Chapter 3 : Chapter 3 :

Key techniques in chemical Key techniques in chemical analysis of foodanalysis of food

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Classical methodsClassical methodsTitrimetric analysisGravimetric procedureSolvent extractionRefractometry

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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

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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)

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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

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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

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Gravimetric proceduresGravimetric proceduresWeight of food constituent is

measured after appropriate treatment◦moisture◦ash◦total dietary fiber

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Solvent extraction methodsSolvent extraction methodsConstituents of food extracted by

non-polar solvents◦used for fat content determination

solvent separated solvent removed residue weighed

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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

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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

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Colorimetry (absorptimoter)Colorimetry (absorptimoter)

Efficiency of milk pasteurization;◦substrate hydrolyses (alkaline

phosphate enzyme) to a yellow end product

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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

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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

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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

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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

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Pertin NIRPertin NIR

Pour Strike off excess Place dish Press ”Analyze” Results in 6 seconds

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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)

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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

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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

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Atomic absorption Atomic absorption spectrophotometerspectrophotometer

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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

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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)

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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

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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

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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)

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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

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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

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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

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Electrophoresis Electrophoresis (1 Dimension)(1 Dimension)

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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

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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

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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

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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)

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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

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