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Chapter 3 : Chapter 3 :
Key techniques in chemical Key techniques in chemical analysis of foodanalysis of food
1
Classical methodsClassical methodsTitrimetric analysisGravimetric procedureSolvent extractionRefractometry
2
Titrimetric assayTitrimetric assayVolume of a solution of known
concentration (standard) required to completely react with a solution (food) of unknown concentration
Stoichiometric point◦estimated by change in colour of
indicator chemicalAcid-base titration’sRedox titration’sPrecipitation titration’s
3
Acid-base titration'sAcid-base titration'sMeasure of Titratable Acidity (TA) of
milk by using standard sodium hydroxide in the presence of (0.5%) phenolphthalein (dye).
CH3CH(OH)COOH + NaOH CH3CH(OH)COONa + H2O
◦endpoint faint pink colour (pH 8.5)The actual point of colour change
known as the end point may not represent the stoichiometric point (titration error)
4
Titratable acidity apparatusTitratable acidity apparatus
5Nielsen, 2003 p219
Redox titrationRedox titrationTwo half reactions one reduction,
one oxidationExample: determination of sulphur
dioxide in foods◦sulphur dioxide is oxidised and iodine
reduced; SO2 + H2O SO3 + 2H+ + 2e-
SO3 + H2O H2SO4
I2 + 2e- 2I-
Summary: SO2 + I2 + 2H2O 2I- + 2H+ + H2SO4
◦end point starch indicator is purple colour
6
Precipitation titrationsPrecipitation titrationsDetermine salt in cheese and butterReaction of salt in food with
standard silver nitrate
AgNO3 + NaCl AgCl + NaNO3
◦Un-reacted AgNO3 is titrated with potassium thiocyanate using Fe3+ salt as indicator
AgNO3 + KCNS AgCNS + KNO3
◦endpoint silver ions react with the Fe3+ indicator to produce reddish-brown precipitate when all salt has reacted
7
Gravimetric proceduresGravimetric proceduresWeight of food constituent is
measured after appropriate treatment◦moisture◦ash◦total dietary fiber
8
Solvent extraction methodsSolvent extraction methodsConstituents of food extracted by
non-polar solvents◦used for fat content determination
solvent separated solvent removed residue weighed
9
Instrumental & modern approaches Instrumental & modern approaches to food analysis - spectroscopic to food analysis - spectroscopic methodsmethodsInteraction 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”
10http://www.globescientific.com/consumables/spec_cuv.jpg
http://www.chem.brandeis.edu/chem18/images/spectrophotometer.jpg
Spectrophotometric error & Spectrophotometric error & correctionscorrections
11
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
))
Radiation is energy that contains both electrical & magnetic properties, therefore electromagnetic◦ultraviolet 10 - 400 nm
ultraviolet spectroscopy
◦visible 400 - 700 nm visible spectroscopy
12
Colorimetry (absorptimoter)Colorimetry (absorptimoter)
Efficiency of milk pasteurization;◦substrate hydrolyses (alkaline
phosphate enzyme) to a yellow end product
13
uv/visible uv/visible spectrophotometryspectrophotometry (cont)(cont)Phosphorus determination◦reacting with ammonium molybdate to
produce yellow colourReducing sugar determination
◦reacting with dinitrosalicylic acid to produce reddish brown colour
14
Infra-red spectrophotometryInfra-red spectrophotometryAbsorbtion 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
15
Infra-red spectrophotometryInfra-red spectrophotometry-Mid infra-red instruments -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
calibration of equipment is required using data from standard analysis methods
16
Infra-red spectrophotometryInfra-red spectrophotometry-Near infra-red instruments-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
17
Pertin NIRPertin NIR
Pour Strike off excess Place dish Press ”Analyze” Results in 6 seconds
18
FluorimetryFluorimetry
Compounds first absorb UV light and then immediately re-emit light at a longer wavelength
Electrons excited from low energy levels to higher then decay to an intermediate
Used to measure florescent and florescent derivative food components such as riboflavin and thiamin respectively◦used with chromatographic methods such as
high performance liquid chromatography (HPLC)
19
Flame photometryFlame photometryAlkali metals heated in flame produce
characteristic colour (Lithium, Na and K)Electrons excited to higher energy
wavelengths and release energy as light when they fall back to lower levels
Can be used to quantify nutritionally important alkali earth metals (Ca, Br & Mg)
Number of elements estimated is limited due to lack of sensitivity
20
Atomic absorption Atomic absorption spectrophotometry (AAS)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
21
Atomic absorption Atomic absorption spectrophotometerspectrophotometer
22
ChromatographyChromatographyA 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
23
General categories of General categories of chromatographic methodschromatographic methods
Planar chromatography◦paper chromatography ◦thin layer chromatography (TLC)
Column chromatography◦gas chromatography (GC)◦liquid & high performance liquid
chromatography (LC & HPLC)
24
Separation principlesSeparation 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
25
Paper & Thin Layer Paper & Thin Layer
Chromatography (TLCChromatography (TLC))Liquid-solid adsorption chromatographyPaper uses vicinal water bound to
cellulose as hydrophilic stationary phaseTLC 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 platesSeparated components identified &
characterised by Rf values
Rf = distance moved by component distance moved by solvent
26
27
Gas chromatographyGas chromatography
28Nielsen, 2003 p486
Gas chromatographyGas chromatographyImportant especially for fat
and oil analysisGas mobile phase nitrogen or
helium flowing through a heated insulated column at from 60C to over 200C
Capillary column (few mm in diameter and many meters in length) contains stationary phase (silicon)
29
Detectors for GCDetectors for GCFlame 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
30
Liquid chromatographyLiquid chromatography-Normal-phase & reverse-phase -Normal-phase & reverse-phase HPLCHPLCUsed 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
31
Liquid chromatographyLiquid chromatography
32)
ElectrophoresisElectrophoresisBased on principal that ions are
attracted to electrode of opposite charge in an electric field
Can separate mixture of components into bands by their relative attraction to anode and cathode
Separation depends on relative anionic or cationic nature of components
Strongly influenced by pH and ionic strength of separation medium
33
Electrophoresis Electrophoresis (1 Dimension)(1 Dimension)
34
What affects protein What affects protein movement in movement in electrophoresiselectrophoresisProtein positive / negative charge◦protein is negatively charged if solution pH
is above its pI, a protein is positively charged if solution pH is below its pI.
The higher the voltage and stronger the charge on the protein, the greater the migration within the filed
Molecular size and shape (stokes radius) affect migration distance within gel◦smaller matrix pore size will decrease
mobility35
Immunochemical methods of Immunochemical methods of food analysisfood analysis
Based on reversible and non-covalent binding of antigen to antibody
Rapid, low cost, easy, accurate, sensitive, only require small sample, no special equipment required
Best known is Enzyme-Linked Immuno-Sorbent Assay (ELISA)◦competitive (two antigens & one
antibody)◦non-competitive (two antibodies & one
antigen)36
37
Competitive ELISACompetitive ELISAWalls of multi-well test plate coated with
competitive antigenAntibody with bound enzyme and sample
to be analysed addedDuring incubation antibody can bind either
◦ to competitive antigen on walls of test plate OR◦ to antigen in sample
Increase antigen in sample leads to;◦ increased binding of antibody to sample
antigen◦decreased binding of antibody to competitive
antigen on well walls
38
Competitive ELISACompetitive ELISA (cont)(cont)
Wells rinsed out leaving only competitive antigen / antibody complex on well walls
Colour developed by adding enzyme substrate to well then measured spectrophotometrically
Colour produced proportional to antigen content of sample◦ antigen in sample colour intensity◦ antigen in sample colour intensity
39
Enzymatic determination of Enzymatic determination of food components-glucose as an food components-glucose as an exampleexampleStarch gelatnisationStarch solution hydrolysed by -
amylaseGluco-amylase converts fragments
into glucoseGlucose specifically oxidised by
enzyme glucose oxidase to produce hydrogen peroxide
glucose oxidase
-D-glucose + 02 -gluconolactone + H2O2 40
Enzymatic determination of food Enzymatic determination of food components-glucose as an examplecomponents-glucose as an example
In presence of second enzyme, peroxidase, the hydrogen peroxide produced reacts with the dye -diansidine to produce yellow colour
peroxidase
H2O2 + -diansidine dye H20 + oxidised dye
(colourless) (yellow colour)
41
Enzymatic determination of food Enzymatic determination of food components-glucose as an components-glucose as an exampleexample Absorbance read at 420 nmGlucose standard curve used to
estimate glucose content of sample
42