NMR - A Non destructive Food

Evaluation Technique








Ramesh. V

Ph.D Scholar
Dairy Chemistry Division
NATI ONAL DAI RY RESEARCH I NSTI TUTE
HARYANA-132 001
Outline
Introduction
Nuclear Resonance Spectroscopy?
Basic Principle of NMR
Nuclear Resonance
Spin Relaxation
Chemical shift
Use of Standard
Chemical Shift Scale
Solvents used
Instrumentation
NMR Imaging
Basic Principle (Frequency encoding)
Advantages and Disadvantages
Applications
Conclusion











Introduction

LABORATORY ANALYSIS OF
FOOD QUALITY
INVOLVES
O Extensive Preparation procedures, during which foods are
severely disturbed by size reducing, Deforming or
dilution steps.
O Unlike the way in which food consumers evaluate the
food produces (Consumed while their integrity is
generally intact)

One may therefore ask :

` Are the current chemical and physical methods for
food quality measurement reliable ?
+Naturally it would be desirable to analyse food quality
in a completely nondestructive and noninvasive way.
NMR Technique
Edward Purcell at Harvard and Felix
Bloch at Stanford in 1946
Nuclear Magnetic Resonance (NMR)
Technique

Include NMR Spectroscopy and NMR imaging or
more frequently termed magnetic resonance imaging
(MRI).

Can be used to nondestructively and non invasively
study the chemical and physical properties and
phenomena, anatomical structure, and dynamic
processes of food materials and products.
Nuclear Magnetic Resonance (NMR)
Technique
O Measures the absorption of electromagnetic radiation in
the radiofrequency region of roughly 4MHz to 750MHz.
O Nuclei of the atom is involved rather than outer electrons
as in case of UV, Visible and IR Spectroscopy.
O In order to cause nuclei to absorb radiation it is necessary
to expose the sample containing nuclei of interest to a
magnetic field.
O The magnetic field has the effect of developing the energy
states required for absorption to occur.
Basic Principles of NMR
Spectroscopy
O Based on the Principle of Resonance.
O Nuclei posses the property called spin are
considered as spinning about an axis
O Spinning generates a magnetic field, known as
magnetic moment.
O Like magnet bar, this magnetic moment has a
north and a south pole called a nuclear
magnetic dipole.
(Roger Raun,2006)
Spin quantum number I 0 = Net spin associated with the
protons and neutrons (odd mass or odd atomic number)
(Susanta Das,2004)
12
C,
16
O,
32
S (Spin quantum number = 0)
O When the nucleus is
placed in a static
magnetic field, it will
interact with the field
via its dipole.
O The dipole will tend to
align with the field
much as a compass
needle aligns with the
earths magnetic field

Basic Principles of NMR
Spectroscopy (Continue..)

Basic Principles of NMR
Spectroscopy (Continue..)

Orientations of the magnetic
dipoles in a static magnetic field
are different depending on their
magnetic quantum number.
Some nuclei align themselves in
either the same direction or the
opposite direction as the field.
Alignment of the dipoles is
determined by whether the nuclei
precession is in a clockwise or
counter clock wise direction.

A nucleus that has its spin
aligned with the field will have
a lower energy (highly
favourable energy state for the
spins) and more stable than
when it has spin aligned in the
direction opposite to the field
(highly unfavourable energy
state).


Basic Principles of NMR
Spectroscopy (Continue..)


o If an RF pulse is now applied to the
aligned spins, they can be made to
flip into the unfavourable (higher)
energy state. This can only be
achieved if the rf pulse is at the
resonance frequency (in MHz) of the
nuclei being observed

Nuclear Magnetic Resonance
Basic Principles of NMR
Spectroscopy (Continue..)
Resonance Frequency of Different
Nuclei
o The resonance frequency will be different for different nuclei
(i.e. the frequency is not the same for hydrogen as that of
carbon) and is also dependent upon the strength of the
magnetic field.
+The nuclear spins are held in this unfavourable state only
when the RF pulse is on. As soon as the pulse ends they
flip back to the energy favourable (lower) state (Relaxation
Process)
+There are two relaxation processes: Spin-lactice and Spin
Spin
+Spin lactice: Nuclei in the upper energy state lose energy
to there surrounding (T
1
) : Increase with mobility
+Spin-Spin : A nucleus in the upper energy state transfers its
energy to the nucleus of lower energy state(T
2
)

Spin Relaxation Process
As soon as the spins flip back to the lower energy
state they must lose the extra energy (supplied by the
RF pulse) they obtained when flipped to the high
energy state. This is achieved by emitting RF
frequencies (Measured)

These emitted frequencies will be slightly different
from the original resonance frequency.

The differences are as a result of the differing
chemical environments of the sample atoms and the
number of spins associated with each nuclei
(Chemical shift).

Continue
Chemical shift ()
OWhen an atom is placed in a magnetic field, its
electrons circulate about the direction of the applied
magnetic field.
OThis circulation causes a small magnetic field at the
nucleus which opposes the externally applied field
OThe magnetic field at the nucleus (the effective field)
is therefore generally less than the applied field by a
fraction

Shielding of the nuclei
by electrons
Chemical Shift Scale
In an NMR experiment the slight differences in
frequencies emitted by nuclear spins in a sample arise
from differences in their chemical environment. These
differences have to be presented on an accepted,
standardised scale. This is partly achieved by using a
reference compound.
For
1
H and
13
C NMR the frequency measurements are
made with respect to the reference.
Compound tetramethylsilane (TMS). All frequency
measurements are then given in terms of their frequency
(in Hz) relative to TMS.


It has 12 hydrogen
atoms all of which are
in exactly the same
environment
The electrons in the C-H bonds are closer to the hydrogens in
this compound than in almost any other one.
Hydrogen nuclei are the most shielded from the external
magnetic field - need to increase the magnetic field by the
greatest amount to bring the hydrogens back into resonance
Peak at Zero Hydrogen atoms of TMS
Chemical Shift Scale (Continue.)
Chemical shift scale () and has units of ppm

= Peak position in Hz (relative to TMS)
Spectrometer frequency in MHz


NMR chemical shifts is dealt with ppm

Chemical Shift Scale (Continue.)
- CH
3
groups have a lower chemical shift than -CH
2
groups.
- Protons attached to C=C double bonds tend to be in the 5 to
7.5 region.
- Protons that are attached to benzene rings (aromatic
protons) tend to be in the 6 to 9 region.
- Protons attached to aldehydes /carboxylic acids are higher
still, 10 to 13 .
- Protons that are near neighbours to electronegative atoms
(O or Br for example) will be at a larger than that
expected for the same proton not neighbouring an
electronegative atom.
eg., -O -CH2 = 3.5 - 4.5, whereas -C -CH2 = approx. 1.5.

Chemical shift () of proton attached to
different groups
Instrumentation
(Joachim, 2004)
In a simple analogy, the spectrometer acts just like
a radio, in that you can tune into the stations
(nuclei) you want to listen to (observe) without
getting all stations at once (Nuclei -
13
C,
1
H,
31
P,
23
Na,
14
N,
13
C)



NMR is just like Radio !
OIt is important that the solvent itself doesn't contain any
simple hydrogen atoms, because they would produce
confusing peaks in the spectrum.
OSolvent such as tetrachloromethane, CCl
4
, which doesn't
contain any hydrogen, or solvent in which any ordinary
hydrogen atoms are replaced by its isotope, deuterium -
for example, CDCl
3
instead of CHCl
3
.
ODeuterium atoms have sufficiently different magnetic
properties from ordinary hydrogen that they don't
produce peaks in the area of the spectrum that we are
looking at.
Solvents for NMR spectroscopy
NMR Imaging
Basic principles of
obtaining spatial
information using
frequency
encoding technique
(Roger and Chen, 2003)
Gradient
Magnetic Field
Projection Reconstruction Imaging
Backprojection
reconstruction
is an extension
of the frequency
encoding

` NMR methods distinguish between states of
matter and different chemical species
` NMR can provide one- two- and three-
dimensional images of a sample; image
projections in any direction are possible.
` In many other imaging techniques a probe beam
is directed at a specific angle through the sample
thereby restricting the regions of the sample that
can be imaged.

Advantages of NMR
Disadvantages of NMR

Less Sensitive
Test sample should be pure, non-paramagnetic and
should not be a mixture of many compounds.
Inability to study ferromagnetic materials and samples
containing significant amounts of paramagnetic species.

Applications of NMR

O Structural elucidation of most of the organic compounds-
natural products separated from plants etc
O NMR technology is extremely versatile and can serve as
an instrument for basic R&D, applied R&D and process
monitoring.
O Quality Control in Industries where purity of a product is
maintained
O To analyze composition of food products

O To detect structural modifications caused by appropriate
processes or process conditions most initial and final food
products are disperse multi-phase systems, like pasta,
dairy and chocolate products
O The structure is correlated with various macroscopic
properties like the storage/ageing-behaviour
(shelflife:microbiological stability), the tendency to
phase-separation [e. g. syneresis, sedimentation, physical
stability, firmness/crispness]

O To determine the distribution of local moisture
(generally: fluid phase) or of the solid concentration (for
example in a suspension) in a sample or of products, in
general, in apparatus containing one-component or multi-
component systems

(Gerhards 1994)
O To examine the chemical and physical characteristics
of meat, fish, dairy products, vegetables, fruits, juices,
pastries, cheese, wine and emulsified materials
O Specific properties that can be measured include
freeze-thaw, percent alcohol, ripening, rheology,
water content, state of water, quantitation of fat and
water, saturated versus unsaturated fatty acid content,
adulteration and ripening.
O Raw fruit can be graded to determine if it should be
sold as fruit, processed into juice or simply rejected
(Martin et al., 1996)
MR Image
Short T
2
OAmong relevant nuclei
1
H,
13
C and
31
P have been
extensively used in the dairy industry-milk fat lipids can
be analysed from either
1
H or
13
C spectra, the latter
giving better resolution.
OComposition of milk fat-providing quantitative data on
the relative molar fractions of oleic, palmitic, butyric
acids and triglycerides
OPositional isomers of triglycerides have great nutritional
importance and, because of the structural sensitivity of
NMR, distribution of acyl groups between sn-1(3) and
sn-2 positions in milk fat triglycerides is possible



Analysis of Composition
O
31
P-NMR has been applied to the analysis of milk and milk
fractions allowing the simultaneous analysis of
phospholipids, phosphorylated carbohydrates, inorganic
phosphate (Pi), phosphoserine (PSer) and other
phosphorylated compounds from a single spectrum

O Quantitative analysis on the PSer resonance has been
useful to determine the degree of phosphorylation in
super- and dephosphorylated casein

Determination of Casein Content in
bovine milk by
31
P NMR
OQuantitation of area under the resonance belonging to
Ser P Using Methylenediphosphoric acid as internal
standard.

O800l milk+10 l Standard+100 l 0.5M EDTA. pH
was adjusted to 9.5 with NaOH. Centrifuged 88000g at
10C for 30 min-upper layer discarded and bottom
layer- injected into NMR instrument.

(Belloqui and Ramis,2002)
NMR Spectra for Ser P in Milk
(Belloqui and Ramis,2002)

Studies on the physical state of dairy
components

Physical characterization of milk fat
O One of the main applications of NMR to dairy products
has been the evaluation of the physical properties of milk
fat.
O The use of
1
H-NMR has proved to be an excellent tool
for the determination of the proportion of liquid fat
relative to solid fat.
O The different mobility between the solid and liquid
fractions provide the basis for the determination. Solid
fat, having less mobility shows rapid relaxation and a
wide signal, while that from liquid fat is sharper. Both
signals can be separated

Physical state of water

OWhen water binds other components in solution, its
mobility changes. Because of this, the measurement of
water-T
2
has been used to evaluate the state of water in
dairy samples.
OWater relaxation measurements can provide Similar
information to that from sorption isotherms about the
rehydration of food systems.
OIt has been applied for the evaluation of rehydration of
caseins,caseinates and milk powder.
OGelation or coagulation processes lead to water trapped into
the network, processes that involve changes in water
mobility-relaxation phenomena in protein gels

T
2
Relaxation and Mobility-Acidified
milks
(Salomonsen,2005)

Structural characterization of molecules
from dairy origin

Changes in conformational and aggregation
state of milk proteins
Partial view of a 2D-1H-NMR spectrum
of -lactoglobulin,dissolved in H
2
O at pH
2, before (a) and after (b) heating for 8
hours at 55C.

Four small peaks, and arise because the
1H of the NH
and (CH) groups ``comunicate'' to each
other, when they belong
to the same residue.

Observe that a portion of the resonances
disappear upon heating, as NH groups
become deuterated, due to the
unfolding of the protein, which allows
for exchange with the solvent

Chemical shifts belonging to NH groups
CH
(Belloque and Smith,1998)
Aggregation of Proteins
(Lambelet and Berrocal,1992)
Conclusion

NMR is a non-invasive and extremely versatile method
to measure the physical properties of organic and
inorganic materials. This technique can simultaneously
characterize the chemical as well as structural
composition and is thus well suited for industrial R&D
and process monitoring applications.
However efforts needed to address the relationships
between NMR properties and sensory quality attributes.
Need to develop affordable NMR hardware and more
sophisticated analysis software.