Applicazioni della spettroscopia NMR in fase

solida e semisolida a problematiche agro-

alimentari ed ambientali
Pellegrino Conte
Dipartimento di Ingegneria e Tecnologie Agro-Forestali (DITAF)
pellegrino.conte@unipa.it
preparation
pulse
acquisition
Direct detection
CSA, strong dipolar couplings
preparation
pulse
acquisition
13
C
1
H
decoupling
Elimination of the strong dipolar couplings
Elimination of the Chemical Shift Anisotropy (CSA)
) 1 cos 3 (
2
2
3

r
S I
S
h
Magic angle spinning (MAS)
= = = =
Sensitivity enhancement by cross polarization
Cross polarization
pulse
acquisition
13
C
1
H
decoupling
Contact
Time (CT)
Contact
Time (CT)
Static, no decoupling
Static, decoupling
MAS, no decoupling
MAS, decoupling and/or CP
Uso della Spettroscopia NMR sui solidi: MAS
2.5 mm
4 mm
7 mm
7 mm, 5000 Hz 4 mm, 13000 Hz
Uso della Spettroscopia NMR sui semisolidi: HRMAS
Uso della Spettroscopia NMR sui semisolidi: HRMAS
Aumento della sensibilit e della risoluzione
Analisi di sostanza organica naturale
Definition of Natural Organic Matter (NOM)
The decomposition of plant, animal
and microbial material in soil and
water produces a variety of complex
organic molecules, collectively called
natural organic matter. These
compounds play many important
roles in the environment.
Roles of Natural Organic Matter (NOM)
NOM is ubiquitous in soils, waters
and sediments.
In agriculture, NOM is important
because of its positive effects on
the structure, water retention
and nutrient properties of soils.
Roles of Natural Organic Matter (NOM)
NOM also interacts with metal ions
and minerals to form complexes of
widely differing chemical and
biological nature.
Solubility, mobility and toxicity of
many trace metals are strongly
correlated with the concentration of
natural organic matter in soil and
water.
Roles of Natural Organic Matter (NOM)
NOM interacts with organic
pollutants (i.e. polycyclic aromatic
hydrocarbons, PAH) to reduce their
environmental availability and toxicity
Roles of Natural Organic Matter (NOM)
Natural organic matter creates
problems for the water supply
industry. It is required its removal in
order to minimize water color and
eliminate formation of potentially
harmful chemical byproducts as a
result of chlorination.
Through a process called "bio-
fouling," natural organic matter is
also a major culprit in degrading the
performance of membrane filtration
systems used for water purification
and desalination.
Nature of Natural Organic Matter (NOM)
Persistent and universal natural
organic matter molecules are in the
environment, they are little
understood.
Natural organic matter has no unique
structure or composition, cannot be
crystallized and is extremely difficult
to characterize.
Structure proposed by Flaig (1960)
Structure proposed by Stevenson (1982)
Structure
proposed by
Stein (1997)
Structure proposed by
Schulten (1993)
Nature of Natural Organic Matter (NOM)
Natural Organic Matter is a complex
mixture of several different self-
aggregating compounds forming
supramolecular aggregates.
The size of each molecular system
within NOM does not exceed 2000
Da.
The new view (by Piccolo, 2001)
Weak dispersive forces are mainly
involved in the conformational
stabilization of NOM
Saccharides
Peptides
Alkyl chains
Aromatic systems
Cations
Importance of NMR in characterizing NOM
Solid state NMR
(CPMAS 13C-NMR)
Liquid state NMR
(13C-NMR)
Hydrophobic core
Hydrophilic moiety
Water
Study of humic substances by solid state NMR spectroscopy
ppm (t2)
-200 -100 0 100 200 300 400
(
(
(
(
(

|
|
|
|
|

\
|

|
|

\
|

|
|

\
|

(
(

CH
CH
CH
T
CT
H T
T
H T
CT
H T
T
I t I
) (
1
exp 1
) (
exp
) (
1 ) (
1
1
1
1
0

T
CH
: 1. fast local motions high T
CH
values; 2. high amount of protons
low T
CH
values
T
1
(H): proton spin lattice in the rotating frame; fast local motions
shorterT
1
(H) values. High proton concentrationfaster spin diffusion with
shorter T
1
(H) values.
Spin diffusion in natural organic matter
SO
4
-
1
2
3
4
5
6
7
8
9
10
11
12
OH
O
OH O
CH
3
1
2
3
4
5
6
7
8
9
10
CH
3
CH
3
CH
3
CH
3
C H
3
C H
3
Hexamethylbenzene (HMB)
Sodium dodecylsulphonate (SDS)
Ferulic Acid
1
2
3
4
5
O
O H
O
6
OH
O
C H
2
7
O
O
O
*
*
n

Carboxymethylcellulose sodium salt (CMC)
Chemical Shift (ppm)
-200 -100 0 100 200 300 400
HMB
CMC
SDS
Ferulic Acid
Quaternary Aromatic C
SSB SSB
Methyl C
COO
-
C1
C2-C6
C1
C2-C9
C10
C11
C12
C9
C3-C4
C7
SSB
C1-C2
C5, C6, C8
C10
OH
O
OH O
CH
3
1
2
3
4
5
6
7
8
9
10
SO
4
-
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
O
O H
O
6
OH
O
C H
2
7
O
O
O
*
*
n

CH
3
CH
3
CH
3
CH
3
C H
3
C H
3
Effect of spin diffusion mediated
by water
C H
3
CH
3
CH
3
CH
3 C H
3
C H
3
CH
3
CH
3
C H
2
CH
3
CH
3
OH
O H
CH
3
CH
3
O H
OH
O H
OH
SO
4
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
CH
3
C H
3
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
SO
4
C H
3
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
C
H
2
SO
4
O
H
H
O
H
H
C H
3
CH
3
CH
3
CH
3 C H
3
C H
3
CH
3
CH
3
C H
2
CH
3
CH
3
OH
O H
CH
3
CH
3
O H
OH
O H
OH
O
H
H
O
H
H
CH
3
CH
3
C H
2
CH
3
CH
3
OH
O H
CH
3
CH
3
O H
OH
O H
OH
O
H
H
O
H
H
O
H
H
Interactions between natural organic matter and heavy metals
(i.e. Fe(III))
Measurements of the relaxation: VCT experiments
(
(
(
(
(

|
|
|
|
|

\
|

|
|

\
|

|
|

\
|

(
(

CH
CH
CH
T
CT
H T
T
H T
CT
H T
T
I t I
) (
1
exp 1
) (
exp
) (
1 ) (
1
1
1
1
0

Measurements of the relaxation
Paramagnetic species reduce relaxation times,
thereby favouring reduction of NMR sensitivity.
In order to increase sensitivity and allow a
quantitative evaluation of solid state NMR spectra
paramagnetism must be reduced.
The main paramagnetic ion in soils is Fe
3+
.
A Fe/C<1 is needed to obtain reliable solid state
NMR spectra.
What does it mean?
(ppm)
-40 -20 0 20 40 60 80 100 120 140 160 180 200 220 240
Content of a chemical group
I
i
/I
i
x 100
Quantitative assessment of CPMAS
13
C-NMR spectroscopy
Standards Humic substances
Solid state NMR in the differentiation of NOM of different origin
Pure Lignites
Coal
Lignites
treated with
gluconic acids
Fulvic acids
Humic acids
Analisi di suoli
Solid state NMR in the analyses of bulk soils
A Chinese soil
An Italian soil
An Australian soil
Studio della degradazione dei contaminanti organici
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)
Il C1 del fenantrene risuona intorno a
125 ppm
In presenza di un acido umico e dopo 89
giorni di incubazione, il carbonio
marcato si osserva non solo a 125 ppm
ma 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 quali
la 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
I processi di umificazione possono
essere utilizzati per la bonifica di suoli
contaminati
Pi in particolare lavaggio di suoli
contaminati da IPA con sostanze
organiche naturali pu essere un
efficace metodo per la bonifica rapida
di siti inquinati
Analisi di sistemi agroalimentari
Analyses of cheeses by 1H-HRMAS spectroscopy
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
Analyses of fruit by 1H-HRMAS spectroscopy
grapes
bananas
peaches
mangoes
Studio dei processi di germinazione
Analyses of Radish seeds during germination
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
C
o
n
t
e
n
t

(
%
)
0h
24h
48h
72h
96h
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
C
o
n
t
e
n
t

(
%
)
0h
24h
48h
96h
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
C
o
n
t
e
n
t

(
%
)
0h
24h
48h
72h
96h
Analyses of fatty acids by GC-MS
CONCLUSIONS
1. Solid state NMR spectroscopy is a very powerful tool to analyze
hardly soluble natural organic matter
2. It is the only way to obtain quantitative information on the chemical
nature of NOM
3. It provides information on the chemical nature of organic matter
directly in bulk soils
4. It can be used to study the interactions between NOM and the
inorganic moieties of soils as well as the interactions with
environmental pollutants
5. High Resolution MAS spectroscopy is very useful in analyzing
semisolid organic matter. It combines the advantages of solid state
NMR with those of the liquid state technique.
6. HRMAS is particularly powerful for the analyses of foods and plant
tissues.