AS226 / AS246

DR AISHAH
 BASIC SPECTROMETRY
 Spectrometry - Involves the study of electromagnetic radiation (EMR) and its
application.
 A technique of analysis that uses the interaction between the energy from
EMR with matter/sample - to identify elements and compounds and to
determine their quantities.

FST606 Instrumental Analysis of Foods Dr Aishah

 Wavelength Description Associated energy change Instrument
(nm)
10-3 – 10-2 gamma rays nuclear emissions from radioactive
substances
10-2 - 10 x-rays inner-shell electronic transitions
10 - 400 ultraviolet valence electron transitions Uv-visible
spectrophotometer,
400 – 700 visible valence electron transitions
800 – 2500 near infrared molecular vibrations Infrared spectrometer
(eg. FTIR, NIR etc)
2500 – 15000 infrared molecular vibrations
106 – 107 microwaves molecular rotations
107 – 108 radio nuclei spin orientation Nuclear Magnetic
frequency Resonance spectrometer
(NMR, Pulsed-NMR)

• Covers wide range of frequencies/wavelengths – different instrument needed for

certain range.

FST606 Instrumental Analysis of Foods Dr Aishah

 NUCLEAR MAGNETIC RESONANCE (NMR)

 Important tool for determination of organic molecular structure.

 Based on absorption of EMR in radio-frequency (Rf) region between 4 to
900 MHz.
 1H-NMR spectroscopy: number and types of H atoms within a molecule.
 13C-NMR spectroscopy: number and types of C atoms within a molecule.

General Limitation:
 Usually required pure compounds.
 Background interferences become limitation with very dilute samples.
 Cannot distinguish among magnetically equivalent monomers and dimers.

FST606 Instrumental Analysis of Foods Dr Aishah

 Basic NMR

 Method arises from the fact that nucleus have a property known as spin
(assume nuclei rotate on an axis).
 All nuclei with odd atomic and mass numbers can spin are NMR active, eg. 1H,
13C, 15N, 19F, 3H (tritium), 11B, 17O, 23Na, 27Al, 29Si, 31P, 33S, 35S, 35Cl, 57Fe, 119Sn,
195Pt and 205Tl.

 Nuclei with even atomic numbers are NMR inactive, eg. 16C, 12C, 14N or
nucleus with atomic mass = atomic number → have no NMR signal.

FST606 Instrumental Analysis of Foods Dr Aishah

  When no magnetic field present, the nuclear spins are oriented randomly
(pointing in every possible direction).
 When nuclei are placed in magnetic field, these nuclei will either align with
(α spin; +½) or align against (β spin; -½) with the applied magnetic field.
 Nuclei that align with magnetic field are slightly lower in energy than those
that align against.

FST606 Instrumental Analysis of Foods Dr Aishah

  Radiofrequency (Rf) effects a transition between the α and β spin energy levels.
 When Rf that hits the sample has the exact energy of the energy gap between
the α and β spins (ΔE) - light is absorbed by the nuclei.
 Energy from light absorption flips the α spins into β spins - a process call spin
flipping.
 When a nucleus absorbs lights and flips its spin – the nucleus is said to be in
resonance.

FST606 Instrumental Analysis of Foods Dr Aishah

  High frequency light absorption indicates large energy gap between α and β
states.
 Low frequency light absorption indicates a small energy gap.

Electron Shielding
 Nucleus is surrounded by electrons – lower energy/frequency required for
spin to flips if a nucleus has less electron density around it.
 For H-NMR spectrum (range of 0 – 10 ppm): a peak close to 10 ppm
indicate a highly shielded H (electron rich) and a peak close to 0 ppm
indicate a highly deshielded H (electron poor).

FST606 Instrumental Analysis of Foods Dr Aishah

 NMR Instrument

- Need temperature-controlled room, away from traffic flow and free from
vibration.

FST606 Instrumental Analysis of Foods Dr Aishah

 Sample Preparation

Instrumentation Place sample tube inside magnetic

chamber (probe)
1. Sample tube
2. Sample chamber (probe)
Spinning of sample
3. Magnet - nuclei align with/against the
magnetic field
4. Input Rf (Rf oscillator)
5. Rf detector
Energy difference (ΔE) measured by
6. Data processor. detector

Display spectrum plot (intensity of light

absorption vs frequency)

FST606 Instrumental Analysis of Foods Dr Aishah

 Sample Preparation
 Sample tube: thin glass/quartz (0.5 cm x 18 cm) - fragile and expensive.
 Choose appropriate solvent:
- can dissolve most organic compounds

- should not have NMR absorption peaks / should not contain any protons.

 Common solvents use CCl4, deuterated water D2O, deuterated CDCl3,

benzene-D6, DMSO4 - highly toxic and carcinogenic.
 For spinning samples and lifting samples in and out of the probe – use
pneumatic system using clean and dry compressed air.

FST606 Instrumental Analysis of Foods Dr Aishah

• Samples are dissolved in solvents free of 1H atoms, e.g.
CCl4, CDCl3.
CH3
• A small amount of TMS
(tetramethylsilane) is added to calibrate CH3 Si CH3
the spectrum.
CH3
• It is used because:
• its signal is away from all the others
• it only gives one signal
• it is non-toxic
• it is inert
• it has a low boiling point so is easy to remove
 Cross-section of an NMR

FST606 Instrumental Analysis of Foods Dr Aishah

 a) Probe (sample chamber)
 Consist of coil of wire surrounding the space between the probe.
 Rf oscillator will apply constant Rf and function to tune for selected
resonance frequency, and to deliver magnetic field to sample.
 Function of Rf detector: to detect Rf emission from excited nucleus
(signal detector).

FST606 Instrumental Analysis of Foods Dr Aishah

 b) Magnet
 The heart of NMR instrument.
 Sensitivity and Resolution of NMR instrument critically dependent on
strength and quality of magnet - increase with the increase in magnetic
field strength.
 3 types of magnets:
a) Permanent magnet (field strength 0.7, 1.4 and 2.1 T correspond to
30, 60 and 90 MHz frequencies) - not ideal for extended periods of
data accumulation.
b) Conventional electromagnet - seldom use, big size.
c) Superconducting solenoids - use in modern high resolution
instrument, magnetic field strength 23 T, correspond to 1 GHz
frequencies - produce strong, homogeneous magnetic field.

FST606 Instrumental Analysis of Foods Dr Aishah

  Superconducting Magnet

- high stability, low operating cost, simple and small size.

- solenoid wound with superconducting wire (niobium-tin or niobium-

titanium wire).
- coil must be maintained at certain temperature → immersed coil in liquid
He then housed in a cryostat (surrounded by liquid nitrogen).
- He dewar flask (filled every few mths), outer liquid N dewar (filled once a
week).

FST606 Instrumental Analysis of Foods Dr Aishah

 c) Data Processor
 Displays and plot spectrum.
 Magnetic field strength (chemical
shift) vs absorption intensity.

FST606 Instrumental Analysis of Foods Dr Aishah

 Chemical Shift (δ)
 Define as:
Resonance freq. of sample nucleus – resonance freq. of reference nucleus (Hz)
spectrometer freq. (MHz)

 Expressed in ppm (not concentration).

 Reference compound: TMS (tetramethylsilane) – provide agreed upon
chemical shift scales for all spectrometers.
 1H-NMR spectrum: δ value from 0 to 10 ppm.
 13C-NMR spectrum: δ value from 10 to 200 ppm.
FST606 Instrumental Analysis of Foods Dr Aishah
FST606 Instrumental Analysis of Foods Dr Aishah
 H-NMR (Proton-NMR)

How many signal to expect?

• when molecule are chemically
equivalent (by symmetry
consideration) - same chemical
shift.
- single absorption peak in
NMR spectrum or single δ
value.

FST606 Instrumental Analysis of Foods Dr Aishah

 Quantitative Interpretation

 Area under peak proportional to number of H generating the peak.

 Height of integral line ≠ number of H (gives “relative number” of each type of
H).
 Peak integration value: 58.117, 21.215 and 33.929. Step1: Find ratios by
dividing with smallest number.
58.117 / 21.215 = 2.74
21.215 / 21.215 = 1.00
33.929 / 21.215 = 1.60
 Step 2: Identify chemical shift (Use Table/Data base) - If peak at 5.1 ppm
caused by 2 H → calculate true ratios by multiplying each figure by 2 and
round off → actual ratios 5 : 2 : 3.
FST606 Instrumental Analysis of Foods Dr Aishah
FST606 Instrumental Analysis of Foods Dr Aishah
• In a spectrum, there is one signal for each set of
equivalent H atoms.
• The intensity of each signal being proportional to the
number of equivalent H atoms it represents.
CH3 CH2 CH3 2 sets of equivalent H’s: ratio 6:2 (3:1)

Br

CH3 CH CH2 CH3 4 sets of equivalent H’s: ratio 3:1:2:3

Br

CH3 CH CH2 CH2 CH3 5 sets of equivalent H’s: ratio 3:1:2:2:3

CH3

CH3 C CH2 CH3 4 sets of equivalent H’s: ratio 6:1:2:3

OH
 For each of the following compounds, predict the
number of signals and the relative intensity of the
signals.

a) methylpropene f) ethyl propanoate

b) propene g) 1,2-dibromopropane
c) 2-chloropropane h) dimethylethyl
d) propanone propanoate
e) methylamine i) but-2-ene

FST606 Instrumental Analysis of Foods © www.chemsheets.co.uk A2 032 Dr Aishah

17-Jul-12
 CH3 O

CH2 C CH3 CH3 C CH3

2 signals: ratio 6:2 (3:1) 1 signal

CH2 CH CH3 CH3 NH2

3 signals: ratio 2:1:3 2 signals: ratio 3:2

Cl O

CH3 CH CH3 CH3 CH2 C O CH2 CH3

2 signals: ratio 6:1 4 signals: ratio 3:2:2:3

© www.chemsheets.co.uk A2 032 17-Jul-12

Br Br O CH3

CH2 CH CH3 CH3 CH2 C O C CH3

3 signals: ratio 2:1:3 3 signals: ratio 3:2:9 CH3

CH3 CH CH CH3
2 signals: ratio 6:2 (3:1)

© www.chemsheets.co.uk A2 032 17-Jul-12

 Signal splitting (n+1 rule)
 H atoms resonance frequency can be affected by magnetic fields of other H
atoms close by.
n = number of H atoms equivalent to each other on adjacent C atoms.

FST606 Instrumental Analysis of Foods Dr Aishah

 Example of splitting pattern

FST606 Instrumental Analysis of Foods Dr Aishah

 Information provide by H-NMR spectrum
 From chemical shifts of each signal – derive info on type of H atoms
(different proton resonances (chemical shift) - type of H present).
 From number of signals – determine max number of sets of equivalent H
atoms (relative numbers of different type of H - chemical structure).
 By integrating signal areas – determine relative numbers of H atoms
(intensity of proton lines – area proportional to number of spins).
 From multiplicity or coupling pattern – number of H atoms on neighboring C
atoms (info about bonding patterns) .

FST606 Instrumental Analysis of Foods Dr Aishah

Coupling / Splitting / Multiplicity

8 7 6 5 4 3 2 1 0
 chemical shift

Number of H’s next door +1

© www.chemsheets.co.uk A2 032 17-Jul-12
 n+1 O
0 H next door
singlet (s)

1 H next door
CH3 CH2 C CH3
doublet (d)

2 H next door
triplet (t)

3 H next door
quartet (q)

more H next door

multiplet (m) 8 7 6 5 4 3 2 1 0
 chemical shift
© www.chemsheets.co.uk A2 032 17-Jul-12
 signal singlet doublet triplet quartet

appearance

number of
1 2 3 4
lines

number of H’s
0 1 2 3
next door

relative size 1:1 1:2:1 1:3:3:1

© www.chemsheets.co.uk A2 032 17-Jul-12

 Br Br

CH3 C CH CH3

Br

CH3 CH2 CH3

CH3 O

CH3 CH C O CH2 CH3

FST606 Instrumental Analysis of Foods © www.chemsheets.co.uk A2 032 Dr Aishah
17-Jul-12
 CH3 CH2 CH2 CH3

8 7 6 5 4 3 2 1 0
 chemical shift
© www.chemsheets.co.uk A2 032 17-Jul-12
 CH3 CH2 CH2 CH2 CH3

O O

CH3 C CH2 CH2 C CH3

O O

CH3 C CH2 CH2 C CH2 CH3

FST606 Instrumental Analysis of Foods © www.chemsheets.co.uk A2 032 Dr Aishah
17-Jul-12
 Number of H’s next door +1

But you don’t couple to

• H’s that are equivalent
• H’s on O’s

FST606 Instrumental Analysis of Foods © www.chemsheets.co.uk A2 032 Dr Aishah

17-Jul-12
 CH3 CH2 OH

8 7 6 5 4 3 2 1 0
 chemical shift
© www.chemsheets.co.uk A2 032 17-Jul-12
Number of signals how many different sets of
equivalent H atoms there are

Position of signals information about chemical

environment of H atom

Relative intensities gives ratio of H atoms for peaks

Splitting how many H atoms on adjacent C

atoms

© www.chemsheets.co.uk A2 032 17-Jul-12

 For each of the following compounds, predict the
number of signals, the relative intensity of the
signals, and the multiplicity of each signal.

a) methylpropene f) ethyl propanoate

b) propene g) 1,2-dibromopropane
c) 2-chloropropane h) dimethylethyl
d) propanone propanoate
e) methylamine i) but-2-ene

FST606 Instrumental Analysis of Foods © www.chemsheets.co.uk A2 032 Dr Aishah

17-Jul-12
 CH3 O

CH2 C CH3 CH3 C CH3

2 signals: ratio 6 : 2 (3 :1) 1 signal
s s

CH2 CH CH3 CH3 NH2

3 signals: ratio 2 : 1 : 3 2 signals: ratio 3 : 2
d m d t q

Cl O

CH3 CH CH3 CH3 CH2 C O CH2 CH3

2 signals: ratio 6 : 1 4 signals: ratio 3 : 2 : 2 : 3
d m t q q t

© www.chemsheets.co.uk A2 032 17-Jul-12

Br Br O CH3

CH2 CH CH3 CH3 CH2 C O C CH3

3 signals: ratio 2 : 1 : 3 3 signals: ratio 3 : 2 : 9 CH3

d m d t q s

CH3 CH CH CH3
2 signals: ratio 6 : 2 (3 :1)
d q

© www.chemsheets.co.uk A2 032 17-Jul-12

 Carbon-NMR (13C-NMR)
• Use similar principle as H-NMR except for peaks appear within chemical
shift range of 10 to 200 ppm.

C-NMR spectra for n-butylvinylether C-NMR spectra for p-ethoxybenzaldehyde

FST606 Instrumental Analysis of Foods Dr Aishah

FST606 Instrumental Analysis of Foods Dr Aishah
FST606 Instrumental Analysis of Foods Dr Aishah
FST606 Instrumental Analysis of Foods Dr Aishah
 Carbon-NMR (13C-NMR)

 A great advantage of Carbon NMR is that the NMR

experiment can be run in two different modes.
 a) "off-resonance decoupling" results in the splitting
of each signal for each carbon atom in the molecule by
the hydrogen atoms attached to it
 deduced simply by counting the peaks of the multiplet
and subtracting 1.
 b) “proton decoupling" results in the removal of
proton from each carbon

FST606 Instrumental Analysis of Foods Dr Aishah

 Carbon NMR

FST606 Instrumental Analysis of Foods Dr Aishah

 Carbon NMR

FST606 Instrumental Analysis of Foods Dr Aishah

 Combination of H-NMR
and C-NMR (2D)

• Use by chemist to determine

molecular structure of a
compound.

FST606 Instrumental Analysis of Foods Dr Aishah

 APPLICATIONS OF NMR SPECTROSCOPY

 Proof of structure includes conforming structures of reaction products,

differentiating isomers, determining stereochemistry & conformational
analysis.
 Polymer structure study: identify & quantify monomer sequence
distributions, extent of branching, cross-linking, crystallinity, molecular
motions & copolymer composition.
 Determine structure of high MW biomolecules such as proteins and DNA in
solution.

FST606 Instrumental Analysis of Foods Dr Aishah

 Chemical shifts of protons in
various structures and
function groups

FST606 Instrumental Analysis of Foods Dr Aishah

 Chemical shifts for 13C in
various structures and
functional groups

FST606 Instrumental Analysis of Foods Dr Aishah

 Next Topic:
Basic Food Rheology

FST606 Instrumental Analysis of Foods Dr Aishah