Chapter 1 Introduction

monitor food composition to ensure quality and safety of food supply

a part of quality management programme

analysis of chemical composition and physical properties of food

depends on proper selection and preparation of food sample&appropiatecalculations and enterpretationof data

analytical method:use of official method in analysis

Sample Preservation

to prevent changes in food composition and degradation

change in food composition

evaporation

enzymic action

action of mirobrobes

should store in the condition that prevent degradation

sample characteristic

light sensitive sample

wrapped in aluminium foil

placed in opaque container

unsaturated lipid component

under nitrogen

use antioxidant to retard lipid oxidation

frozen storage

enzymic action

heat treatment

low temperature

mirobial growth

freezing

drying

using chemical preservative

Sampling

sampling

sample preparation

analysis

an ideal sample of analysis should be identical/representing the intrinsic properties with the bulk properties of material

analysis of sample should be performed 3 times(triplicate)

Accuracy

how close experiment measure is to the correct/true value of an analyzed

innacuracy inherent in the procedure

effect of other substances in the analyzed product

Precision

how close replicate measures are indicates reproducibility

Repeatability(reproducibility)

precision under repeatability conditions

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

reproducibility: test results are obtained with the same method on identical test items in different lab,with different operators using different equipment

Standard reference/internal control sample

%Relative error

Standar deviation

Coefficient of variation

Standard curves

Principles

the sample is heated under specified conditions(evaporation of water from sample) until constat weight and calculation of moisture is based on loss of weight

the thermal energy used to evaporate the water from a food sample can be provided directly or indirectly

the moisture content value obtained is highly influenced by:

time and temperature of drying

type of oven used and condition of oven

convection and force draft oven

vacuum oven

microwave oven

infrared(IR)drying

type of sample

Advantage

precise

relatively cheap

easy to use

officially approved for many applications

many sample can be analyzed simultaneously

Disadvantages

Destructive

Unsuitable for some type of food

time consuming

Dean and Stark Method

Principles

a known weight of food is placed in a flask with oganic solvent;eg xylene or toluene

insoluble with water

have a higher boiling point than water

less dense than water

safe to use

the flask containing the sample&the organic solvent is attached to a condenser

the water vapour then condensed&collected in graduated collection tube

volume of water produced by distillation read from a tube and serves as a function of the total weight of food sample

Advantages

suitable for application for food with low moisture content

applied for food containning volatile olls such as herbs or spices

equipment cheap,east to set up&operate

officially approved for many applications

Disadvantages

destructive

relatively time consuming

used of flammable solvents

not applicable for some type of food

reading the meniscus of receiving tube to determine the volume of water may be less accurate than using weight measurement

solubility of water in the distillation liquid

incomplete evaporation of water(underestimation of moisture content)

distillation of water soluble component

Karl-Fischer Titration

Principles

based on the fundamental reaction involving reduction of iodine by sulphur dioxide in the presence of water

any water remains in the sample react with iodine&produce colourless solution

once the water has been used up,addtional iodine observed as dark red-brown as an end point

the volume of iodine required to titrated the water can be related to moisture content

the above reaction has been modified by adding solvent;C5H5N

the reagent for Karl-Fischer titration consist of iodine,pyridine,sulphur dioxide and methanol

std reagent for general work using methanolic solution: 1 iodine:3 SO2: 10 pyridine

in KFR volumetric reaction,the reagent is added directly as a titrant if the water in the sample accessible

if it inaccessible to the reagent,the moisture the extracted from the food with appropiate solvent such as methanol

then methanol extract is titrated with KFR

before amount of water found in a food sample can be determined,a KFR water equivalent(KFReq) must be determined

iodine and SO2 in the appropiate form are added to the sample in a closed chamber protected from athmospheric moisture

excess I2 that cannot react with water can be determined visually

the end point colour is dark red brown

applicable for sample with moisture content 0.03% or more

if the sample containning low levels of moisture,colourimetric is ideal procedure

procedure

sample is put in an extraction ceric thimble,and the solvent is added into the boiling flask

sample required removal of moisture(vacuum or dried) and ground to small particle size prior to analysis

allow the solvent from a boiling flask to continuously flows over the sample that held in a ceramic thimble

solvent:hexane,pet-ether,diethyl ether

after completion of extraction(4 hrs or more),the solvent is evaporated from extraction flask(air drying overnight followed by brief oven-drying),and the fat remaining in the flask is weighed

Advantages

faster

more efficient than Soxhlet extraction method

Disadvantages

channeling of solvent may occur(inefficient/incomplete extraction)

the solvent may preferentially take certain routes through the sample

removes mainly non-polar lipids from sample

commonly used method to increase the efficiency of lipid extraction from food

sample preparation

high moisture food > 10% requires pre dryingsample under vacuum <100 mm Hg

at low temperature(40-50 degree celcius overnight or 95-100 degree celcius for 5 hrs)

sample is ground into small particles&placed in a porous thimble

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

the flask is heated,the solvent evaporates,move up into the condenser(converted into a liquid&drips back into the extraction chamber containing sample)

the solvent allow to buid up in the extraction chamber for 5-10 mins&completely surrounds the sample,then siphons back into the boiling flask

provide soaking effect to the sample(provide more complete extraction of fat from the sample)

avoid channeling of the solvent

as the solvent passed through the sample,it extracts the lipids&carries them into the flask

lipid remains in the flask due their low volatility

extraction is carried out 4hrs or until 16 hrs

in the end extarction process,the flask(containing solvent+lipid)is removed,the solvent is eveporated,dry the flask with extract fat in air oven,cool in dessicator and weigh

Principles

for analysis of fat in milk

to determine fat in cream,sweetened condense milk products,ice-cream,frozen desserts and other dairy based products

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

the extracted fat is dried into a constant weight and expressed as a percent of fat by weight

sample preparation

does not require prior removal moisture from sample

sample is brought to 20 degree celcius

homogenous sample is prepared by mixing and and inverting the sample bottle or by pouring back&forth between cean beakers

promptly weigh/measure the test portion

function of reagents

alcohol:precipitates protein;prevent possible gel formation

ammonia:neutralize sample and dissolve protein

pet-ether:remove moisture from ethyl ether extract and dissolves more non-polar lipid(reduce propotion of water and non-fatty soluble substances e.g;lactose

Principle

H2SO4 is added into a known amount of milk in Babcock bottle

the mixture then shaken until homogenous,centrifuged,and submerged into water at 63 degree celcius

sulphuric acid:digest protein,generates heat and release the bound fat from the sample

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

the fat is measured volumetrically,but the result is expressed as a% fat by weight

Principle

H2SO4 is added to known amount of milk in a Gerber tube for digestion of protein and CHO,release fat and mantain the fat in liquid state by generating heat

the amyl alcohol is added into mixture to give a clear homogenous fat column and the tube is carefully inverted

the tube is centrifuged and incubated in waterbath at 60-63 degree celcius for 5 min

the fat contain was read directly from the garduated tube

principles

detergent react with protein to form protein-detergent complex to break up emulsions&release fat

milk is pipette into babcock bottle

an ionic detergent(dioctyl sodium phospate) is added (to disperse the protein layer that stabilize the fat)to liberate fat

then the strong hydrophilic polyoxyethelene detergent,sorbitan monolaurate is added to separate fat from other food components

the percent of fat sis measured volumetrically and expressed as percent fat

principles

involves reaction with peptide linkages

cupric ion complexed with peptide bonds

the absorbance of the colour produced is read at 540nm

the colour intensity(absorbance)is propartional to the protein content of the sample

advantages

rapid test(analysed can be completed within 30 mins)

colour derivations encountered less frequently than other method

very few substances other than proteins in food interfere with the biurete reaction

does not detect nitrogen from non-peptide or non-protein sources

disadvantages

relatively low sensitivity comparaed to other UV-vis method

not an absolute method:colour must be standardized against known protein(BSA) or against Kjedahl nitrogen method

opalescence could occur in the final solution with presence of high levels of lipid or CHO

Principles

protein and other organic food component is digested with sulphuric acid in the presence of catalysts

total organic nitrogent is converted into ammonium sulphate

the digest is neuralized with alkali and distilled into boric acid solution

borates anions are formed&titrated with standardized acid-converted to nitrogen in the sample

result-represent crude protein content

Function of reagent

concentrated sulphuric acid:for digestion of protein and other food component,with the presence of catalysts to complete oxidation and conversion of total organic nitrogen to ammonium sulphate

potasium sulphate:increase the boiling point of sulphuric acid and accelerate digestion mixture to shorten the reaction

copper(II)sulphate:t as catalyst and convert organic nitrogen present to ammonium sulphate

Boric acid:for distillation of ammonia which contain methylene blue;borate ion formed is proportional to the amount of nitrogen

advantages

applicable for all types of food

relatively simple

inexpensive

accurate and good reproducibility

official method for for crude protein content

disadvantages

does not give a measure of a true protein-measure total oganic nitogen

different protein need different correcting factor

time consuming

corrosiv reagent

principles

combines biuret reagent with another reagent;Follin-Ciocalteau phenol reagent);which reacts with tyrosine and trytophan residue in proteins

the reaction give bluish colour;the absorbance is read at: 750 nm(high sensitivity for lowprotein concentration) or 500 nm low sensitivity for high protein concentrattion)

advantages

very sensitive

less affected by turbidity of the sample

more specific than most other methods

relatively simple

disadvantages

colour variest proteins to a greater extent than biuret method

colour is not strictly proportional to protein concentration

the reaction is interferes varying degrees of sucrose,lipids,monosaccharides,etc.

the reaction is interferes wth high concentration of reducing sugars,ammonium sulphate and sulphydryl compounds

Principles

protein containing sample is mixed with a known excess amount of anionic(negative charge) dye in a buffered solution(so that protein are positively charged)

protein bind the dye,to form an insoluble complex(electrostatic attraction between the molecules)

the amount of unbound soluble dye is determined by measuring it absorbance

the nionic sulphonic acid dye,including acid orange 12,orange G and amido black 10B bind cationic groups of the basic amino terminal group of the proteins

unbound dye is inversely related to the protein contain of the sample

Advantages

rapid,inexpensive,relatively accurate

may be use to estimate changes in available lysine contain of cereal product during processing

no corrosive ragents

does not measure non-protein nitrogen

more precise than Kjedahl

Disadvantages

not sensitive;mg quantities of protein are required

protein differs in basic amino acid content,so differ in dye-binding capacity

non-protein binding dye;eg starch and/protein(i.e clcium or phospate)-cause error

principles

based on reaction of reducing sugar with a solution of copper sulphate

followed by reaction with alkaline tartrate

mixture are boile for a specific time

followed by addition of methylene blue(as an indicator)

coloured solution is titrated until the decolouration of the indicator

advantages

determination of sugar and honey and other high reducing sugar content

disadvantages

the reaction is not stoichiometric

result depend on the precise reaction times,temperature&reagent concentration

cannot distiguish between different typs of reducing sugar

cannot directly determine the concentration of reducing sugar

susceptible to interference of molecules that act as reducing agent

principles

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

cuprous oxide is treated with arsenomolybdate reagent

reduction of arsenomolybdate complex produces an intense,stable blue-coloured solution

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

required preparation of standard curve

principles

involving oxidation of the CHO in the presence of heat and all excess of copper sulphate and alkaline tartrate

under carefully controlled conditions

leads to the formation of a copper oxide precipitate

amount of precipitate formed is directly related to the concentration of reducing sugar in the sample

advantage

more reproducible

accurate

disadvantage

same disadvantages as Lane-Eynon method

polarimetry

refractive index

specific gravity/density

infrared

Gravimetrically

weighing the mass of an undigestable polysaccharide remains

digestable CHO,lipid&protein ar selectively solubilized by chemical/enzymes

undigestible materials are tehn collected by filtartion,and the fibre residue is quantitated gravimetrically

Chemically

digestible CHO are removed by enzymatic digestion,fibre components are hydolyzed by acid,and monosaccharide are measured

the sum of monosaccharide in the acid hydrolysate represents fibre

principles

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

duplicate of dry,defated food sample is enzymatically digested with alpha amylase,amyloglucosidase and protease to break down the starch and protein

total fibre content-adding 95% ethanol to the solution

the solution then filtered and fibre collected

insoluble fibre is collected by filtration;soluble fibre is precipitated by bringing the filtrate nd collected by filtration

adavantages

suitable for routine fibre analyses for research,egisatio and labelling purpose

can be use to determine fibre content in all food

disadvantage

greatly overestimate the fibre with ahigh content of simple sugar

principles

digestible CHO,lipid,and protein are selectively solubilize by chemical and/or enzyme

indigestible materials are then collected by filtration,and the fibre residue is quantitated gravimetrically

crude fibre measures cellulose&lignin,but does not determine hemicellulose&hydrocolloid

disadvantages

measures variable amounts of cellulose and sample,but the hemicellulose,pectins,and the hydrocolloid are solubilized and not detected

the particles size is important,the finer the material is ground,the lower the determined crude fibre content

filtering each digestion must bbe completed within a given time;delays in filteer acid or alkali digestion generally lower the results

principle

defatted food sample is heated in water(to gelatinez starch)

enzyme then added(to digest starch and protein)

pure ethanol is added to precipitate fibre,which is separated from the digest by centrifugation,then washed&dried

fibre is hydrolize using concentrated H2O4 solution to break down starch into monosaccharide

conc. of monosaccharide is determined colourimetrically or chromatographycally

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

application

to determine fibre content in most food(with low content of lignin)

fibre is equal to the sum of all non-starch monosaccharide plus lignin

allow estimation of resistant starch

principles

free sugar&lipids are extracted with ethanol&hexane

starch is removed by enzymatic digestion

insoluble fibre is separated from soluble fibre

fibre fractions are hydrolize with H2SO4 and sugar content of the acid hydrosates is determine

lignin is determined gravimetrically

fibre:monosaccharide lignin

applications

measuring dietary fibre

provide most accurate estimateof fibre for wide range of food

suitable for research,legislation,and labelling purpose

principles

sample weighed into a dish

the organic matter is burn off without flamming and heated either for a fixed period of time or to constant weight

the residue must be free from carbon

the dish containning residue is cooled in dessicator and the amount of total ash is determine by weighing

crucible selection

Quartz crucible

Porcelain crucible

Steel crucible

Platinum crucible

advantages

safe method

requires no added reagents or blank substraction

large number of crucibles can be handled at once

resultant ash can be used for for other analyses eg;acid insoluble ashes and water soluble and insoluble ash

requires little attention,not labour intensive

disadvantages

time consuming(12 hrs-18hrs,or overnight)

interaction between mineral components and crucibls

procedures

ash is diluted with distilled water,then heated nearly boiling

the resulting solution is filtered and washed severals time with hot distilled water

dry and reash the filter paper in murffle furnace at least 30 mins until constant weight achieved

the weight remaining represents the amoundt of insoluble ash

calculate soluble ash by substrating insoluble ash from total ash or dry the filtrate,re ash and weigh

procedures

add 10% of HCl total ash or H20 insoluble ash

cover the boil the ash for 5 mins

then,filter on ashless filter paper and washed several time with hot distilled water

the filter paper+residue is dried and re-as at least for 30 minutes(until constant weight)

was weighed and calculate the percentage

principle

oxidation of organic substances by strong acid(HNO3) oxidizing agent,perchloric acid(HCIO4)

sample solution is heated up slowly up to 350 degree celcius until organic matter is completely digested(leaving only mineral oxides in solution) and HNO3 is almost evaporated

boiling of sample solution is continued until the solution become colurless or light in colour

solution is cooled,and 50% of HCl is added and diluted with distilled,deionized water

advantages

mineral usually stay in solution

little/no loss from mineral volatilization(lower temp is used)

rapid than dry ashing

disadvantages

hazardous;required fum hood,hot plate,long tongs and safety equipments

corrosive reagents

small numbers of sample can be handled at one time

require special perchloric acid hoods(with wash down capabilities to protect from explosion)

principles

sample is placed into a glass chamber,sealed and vacuum is applied

small flow of oxygen

air is introduced into the system while mantaining the specific minimum vacuum

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

combustion products which are completely dissociated are carried away in the gas stream

variable power frequency adjusts the rate of incineration

advantages

less chances of losing trace elements by volatilation

low temp less than or equal to 150 degree celcius to preserve microscopic&structural components

equipment of choice for volatile salts

utilization of oxygen as sole reagent

disadvantages

small sample capacity

relatively expensive equipment

procedure

place ash(total/water insoluble ash) in platinum dish

add 0.1 N HCl and warm on a steam bath

cool and transfer to Erlenmeyer flask,titrate HCl 0.1 N NaOH using methyl orange as indicator

express the result as mL OF 1N acid/100g sample

colorimetric method

principle

measure the unstable colour at A620nm that results from reaction between Vitamin A and antimony trichloride(SbCl3)

the intensity of blue colour proportional to the amount of retinol in food sample

the intensity of blue coloured against the set of known standards

the colour reaction does not differentiate between retinol isomers and retinol esters

HPLC method

involve chromatographic separation and quantitative determination at 325 nm

principle(Thiochrome Fluorometric method)

the material to be examined is digested with sulphuric acid and subsequently treated with a phosphatase preparation

the thiochrome resulting from oxidation with ptassium ferricyanide/hydrogen peroxide in alkaline solution is extracted with isobutyl alcohol

the intensity of the blue fluorescent of is proportional to the thiamine concentration

the intensity of the fluorescence of isobutyl alcohol extract is compared with that standard soltion

the intensity of fluorescent is measured

2,6-dichlorophenolindophenol titrimetric method

principle

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

it measures the decolourization of 2,6-dichlorophenolindophenol dye by ascorbic acid

at the end point,excess of unreduced dye is rose pink acid solution lasting at least 10 sec

Fluorometric method

principle

measure both ascorbic acid and o-phenylenediamine formed a fluorescent quinoxaline compound

the fluorescent compound intensity proportional to vitamin C content

Colorimetric method

principle

involve reaction between niacine and cynogen bromide

form coloured compound with intensity proportional to niacine concentration

critical: toxicity of cynogen vromide;the analysis must be carried out under fume hood

the result is express as microgram niacine/g sample

HPLC

same as outline for the vitamine determination

however,a common extract of vitamin is concentrated and seperated by HPLC