ex=243) 99.98 -0.92Em. (ex=455) 99.81 -0.93Ex. (em=500) 97.36 -0.95

13C-NMR 24.73 -0.95OFR (EPR) 64.61 -0.88

PC Spectroscopy Variance (%) R

Results and Discussion

250 300 350 400 450 500 550 600 650

0

( nm)

Exc. em

= 500 nm ( 2.57%) Em.

exc = 450 nm ( 0.02%)

Em. exc

= 240 nm ( 0.16%)

Loadings with red shift

Results and Discussion

240 210 180 150 120 90 60 30 0

13C Chemical Shift ( ppm)

Loadings of 1a PC ( 67%)

Long chain AlkylLignin

240 210 180 150 120 90 60 30 0

13C Chemical Shift ( ppm)

Loadings of 2a PC

Results and DiscussionCOO O-Alk.di-O-Alk.

Results and Discussion

CT1

MT1 NT1

CT2

MT2 NT2

CT3

MT3 NT3

CT4

CM4

PD4

-0.002

-0.001

0.000

0.001

0.002Factor Scores of 1.a PC ( NMR)

Fact

or S

core

s (

a.u.

)

• 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

Results and Discussion

343 344 345 346 347 348

Magnetic Field ( mT)

Minimum Tillage 0-5 cm

Results and Discussion

343 344 345 346 347 348

Magnetic Field ( mT)

Minimum Tillage 0-5 cm

Load. of 1.a PC ( 65%)

Load. of 2.a PC ( 25%)

The signal of OFR is due at least two paramagnetic centers

Results and Discussion

343 344 345 346 347 348

Magnetic Field ( mT)

Minimum Tillage 0-5 cm

Load. of 1a PC ( 65%)

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

Results and Discussion

343 344 345 346 347 348

Magnetic Field ( mT)

Minimum Tillage 0-5 cm

Load. of 2a PC ( 25%)

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

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

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

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.

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)