There are some analytical methods used in the Food Industry

1. ANALYTICAL TECHNIQUE
IN FOOD BIOCHEMISTRY
Presented by :
Rishikesh Conhye
Abhishek Chiniah
Ludovic Sophie

2. LIST OF TECHNIQUES USED IN FOOD
BIOCHEMISTRY THAT WE WILL PRESENT
TODAY
 KJEDAHL METHODS
 DUMAS METHOD
 SPECTROSCOPY
 TITRATION
 CHROMATOGRAPHY
 SOLVENT EXTRACTION

3. KJEDAHL METHODS

4. DEFINITION
 Method for the quantitative determination of nitrogen in
chemical substances developed by Johan Kjeldahl in 1883.

5. PRINCIPLE
 1. The organic compounds is digested with strong sulfuric acid in the
presence of catalysts(usually potassium suphate to increase boiling point)
while heating.
 2. The total organic N is converted to ammonium sulphate.
 3. The digested sol’n is ddigested with abundant alkali. Here, the N is
converted to ammonium hydroxide, and then being distilled into a boric
acid solution and converted to ammonium borate.
 4. Ammonium borate is titrated with strong acid.
 5. N content in proteins is averagely 16%.

6. MECHANISM
 1. Digestion
 NCOC + H2SO4 （NH4）2SO4 + CO2 + SO2 +
H2O
 2. Neutralization &distillation
 2NaOH +（NH4）2SO4 2NH3↑+Na2SO4 + 2H2O
 3. Absorption by boric acid :
 2NH3 + 4H3BO3 （NH4）2B4O7 + 5H2O
 4. Titration by strong acid
 （NH4）2B4O7 + 5H2O + 2HCl 2NH4Cl + 4H3BO3

7. APPARATUS USED IN KJEDAHL

9. IMPORTANT NOTES
 1. Amount of protein sample and reagents used should be
proportional.
 2. All the working solution should be prepared with ammonia-free
distilled water
 3. Mildly heating When digestion, so that no sample to spatter onto
flask wall.
 4. Rotate the flask while digestion.
 5. Antifoam (silica oil) should be added if necessary.
 6. 30% hydrogen peroxide can accelerate the digestion.
 7. At the end of fully digestion, the solution should be clear light-blue
or greenish.

10.  8. Digestion should be carried out in a ventilating cabinet.
 9. The distillation apparatus should be connected well before adding alkali
into digested solution.
 10. Abundant alkali should be added until there are red copper hydroxide
formed.
 11. Absorption solution should be less than 40 deg.C throughout the
absorption. Cold water bath is a good choice to lower the temperature.
 12. Indicating paper should be used to help for the determination of
distillation terminus.
 13. Indicators of methylene blue and methyl red should be added to
absorption bottle before carrying on the distillation.

11. DUMAS METHOD

12. DEFINITION
 Is a method for the quantitative determination of nitrogen in
chemical substances based on a method first described by
Jean-Baptiste Dumas in 1826.

13. PRINCIPLE
 The method consists of combusting a sample of known mass in a
high temperature (about 900°C) chamber in the presence of
oxygen.
 This leads to the release of carbon dioxide, water and nitrogen.
 The gases are then passed over special columns(such as
potassium hydroxide aqueous solution) that absorb the carbon
dioxide and water.

14.  A column containing a thermal conductivity detector at the end
is then used to separate the nitrogen from any residual carbon
dioxide and water and the remaining nitrogen content is
measured.
 The instrument must first be calibrated by analyzing a material
that is pure and has a known nitrogen concentration.
 The measured signal from the thermal conductivity detector for
the unknown sample can then be converted into a nitrogen
content

15. MECHANISM

16. APPARATUS USED IN DUMAS

17. ADVANTAGE OF DUMAS
 Fast and fully automated.(results in minutes not in hours)
 No hazardous and harmful reagents
 Large concentration range
 High precision
 Easy installation
 Lower price per analysis

18. SPECTROSCOPY METHODS

19. SPECTROSCOPY METHODS
 Spectroscopic methods are highly desirable for analysis of food
components because
 they often require minimal or no sample preparation,
 provide rapid and on-line analysis,
 and have the potential to run multiple tests on a single sample.
 These advantages particularly apply to nuclear magnetic
resonance (NMR), infrared (IR), and near-infrared (NIR)
spectroscopy.
 Additionally, UV–VIS spectroscopy, fluorescence and mid-infrared
(MIR) and Raman spectroscopy are used in the food quality
monitoring.

20. UV-VIS SPECTROSCOPY
 Absorption spectroscopy in the UV–VIS region is based on
the Lambert-Beer’s law, expressed by the following equation
 A = Ɛlc
 where ε – extinction molar coefficient; c– molar concentration
of
 substance; l– thickness of the sample (cm)

21. SPECTRUM OF UV-VIS
 Radiation is energy that contains
both electrical & magnetic
properties, therefore
electromagnetic
 ultraviolet 10 - 400 nm
 ultraviolet spectroscopy
 visible 400 - 700 nm
 visible spectroscopy

22. USES
 Phosphorus determination
 reacting with ammonium
molybdate to produce yellow
colour
 Reducing sugar determination
 reacting with dinitrosalicylic acid to
produce reddish brown colour
 To examine the quality of edible oils
regarding a number of parameters
including the anisidine value.
Anisidine value is a measurement of
the level of fats oxidation, and is
used for the assessment of poorer
quality oils.

23. INFRA-RED SPECTROPHOTOMETRY
 The IR range is divided into the following three
 near-infrared (NIR; 780 nm – 5 μm),
 mid-infrared (MIR; 5 – 30 μm) and
 far-infrared(FIR; 30 – 1000 μm).
 Absorbtion of radiation 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

24. NEAR INFRA-RED
 Near infra-red (NIR) 780 nm – 5 μm
 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

25. MID INFRA-RED
 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

26. FAR-INFRARED
 compounds containing halogen atoms, organometallic
compounds and inorganic compounds absorb in the far-
infrared and torsional vibrations and hydrogen bond stretching
modes are found in this region

27. 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)

28. 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

29. 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

30. COLORIMETRY (ABSORPTIMETER)
 Efficiency of milk pasteurization;
 substrate hydrolyses (alkaline phosphate enzyme) to a yellow end
product

31. 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

32. TITRATION METHODS

33. 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 titration’s
 Redox titration’s
 Precipitation titration’s

34. 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)

35. TITRATABLE ACIDITY APPARATUS
Nielsen, 2003 p219

36. 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

37. 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

38. HPLC
High performance Liquid Chromatography

39. HPLC APPLICATIONS
 Sugars: Glucose, Fructose, Maltose and other saccharides
 Cholesterol and sterols
 Dyes and synthetic colours
 Steroids and flavanoids
 Aspartame and other artificial sweeteners
 Fat soluble vitamins (A,D,E and K)
 Analysis of proteins

40. GENERAL TERMS USED IN
CHROMATOGRPHY
 Several terms that must be known for Chromatography:
 The mobile phase is the phase that moves in a definite direction
 The retention time is the characteristic time it takes for a
particular analyte to pass through the system
 The stationary phase is the substance fixed in place for the
chromatography procedure
 The analyte is the substance to be separated during
chromatography

41. SCHEMATIC DRAWING OF
APPARATUS

43.  The sample is pumped in small volume at high pressure in the
HPLC column.
 The sample is retarded by the interaction with the stationary
phase as it traverses the length of the column
 The sample is then passed through a detector at the end of the
column
 The separation of component is due to Adsorption process
 The different component of the solution passes by the detector
and a chromatogram is obtained

44.  Adsorption is the forming some of bonds to the surface of one
substance to another one
 Retardation time is different due to:
 Solubility of components in the solvent
 Strength of bonds formed on the stationary phase
 the pressure used (because that affects the flow rate of the
solvent)
 the temperature of the column

45.  These separated components are detected at the exit of the
column
 The output will be recorded as a series of peaks
 Each one representing a compound in the mixture passing
through the detector
 The quantity of the substance can also be determine
 The area under the peak is proportional to the amount of
substance which has passed the detector

46. IN THE DIAGRAM, THE AREA UNDER THE
PEAK FOR Y IS LESS THAN THAT FOR X. THIS
IS BECAUSE THERE IS LESS Y THAN X IN THE
MOBILE PHASE

47. SOLVENT EXTRACTION
(For analysis of Lipids)

48.  Solvent extraction technique is one of the most commonly used
methods of isolating lipids from foods
 Used to determine total lipid content in food
 Use the principle of solubility of lipids in organic compounds
 Different solvent can be used, for example Ethyl ether, petroleum
ether, pentane and hexane
 Efficiency of solvent extraction depends upon polarity of the lipids
present
 Not all lipids are extracted using only 1 organic solvent

49.  Polar lipids such as phospholipids is more soluble in polar solvents for example
alcohols
 Non-polar lipids such as triacylglycerol are more soluble in non-polar solvents
such as hexane
 Thus the total lipid content determined by solvent extraction depends on the
nature of the organic solvent used
 The total lipid content determined using one solvent may be different from
that determined using another solvent
 The solvent should be inexpensive, low boiling point, be non-toxic and be
nonflammable

50.  Drying sample. Many organic solvents cannot easily penetrate
into foods containing large quantity of water
 Particle size reduction. Dried samples are finely ground. Grinding
is often carried out at low temperatures.
 Acid hydrolysis. Some foods contain lipids that are combined with
proteins (lipoproteins) or polysaccharides (glycolipids). It is done
by heating it for 1 hour in the presence of 3N HCl acid.

51. BATCH SOLVENT EXTRACTION
 It is done mixing the sample and the solvent in a suitable
container, e.g., a separatory funnel
 The container is shaken vigorously and the organic solvent and
aqueous phase are allowed to separate (either by gravity or
centrifugation)
 The aqueous phase is decanted and left aside
 The solvent is evaporated
 The concentration of lipid in the solvent is determined by
measuring the mass of lipid remaining: %Lipid =
100 x (Mlipid/Msample)

52. BATCH SOLVENT EXTRACTION
 The procedure is repeated using the aqueous phase to improve
efficiency of extraction
 All the solvent fractions would be collected together and the
lipid determined by weighing after evaporation of solvent
 The efficiency of the extraction of a lipid by a solvent can be
quantified by an equilibrium partition
coefficient, K = csolvent/caqueous
 The higher the partition coefficient the more efficient the
extraction process

53. SEMI-CONTINUOUS SOLVENT
EXTRACTION
 Soxhlet method is most commonly used
 The source material containing the compound to be extracted is
placed inside the thimble.
 The thimble is loaded into the main chamber of the Soxhlet
extractor.
 The extraction solvent to be used is placed in a distillation flask.
 The flask is placed on the heating element.
 The Soxhlet extractor is placed atop the flask.
 A reflux condenser is placed atop the extractor

54. SEMI-CONTINUOUS SOLVENT
EXTRACTION

55. ACCELERATED SOLVENT EXTRACTION
 The efficiency of solvent extraction can be increased with an
higher temperature and pressure than are normally used
 The effectiveness of solvent extraction increases as its
temperature increases
 pressure must also be increased to keep the solvent in the liquid
state.
 This reduces the amount of solvent required to carry out the
analysis

56. REFERENCES
 http://people.umass.edu/~mcclemen/581Proteins.html
 http://people.umass.edu/~mcclemen/581Lipids.html
 http://people.umass.edu/~mcclemen/581Carbohydrates.html
 https://books.google.mu/books?id=nAugAPE8aNIC&pg=PA26&lpg=PA26&d
q=analytical+technique+in+food+biochemistry&source=bl&ots=36DLmYvfPa
&sig=aE45MNDsNCUWRCsgGg6NCjCkaNM&hl=en&sa=X&ei=tl4pVZawM8au
UeTPg7gD&ved=0CFIQ6AEwBw#v=onepage&q=analytical%20technique%20
in%20food%20biochemistry&f=false
 http://people.umass.edu/~mcclemen/581Lipids.html
 http://www.nacalai.co.jp/global/cosmosil/pdf/food_additive_analysis.pdf
 http://www.bmj.com/content/299/6702/783

57. THANK YOU