NMR Slides Final

8/12/2019 NMR Slides Final

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NMR - A Non destructive FoodEvaluation Technique

Ramesh. V

Ph.D Scholar

Dairy Chemistry Division

NATIONAL DAIRY RESEARCH INSTITUTE

HARYANA-132 001


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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


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Introduction

LABORATORY ANALYSIS OF

FOOD QUALITY

INVOLVES

Extensive Preparation procedures, during which foods are

severely disturbed by size reducing, Deforming or

dilution steps.

Unlike the way in which food consumers evaluate the

food produces (Consumed while their integrity is

generally intact)


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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


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Basic Principles of NMR

Spectroscopy

Based on the Principle of Resonance.

Nuclei posses the property called spin are

considered as spinning about an axis

Spinning generates a magnetic field, knownas

magnetic moment.

Like magnet bar, this magnetic moment has a

north and a south pole called a nuclear

magnetic dipole.

(Roger Raun,2006)


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Spin quantum number I 0 = Net spin associated with the

protons and neutrons (odd mass or odd atomic number)

(Susanta Das,2004)12C, 16O, 32S (Spin quantum number = 0)


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When the nucleus is

placed in a static

magnetic field, it will

interact with the fieldvia its dipole.

The dipole will tend to

align with the field

much as a compass

needle aligns with the

earthsmagnetic field


Spectroscopy (Continue..)


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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.


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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 energystate).




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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




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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.


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The nuclear spins are held in this unfavourable state onlywhen 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 (T1) : Increase with mobility

Spin-Spin :A nucleus in the upper energy state transfers its

energy to the nucleus of lower energy state(T2

)

Spin Relaxation Process


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As soon as the spins flip back to the lower energystate 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


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Chemical shift ()

When an atom is placed in a magnetic field, its

electrons circulate about the direction of the applied

magnetic field.

This circulation causes a small magnetic field at thenucleus which opposes the externally applied field

The 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


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Chemical Shift Scale

In an NMR experiment the slight differences infrequencies 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 1H and 13C 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.


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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 ZeroHydrogen atoms of TMS

Chemical Shift Scale (Continue.)


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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.)


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CH3groups have a lower chemical shift than -CH2groups.

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 todifferent groups


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Instrumentation

(Joachim, 2004)


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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 - 13C, 1H, 31P,23Na, 14N, 13C)

NMR is just like Radio !


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It is important that the solvent itself doesn't contain anysimple hydrogen atoms, because they would produce

confusing peaks in the spectrum.

Solvent such as tetrachloromethane, CCl4, which doesn't

contain any hydrogen, or solvent in which any ordinaryhydrogen atoms are replaced by its isotope, deuterium -

for example, CDCl3instead of CHCl3.

Deuterium atoms have sufficiently different magneticproperties from ordinary hydrogen that they don't

produce peaks in the area of the spectrum that we are

looking at.

Solvents for NMR spectroscopy


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NMR Imaging

Basic principles ofobtaining spatial

information using

frequency

encoding technique(Roger and Chen, 2003)


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Gradient

Magnetic Field

Projection Reconstruction Imaging

Backprojection

reconstructionis an extension

of the frequency

encoding


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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


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Disadvantages of NMR

Less Sensitive

Test sample should be pure, non-paramagnetic andshould not be a mixture of many compounds.

Inability to study ferromagnetic materials and samples

containing significant amounts of paramagnetic species.


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Applications of NMR

Structural elucidation of most of the organic compounds-

natural products separated from plants etc

NMR technology is extremely versatile and can serve as

an instrument for basic R&D, applied R&D and processmonitoring.

Quality Control in Industries where purity of a product is

maintained

To analyze composition of food products


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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

The structure is correlated with various macroscopic

properties like the storage/ageing-behaviour

(shelflife:microbiological stability), the tendency tophase-separation [e. g. syneresis, sedimentation, physical

stability, firmness/crispness]

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)


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To examine the chemical and physical characteristics

of meat, fish, dairy products, vegetables, fruits, juices,

pastries, cheese, wine and emulsified materials

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.

Raw fruit can be graded to determine if it should be

sold as fruit, processed into juice or simply rejected

(Martin et al., 1996)


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MR Image

Short T2


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Among relevant nuclei 1H, 13C and 31P have been

extensively used in the dairy industry-milk fat lipids can

be analysed from either 1H or 13C spectra, the latter

giving better resolution.

Composition of milk fat-providing quantitative data onthe relative molar fractions of oleic, palmitic, butyric

acids and triglycerides

Positional 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


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31P-NMR has been applied to the analysis of milk and milkfractions allowing the simultaneous analysis of

phospholipids, phosphorylated carbohydrates, inorganic

phosphate (Pi), phosphoserine (PSer) and other

phosphorylated compounds from a single spectrum

Quantitative analysis on the PSer resonance has been

useful to determine the degree of phosphorylation in

super- and dephosphorylated casein


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Determination of Casein Content in

bovine milk by 31P NMR

Quantitation of area under the resonance belonging to

Ser P Using Methylenediphosphoric acid as internal

standard.

800l 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 bottomlayer- injected into NMR instrument.

(Belloqui and Ramis,2002)


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NMR Spectra for Ser P in Milk

(Belloqui and Ramis,2002)


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Studies on the physical state of dairy

components

Physical characterization of milk fat One of the main applications of NMR to dairy products

has been the evaluation of the physical properties of milk

fat.

The use of 1H-NMR has proved to be an excellent tool

for the determination of the proportion of liquid fat

relative to solid fat.

The different mobility between the solid and liquidfractions 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


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Physical state of water

When water binds other components in solution, itsmobility changes. Because of this, the measurement ofwater-T2 has been used to evaluate the state of water indairy samples.

Water relaxation measurements can provide Similarinformation to that from sorption isotherms about therehydration of food systems.

It has been applied for the evaluation of rehydration of

caseins,caseinates and milk powder.Gelation or coagulation processes lead to water trapped into

the network, processes that involve changes in watermobility-relaxation phenomena in protein gels

T R l ti d M bilit A idifi d


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T2Relaxation and Mobility-Acidified

milks

(Salomonsen,2005)


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Structural characterization of molecules

from dairy origin

Ch i f ti l d ti


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Changes in conformational and aggregation

state of milk proteins

Partial view of a 2D-1H-NMR spectrum

of -lactoglobulin,dissolved in H2O at pH

2, before (a) and after (b) heating for 8

hours at 55C.

Four small peaks, and arise because the

1H of the NHand (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 theunfolding of the protein, which allows

for exchange with the solvent

Chemical shifts belonging to NH groups

CH

(Belloque and Smith,1998)


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Aggregation of Proteins

(Lambelet and Berrocal,1992)


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Conclusion

NMR is a non-invasive and extremely versatile methodto measure the physical properties of organic andinorganic materials. This technique can simultaneouslycharacterize the chemical as well as structural

composition and is thus well suited for industrial R&Dand process monitoring applications.

However efforts needed to address the relationshipsbetween NMR properties and sensory quality attributes.

Need to develop affordable NMR hardware and moresophisticated analysis software.


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NMR Slides Final