Analytical Chemistry

1. NUT 3315
Instrumental and Modern
methods in food
analysis
1

2. 2
Spectroscopy
Utilises the Absorption and Emission
of electromagnetic radiation by atoms
Absorption:
Low energy electrons absorb energy to move to higher energy
level
Emission:
Excited electrons return to lower energy states

3. 3
Absorption v. Emission
Ground State
1st
2nd
3rd
Energy is absorbed as
electrons jump to higher
energy levels
Energy is emitted by
electrons returning to
lower energy levels
Excited
States

4. 4
The Spectroscopic Techniques are based on the fact that
Light absorbed
is directly proportional to the
Concentration
of the absorbing component.
(Absorption)

5. 5

6. Properties of electromagnatic
radiation
 They are characteristics of energy
changes.
 They originate from atomic or
molecular processes.
 They have wave like characteristics.
 They are particulate and quantum
nature.
6

7. spectroscopic methods
 Interaction between electromagnetic
radiation and atoms or molecules in food
 Measure radiation emitted or absorbed
◦ absorption based on Beer-Lambert Law
“amount of light absorbed by a solution is
proportional to the concentration and length
of the solution”
7
http://www.globescientific.com/consumables/spec_cuv.jpg
http://www.chem.brand
eis.edu/chem18/images
/spectrophotometer.jpg

8. Spectrophotometric error &
corrections
8
Error Reduce or eliminated error
Radiation reflected
absorbed by sample holder
Use cuvettes of appropriate
quality
Sample solvent may absorb
radiation
Use blank sample
Sample may associate or
disassociate
None
Wavelength of incident light
not strictly monochromatic
Set wavelength to that of
maximum absorption

9.  Violet: 400 - 420 nm
 Indigo: 420 - 440 nm
 Blue: 440 - 490 nm
 Green: 490 - 570 nm
 Yellow: 570 - 585 nm
 Orange: 585 - 620 nm
 Red: 620 - 780 nm

10. )
 Radiation is energy that contains both
electrical & magnetic properties,
therefore electromagnetic
◦ ultraviolet 10 - 400 nm
 ultraviolet spectroscopy
◦ visible 400 - 700 nm
 visible spectroscopy
◦ IR
10

11. 11
Regions of the Electromagnetic Spectrum
Light waves all travel at the same speed through a vacuum but
differ in frequency and, therefore, in wavelength.

12.  UV & V- is result of exitation of
bonding electrons, (valency electrons)
12

13. Colorimetry (absorptimoter)
 Efficiency of milk pasteurization;
◦ substrate hydrolyses (alkaline phosphate
enzyme) to a yellow end product
13

14. uv/visible spectrophotometry (cont)
 Phosphorus determination
◦ reacting with ammonium molybdate to produce
yellow colour
 Reducing sugar determination
◦ reacting with dinitrosalicylic acid to produce
reddish brown colour
14

15. 15
UV Spectroscopy
II. Instrumentation and Spectra
A. Instrumentation
1. The construction of a traditional UV-VIS spectrometer – sample
handling, irradiation, detection and output are required
2. Here is a simple schematic that covers most modern UV
spectrometers:
sample
reference
detector
I0
I0 I0
I
log(I0/I) = A
200 700
l, nm
monochromator/
beam splitter optics
UV-VIS sources

16. 16
Use specific wavelengths of light that solution is known to
MAXIMALLY absorb generated by
hydrogen, deuterium of mercury vapour lamps (uv)
Split into specific wavelengths using prism or diffraction
grating
Focussed beam by lens
Split into two by beam splitter
Passes through sample and control (blank)
Unabsorbed light of the wavelength being used passes
through sample and control and is focussed on detector by
beam chopper
Detector converts light energy to electrical energy that
calculates an absorbance figure based on Beer-Lambert
law of sample compared to blank
Blank is used to correct for absorbance by cuvette and
solvent and other components so should be identical to
sample except not containing compound of interest

17. Infrared Spectroscopy
a wavelength range from 2,500 to 16,000 nm,
with a corresponding frequency range
from 1.9*1013 to 1.2*1014 Hz.

18. Infra-red spectrophotometry
 Absorbtion of radiation
(2500-15000 nm) at specific
wavelengths
◦ by bonds in compounds due to
molecular vibrations
 at correct frequency transition
occurs from the ground state to
vibrational excited state
◦ radiation absorbed is
proportional to the number of
similar bonds vibrating
 Sample tested may be
opaque & solid
18

19.  Infrared spectroscopy exploits the fact
that molecules have specific
frequencies at which they rotate or
vibrate corresponding to discrete
energy levels (vibrational modes).
 The frequency of the vibrations can be
associated with a particular bond type;
◦ the strength of the bond,
◦ the mass of the atoms at either end of it
19

20. Infra-red spectrophotometry
-Mid infra-red instruments
 Used for routine analysis of large
numbers of samples of one type of food
eg. milk
◦ 3480 nm for fat (CH2)groups
◦ 5723 nm for fat (C=O) groups
◦ 6465 nm for protein (N-H) groups
◦ 9610 nm for lactose (C-OH) groups
◦ 4300 nm for water (H-O-H) groups
20

21. Infra-red spectrophotometry
-Near infra-red instruments
 Near infra-red (NIR) 800-2500 nm
◦ absorbtivity 10-1000 times less than mid
infra-red bands
◦ penetrate deeper giving more representative
sample
◦ complex calibration is required using
sophisticated statistical techniques
◦ of particular importance in the wheat
industry for measurement of grain hardness,
protein and moisture levels
21

22. Pertin NIR
 Pour
 Strike off excess
 Place dish
 Press ”Analyze”
 Results in 6 seconds 22

23. 23

24. 24

27. Typical Infrared Absorption Frequencies
Stretching Vibrations Bending Vibrations
Functional
Class
Range (nm) Intensity Assignment Range (nm) Intensity Assignment
Alkanes 2850-3000 str CH3, CH2 & CH
2 or 3 bands
1350-1470
1370-1390
720-725
med
med
wk
CH2 & CH3 deformation
CH3 deformation
CH2 rocking
Alkenes 3020-3100
1630-1680
1900-2000
med
var
str
=C-H & =CH2 (usually sharp)
C=C (symmetry reduces intensity)
C=C asymmetric stretch
880-995
780-850
675-730
str
med
med
=C-H & =CH2
(out-of-plane bending)
cis-RCH=CHR
Alkynes 3300
2100-2250
str
var
C-H (usually sharp)
C@C (symmetry reduces intensity)
600-700 str C-H deformation
Arenes 3030
1600 & 1500
var
med-wk
C-H (may be several bands)
C=C (in ring) (2 bands)
(3 if conjugated)
690-900 str-med C-H bending &
ring puckering
Alcohols &
Phenols
3580-3650
3200-3550
970-1250
var
str
str
O-H (free), usually sharp
O-H (H-bonded), usually
broad
C-O
1330-
1430
650-770
med
var-wk
O-H bending (in-plane)
O-H bend (out-of-plane)
Amines 3400-3500 (dil. soln.)
3300-3400 (dil. soln.)
1000-1250
wk
wk
me
d
N-H (1°-amines), 2 bands
N-H (2°-amines)
C-N
1550-
1650
660-900
med-
str
var
NH2 scissoring (1°-amines)
NH2 & N-H wagging
(shifts on H-bonding

28. Aldehydes &
Ketones
2690-2840(2 bands)
1720-1740
1710-1720
1690
1675
1745
1780
med
str
str
str
str
str
str
C-H (aldehyde C-H)
C=O (saturated
aldehyde)
C=O (saturated
ketone)
aryl ketone
a,b-unsaturation
cyclopentanone
cyclobutanone
1350-
1360
1400-
1450
1100
str
str
me
d
a-CH3 bending
a-CH2 bending
C-C-C bending
Carboxylic Acids
& Derivatives
2500-3300 (acids)
overlap C-H
1705-1720 (acids)
1210-1320 (acids)
1785-1815 ( acyl halides)
1750 & 1820 (anhydrides)
1040-1100
1735-1750 (esters)
1000-1300
1630-1695(amides)
str
str
med-
str
str
str
str
str
str
str
O-H (very broad)
C=O (H-bonded)
O-C (sometimes 2-
peaks)
C=O
C=O (2-bands)
O-C
C=O
O-C (2-bands)
C=O (amide I band)
1395-
1440
1590-
1650
1500-
1560
me
d
me
d
me
d
C-O-H bending
N-H (1¡-amide) II
band
N-H (2¡-amide) II
band
Nitriles
Isocyanates,Isoth
iocyanates,
Diimides, Azides
& Ketenes
2240-2260
2100-2270
med
med
C@N (sharp)
-N=C=O, -N=C=S
-N=C=N-, -N3, C=C=O

29. 29

30. 30

31. Atomic absorption
spectrophotometry (AAS)
 Atoms of metal in atomised sample
absorb energy from radiation at
characteristic excitation wavelengths
 Reduction in intensity of applied radiation
is proportional to the concentration of the
element present
31

32. Atomic absorption
spectrophotometer
32

33. 33

34. Chromatography
 A separation technique to identify and
quantify chemical components based on
interaction between:
◦ the mixture to be separated known as sample
or solute
◦ a solid phase known as stationary phase (eg.
paper, thin-layer or column)
◦ a mobile phase known as the solvent
34

35. General categories of chromatographic
methods
 Planar chromatography
◦ paper chromatography
◦ thin layer chromatography (TLC)
 Column chromatography
◦ gas chromatography (GC)
◦ liquid & high performance liquid
chromatography (LC & HPLC)
35

36. Separation principles
 The principle approaches to
separation of solute are:
◦ Adsorption onto adsorbent polar solid
phase (silica & alumina) using non-polar
solvent
◦ Partition onto inert solid phase by
solubility in mixture of polar and non-polar
solvents
◦ Ion-exchange by ionic constituents on
ionic solid phase (silica & polystyrene) in
aqueous buffer
◦ Gel filtration by size and shape through
hydrated gel in aqueous solvent 36

37. Paper & Thin Layer Chromatography
(TLC)
 Liquid-solid adsorption chromatography
 Paper uses vicinal water bound to cellulose as
hydrophilic stationary phase
 TLC uses wide range of materials to separate by
any of the afore mentioned separation principles
◦ thin layer of sorbent (silica gel alumina) bound to an inert
support such as glass plates
 Separated components identified &
characterised by Rf values
Rf = distance moved by component
distance moved by solvent
37

38. Gas chromatography
38
Nielsen, 2003 p486

39. Gas chromatography
 Important especially for fat and oil
analysis
 Gas mobile phase nitrogen or
helium flowing through a heated
insulated column at from 60C to
over 200C
 Capillary column (few mm in
diameter and many meters in
length) contains stationary phase
(silicon)
39

40. Detectors for GC
 Flame ionisation detector
◦ detector adds H2 to column effluent
◦ mixture passes through jet and burned in air
◦ generates ions and free electrons
◦ produces current flow between 2 electrodes
that is proportional to the amount of material
present
40

41. Liquid chromatography
-Normal-phase & reverse-phase HPLC
 Used to analyse sugars, lipids, vitamins,
preservatives and antioxidants
◦ combination of separation methods;
 partition, gel-filtration, ion exchange
◦ detection by;
 refractive index = sugars
 UV absorbance detectors = preservative, antioxidants
 Normal or straight phase
◦ polar stationary phase, non-polar mobile phase
 Reverse-phase (higher use)
◦ non-polar stationary phase, polar mobile phase
41

42. Liquid chromatography
42
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