Chapter 3 : Key techniques in chemical analysis of food

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Chapter 3 Key techniques in chemical analysis
of food
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Classical methods

• Titrimetric analysis
• Gravimetric procedure
• Solvent extraction
• Refractometry

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Titrimetric assay

• Volume 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
  chemical
• Acid-base titrations
• Redox titrations
• Precipitation titrations

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Acid-base titration's

• Measure 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 apparatus
Nielsen, 2003 p219
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Redox titration

• Two half reactions one reduction, one oxidation
• Example 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 titrations

• Determine salt in cheese and butter
• Reaction 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 procedures

• Weight of food constituent is measured after
  appropriate treatment
• moisture
• ash
• total dietary fiber

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Solvent extraction methods

• Constituents 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 to food analysis
- 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

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Spectrophotometric error corrections
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

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)

• 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)

• Efficiency of milk pasteurization
• substrate hydrolyses (alkaline phosphate enzyme)
  to a yellow end product

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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

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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

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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 (CO) 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 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

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Pertin NIR

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

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Fluorimetry

• 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 photometry

• Alkali 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 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 spectrophotometer
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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

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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)

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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

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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

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Gas chromatography
Nielsen, 2003 p486
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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)

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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

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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

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Liquid chromatography
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Electrophoresis

• Based 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 (1 Dimension)
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What affects protein movement in electrophoresis

• Protein 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 mobility

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Immunochemical methods of food 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)

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Competitive ELISA

• Walls of multi-well test plate coated with
  competitive antigen
• Antibody with bound enzyme and sample to be
  analysed added
• During 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 ELISA (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 food
components-glucose as an example

• Starch gelatnisation
• Starch solution hydrolysed by ?-amylase
• Gluco-amylase converts fragments into glucose
• Glucose specifically oxidised by enzyme glucose
  oxidase to produce hydrogen peroxide
• glucose oxidase
• ?-D-glucose 02
  ?-gluconolactone H2O2

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Enzymatic determination of food
components-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
components-glucose as an example

• Absorbance read at 420 nm
• Glucose standard curve used to estimate glucose
  content of sample