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