ANALYTICAL TECHNIQUE
IN FOOD BIOCHEMISTRYPresented by :
Rishikesh Conhye
Abhishek Chiniah
Ludovic Sophie
LIST OF TECHNIQUES USED IN FOOD
BIOCHEMISTRY THAT WE WILL PRESENT
TODAY
KJEDAHL METHODS
DUMAS METHOD
SPECTROSCOPY
TITRATION
CHROMATOGRAPHY
SOLVENT EXTRACTION
KJEDAHL METHODS
DEFINITION
Method for the quantitative determination of nitrogen in
chemical substances developed by Johan Kjeldahl in 1883.
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%.
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
APPARATUS USED IN KJEDAHL
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.
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.
DUMAS METHOD
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.
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.
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
MECHANISM
APPARATUS USED IN DUMAS
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
SPECTROSCOPY METHODS
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.
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)
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
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.
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
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
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
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
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)
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
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
COLORIMETRY (ABSORPTIMETER)
Efficiency of milk pasteurization;
substrate hydrolyses (alkaline phosphate enzyme) to a yellow end
product
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
TITRATION METHODS
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
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)
TITRATABLE ACIDITY APPARATUS
Nielsen, 2003 p219
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
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
HPLC High performance Liquid Chromatography
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
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
SCHEMATIC DRAWING OF
APPARATUS
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
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
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
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
SOLVENT EXTRACTION(For analysis of Lipids)
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
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
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.
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)
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
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
SEMI-CONTINUOUS SOLVENT
EXTRACTION
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
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&dq=analytical+technique+in+food+biochemistry&source=bl&ots=36DLmYvfPa&sig=aE45MNDsNCUWRCsgGg6NCjCkaNM&hl=en&sa=X&ei=tl4pVZawM8auUeTPg7gD&ved=0CFIQ6AEwBw#v=onepage&q=analytical%20technique%20in%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
THANK YOU
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