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