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TRANSCRIPT
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Applicazioni della spettroscopia NMR in fasesolida e semisolida a problematiche agro-
alimentari ed ambientali
Pellegrino Conte
Dipartimento di Ingegneria e Tecnologie Agro-Forestali (DITAF)
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preparation
pulse
acquisition
Direct detection
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CSA, strong dipolar couplings
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preparation
pulse
acquisition13C
1Hdecoupling
Elimination of the strong dipolar couplings
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Elimination of the Chemical Shift Anisotropy (CSA)
)1cos3(2
2
3
r
SI
S
h
Magic angle spinning (MAS)
= = = =
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Sensitivity enhancement by cross polarization
Cross polarization
pulse
acquisition13C
1Hdecoupling
ContactTime (CT)
ContactTime (CT)
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Static, no decoupling
Static, decoupling
MAS, no decoupling
MAS, decoupling and/or CP
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Uso della Spettroscopia NMR sui solidi: MAS
2.5 mm
4 mm
7 mm
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7 mm, 5000 Hz 4 mm, 13000 Hz
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Uso della Spettroscopia NMR sui semisolidi: HRMAS
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Uso della Spettroscopia NMR sui semisolidi: HRMAS
Aumento della sensibilit e della risoluzione
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Analisi di sostanza organica naturale
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Definition of Natural Organic Matter (NOM)
The decomposition of plant, animaland microbial material in soil andwater produces a variety of complex
organic molecules, collectively callednatural organic matter. Thesecompounds play many importantroles in the environment.
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Roles of Natural Organic Matter (NOM)
NOM is ubiquitous in soils, watersand sediments.
In agriculture, NOM is importantbecause of its positive effects onthe structure, water retentionand nutrient properties of soils.
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Roles of Natural Organic Matter (NOM)
NOM also interacts with metal ionsand minerals to form complexes ofwidely differing chemical and
biological nature.
Solubility, mobility and toxicity ofmany trace metals are stronglycorrelated with the concentration of
natural organic matter in soil andwater.
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Roles of Natural Organic Matter (NOM)
NOM interacts with organicpollutants (i.e. polycyclic aromatic
hydrocarbons, PAH) to reduce theirenvironmental availability and toxicity
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Roles of Natural Organic Matter (NOM)
Natural organic matter createsproblems for the water supplyindustry. It is required its removal inorder to minimize water color andeliminate formation of potentiallyharmful chemical byproducts as aresult of chlorination.
Through a process called "bio-fouling," natural organic matter is
also a major culprit in degrading theperformance of membrane filtrationsystems used for water purificationand desalination.
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Nature of Natural Organic Matter (NOM)
Persistent and universal naturalorganic matter molecules are in theenvironment, they are littleunderstood.
Natural organic matter has no uniquestructure or composition, cannot becrystallized and is extremely difficultto characterize.
Structure proposed by Flaig (1960)
Structure proposed by Stevenson (1982)
Structureproposed byStein (1997)
Structure proposed bySchulten (1993)
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Nature of Natural Organic Matter (NOM)
Natural Organic Matter is a complexmixture of several different self-
aggregatingcompounds formingsupramolecular aggregates.
The size of each molecular systemwithin NOM does not exceed 2000Da.
The new view (by Piccolo, 2001)
Weak dispersive forcesare mainlyinvolved in the conformationalstabilization of NOM
Saccharides
Peptides
Alkyl chains
Aromatic systems
Cations
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Importance of NMR in characterizing NOM
Solid state NMR(CPMAS 13C-NMR)
Liquid state NMR(13C-NMR)
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Hydrophobic core
Hydrophilic moiety
Water
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Study of humic substances by solid state NMR spectroscopy
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ppm (t2)-200-1000100200300400
=
CH
CH
CH
T
CTHT
T
HT
CT
HT
TItI
)(1
exp1)(
exp)(
1)(1
1
1
1
0
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TCH: 1. fast local motionshigh TCHvalues;2. high amount of protonslow TCHvalues
T1(H):proton spin lattice in the rotating frame; fast local motionsshorterT1(H) values. High proton concentrationfaster spin diffusion withshorter T1(H) values.
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Spin diffusion in natural organic matter
SO4-
12
34
56
78
910
1112
OH
O
OHO
CH3
12
34
5
6
78
9
10
CH3
CH3
CH3
CH3
CH3
CH3
Hexamethylbenzene (HMB)
Sodium dodecylsulphonate (SDS)
Ferulic Acid
12
3
45
O
OH
O
6
OH
O
CH2
7O
O
O
*
*
n
Carboxymethylcellulose sodium salt (CMC)
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Chemical Shift (ppm)
-200-1000100200300400
HMB
CMC
SDS
Ferulic Acid
Quaternary Aromatic C
SSB SSB
Methyl C
COO-C1
C2-C6
C1
C2-C9 C10
C11
C12
C9C3-C4
C7
SSB
C1-C2
C5, C6, C8
C10
OH
O
OHO
CH3
12
34
5
6
78
9
10
SO4-
12
34
56
78
910
1112
12
3
45
O
OH
O
6
OH
O
CH2
7O
O
O
*
*
n
CH3
CH3
CH3
CH3
CH3
CH3
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Effect of spin diffusion mediatedby water
CH3
CH3
CH3
CH3CH
3
CH3
CH3
CH3 CH2CH
3
CH3
OHOH
CH3
CH3
OH
OH
OH
OH
SO4
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
CH3
CH3
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
SO4
CH3
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
C
H2
CH
2
SO4
O
H
HO
H
H
CH3
CH3
CH3
CH3CH
3
CH3
CH3
CH3
CH2
CH3
CH3
OHOH
CH3
CH3
OH
OH
OH
OH
OH
H
OH
H
CH3
CH3
CH2
CH3
CH3
OHOH
CH3
CH3
OH
OH
OH
OH
OH
H
O
H
H
OH H
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Interactions between natural organic matter and heavy metals(i.e. Fe(III))
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Measurements of the relaxation: VCT experiments
=
CH
CH
CH
T
CTHT
T
HT
CT
HT
TItI
)(1
exp1)(
exp)(
1)(1
1
1
1
0
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Measurements of the relaxation
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Paramagnetic species reduce relaxation times,
thereby favouring reduction of NMR sensitivity.
In order to increase sensitivity and allow aquantitative evaluation of solid state NMR spectra
paramagnetism must be reduced.
The main paramagnetic ion in soils is Fe3+.
A Fe/C
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What does it mean?
(ppm)
-40-20020406080100120140160180200220240
Content of a chemical groupI
i/I
ix 100
Quantitative assessment of CPMAS 13C-NMR spectroscopy
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Standards Humic substances
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Solid state NMR in the differentiation of NOM of different origin
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Pure Lignites
Coal
Lignitestreated withgluconic acids
Fulvic acids
Humic acids
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Analisi di suoli
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Solid state NMR in the analyses of bulk soils
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A Chinese soil
An Italian soil
An Australian soil
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Studio della degradazione dei contaminanti organici
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Spettro CPMAS 13C NMR di un acido
umico da suolo addizionato di
fenantrene marcato al C1
Spettro CPMAS 13C NMR dellacido
umico da suolo
Spettro differenza (b)-(a)
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Il C1 del fenantrene risuona intorno a125 ppm
In presenza di un acido umico e dopo 89
giorni di incubazione, il carbonio
marcato si osserva non solo a 125 ppmma anche a 63, 73, 135, 150, e 190 ppm
La presenza di nuovi segnali indica che
il fenantrene si degradato. La
combinazione tra NMR e tecniche qualila MS consentono di concludere che i
residui di fenantrene si legano
stabilmente alla sostanza organica
naturale
Durante i processi di umificazione
contaminanti organici come gli IPA
perdono la loro tossicit perch
diventano essi stessi parte integrante
della sostanza organica naturale
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I processi di umificazione possonoessere utilizzati per la bonifica di suoli
contaminati
Pi in particolare lavaggio di suoli
contaminati da IPA con sostanzeorganiche naturali pu essere un
efficace metodo per la bonifica rapida
di siti inquinati
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Analisi di sistemi agroalimentari
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Analyses of cheeses by 1H-HRMAS spectroscopy
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soft Kraft
sheep aged mouldy
sheep aged
cow mozzarella
buffalo mozzarella
asiago
parmigiano
-1.5
-1
-0.5
0
0.5
1
-3 -2 -1 0 1 2 3
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Analyses of fruit by 1H-HRMAS spectroscopy
grapes
bananas
peaches
mangoes
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Studio dei processi di germinazione
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Analyses of Radish seeds during germination
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0
10
20
30
40
50
60
70
80
90
100
8-6 ppm 6-5 ppm 4.5-3.0
ppm
3.0-2.5
ppm
2.5-0 ppm
Content(%) 0h
24h
48h
72h
96h
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0
5
10
15
20
25
30
35
40
8-6 ppm 6-5 ppm 4.5-3.0
ppm
3.0-2.5
ppm
2.5-0 ppm
Content(%)
0h
24h
48h96h
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0
10
20
30
40
50
60
70
80
90
8-6 ppm 6-5 ppm 4.5-3.0
ppm
3.0-2.5
ppm
2.5-0 ppm
Cont
ent(%) 0h
24h
48h
72h
96h
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Analyses of fatty acids by GC-MS
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CONCLUSIONS
1. Solid state NMR spectroscopy is a very powerful tool to analyzehardly soluble natural organic matter
2. It is the only way to obtain quantitative information on the chemicalnature of NOM
3. It provides information on the chemical nature of organic matterdirectly in bulk soils
4. It can be used to study the interactions between NOM and theinorganic moieties of soils as well as the interactions withenvironmental pollutants
5. High Resolution MAS spectroscopy is very useful in analyzing
semisolid organic matter. It combines the advantages of solid stateNMR with those of the liquid state technique.
6. HRMAS is particularly powerful for the analyses of foods and planttissues.