by Selma Philene 6 years ago
690
Food Analysis (FST306)
by Selma Philene 6 years ago
690
However, in this method, a common extract of the vitamin is concentrated and separated by HPLC.
The extraction procedure are the same as outlined for the vitamin determination.
COLOURIMETRIC METHOD
The result expressed as μg niacin / g sample
Critical : toxicity of cyanogen bromide, the analysis must be carried out under fume hood.
Involves a reaction between niacin (nicotinic acid) and cynogen bromide, forms a coloured compound with an intensity proportional to niacin concentration (under proper conditions)
THIOCHROME FLUOROMETRIC METHOD
PRECAUTION
Thiamine is sensitive to heat especially at alkaline pH.
The analytical steps starting from oxidation of thiamine until flourescent measurement need to be carried out rapidly and precisely according to the instructions.
Thiochrome is light sensitive, therefore, analysis should be performed under subdued light.
METHOD
The intensity of the blue fluorescence of the isobutyl alcohol extract is compared with that of the standard solution.
The intensity of fluorescence is measured.
The intensity of the blue fluorescence proportional to the thiamine concentration.
The thiochrome resulting from oxidation with potassium ferricyanide/hydrogen peroxide in alkaline solution is extracted with isobutyl alcohol.
In order to free the thiamine from the natural ester and protein bonds, the material to be examined is digested with sulphuric acid and subsequently treated with a phosphatase preparation.
FLUOROMETIC METHOD
The flourescent compound intensity proportional to the vitamin C content.
Reaction between dehydroascorbic acid and o-phenylenediamine formed a fluorescent quinoxaline compound.
Ascorbic acid oxidised to dehydroascorbic acid upon reaction with o-phenylenediamine.
This method measures both ascorbic acid and dehydroascorbic acid.
2,6-DICHLOROPHENOLINDOPHENOL TITRIMETRIC METHOD
L- dehydroascorbic acid can be determined by first converting it to L-ascorbic acid with a suitable reagent.
This method is not suitable for highly coloured products (for example coloured fruit juices) because of difficulty of determining the endpoint during titration.
At the endpoint, excess of unreduced dye is rose pink in acid solution lasting at least 10 sec.
It measures the decolourization of 2,6-dichorophenolindophenol dye by ascorbic acid.
L-ascorbic acid is oxidizes to dehydroascorbic acid by indicator dye.
HPLC METHOD
This method involve chromatographic separation and quantitative determination at 325 nm
COLORIMETRIC METHOD
PRECAUTIONS
Therefore, need to use low actinic glassware/cover glassware with aluminium foil, nitrogen or vacuum, avoid excessively high temperature and use antioxidant at the onset of procedure.
Vitamin A sensitive to UV light, air, prooxidant, high temperature and moisture.
The colour reaction does not differentiate between retinol isomers and retinol esters.
The intensity of blue colour is measured against the set of known standards.
The intesity of blue colour proportional to the amount of retinol in food sample.
It measures the unstable colour at A620nm that results from reaction between Vitamin A and antimony trichloride (SbCl3)
Indicate adulteration of the food with minerals
To measure acid-base balance of food
3) Express the result as mL of 1N acid / 100g sample.
2) Cool and transfer to Erlenmeyer flask, titrate HCl with 0.1N NaOH using methyl orange as indicator.
1)Place ash (total / water-insoluble ash) in platinum dish. Add 0.1N HCl and warm on a steam bath.
4) The acid insoluble ash was weighed and calculate the percentage.
3) Then, filter on ashless filter paper and washed several times with hot distilled water. The filter paper + residue is dried and re-ash for at least 30 min. (until constant weight).
2) Cover and boil the ash for 5 min.
1) Add 10% HCl to total ash or H2O-insoluble ash.
3) Calculate soluble ash by subtracting insoluble ash from total ash, or, dry the filtrate, re-ash and weigh.
2) Dry and re-ash the filter paper in muffle furnace at least 30 min. until constant weight is achieved. The weight remaining represents the amount of insoluble ash.
1) Ash is diluted with distilled water, then heated to nearly boiling, the resulting solution is filtered and washed several times with hot distilled water.
Relatively expensive equipment
Small sample capacity.
Utilization of O2 as sole reagent.
Equipment of choice for volatile salts.
Low temp. (less than 150'C) preserve microscopic & structural components.
Less chances of losing trace elements by volatilization.
3) Variable power frequency adjusts the rate of incineration
2) Small flow of O2 / air is introduced into the system while maintaining the specific minimum vacuum.
Combustion products which are completely dissociated are carried away in the gas stream.
Electromagnetic radio frequency generator is activated to control the rate of incineration, excites the gas molecules and dissociates it into chemically active atoms and molecules.
1) Sample is placed into a glass chamber, sealed and vacuum is applied.
4. Requires special perchloric acid hoods (with wash-down capabilities to protect from explosion).
3. Small numbers of samples can be handled at one time.
2. Corrosive reagents.
.1. Hazardous.
Requires
safety equipments.
long tongs and
hot plate,
fume hood,
3. Rapid than dry ashing
2. Little / no loss from mineral volatilization (lower temp. is used)
1. Minerals usually stay in solution.
4. Solution is cooled, and 50% of HCl is added and diluted with distilled, deionized water.
3. Boiling of sample solution is continued until the solution become colourless or light in colour.
2. Sample solution is heated up slowly up to 350'C until organic matter is completely digested (leaving only mineral oxides in solution) and HNO3 is almost evaporated.
1. Oxidation of organic substances by strong acid (HNO3) and oxidizing agent, perchloric acid (HClO4).
used in the preparation of samples for specific mineral analysis, samples with high fat content (meat & meat products) and metallic poisons.
iii.Interactions between mineral components and crucibles.
ii. Loss of volatile elements at high temp.
Zn.
Ni,
Hg,
Pb,
Fe,
Cu,
i. Time consuming (12 – 18 hrs, or overnight).
v. Requires little attention, not labour intensive.
iv. Resultant ash can be used for other analyses
e.g.
water soluble and insoluble ash.
acid insoluble ash,
iii. Large number of crucibles can be handled at once.
ii. Requires no added reagents or blank subtraction.
i. Safe method.
iv.The food sample is weighed before and after ashing to determine the concentration ash present.
iii. Most minerals are converted to
silicates.
chlorides or
phosphates,
sulfates,
oxides,
ii. Water & other volatile materials are vaporized, organic substances are burned in the presence of the O2 in air to CO2, H2O and N2.
i. Incineration at high temp. with muffle furnace (525'C or higher)
The dish containing the residue is cooled in desiccator and the amount of total ash is determined by weighing.
The residue must be free from carbon.
In dry ashing, sample is weighed into a dish, the organic matter is burned off without flaming and heated either for a fixed period of time or to constant weight.
Require high degree of
thus not routinely used for analyses & labeling purpose.
costly equipment,
time commitment, and
analytical skill,
Suitable for
and labeling purpose.
legislation,
research,
Measuring dietary fibre, provides most accurate estimate of fibre for wide range of foods
Total fibre = Soluble fibre + Insoluble fibre
6) Lignin in insoluble fraction is not hydrolyzed by acid but remains as insoluble complex which is removed by
dried & weighed.
washed,
5) Both fractions are then mixed with concentrated H2SO4 to hydrolyze cellulose and non-cellulose polysaccharides, and mono- concentration are analyzed
4) Soluble residue precipitated from solution by
removed by filtration,
washed and dried
collected,
adding ethanol,
3) Insoluble residue is removed by
dried.
washed with ethanol & acetone,
filtration,
centrifugation,
2) Resultant solution contains mixture of soluble & insoluble fibre, which is separated by centrifugation.
1) Dry ground food suspended in 80% ethanol to remove free sugars.
Starch digestion is completed by incubating with enzymes
Lipids are extracted with hexane.
Fibre = Monosaccharides + Lignin
Fibre fractions are hydrolyzed with H2SO4, and sugar content of the acid hydrolysates is determined.
Lignin is determined gravimetrically
Starch is removed by enzymatic digestion and insoluble fibre is separated from soluble fibre.
Free sugars & lipids are extracted with ethanol & hexane.
Fibre is equal to the sum of all non-starch monosaccharides plus lignon
Allow estimation of resistant starch
Suitable method for determining fibre content in most foods
low content of lignin
Fibre is hydrolyzed using concentrated H2SO4 solution to break down starch into mono-.
Pure ethanol is added to precipitate fibre
separated from the digest by centrifuguration, them is washed & dried
Enzymes are then added (to digest starch & proteins)
Defatted food sample is heated in water (to gelatinize starch)
DISADVANTAGE
Greatly over estimate the fibre content with a high content of simple sugars
APPLICATION
Used to determine fibre content in all foods
Suitable for routine fibre analysis for research, legislation and labeling purpose
to determine its mass by weighing
To isolate the fraction of interest be selective precipitation
Filtering each digestion
must be completed within given time
delays in filtering after acid or alkali digestion generally lower the results
The particle size
the finer the material is ground, the lower the determined crude fibre content.
The method measures variable amounts of the cellulose and lignin in the sample
are solubilized and not detected
hemicelluloses
2) Insoluble residue is collected by
cooled and weighed
to correct for mineral contamination of the fibre residue
ashed
weighed
dried
filtration
1) Crude fibre is determined by sequential extraction of defatted sample with 1.25% H2SO4 and 1.25% NaOH (sample is digested with H2SO4, to hydrolyze CHO & protein, and digestiom with NaOH to saponifyfatty acids)
Crude fibre measures cellulose & lignin
does not determine
hydrocolloids
pectins
hemicellulose
Indigestible materials are
the fibre residue is quantified gravimetrically
collected by filtration
Digestible
Protein
selectively solubilized by chemical and, or enzymes
Lipid
CHO
Performed with simple hand-held intrument
Method is quick & simple to carry out
require only one or two drops of sample
gives direct reading
RI of a substance depends on
Wavelength of light
Temperature
Concentration
When electromagnetic radiation passes from one medium to another, it can change direction
refracted
bent
RI (n) of a substance is the ratio of light velocity in a vacuum to its velocity of a substance
Polarimetry method unable to analyzed mixtures of CHO
Angle of polarization proportional to the concentration of optically active molecules in solution
Plane polarized light passed through solution exhibiting optical activity
Asymmetric carbon atoms have the ability to rotate plane of polarization of polarized light
Applicable for samples containing low concentrations
Require preparation of standard curve
The absorbance of the solution is determined at either 500nm or 520nm against standard
Same disadvantages as Lane-Eynon method
ADVANTAGES
More reproducible and accurate
Amount of precipitate formed is directly related to the concentration of reducing sugar in the sample
Involving oxidation of the CHO in the presence of heat and an excess of copper sulfate and alkaline tartrate, under carefully controlled conditions
DISADVANTAGES OF LANE-EYNON METHOD
Cannot directly determine the concentration of non-reducing sugar
Cannot distinguish between different types of reducing sugar
Results depends on
reagent concentrations
temperature
precise reaction times
The reaction is not stoichiometric
Determinations of reducing sugars in honey and other high-reducing sugar syrups
PROCEDURES
Air excluded from reaction mixture by keeping liquid boiling throughout titration process
CHO solution in a burette is titrated into a flask containing
methylene blue indicator
known amount of boiling copper sulfate solution and
Coloured solution is titrated until decolouration of the indicator
Mixture is boiled for a specific time,
followed by addition of methylene blue (indicator)
Based on the reaction of reducing sugar with a solution of copper sulfate followed by reaction with alkaline tartrate
DISADVANTAGES OF DYE BINDING
Non-protein components bind dye and causes error
Proteins differ in basic amino acid content
Not sensitive
mg quantities of proteins are required
ADVANTAGES OF DYE BINDING
No corrosive reagents
Relatively accurate
Rapid
May be used to estimate changes in available lysine content of cereal products
The amount of unbound soluble dye is determined by measuring its aborbance
Proteins bind the dye
to form an insoluble complex (electrostatic attraction between the molecules)
Protein-containing sample is mixed with a known excess amount of anionic ( negatively charged) dye in a buffered solution (proteins are positively charged)
DISADVANTAGES OF LOWRY METHOD
Reaction is interfered with high conc. of
sulfhydryl compounds
ammonium sulfate
reducing sugar
The reaction is interfered with varying degrees of
monosaccharides
lipids
sucrose
Colour is not strictly proportional to protein concentration
Colour varies with different proteins to a greater extent than Biuret method
ADVANTAGES OF LOWRY METHOD
More specific than most other methods
Less affected by turbidity of the sample
Very sensitive
The absorbance of the solution is read at 650nm
3) Freshly prepared Folin reagent is
incubated
mixed
added,
2) Biuret reagent is added to the diluted sample and incubated at room temperature for 10 min.
1) Proteins to be analyzed is diluted to an appropriate range (20 to 100mg)
The reaction gives a bluish colour; absorbance is read
500nm
low sensitivity for high protein concentration
750nm
high sensitivity for low protein concentration
Lowry method combines biuret reagent with another reagent (Folin-Ciocalteau phenol reagent)
reacts with tyrosine and trytophan residues in proteins
DISADVANTAGES OF BIURET METHOD
Opalescene could occur in the final solution with the presence of high levels of lipid or CHO
Not an absolute method
colour must be standardized against known protein (BSA) or against Kjedahl nitrogen method
Relatively low sensitivity compared to other UV-vis methods
ADVANTAGES OF BIURET METHOD
Does not detect nitrogen from non-peptide or non-protein sources
Very few substances other than proteins in foods interfere with biuret reaction
Rapid test
Colour derivations encountered less frequently than other method
3) The absorbance of the mixture solution is read at 540nm against blank reagent
2) The mixture is allowed to stand at room temperature for 15-30 min.
1) Biuret reagent is mixed with a portion solution of the sample. the reagent includes
potassium sodium tartrate
sodium hydroxide (NaOH)
copper sulfate
Cupric ions (Cu2+) complexed with peptide bonds under alkaline conditions and produced a violet-purplish colour
The colour intensity (absorbance) is proportional to the protein content of the sample
The absorbance of the colour produced is read at 540nm
Biuret method Involves a reaction with peptide linkages
METHODS
requires standard curves, relating the absorbance at a particular wavelength to the concentration of the specific protein
to measure protein concentration
Does not give a measure of the true protein
measures total organic nitrogen
Different proteins need different correction factors
Corrosive reagent
ADVANTAGES OF KJEDAHL METHOD
Accurate and good reproducibility
Relatively simple
Inexpensive
Applicable to all types of food
A conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration.
A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen
Titration of the ammonium borate formed with standard sulfuric / hydrochloric acid
using suitable indicator
to determine to end-point of the reaction
Distillation of diluted digest
Neutralization of diluted digest
Digestion by heating with sulfuric acid with the addition of a catalyst
Borate anions are formed & titrated with standardized acid
converted to nitrogen in the sample
Total organic nitrogen is converted to aluminium sulfate
the digest is neutralized with alkali and distilled into boric acid solution
Proteins and other organic food components in a sample are digested with sulfuric acid in the presence of catalysts
To determine fat in milk
The percent (%) fat is measured volumetrically and expressed as percent fat
Strong hydrophilic non-ionic polyoxyethylene detergent, sorbitan monolaurate is added to separate fat from other food components.
An anionic detergent (dioctyl sodium phosphate) is added ( to disperse the protein layer that stabilizes the fat) to liberate fat.
Milk is pipetted into a Babcock test bottle.
Detergents react with protein to form
protein-detergent complex to
release fat
break up emulsions
Isoamyl alcohol generally
prevents charring of sugar
reduces the effect of sulfuric acid
improves the fat separation
Comparable to Babcock method
faster
simpler
Wider application to a variety of dairy products
The tube / butyrometer is centrifuged and incubated in waterbath at 60-63'C for 5 min.
The amyl alcohol is added into the mixture to give a clear, homogenous fat column and the tube , butyrometer is carefully inverted.
Sulfuric acid(H2SO4) of specific gravity is added to a known amount of milk in
a Gerber tube or butyrometer
for digestion of protein and CHO, releases fat and maintains the fat in liquid state by generating heat
APPLICATIONS
not applicable to products containing chocolate or added sugar
due to charring of chocolate & sugars by sulfuric acid
does not determine phospholipids in the milk products
common method used to determine fat content in milk
The fat is measured volumetrically, but the result is expressed as percent (%) fat by weight
Subsequent centrifugation and addition of hot water isolate fat for quantification in the graduated portion of test bottle
The mixture is shaken until homogenous, centrifuged and submerged into water at 63'C
In the method, sulfuric acid (H2SO4) is added to a known amount of milk in Babcock bottle
ADVANTAGES OF MOJONNIER METHOD
to determine fat content in flour
2) The extractions are repeated three(3) times
the fat-containing solvents (from repeated extraction) are pooled
solvent is removed by evaporation
the weight of the fat is measured to determine the fat content
carried out in flasks designed to facillitate decanting of fat-containing organic solvent (top layer) from aqueous layer (bottom layer) during extraction
1) Involving release of bound fat by alkaline digestion with
followed by discontinuous extraction of fat using
ethyl ether & pet-ether
addition of ethanol
ammonium hydroxide,
Sample id brought to 20'C, homogenous sample is prepared by mixing and inverting the sample bottle or by pouring back and forth between clean beakers.
Does not require prior removal of moisture from the sample
The extracted fat is dried to a constant weight and expressed as percent (%) fat by weight.
Fat is extracted with a mixture of ethyl ether & pet-ether in a Mojonnier flask
at the end extraction process
cool in dessicator and weigh
dry the flask with extracted fat in an air-oven,
the solvent is evaporated,
the flask (containing solvent + lipid) is removed,
extraction is carried out for 4 hours or until 16 hours.
as the solvent passes through the sample, it extracts the lipids and carries them into the flask.
the solvent is allow to build up in the extraction chamber for 5-10 min. & completely surrounds the sample, then siphons back to the boiling flask.
the flask is heated
moves up into the condenser
solvent evaporates
thimble is placed in an extraction chamber, which suspended above a flask containing the solvent and below the condenser.
sample is ground into small particles & placed in a porous thimble
high moisture food requires pre-drying sample under vacuum at low temperature.
Commonly used method to increase the efficiency of lipid extraction from food
The method removes mainly non-polar lipids from sample
DISADVANTAGES OF GOLFISCH METHOD
Channeling of the solvent may occur (inefficient/ incomplete extraction)
ADVANTAGES OF GOLFISCH METHOD
Faster and more efficient extraction method than Soxhlet extraction method
after completion of extraction (4 hours or more),
the fat remaining in the flask is weighed
solvent is evaporated from extraction flask (air-drying overnight)
ground to small particle size prior to anaysis
sample require removal of moisture (vacuum oven dried)
PRINCIPLE
Sample is put in an extraction ceramic thimble, and the solvent is added into the boiling flask
KARL-FISCHER TITRATION
DISADVANTAGES
Incomplete water extraction
ADVANTAGES OF KARL-FISCHER TITRATION
Suitable for low-moisture foods that are sensitive to decomposition or volatilization under vacuum or high temperature
foods with high volatile oils
sugar-rich foods
Low moisture food products
In Karl-Fisher volumetric titration
If sample containing low levels of moisture, coulometric titration is used
If the moisture is inaccessible to the reagent
Methanol extract is titrated with KFR
the moisture is extracted from the food with an appropiate solvent.
KFR reagent is added directly as the titrant if the water in the sample is accessible
The reagent for Karl-Fischer (KFR) titration method consists of
methanol
sulfur dioxide
pyridine
idone
Any water remains in the sample with iodine & produces colourless solution
The fundamental reaction involving reduction of iodine by sulfur dioxide (SO2) in the presence of water
DISADVANTAGES OF DISTILLATION METHOD
Not applicable for some types of food
Distillation of water-soluble components
Incomplete evaporation of water
Involving the use of flammable solvents
Relatively time-consuming
ADVANTAGES OF DISTILLATION METHOD
Officially approved for many applications
Equipment
easy to setup & operate
cheap
Suitable for application to food containing volatile oils
Suitable for application to foods with low-moisture contents
Distillation procedure
Dean and Stark Method
PROCEDURE
4) Volume of water produced by distillation is read from the tube and serves as a function of the total weight of food sample
3) The water vapours are then condensed & collected in graduated collection tube
2) The flask containing the sample & the organic solvent is attached to a condenser.
1) A known weight of food is placed in a flask with an organic solvent
toluene
Reflux distillation
Provide better accuracy & precision
Adverse chemical reactions can be reduced by
using a solvent with a lower boiling point
During heating
water and immiscible solvent distills off together at a constant ratio and frequently at a lower temperature than the boiling point of both components
Uses
solvent more dense than water
Tetrachloroethylene
solvent less dense than water
xylene
Toluene
Direct distillation (Dean & Stark Method)
Method with immiscible solvents with higher boiling point than water
The water that distills off condenses and is collected in a suitable measuring cylinder
The sample is suspended and heated in mineral oil/ liquid with a flash point well above the boiling point for water
2) Loss thermal decomposition of some foods
1) Involves
4) measuring the volume of water
3) collecting the mixture that distills off in a collecting vessel or trap
2) the distilled water is condensed
1) co-distilling the water in a food sample with a high boiling point solvent that is immiscible in water
DISADVANTAGES OF OVEN DRYING METHOD
Unsuitable for some types of food
Time consuming
Destructive
ADVANTAGES OF OVEN DRYING METHOD
Many samples can be analyzed simultaneously
Easy to use
Relatively cheap
Precise
DEVICES FOR OVEN DRYING
Infrared (IR) Drying
Microwave Oven
Vacuum Oven
Convection & Forced Draft Oven
PRINCIPLES
3) The moisture content value obtained by
Type of sample
Type of oven used & conditions in the oven
Time & temperature of drying
2) The thermal energy used to evaporate the water from a food sample can be provided directly or indirectly
1) The sample is heated under specified conditions (evaporation of water from sample)
until constant weight & calculation of moisture is based on loss of weight
test results are obtained with the same method on identical test itams, in different lab, with different operators using different equipment.
test results are obtained with the same method on identical test items, in the same lab, by the same operator
the magnitude of change of measuring device (instrument) with changes in compound concentration
how close the replicate measures indicates reproducibility
the experimental measure is close to the true value of an analyzed substance
a particular analytical method
Using chemical preservatives
Example
formaldehyde
sodium benzoate
sorbic acid (sorbate)
drying
freezing
Enzymic inactivation
store the sample at low temperature (-20'C to -30'C)
using heat treatment
Stored under nitrogen
frozen storage of the samples
use antioxidant
Wrapped in aluminium foil
placed in an opaque container
Sample characteristics
Microbial growth
Enzymatic action
Unsaturated lipid components in food sample (lipid oxidation)
Light sensitive samples
analysis to be performed
storage period and conditions
nature of food
expected contamination
The best practice
using bowl cutters for material disintergration
blended
grounded
Comminuted
by grinding while frozen
After separation from pit, pulped using
food chopper
blender
Grated to fine granular condition
after trimming and complete deboning
the flesh is ground through
food chopper 3 times
mixed thoroughly after ground
Cut into slices of 2cm to 3cm thickness, dried until crisp and gold
shaken during softening
softened in water bath
Liquid and solid portion are separated into solid & liquid portion
by draining through the sieve
