Stefano Mammi

Dipartimento di Scienze Chimiche

Università di Padova

stefano.mammi@unipd.it

Risonanza Magnetica Nuclearequalche principio e molte applicazioni

NMR in Everyday Life

Risonanza Magnetica Nucleare…la disgrazia di un nome corretto…

NUCLEARE: Assolutamente vietato!!!

Risonanza Magnetica Nucleare…la disgrazia di un nome corretto…

NUCLEARE: Assolutamente vietato!!!

MAGNETICA: Molto pericoloso…

Risonanza Magnetica Nucleare…la disgrazia di un nome corretto…

NUCLEARE: Assolutamente vietato!!!

MAGNETICA: Molto pericoloso…

RISONANZA: Speriamo che faccia bene…

Protons (and other Nucleons) Have Spin

Spin up Spin down

Each Spinning Proton is Like a “Mini-Magnet”

Spin up Spin down

N

S

N

S

Principles of NMR

B0

M0

∆E = γhB0 = hω0 = hν0

kT

E

eP

P∆−

α

β =

M0 = Σµ = Mz

S

N

Principles of NMR

hν

Low Energy High Energy

SS

N N

L’esperimento FT-NMR

equilibrazione

impulso a 90º

osservazione dei segnali

Esperimento(t)

Analisi

dei Dati

trasformata di Fourier

dominio dei tempi (t)

FID

8 7 6 5 4 3 2 1 0

Le osservabili NMR: Chemical ShiftO

C6H5−CH2−O−C−CH3

H H

H

H

H

H|

— C —|H

H|

— C —H|H

TMS

6

0

TMS 10)ppm( ×ωω−ω=δ

N

eBo

σBo

Le osservabili NMR: Accoppiamento Scalare e Dipolare

�L’accoppiamento fra nuclei fornisce

informazioni sulla struttura

attraversolegami

attraversolo spazio

Le osservabili NMR:

Larghezza di riga (Rilassamento)

ν/2 (Hz) = 1/T2 (s-1)

8 7 6 5 4 3 2 1 08 7 6 5 4 3 2 1 0

Le osservabili NMR: Integrazione

O

C6H5−CH2−O−C−CH3

TMS

52

2132

CH3 S

O

O

NF

CH3

Which Elements or Molecules are NMR Active?

• Any atom or element with an odd number of neutrons and/or an odd number of protons

• Any molecule with NMR active atoms

• 1H - 1 proton, no neutrons, AW = 1

• 13C - 6 protons, 7 neutrons, AW =13

• 15N - 7 protons, 8 neutrons, AW = 15

• 19F - 9 protons, 10 neutrons, AW = 19

• 31P - 15 protons, 16 neutrons, AW = 31

Typical Applications of NMR:1 - Structural (chemical) elucidation

* Natural product chemistry

* Synthetic organic chemistry

- analytical tool of choice of synthetic chemists

- used in conjunction with MS and IR

2 - Study of dynamic processes

* reaction kinetics

* study of equilibrium (chemical or structural)

3 - Structural (three-dimensional) studies

* Proteins, Protein-ligand complexes

* DNA, RNA, Protein/DNA complexes

* Polysaccharides

4 - Metabolomics

5 - Drug Design

* Structure Activity Relationships by NMR

6 - Medicine – MRI, fMRI

7 - Solid State NMR

* Materials

* Structural biology

* Pharmaceutical analysis

8 - TD-NMR

MRI images of the Human Brain

NMR Structure of MMP-13 complexed to a ligand

O

O

O

O

OH

OO

O

HO

NH

OH

OO

O

O

Taxol (natural product)

PARAMETRI CHE INFLUENZANO IL RAPPORTO D/H

NEI COMPOSTI VEGETALI

Composizione isotopica dell'acqua di faldaeffetto latitudine

ciclo

planetario

dell'acqua

effetto geografico - continentale

effetto altitudine

Evapotraspirazione fogliareeffetto climatico

(temperatura - umidità relativa)

bietola, fragola ( 91 ÷ 93 ppm )

piante a ciclo C3 mele, pere, ciliegie ecc

( 96 ÷ 108 ppm ) ( 95 ÷ 101 ppm )

Provenienza vegetaledell’alcool vite

( 98 ÷ 108 ppm )

piante a ciclo C (canna da zucchero, mais)4 ( 110 ÷ 115 ppm )

Site-Specific Natural Isotope Fractionation-

Nuclear Magnetic Resonance (SNIF-NMR)

HOCH2CH2D (I)

HOCHDCH3 (II)

DOCH2CH3 (III)

Metabolomics in terms of ‘Systems Biology’

What can happen

What appears to

be happening

What makes it

happen

What has happened

and is happening

Phenotype

Universal quantitative NMR analysis of complex natural samples

Plasma,

Urine,

Saliva,

CSF,

…

Drugs, NPS,

Oils, Fuels,

Explosives,

…

Current Opinion in Biotechnology 2014, 25:51–59

1234567ppm

hippurate urea

allantoin creatininehippurate

2-oxoglutarate

citrate

TMAO

succinatefumarate

water

creatinine

taurine

1234567ppm

-25

-20

-15

-10

-5

0

5

10

15

20

25

-30 -20 -10 0 10

PC1

PC2

Metodi

quantitativi

Metodi chemiometrici

(fingerprinting e pattern recognition)

• Identificare e quantificare quanti più metaboliti possibile

(o una selezione)

• Usare l’intero spettro come fingerprint

(analisi statistica)

Due Approcci alla Metabolomica

Application of NMR to the study of olive oils

CH2

CH

CH2

O

O

O

O

R"

O

RO

R'

Adulterazione di Olio E.V. d’Oliva

con Altri Oli Vegetali

Olio di nocciola

Olio E.V. d’oliva

Mannina, Sobolev, Segre,

Spectroscopy Europe 15 (2003) 6-14

8.70 8.65 8.60 8.55

Chemical Shift ( ppm)

8.70 8.65 8.60 8.55

Chemical Shift ( ppm)

Histamine in Cheese and Fish

Health and Hygiene Indicator

J. Agric. Food Chem. 2009, 57, 2647–2652

negative

HMF in HoneyO

OOH

Targeted-qNMR

Freshness and preservation state

IndicatorMagn. Reson. Chem. 2009, 47, 868–872

positive

LoQ 2 mg/kg

Caffeine in Saliva

Talanta 2015, 140, 36–41

Targeted-qNMR

Caffeine and Other Purine Alkaloids

Caffeine Theobromine Theophylline

Caffeine Biosynthesis

Ashihara et al., 2013, Natural Products, pp 953-975

Caffeine Metabolism

Fig. 3. Zoom of the aliphatic region of the spectra of saliva extracts at different times after caffeine intake

Schievano et al. Talanta 140 (2015) 36–41

Caffeine Metabolism

Schievano et al. Talanta 140 (2015) 36–41

Fig. 4. Experimentation (black diamond = capsule, gray squares = espresso coffee).

Caffeine Metabolism

Identification of Robusta Coffee in Blends

JAFC 2014, 62, 12309−12314

• Grows between 600 and 2000 m

elevation

> Sweet

> Aromatic

< Caffeine

• More popular and 40% more

expensive

• Grows between 0 and 1000 m

elevation

• The plant is more resilient

> Astringent

> Full-bodied

< Delicate

• More economic

COFFEE

Coffea Arabica Coffea Canephora

var. Robusta

DIN 10779 Method

→ Grinding of the beans

→ Soxhlet extraction with tBME for 5 hours

→ Saponification

→ Extraction of the non-saponifiable fraction with tBME (up to 1-2

days)

→ Anhydrification and filtration

→ Solvent evaporation

→ HPLC analysis with UV-vis detector (about 45 minutes)

→ Limit of quantification: down to 2% of Robusta in the blend

At least 2 days are necessary

→ Grinding of the beans in liquid nitrogen

→ Extraction of 0.15 g ground coffee with 1.5 mL CDCl3

→ Filtration (directly in the NMR tube)

→ 1H-NMR analysis (about 25-50 minutes)

Sample in CDCl3

DMF in D2O

Total time: < 1 hr

NMR Method

Green

Roasted

JAFC 2014, 62, 12309−12314

16-OMC is a marker of Robusta coffee

JAFC 2014, 62, 12309−12314

LoD 5 mg/kg

LoQ 20 mg/kg< 0.9% of Robusta in the blend

1234567ppm

hippurate urea

allantoin creatininehippurate

2-oxoglutarate

citrate

TMAO

succinatefumarate

water

creatinine

taurine

1234567ppm

-25

-20

-15

-10

-5

0

5

10

15

20

25

-30 -20 -10 0 10

PC1

PC2

Metodi

quantitativi

Metodi chemiometrici

(fingerprinting e pattern recognition)

• Identificare e quantificare quanti più metaboliti possibile

(o una selezione)

• Usare l’intero spettro come fingerprint

(analisi statistica)

Due Approcci alla Metabolomica

Spectral integration and construction of the data matrix

bin1 bin2 bin3 bin4 bin5 bin… bin250 bin…

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample ….

Sample 520

Sample …

variables

samples

Principal Component Analysis (PCA)

x

y

x

yx

y

x

y

PC3

PC2

PC1

e.g. Principal Component Analysis

-0.1

0.0

0.1

0.2

-0.1 0.0 0.1 0.2

p[2

]

p[1]

Loading plot di PCA su acacia castagno

R2X[1] = 0.465243 R2X[2] = 0.0857414

[2.68 .. 2

[2.80 .. 2

[2.84 .. 2

[3.00 .. 3

[3.08 .. 3

[3.28 .. 3

[3.48 .. 3

[3.92 .. 3

[3.96 .. 4

[4.00 .. 4

[4.40 .. 4

[4.44 .. 4

[4.48 .. 4

[4.56 .. 4

[4.60 .. 4

[4.72 .. 4

[5.04 .. 5[5.08 .. 5

[5.24 .. 5

[5.40 .. 5

[5.44 .. 5[5.52 .. 5

[5.60 .. 5

[5.76 .. 5

[5.88 .. 5[5.92 .. 5

[6.00 .. 6[6.04 .. 6

[6.08 .. 6

[6.16 .. 6[6.20 .. 6

[6.24 .. 6

[6.28 .. 6 [6.44 .. 6

[6.48 .. 6

[6.52 .. 6

[6.56 .. 6

[6.84 .. 6

[6.88 .. 6

[6.92 .. 6

[6.96 .. 7

[7.00 .. 7[7.04 .. 7

[7.44 .. 7

[7.48 .. 7

[7.60 .. 7

[7.76 .. 7

[7.92 .. 7[7.96 .. 8

[8.00 .. 8

[8.24 .. 8[8.28 .. 8

[8.32 .. 8

[8.36 .. 8

[8.40 .. 8

[8.44 .. 8

[8.48 .. 8[8.52 .. 8

[8.56 .. 8

[8.60 .. 8

[8.68 .. 8

[8.72 .. 8

[8.76 .. 8[8.80 .. 8

[8.84 .. 8

[8.88 .. 8

[8.96 .. 9

[9.12 .. 9

[9.20 .. 9[9.36 .. 9

[9.40 .. 9

[9.60 .. 9

[9.64 .. 9

[9.76 .. 9

[9.84 .. 9

[10.44 ..

[11.04 ..

[11.20 ..

[11.60 ..

[11.72 ..

[11.76 ..

[11.80 .. [12.04 ..

[12.64 ..

[12.72 .. [12.84 ..

SIMCA-P 11 - 11/30/2010 11:47:45 AM

-4

-3

-2

-1

0

1

2

3

4

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

t[2

]

t[1]

Score plot di PCA su acacia castagno

R2X[1] = 0.465243 R2X[2] = 0.0857414

Ellipse: Hotelling T2 (0.95)

acacia

castagno

SIMCA-P 11 - 11/30/2010 11:48:11 AM

Samples Variables

PCA is able to extract the maximum possible information present in a

set of multivariate data, summarizing them in few linear combinations

of the variables themselves.

”Unsupervised” Method of Analysis

Principal Component Analysis (PCA)

Discriminant Analysis (PLS-DA, O-PLS-DA)

Models are generated that predict the class to which

unknown samples belong

”Supervised” Methods of Classification

11 10 9 8 7 6 5 4 3 2 ppm

8.59.09.5 ppm

4.64.74.84.9 ppm

0.70.80.9 ppm

Spettro 1H NMR

(componenti minoritari)

Garda

Veneto

VR BS

Fingerprinting

DOCG Conegliano-Valdobbiadene

MICRO-TERROIR origin of Prosecco wine

Alpine farmsFactories

Determination of the PRODUCTION CHAIN

of Asiago cheese

JAFC 2008 56, 7208-7214

Authentication of the botanical origin of honey

JAFC 2016, 64, 3645–3652

Fast

motion

Intermediate

motion

Slow

motion

CSF

White MatterGray Matter

Magnetic Resonance Imaging

Bruker Minispec

NMR Signal Shape in the Presence of Solid and Liquid

time

sig

na

l a

mp

litu

de

90°pulse

T2 (solid) ≈ 10 µs

T2 (liquid) ≈ 100 ms

LS

STS

dir +⋅= 100

)(%

S

L

Standard QC/QA Methods

AOCS Cd 16b-93 American Oil Chemists' Society standard method for

Solid Fat Content (SFC) by pulsed NMR

IUPAC Norm 2.150 International Union of Pure and Applied Chemistry

standard method for Solid Fat Content (SFC) by pulsed

NMR

ISO DIS/10565 International Standards Organization standard method

for oil and moisture in oilseeds by pulsed NMR

AOCS Ak 4-95 American Oil Chemists' Society standard method for oil

and moisture in oilseeds by pulsed NMR

ISO CD 10632 - OILSEED RESIDUES - Simultaneous Determination of

Oil and Water Contents - Method using pulsed NMR

Oil and Water Content in Oilseeds

(and in many other matrices…)

∝ Water

∝ Oil

Moisture and Oil Content in Oilseeds

60

65

70

75

80

85

90

95

100

30 35 40 45 50Fat Content in [%]

NM

R S

ign

al

/ M

ass

20 40 60 80 1000

% Moisture by Oven Method

Moisture Content in Green Coffee Beans

Effect of hydration by manual addition of either H2O (solid line) or D2O (dashed line) on the T2 distribution of a single dried coffee bean

Venditti et al. Food Biophysics 6 (2011) 321–326

The Bruker ProFiler

Console NMR

preamplificatore

magneterisuonatori

Andamento di B0

e B1

y

z

x

cosa c‘è dentro?

- 40 - 20 0 20 40 60

20

40

60

80

100

120

140

N

S

S

N

B0

B1

Contenuto di grasso

in beni di consumo

fat content of packaged dairy products

Fat Content Calibration

0,1

0,12

0,14

0,16

0,18

0,2

0,22

0,24

0,26

0 5 10 15 20 25 30 35

Fat Content [%]

NM

R-R

ati

o

Elisabetta Schievano

Roberto Lava

Veronica Simionato

Luca AldegheriKatia Guardini

Giampaolo Venditti

Elisa MorelatoChiara Facchin

Ivan Mazzaretto

Emmanuel Ndoum

Cristina MelerClaudia Finotello

Jalal Uddin

Federico Rastrelli

Alessandro BagnoGabriella FavaroFranco Magno

Gabriella PasiniGiulio Cozzi

Gregorio Peron

Stefano Dall’Acqua

Luisa Mannina

Roberto Ferrarini

Michele D’Ambrosio

Roberto Consonni

Luciano Navarini

Matteo Stocchero

Lucia Piana

Uni-PD

Uni-Roma1

Uni-VR

Uni-TN

CNR-MI

s.r.l. BO

Thank you for

your attention!!