Food Analysis

SAMPLE PREPARATION

Sampling Plan :
1) Sampling
2)Sample Preparation
3)Analysis

IDEAL SAMPLE

Identical and representing the intrinsic properties with the bulk properties of the material.

Sufficient number of portion of the whole plant or part of the plant, or cuts of meat or fish flesh

PROCEDURE

Reduction in amount from the total population, simultaneously reduction in particle size to achieve representative portion of the composition mixture
Analysis of a sample shall be performed at least three times (triplicate)

Canned fruit & vegetables – liquid & solid portion are separated into solid & liquid portion by draining through the sieve

Solid fat / butter – softened in water bath & shaken during softening

Bread – cut into slices of 2cm to 3cm thickness, dried until crisp & grind

Fresh fish & meat – after trimming & complete deboning, the flesh ground through a food chopper 3 times and mixed thoroughly after ground

Chocolate & cheese – grated to fine granular condition

Fresh or processed fruit – after separation from pit, are pulped using blender or food chopper

Frozen food product – comminuted by grinding while frozen

Leafy vegetables – grounded / blended. However, the best practice is by using bowl cutters for material disintegration

SAMPLE PRESERVATION

To prevent changes in food composition and degradation

3 changes in food composition:
1. Through evaporation/absorption of moisture
2. Through enzymic action
3. Through microorganism activity

Methods of sample preservation:

Light sensitive sample- Wrapped in aluminum foil / placed in an opaque container

Unsaturated lipid sample- Stored under nitrogen / use antioxidant to retard lipid oxidation / frozen storage of the samples

Enzymic action- Using heat treatment /storage of the sample at low temperature (-20oC to -30oC)

Microbial growth- Freezing / drying / using chemical preservatives e.g. sorbic acid (sorbate), sodium benzoate, formaldehyde; or combination of the three methods

EVALUATION OF ANALYTICAL DATA

Reliability of analysis depends on :
1. Specificity
2. Accuracy
3. Precision
4. Sensitivity

Repeatibility :
obtained with the same method on identical test items in the same lab by the same operator within short interval time

Reproducibility :
obtained with the same method on identical test items in different lab with different operators using different equipment.

DEFINITION OF LIPID

Substances that are soluble in organic solvent but insoluble in water

CLASSIFICATION OF LIPID

Simple lipid

Esters of fatty acid with alcohol

Compound lipid

Phospholipid

glycerols esters of fatty acids, phosphoric acids & other groups containing nitrogen

Cerebrosides lipid

Compounds containing fatty acids, a CHO, and a nitrogen moiety

Sphingolipid

Compounds containing fatty acids, a nitrogen moiety, and phosphoryl group

Derived lipid

Derived from neutral lipids or compound lipids

Contain general properties of lipid

IMPORTANCE OF ANALYSIS

Legal

standard of identity & nutritional labeling law

Health

development of low fat foods

Quantity

shelf life of food product

Processing

processing conditions depends on the total lipid content

SAMPLE PREPARATION

Depends on

Type of food

Type and nature of lipids in food

Type of analytical procedures used

Carried out under an inert atmosphere of nitrogen at low temperature to minimize chemical reaction such as lipid fraction

Preparation of sample

Pre-drying sample

Vacuum oven drying at low temp – increase surface area for better lipid extraction

Importance

Make a sample easier to grind - better lipid extraction

Breaks fat-water emulsions – fat dissolve easily in organic solvent

Helps fat free from the tissues of foods

Particle size reduction

Lipid extraction efficiency from dried foods depends on particle size

sample + solvent are mixed in a high-speed comminuting device = better extraction

To reduce particle size and accelerate fat extraction

Acid hydrolysis

To digest non-polar solvents

Importance

Break both covalently & ionically bound lipids into easily extractable lipid forms

SOLVENT SELECTION

Inexpensive & non-hygroscopic

Non-flammable & non-toxic

Relatively low boiling point

Evaporate readily and leave no residue

completely extract all the lipid components from a food

SOLVENT EXTRACTION

Ethyl ether

Better solvent for fat than pet-ether

Generally expensive than other solvents

Hygroscopic

Forms peroxide

Petroleum ether

Low boiling point fraction of petroleum

Cheaper

More non-hygroscopic

Less flammable than ethyl ether

SOLVENT EXTRACTION METHOD(GOLDFISCH)

Principle

Sample put in an extraction ceramic thimble and the solvent is added into the boiling flask

solvent from boiling flask continuously flows over the sample in ceramic thimble

The solvent carries fat extracted as solvent drips through the sample, is collected back in the boiling flask

After completion of extraction (4 hrs or more), the solvent is evaporated from extraction flask (air-drying overnight & oven-drying briefly), and the fat remaining in the flask is weighed

Advantage

Faster & more efficient extraction method than Soxhlet extraction method

Disadvantage

Incomplete extraction might occur

Calculations

Weight of fat in sample = (beaker + extracted fat) – beaker

% fat on dry weight basis = weight of fat in sample (g) x 100
_____________________
weight of dried sample (g)

SOXHLET METHOD

Principle

removes mainly non-polar lipids from sample

to increase the efficiency of lipid extraction from food

Thimble(sample) placed in an extraction chamber, which suspended above a flask containing the solvent & below the condenser

Flask heated-->solvent evaporates and moves up into condensor-->drip onto sample in extraction chamber

Solvent build up in extraction chamber for 5-10 minutes--> surrounds sample-->siphons back to boiling flask

Provide soaking effects of the sample(better lipid extraction)

Avoid chanelling of the solvent

As the solvent passes through the sample, it extracts the lipids & carries them into the flask

end extraction process: the flask (containing solvent + lipid) is removed--> solvent evaporated--> dry the flask with extracted fat in an air-oven--> cool in dessicator--> weigh

Calculations

% fat on dry weight basis = weight of fat in sample x 100
__________________
weight of dried sample

MOJONNIER METHOD

Official method to determine fat in milk

Can determine cream, sweetened condensed milk products, ice-cream and other dairies product

Principle

Fat is extracted with a mixture of ethyl ether & pet-ether in a Mojonnier flask.

The extracted fat is dried to a constant weight

Sample preparation

Does not require removal of moisture

brought to 20oC,
Homogenous sample-->mixing and inverting the sample bottle/pouring back & forth between clean beakers.

Weigh/measure test portion

Procedure

Release of bound fat by alkaline digestion+ammonium hydroxide+addition of ethanol--> discontinuous extraction of fat using ethyl ether & pet-ether

Repeated 3 times

fat-containing solvents (from repeated extraction) are pooled, solvent+fat-->evaporate-->fat-->weigh-->content determined.

Modified

To determine fat content in flour

Using HCL

Function of reagents

Alcohol – precipitates protein; prevents gel formation

Ammonia – neutralizes acidic sample and dissolves protein

Pet-ether – removes moisture from the ethyl ether extract and dissolves more non-polar lipid

Calculations

% fat = [(wt dish + fat) - (wt dish)] x 100
_____________________
wt of sample taken

BABCOCK METHOD

Principle

H2SO4 + milk in babcock bottle

Mixture shaken until homogeneous-->centrifuged-->submerged in water 63 C

H2SO4 digest protein-->produce heat-->release bound fat from sample

Subsequent centrifugation and addition of hot water isolate fat for quantification in the graduated portion of test bottle

The fat measured volumetrically but result expressed as %weight

Application

Common method to determine fat content in milk.

Does not determine phospholipid in fat milk

Does not recommend for chocolate or added sugar product due to charring of chocolate and sugars by sulphuric acid

Modification

to determine essential oil in flavour extracts and fat in seafood

GERBER METHOD

Principle

H2SO4 added to milk in gerber tube-->digest CHO & protein-->release bound fat from milk & maintain fat in liquid state by generating heat

Amyl alcohol is added into mixture-->give clear homogenous fat column and the tube / butyrometer is carefully inverted.

Centrifuged-->incubated in 60-63 C water bath for 5 minutes

Directly read fromgraduated tube

Applications

Wider application to a variety of dairy products

Simpler and faster than babcock

Isoamyl alcohol improves fat separation, reduces effect of sulfuric acid & prevents charring of sugar

DETERGENT METHOD

Principle

Detergent react with protein to form protein detergent complex to break up emulsions and release fat

Milk pipetted into a Babcock test bottle.
An anionic detergent (dioctyl sodium phosphate) is added to liberate fat.

Then, strong hydrophilic nonionic polyoxyethylene detergent, sorbitan monolaurate added to separate fat from other food components.

The percent fat is measured volumetrically and expressed as percent fat.

Application

To determine fat in milk

Develop because of corrosive properties H2S04 in babcock method

IMPORTANCE OF ANALYSIS

Biological activity determination

Functional properties investigation

Nutritional labeling

Required for

Total protein content
Amino acid composition
Content of a particular protein in a mixture
Nutritive value

KJEDAHL METHOD

Principle

Proteins+other organic food component digested with H2S04+catalyst

Total organic nitrogen converted to ammonium sulfate
The digest neutralized with alkali--> distilled into boric acid solution

Borate anions are formed & titrated with standardized acid – converted to nitrogen in the sample

Result represents crude protein content

1)Digestion--> heating with sulfuric acid+catalyst

convert nitrogen to ammonium sulfate & complete oxidation

converts nitrogen in the food-->ammonia
Other organic matter--> CO2 & H2O

2)Neutralization of diluted digest

digestion flask is connected to a receiving flask by a tube

Solution in digestion flask is made alkaline by addition of NaOH, which converts the ammonium sulfate into ammonia gas

3)Distillation of diluted digest

Ammonia gas liberated from solution-->moves out of the digestion flask into receiving flask (contains excess of boric acid)

The low pH of the solution in receiving flask converts the ammonia gas-->ammonium ion & simultaneously converts the boric acid-->borate ion

4)Titration of the ammonium borate formed with standard sulfuric / hydrochloric acid, using suitable indicator to determine the end-point of the reaction

A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen

A conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration

Calculation

1. Total nitrogen (g) per 100 g food sample
= (titre sample – titre blank) x 1.4 mg N x 100
____________________________________
1000 x sample weight (g)

2. Total protein (g) per 100 g food sample
= total nitrogen x factor for foodstuff analyzed

Function of reagents

Concentrated sulfuric acid

digestion of proteins&other food components, with the presence of catalysts to complete oxidation&conversion of total organic nitrogen to ammonium sulfate

Potassium sulfate

1)Use to increase boiling point of sulfuric acid
2)Accelerate digestion mixture to shorten the reaction

Copper (II) sulfate

1)Act as catalyst
2)Convert organic nitrogen present to ammonium sulfate

Boric acid

1)Use for distillation of ammonia, which contains methylene blue & methyl red
2)Borate ion formed is proportional to the amount of nitrogen

Advantages

1)Applicable to all types of food
2)Relatively simple
3)Inexpensive
4)Accurate and good reproducibility – official method for crude protein content

Disadvantage

1)Does not give a measure of the true protein – measures total organic nitrogen
2)Different proteins need different correction factors
3)Time consuming
4)Corrosive reagent

BIURET METHOD

Principle

Biuret method involves a reaction with peptide linkages

Cupric ions (Cu2+) complexed with peptide bonds under alkaline conditions and produced a violet-purplish colour

The absorbance read at 540 nm

The colour intensity (absorbance) proportional to protein content of the sample

Procedure

Biuret reagent mixed with protein solution of the sample.
Reagent includes:
1.Copper sulfate,
2.NaOH&
3.Potassium sodium tartrate (to stabilize the cupric ion in the alkaline solution)

The mixture is allowed to stand at room temperature for 15 – 30 min.

The absorbance of the mixture solution is read at 540 nm against blank reagent.

Application

1)Determination of protein content in cereals, meat, soybean proteins and as a qualitative test for animal feed

2)The method also is used widely to measure the protein content of isolated proteins

Advantages

1)Rapid test
2)Colour derivations encountered less frequently than other method
3)Very few substances other than proteins in foods interfere with the biuret reaction
4)Does not detect nitrogen from non-peptide or non-protein sources

Disadvantages

1)Relatively low sensitivity compared to other UV-vis methods
2)Not an absolute method : colour must be standardized against known protein (BSA) or against Kjedahl nitrogen method
3)Opalescence could occur in the final solution with presence of high levels of lipid or CHO

LOWRY METHOD

Principle

Lowry method combines biuret reagent with another reagent (Folin-Ciocalteau phenol reagent) ; which reacts with tyrosine& trytophan residues in proteins

The reaction gives a bluish colour&the absorbance is read at :
1)750 nm (high sensitivity for low protein concentration)
2)500 nm (low sensitivity for high protein concentration)

Procedure

Proteins to be analyzed diluted to an appropriate range

Biuret reagent+diluted sample-->incubated at room temp. for 10 min.

Freshly prepared Folin reagent added, mixed and incubated

The absorbance of the solution is read at 650 nm

Application

Widely used in protein biochemistry

Advantages

1)Very sensitive
2)Less affected by turbidity of the sample
3)More specific than most other methods
4)Relatively simple

Disadvantage

1)Colour varies with different proteins to a greater extent than biuret method
2)Colour is not strictly proportional to protein concentration
3)Interfered with varying degrees of sucrose, lipids, monosaccharides, etc.
4)Interfered with high concentrations of reducing sugars, ammonium sulfate, and sulfhydryl compounds

DYE BINDING METHOD

Principle

Protein-containing sample+known excess amount of anionic dye in a buffered solution = protein positively charged

Proteins bind the dye =insoluble complex formed

The amount of unbound soluble dye is determined by measuring its absorbance

Protein + excess dye--->protein-dye insoluble complex + unbound soluble dye

Unbound dye is inversely related to the protein content of the sample

Dye bound = dye initial - dye free

Application

Used to estimate proteins in milk, wheat flour, soy products and meats

Advantages

1)Rapid, inexpensive, relatively accurate
2)May be used to estimate changes in available lysine content of cereal products during processing
3)No corrosive reagents
4)Does not measure non-protein nitrogen
5)More precise than Kjedahl method

Disadvantage

1)Not sensitive; mg quantities of proteins are required
2)Proteins differ in basic amino acid content, so differ in dye-binding capacity
3)Non-protein components bind dye – cause error

DEFINITION OF ASH

inorganic residue remaining after either ignition or complete oxidation of organic matter in a food stuff.

MINERAL CONTENT IN FOOD

Calcium

Phosphorus

Iron

Magnesium

Potassium

Sulfur

Zinc

DRY ASHING METHOD

Principles

to determine total ash and before an elemental analysis for individual minerals.

high-temp. muffle furnace used
temp. between 500 – 600oC.

sample weighed-->organic matter burned off w/o flaming and heated either for a fixed period of time or to constant weight.

The residue must be free from carbon.

Residue cooled in desiccator-->amount of total ash determined by weighing.

1)Incineration at high temp. with muffle furnace (5250C or higher)

2)Water & other volatile materials are vaporized
organic substances--> burned in the presence of the O2 in air to CO2, H2O and N2.

3)Most minerals are converted to oxides, sulfates, phosphates, chlorides or silicates.

4)The food sample is weighed before and after ashing to determine the concentration ash present.

% Ash (dry basis) = Mash x 100
Mdry

Advantages

i.Safe method.
ii. Requires no added reagents or blank subtraction.
iii. Large number of crucibles can be handled at once.
iv. Resultant ash can be used for other analyses e.g. acid insoluble ash, and water soluble and insoluble ash.
v. Requires little attention, not labour intensive.

Disadvantages

i. Time consuming (12 – 18 hrs, or overnight).
ii. Loss of volatile elements at high temp. e.g. Cu, Fe, Pb, Hg, Ni, Zn.
iii.Interactions between mineral components and crucibles.

CRUCIBLE SELECTION

Quartz crucible

Resistant to acid and halogen but not alkali in high temp.

Platinum crucible

Very inert and best crucible but expensive

Steel crucible

resistant to both acids & alkalies, inexpensive, but possible sources of contamination

Porcelain crucible

withstand high temp. (<1200oC), resistant to acids, but can be corroded by alkaline samples, easy to clean, relatively inexpensive, prone to crack with rapid temp. changes.

WET ASHING METHOD

Principles

1. Oxidation of organic substances by strong acid (HNO3) and oxidizing agent, perchloric acid (HClO4).

2. Sample solution--> heated(350oC)-->organic matter digested (leaving only mineral oxides in solution) & HNO3 is almost evaporated.

3. Boiling continue until solution become colourless or light in colour.

4. Solution cooled--> 50% of HCl is added and diluted with distilled, deionized water.

Advantages

1. Minerals usually stay in solution.
2. Little / no loss from mineral volatilization
3. Rapid than dry ashing.

Disadvantages

1. Hazardous. Requires fume hood, hot plate, long tongs and safety equipments.
2. Corrosive reagents.
3. Small numbers of samples can be handled at one time.
4. Requires special perchloric acid hoods (with wash-down capabilities to protect from explosion).

LOW TEMP. PLASMA ASHING

Principles

1.Sample is placed into a glass chamber, sealed and vacuum is applied.

2.Small flow of O2 / air is introduced into the system while maintaining the specific minimum vacuum.

Electromagnetic radio frequency generator is activated to control the rate of incineration-->excites the gas molecules& dissociates it into chemically active atoms and molecules.

Combustion products which are completely dissociated are carried away in the gas stream.

3.Variable power frequency adjusts the rate of incineration.

Advantages

1)Less chances of losing trace elements by volatilization.
2)Low temp. (≤ 150oC) preserve microscopic & structural components.
3)Equipment of choice for volatile salts.
4)Utilization of O2 as sole reagent.

Disadavantage

1)Small sample capacity.
2)Relatively expensive equipment.

WATER SOLUBLE&WATER INSOLUBLE ASH

Procedure

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.
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.
Calculate soluble ash by subtracting insoluble ash from total ash, or, dry the filtrate, re-ash and weigh.

What for?

1)Useful indication of the quality of certain foods e.g. fruit content of preserves and jellies.
2)Lower ash value in water-soluble fraction is an indication that extra fruit is added to fruit or sugar products.

ACID INSOLUBLE ASH

Procedure

Add 10% HCl to total ash or H2O-insoluble ash. Cover and boil the ash for 5 min. 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.

The acid insoluble ash was weighed and calculate the percentage.

ALKALINITY OF ASH

Procedure

1)Place ash (total / water-insoluble ash) in platinum dish. Add 0.1N HCl and warm on a steam bath.
2)Cool and transfer to Erlenmeyer flask, titrate HCl with 0.1N NaOH using methyl orange as indicator.
3)Express the result as mL of 1N acid / 100g sample.

MOISTURE CONTENT

Most commonly measured properties of food materials

IMPORTANCE OF MOISTURE CONTENT

1. Legal and labelling requirements
2.Food Quality
3. Microbial stability
4. Food processing operations

TYPE OF WATER IN FOOD

Free form

-Retains its physical form
-Dispersing agent for colloids
-Solvents for salts
-Easily lost by evaporation

Trapped water

Held within food that are surrounded by physical barrier that prevents water from escaping

Bound water

-Chemically bound
-Does not freeze at low temperature
-Reduced mobility of water

Adsorb water

-Physically bound as a monolayer to food surface constituent

SAMPLE PREPARATION

Source of error:
1. Selection of a representative sample
2. Prevention of changes in the properties of the sample prior to analysis

Ways to overcome

Minimize exposure of sample to atmosphere during grinding

Minimize headspace in sample container

METHODS FOR MOISTURE DETERMINATION

Oven drying

Principles

-Heated under specified conditions until constant weighed achieved and calculate loss of moisture by loss of weight

Thermal energy used to evaporate the moisture can be directly or indirect

Moisture content value obtained depend on

Time and temperature of drying

Type and condition of oven used

Type of sample

Type of devices

Convection and forced draft oven

Weighed samples---> placed in oven(specified time&temperature)--->mass determined or dried until constant mass achieved

Thermal energy: Directly applied via shelf and air

High carbohydrate sample is not suitable

WHY?
1. Sample might undergo chemical changes/ loss of volatile materials other than water
2. Lipid oxidationn
3. Weight gain might occur

Vacuum oven

Dried under reduced pressure for a specified temperature & time.

More complete removal of water and volatiles w/o decomposition

Need dry air purge in addition to temperature & vacuum controls to operate within method definition

Thermal energy : Applied directly via metallic shelf

Air inlet & outlet : Carry out moisture to prevent accumulation of moisture within oven

Microwave oven

Weighed samples---> Placed for a specified time---> Power level & their dried mass is weighed
Alternatively : Weighed samples---> Dried until constant mass reached

Water molecules absorbed microwave energy--> thermally excited--> evaporate

Why samples placed at center & distributed evenly?

To avoid certain portion get burn while other area under processed

Infrared drying

Heat penetrate intro sample being dried to evaporate water from sample

Water molecules thermally excited

To obtain reproducible measurements we must

Control distance between sample and IR lamp

Thickness/dimensions of sample

Calculations

% Moisture (wt / wt) = (wt of wet sample – wt of dry sample) x 100 ______________________________
wt of wet sample

% Total solids (wt / wt) = wt of dry sample x 100
wt of wet sample

Thus, % Total solids = (100 - % Moisture)

Advantage and disadvantage of oven drying method

Advantage

Precise

Relatively cheap

Officially approved for many applications

Many samples can be analyzed simultaneously

Disadvantage

Destructive

Time consuming

Unsuitable for some food

Practical consideration during moisture removal

Moisture removal sometimes best achieved in 2 stage process

High moisture sample-->pre-dried(steam bath) to completing drying in an oven

Why?--> To prevent spattering of sample & accumulation of moisture in an oven

Products such as bread & field-dried grain are often air-dried, then ground and oven-dried

moisture content is calculated from moisture loss at both air & oven-drying steps

Sample dimensions

High surface area, high rate of moisture removal

Decompositon of other food component

Because heat sensitive component in food decomposed causing changes in mass leading to error of moisture content determination

Use suitable time & temperature

Weight gain due to oxidation of unsaturated fatty acid

Decomposition product : C02, C0, CH4, H20

Overestimation/Underestimation of true moisture content

Temperature control

Sample pans

Aluminium pans- Cheap & have high thermal conductivity

Pan covers & lids- Control/prevent/spattering of sample

Handle pans with tongs because fingerprints can contribute to mass of a sample

Sample--> Dried in oven--> store in dessicator
-To ensure no residual moisture is attached to them

Clumping & surface crust formation (Sand pan technique)

Add sand or other inert materials to prevent clumping of food

1.To prevent formation of surface crust
2.To disperse the sample

Distillation

Principles

Involves co-distilling water in food samples with high b.p solvent that immiscible in water

Distilled water is condense--> Mixture that distills off collected in a collecting vessel--> Volume of water measured

Less thermal decomposition of some foods

Procedure

Direct distillation ( Dean & Stark)

Sample suspended & heated in mineral oil/liquid with a flash point well above b.p for water---> water that distills off condenses & collected in a measuring cylinder

Reflux distillation

Uses either solvent less dense than water or solvent more dense than water

During heating,water and immiscible solvents distills off together at a constant ratio at a lower temperature than the b.p of both components

Use lower b.p solvent to reduce chemical reactions (distillations times increase)

Better accuracy & precision than oven drying methods especially for low moisture sample

Applications

Suitable for low moisture analysis for low moisture samples such as cheese, spices, oils

Dean & Stark method

Known weight of food placed in flask with an organic solvent

Toluene, xylene

-Insoluble in water
-High b.p
-Less dense than h20
-safe to use

Flask connected to condenser. Water vapour condensed & collected in graduated collection tube

Volume of water produced = total weight of food sample

Potential Sources Of Error With Distillation

Formation of emulsions between water & solvent

Solution:
Allow apparatus to cool after distillation completed & before reading the amount of moisture

Water droplets adhere to the inside of glassware

Solution:
Use clean glassware

Thermally labile component decomposed with production of water at elevated temperature used

Solution:
Discontinue use of method
Find alternative methods

Advantages & Disadvantages of Distillation Method

Advantage

For low moisture food

For food contain volatile oil

Cheap equipment, easy to setup & operate

Officially approved for many applications

Disadvantage

Destructive

Time consuming

Involve flammable solvents

Some types of food are not applicable

Incomplete evaporation of water

Solubility of water in the distillation liquid

Less accurate of reading than using weight measurement

Chemical Method : Karl Fischer Titration

Principles

Reduction of iodine by S02 in the presence of water

Water remains + I2 --> Colourless solution
Water used up--> Additional I2 observed as--> Dark red-brown (endpoint)

Modified Karl Fischer Titration

C5H5n was added

KF reagent

Methanol

Iodine

Sulfur dioxide

Pyridine

In KF volumetric titration

KFR added directly as titrant if water in sample is accesible

If moisture is inaccesible to reagent--> moisture extracted from food with appropriate solvent (ex: methanol) --> methanol extract titrated with KFR

KFR water equivalent must be determined first before amount of water in food sample can be determined

Iodine & S02 + sample in a closed chamber protected from atmospheric moisture.

Excess I2 that did not react with water determined visually

End point color : Dark-red brown

Volumetric titration applicable for moisture content below or equal 0.03%

Application

Suitable for low moisture food that sensitives to decomposition or volatilization under vacuum or high temperature

1. Low moisture food

2.Sugar rich food

3.Food with high volatile oils

4.Food rich in reducing sugar & protein

5.Food with intermediate moisture levels

Sources of error

1.Incomplete water extraction

2.Atmospheric condition

3.Moisture adhering to walls

4.Interferences from certain food constituents

LANE EYNON METHOD

Principle

Reaction of reducing sugar+solution of copper sulfate followed by reaction with alkaline tartrate

Mixture-->boiled for a specific time+methylene blue (as an indicator)

Coloured solution is titrated until decolouration of the indicator

Procedure

CHO solution in a burette is titrated in into a flask containing known amount of boiling copper sulfate solution (mixed Fehling’s solution) and methylene blue indicator.

Air excluded from reaction mixture by keeping liquid boiling throughout titration process.

Reducing sugars in the solution will react with copper sulfate, converted to insoluble cuprous oxide.

Once all copper sulfate in solution has reacted, indicator change color from blue-->colorless.

Volume of sugar solution required to reach end point recorded.

Application

Determinations of reducing sugars in honey and other high-reducing sugar syrups

Disadvantages

1)The reaction is not stoichiometric – necessary to prepare a calibration curve with a series of standard solutions of known CHO concentration.
2)Results depends on the precise reaction times, temp., & reagent concentrations
3)Cannot distinguish between different types of reducing sugar
4)Cannot directly determine the concentration of non-reducing sugar
5)Susceptible to interference from other types of molecules that act as reducing agents

MUNSON WALKER METHOD

Principle

Involving oxidation of the CHO in the presence of heat and an excess of copper sulfate and alkaline tartrate, under carefully controlled conditions – leads to the formation of a copper oxide precipitate

Amount of precipitate formed=concentration of reducing sugar in the sample

Concentration of precipitate present can be determined

1)gravimetrically (by filtration, drying and weighing)
2)Titrimetrically (be redissolving the precipitate and titrating with a suitable indicator)

Basic conditions (alkaline) are required to keep copper solution as copper hydroxide (Cu+).

The methods depends on the ability of reducing sugar to react with copper solution

Modification

involves the use of an excess alkaline copper citrate with sodium carbonate (base). Following the reduction, excess copper citrate react with excess potassium iodide. Liberation of iodine is titrated with sodium thiosulfate.

Advantages

More reproducible and accurate

Disadvantages

Same disadvantages as Lane-Eynon method

SOMOGYI NELSON METHOD

Principles

The reducing sugar when heated with alkaline copper tartrate reduce the copper, from cupric to cuprous state, thus cuprous oxide is formed

Cuprous oxide is treated with arsenomolybdate reagent (prepared by reacting ammonium molybdate [(NH4)6Mo7O24)] and sodium arsenate (Na2HAsO7) in sulfuric acid). Reduction of arsenomolybdate complex produces an intense, stable blue-coloured solution.

The absorbance of the solution is determined at either 500 or 520nm against standard

Require preparation of standard curve

POLARIMETRY

Principles

Asymmetric carbon atoms have the ability to rotate plane of polarization of polarized light

Plane polarized light passed through solution exhibiting optical activity, it rotated either to left (-) or right (+).

Angle of polarization proportional to the concentration of optically active molecules in solution

Prior to analysis, sample solution must be clarified.

CHO able rotate plane polarized light through an angle of rotation

Concentration is determined from the specific optical rotation value, when no other optically active compounds are present and all other factors are held constant

Concentration of an unknown sample is determined by measuring the angle of rotation and comparing it with a calibration curve

Precaution

1)Solution to be analyzed need to be clarified

2)All reducing CHO display mutarotation between α and ß isomers. If CHO solution is freshly prepared / not equilibrated, error may occur due to the phenomenon. Therefore, CHO solution should be allowed to stand for several hours to establish equilibrium; or add a few drops of ammonia to establish equilibrium rapidly.

Disadvantages

Polarimetry method unable to analyzed mixtures of CHO

REFRACTIVE INDEX

Principle

RI (n) of a substance is the ratio of light velocity in a vacuum to its velocity of a substance.

When electromagnetic radiation passes from one medium to another, it can change direction

RI of a substance depends on :

Concentration
Temperature (T)
Wavelength of light

RI standard measurement are made specific at T (20oC) and wavelength

RI readings are normally expressed as % sugar wt./wt. or alternatively oBrix (g sucrose / 100 g of sample).

In practice, RI of CHO solution is usually measured at a boundary with quartz.

Abbe refractometer – common type of refractometer

Precaution

Do not use ether or acetone to clean off samples from prism because these solvents evaporate quickly and in that process change the temperature.

Applications

1)Method is quick & simple to carry out, gives direct reading and require only one or two drops of sample.
2)Performed with simple hand-held instrument.
3)Analysis of food carbohydrates (total soluble solids) in variety of products

CALCULATION OF CHO BY DIFFERENCES

Total CHO = 100 – (% moisture + % protein + % fat/lipid + % ash)

Disadvantage

1)Inaccurate result for CHO content due to experimental error that may occur during determination of these major food constituents

2)Incomplete digestion / extraction of these major food constituents – inaccurate result for CHO content

3)Does not differentiate between available & non-available CHO. Hence, specific analyses are necessary.

Non digestible carbohydrate

Insoluble component

Traditional insoluble fiber

cellulose, hemicellulose, lignin in wheat and rice

Resistant starch

found in whole grains, pulses, seeds

Soluble component

Traditional soluble fiber

glucans in oats and barley, pentoses in rye

Oligosaccharides

found in pulses, onions, Jerusalem artichoke, garlic

DEFINITION OF CRUDE FIBRE

Residue remaining after a foodstuff has been sequentially treated with solvents, acids and bases

MAJOR COMPONENT OF DIETARY FIBRE

Cell wall polysaccharide

cellulose

hemicellulose

pectins

Non cell wall

Lignin

ACID&ALKALI DIGESTION METHOD

Principles

Digestible CHO, lipid, and protein-->selectively solubilized by chemical and/or enzymes.
Indigestible materials--> collected by filtration
Fibre residue-->quantitated gravimetrically.

Procedures

Crude fibre is determined by:
sequential extraction of defatted sample with H2SO4&NaOH. H2SO4 hydrolyze CHO & protein, and digestion with NaOH to saponify fatty acids

Insoluble residue is collected by filtration, dried, weighed and ashed, cooled and weighed (to correct for mineral contamination of the fibre residue).

DISADVANTAGES

1)The method measures variable amounts of the cellulose and lignin in the sample, but hemicelluloses, pectins, and the hydrocolloids are solubilized and not detected.
The method does not represent any specific compound or groups of compound.

2)The particle size is important, the finer the material is ground, the lower the determined crude fibre content.

3)Filtering each digestion must be completed within a given time; delays in filtering after acid or alkali digestion generally lower the results.

AOAC METHOD

Principles

to isolate the fraction of interest by selective precipitation and then to determine its mass by weighing.

Duplicates of dry, defatted food sample is enzymatically digested with:
α-amylase, amyloglucosidase&protease to break down the starch and protein.

Total fibre content – adding 95% ethanol to the solution.
Solution-->filtered & fibre is collected.

insoluble fibre is collected by filtration. Soluble fibre is precipitated by bringing the filtrate to 78% ethanol and collected by filtration.

Filtered fibre residues are washed with ethanol & acetone, oven dried, and weighed

One duplicate is analyzed for protein determination

One duplicate is incinerated to determine ash content

Application

Suitable for routine fibre analyses for research, legislation and labeling purpose.

The method can be used to determine fibre content in all foods.

Disadvantage

Greatly overestimate the fibre content with a high content of simple sugars

ENGLYST-CUMMING METHOD

Principles

Defatted food sample is heated in water--> Enzyme added

Pure ethanol added-->ppt fibre,
Separated from the digest by centrifugation-->washed-->dried.

Fibre is hydrolyzed using concetrated H2SO4 solution to break down starch into mono-.

Concentration of mono- is determined colourimetrically or chromatographycally.

Mass of fibre in original sample assumed to be equal to the total mono- present

Application

Suitable method for determining fibre content in most foods (with low content of lignin).

Fibre = to the sum of all non-starch monosaccharide+lignin

Allow estimation of resistant starch.

THEANDERR-MARLETT METHOD

Principles

Free sugars & lipids are extracted with ethanol & hexane.

Starch is removed by enzymatic digestion and insoluble fibre is separated from soluble fibre.

Fibre fractions are hydrolyzed with H2SO4 and sugar content of the acid hydrolysates is determined.

Lignin is determined gravimetrically.

Fibre = Monosaccharides + Lignin

Procedure

Dry ground food-->suspend in 80% ethanol-->free sugars remove

Lipids extracted with hexane.

Starch digestion is completed by incubating with enzymes

Resultant solution contains mixture of soluble & insoluble fibre is separated by centrifugation.

Insoluble residue is removed by centrifugation, filtration, washed with ethanol & acetone, dried.

Soluble residue precipitated from solution by adding ethanol, removed by filtration, and then collected, washed and dried.

Both fractions-->mixed with concentrated H2SO4-->hydrolyze cellulose&non-cellulose polysaccharides&mono- concentration are analyzed.

Lignin in insoluble fraction is not hydrolyzed by acid but remains as insoluble complex which is removed by centrifugation, washed, dried & weighed.

Total fibre = Soluble fibre + Insoluble fibre

Applications

provides most accurate estimate of fibre for wide range of foods

Suitable for research, legislation, and labeling purpose.

However, require high degree of analytical skill, time commitment, and costly equipment, thus not routinely used for analyses & labeling purpose.

DEFINITION

Relatively low molecular weight compounds requires by human body and any type of living organisms as a source of nutrient for normal metabolisms.

IMPORTANCE OF ANALYSIS

1)Assuring adequate supply from existing food regimen.

2)Assessment of vitamin bioavailability for its user.

TYPES OF EXTRACTION METHOD

1)ascorbic acid- cold extraction with metaphosphoric acid/acetic acid.

2)Vitamin B1 and B2-boiling or autoclaving in acid and enzyme treatment.

3)Niacin-autoclaving in acid or alkali
Vit. A, E or D - organic solvent extraction, saponification and re-extraction with organic solvents

VITAMIN A

Precautions

Vitamin A sensitive to UV light, air, prooxidant, high temperature and moisture.

Solution

1)Use low actinic glassware/cover glassware with aluminium foil, nitrogen or vacuum
2)Avoid excessively high temperature
3)Use antioxidant

Colorimetric method

Principle

Measures unstable colour at A620nm that results from reaction between Vitamin A+antimony trichloride (SbCl3)

The intesity of blue colour proportional to the amount of retinol in food sample.

The intensity of blue colour is measured against the set of known standards.

The colour reaction does not differentiate between retinol isomers and retinol esters.

HPLC method

This method involve chromatographic separation and quantitative determination at 325 nm

VITAMIN C

Precautions

The vitamin (L-ascorbic & -dehydroascorbic acid) is very susceptible to oxidative deterioration, which enhanced by: 1)High pH
2)Presence of ferric&cupric acid.

Solution

Conduct at low pH and with addition of chelating agent.

2,6-dichlorophenolindophenol titrimetric method

Principle

L-ascorbic acid is oxidizes to dehydroascorbic acid by indicator dye.

Measures the decolourization of 2,6-dichorophenolindophenol dye by ascorbic acid.

At the endpoint, excess of unreduced dye is rose pink in acid solution lasting at least 10 sec.

This method is not suitable for highly coloured products

L- dehydroascorbic acid determined by first converting it to L-ascorbic acid with a suitable reagent.

Fluorometric method

Principle

This method measures both ascorbic acid and dehydroascorbic acid.

Ascorbic acid + o-phenylenediamine--> Dehydroascorbic acid

Dehydroascorbic acid + o-phenylenediamine-->fluorescent quinoxaline compound.

The flourescent compound intensity proportional to the vitamin C content.

THIAMINE (VITAMIN B1)
THIOCHROME FLUOROMETRIC METHOD

Principle

Digested with H2S04 and subsequently treated with a phosphatase preparation to free from natural ester and protein bond

The thiochrome resulting from oxidation with potassium ferricyanide/hydrogen peroxide in alkaline solution is extracted with isobutyl alcohol.

The intensity of the blue fluorescence proportional to the thiamine concentration.

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.

Precaution

1)Thiochrome is light sensitive
2)Thiamine is sensitive to heat especially at alkaline pH.

Solution

1)Analysis performed under subdued light.
2)Steps starting from oxidation of thiamine until flourescent measurement need to be carried out:
-Rapidly&precisely according to the instructions.

NIACIN (VITAMIN B3)
COLOURIMETRIC METHOD

Principle

niacin+cynogen bromide--> coloured compound with an intensity proportional to niacin concentration

Critical : toxicity of cyanogen bromide, the analysis must be carried out under fume hood.

The result expressed as μg niacin / g sample

OTHER METHOD OF VITAMIN ANALYSIS

HPLC

The extraction procedure are the same as outlined for the vitamin determination
Common extract of the vitamin is concentrated and separated by HPLC.