Chapter 8: Vitamin
Vitamin B3(Niacine)
HPLC
however,a common extract of vitamin is concentrated and seperated by HPLC
same as outline for the vitamine determination
Colorimetric method
the result is express as microgram niacine/g sample
critical: toxicity of cynogen vromide;the analysis must be carried out under fume hood
form coloured compound with intensity proportional to niacine concentration
involve reaction between niacine and cynogen bromide
Vitamin C
Fluorometric method
the fluorescent compound intensity proportional to vitamin C content
measure both ascorbic acid and o-phenylenediamine formed a fluorescent quinoxaline compound
2,6-dichlorophenolindophenol titrimetric method
at the end point,excess of unreduced dye is rose pink acid solution lasting at least 10 sec
it measures the decolourization of 2,6-dichlorophenolindophenol dye by ascorbic acid
L-ascorbic acid is oxidizes to dehydroascorbic acid by indicator dye
Vitamin B1(Thiamine)
principle(Thiochrome Fluorometric method)
the intensity of fluorescent is measured
the intensity of the fluorescence of isobutyl alcohol extract is compared with that standard soltion
the intensity of the blue fluorescent of is proportional to the thiamine concentration
the thiochrome resulting from oxidation with ptassium ferricyanide/hydrogen peroxide in alkaline solution is extracted with isobutyl alcohol
the material to be examined is digested with sulphuric acid and subsequently treated with a phosphatase preparation
Vitamin A
HPLC method
involve chromatographic separation and quantitative determination at 325 nm
colorimetric method
the colour reaction does not differentiate between retinol isomers and retinol esters
the intensity of blue coloured against the set of known standards
the intensity of blue colour proportional to the amount of retinol in food sample
measure the unstable colour at A620nm that results from reaction between Vitamin A and antimony trichloride(SbCl3)
Chapter 7:Ash and Minerals
Alkalinity of Ash
express the result as mL OF 1N acid/100g sample
cool and transfer to Erlenmeyer flask,titrate HCl 0.1 N NaOH using methyl orange as indicator
add 0.1 N HCl and warm on a steam bath
place ash(total/water insoluble ash) in platinum dish
Low-Temp. Plasma Ashing
relatively expensive equipment
small sample capacity
utilization of oxygen as sole reagent
equipment of choice for volatile salts
low temp less than or equal to 150 degree celcius to preserve microscopic&structural components
less chances of losing trace elements by volatilation
variable power frequency adjusts the rate of incineration
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
air is introduced into the system while mantaining the specific minimum vacuum
small flow of oxygen
sample is placed into a glass chamber,sealed and vacuum is applied
Wet ashing(Wet Oxidation/Wet Digestion)
require special perchloric acid hoods(with wash down capabilities to protect from explosion)
small numbers of sample can be handled at one time
corrosive reagents
hazardous;required fum hood,hot plate,long tongs and safety equipments
rapid than dry ashing
little/no loss from mineral volatilization(lower temp is used)
mineral usually stay in solution
solution is cooled,and 50% of HCl is added and diluted with distilled,deionized water
boiling of sample solution is continued until the solution become colurless or light in colour
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
oxidation of organic substances by strong acid(HNO3) oxidizing agent,perchloric acid(HCIO4)
Acid insoluble ash
was weighed and calculate the percentage
the filter paper+residue is dried and re-as at least for 30 minutes(until constant weight)
then,filter on ashless filter paper and washed several time with hot distilled water
cover the boil the ash for 5 mins
add 10% of HCl total ash or H20 insoluble ash
Water Soluble and Water insoluble ash
procedures
calculate soluble ash by substrating insoluble ash from total ash or dry the filtrate,re ash and weigh
the weight remaining represents the amoundt of insoluble ash
dry and reash the filter paper in murffle furnace at least 30 mins until constant weight achieved
the resulting solution is filtered and washed severals time with hot distilled water
ash is diluted with distilled water,then heated nearly boiling
Dry Ashing
interaction between mineral components and crucibls
time consuming(12 hrs-18hrs,or overnight)
requires little attention,not labour intensive
resultant ash can be used for for other analyses eg;acid insoluble ashes and water soluble and insoluble ash
large number of crucibles can be handled at once
requires no added reagents or blank substraction
safe method
crucible selection
Platinum crucible
Steel crucible
Porcelain crucible
Quartz crucible
the dish containning residue is cooled in dessicator and the amount of total ash is determine by weighing
the residue must be free from carbon
the organic matter is burn off without flamming and heated either for a fixed period of time or to constant weight
sample weighed into a dish
Chapter 6: Dietary Fibre
Theander-Marlett Method
applications
suitable for research,legislation,and labelling purpose
provide most accurate estimateof fibre for wide range of food
measuring dietary fibre
fibre:monosaccharide lignin
lignin is determined gravimetrically
fibre fractions are hydrolize with H2SO4 and sugar content of the acid hydrosates is determine
insoluble fibre is separated from soluble fibre
starch is removed by enzymatic digestion
free sugar&lipids are extracted with ethanol&hexane
Englyst-Cumming Method
application
allow estimation of resistant starch
fibre is equal to the sum of all non-starch monosaccharide plus lignin
to determine fibre content in most food(with low content of lignin)
principle
mass of fibre in original sample assumed to be equal to the total mono-present
conc. of monosaccharide is determined colourimetrically or chromatographycally
fibre is hydrolize using concentrated H2O4 solution to break down starch into monosaccharide
pure ethanol is added to precipitate fibre,which is separated from the digest by centrifugation,then washed&dried
enzyme then added(to digest starch and protein)
defatted food sample is heated in water(to gelatinez starch)
Acid and alkali digestion method
filtering each digestion must bbe completed within a given time;delays in filteer acid or alkali digestion generally lower the results
the particles size is important,the finer the material is ground,the lower the determined crude fibre content
measures variable amounts of cellulose and sample,but the hemicellulose,pectins,and the hydrocolloid are solubilized and not detected
crude fibre measures cellulose&lignin,but does not determine hemicellulose&hydrocolloid
indigestible materials are then collected by filtration,and the fibre residue is quantitated gravimetrically
digestible CHO,lipid,and protein are selectively solubilize by chemical and/or enzyme
AOAC Method
greatly overestimate the fibre with ahigh content of simple sugar
adavantages
can be use to determine fibre content in all food
suitable for routine fibre analyses for research,egisatio and labelling purpose
insoluble fibre is collected by filtration;soluble fibre is precipitated by bringing the filtrate nd collected by filtration
the solution then filtered and fibre collected
total fibre content-adding 95% ethanol to the solution
duplicate of dry,defated food sample is enzymatically digested with alpha amylase,amyloglucosidase and protease to break down the starch and protein
to isolate the fraction of interest by selective precipitation and then to determine its mass by weighing
Method of Analysis
Chemically
the sum of monosaccharide in the acid hydrolysate represents fibre
digestible CHO are removed by enzymatic digestion,fibre components are hydolyzed by acid,and monosaccharide are measured
Gravimetrically
undigestible materials are tehn collected by filtartion,and the fibre residue is quantitated gravimetrically
digestable CHO,lipid&protein ar selectively solubilized by chemical/enzymes
weighing the mass of an undigestable polysaccharide remains
Chapter 5: Carbohydrate
Physical Method
infrared
specific gravity/density
refractive index
polarimetry
Munson-Walker Method
disadvantage
same disadvantages as Lane-Eynon method
advantage
accurate
more reproducible
amount of precipitate formed is directly related to the concentration of reducing sugar in the sample
leads to the formation of a copper oxide precipitate
under carefully controlled conditions
involving oxidation of the CHO in the presence of heat and all excess of copper sulphate and alkaline tartrate
Nelson-Somogyi method
required preparation of standard curve
the absorbance of solution is determine at either 500 or 520 nm against standard
reduction of arsenomolybdate complex produces an intense,stable blue-coloured solution
cuprous oxide is treated with arsenomolybdate reagent
the reducing sugar when heated with alkaline copper tartrate reduce the copper,from cupric to cuprous state,thus cuprous oxide is form
Lane-Eynon Method
susceptible to interference of molecules that act as reducing agent
cannot directly determine the concentration of reducing sugar
cannot distiguish between different typs of reducing sugar
result depend on the precise reaction times,temperature&reagent concentration
the reaction is not stoichiometric
determination of sugar and honey and other high reducing sugar content
coloured solution is titrated until the decolouration of the indicator
followed by addition of methylene blue(as an indicator)
mixture are boile for a specific time
followed by reaction with alkaline tartrate
based on reaction of reducing sugar with a solution of copper sulphate
Chapter 4: Protein Analysis
Dye Binding Method
non-protein binding dye;eg starch and/protein(i.e clcium or phospate)-cause error
protein differs in basic amino acid content,so differ in dye-binding capacity
not sensitive;mg quantities of protein are required
more precise than Kjedahl
does not measure non-protein nitrogen
no corrosive ragents
may be use to estimate changes in available lysine contain of cereal product during processing
rapid,inexpensive,relatively accurate
unbound dye is inversely related to the protein contain of the sample
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
the amount of unbound soluble dye is determined by measuring it absorbance
protein 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(negative charge) dye in a buffered solution(so that protein are positively charged)
Lowry Method
the reaction is interferes wth high concentration of reducing sugars,ammonium sulphate and sulphydryl compounds
the reaction is interferes varying degrees of sucrose,lipids,monosaccharides,etc.
colour is not strictly proportional to protein concentration
colour variest proteins to a greater extent than biuret method
more specific than most other methods
less affected by turbidity of the sample
very sensitive
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)
combines biuret reagent with another reagent;Follin-Ciocalteau phenol reagent);which reacts with tyrosine and trytophan residue in proteins
Kjedahl Method
corrosiv reagent
different protein need different correcting factor
does not give a measure of a true protein-measure total oganic nitogen
official method for for crude protein content
accurate and good reproducibility
inexpensive
relatively simple
applicable for all types of food
Function of reagent
Boric acid:for distillation of ammonia which contain methylene blue;borate ion formed is proportional to the amount of nitrogen
copper(II)sulphate:t as catalyst and convert organic nitrogen present to ammonium sulphate
potasium sulphate:increase the boiling point of sulphuric acid and accelerate digestion mixture to shorten the reaction
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
result-represent crude protein content
borates anions are formed&titrated with standardized acid-converted to nitrogen in the sample
the digest is neuralized with alkali and distilled into boric acid solution
total organic nitrogent is converted into ammonium sulphate
protein and other organic food component is digested with sulphuric acid in the presence of catalysts
Biuret Method
disadvantages
opalescence could occur in the final solution with 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 comparaed to other UV-vis method
advantages
does not detect nitrogen from non-peptide or non-protein sources
very few substances other than proteins in food interfere with the biurete reaction
colour derivations encountered less frequently than other method
rapid test(analysed can be completed within 30 mins)
the colour intensity(absorbance)is propartional to the protein content of the sample
the absorbance of the colour produced is read at 540nm
cupric ion complexed with peptide bonds
involves reaction with peptide linkages
Chapter 3:Crude Fat
Detergent Method
principles
the percent of fat sis measured volumetrically and expressed as percent fat
then the strong hydrophilic polyoxyethelene detergent,sorbitan monolaurate is added to separate fat from other food components
an ionic detergent(dioctyl sodium phospate) is added (to disperse the protein layer that stabilize the fat)to liberate fat
milk is pipette into babcock bottle
detergent react with protein to form protein-detergent complex to break up emulsions&release fat
Gerber Method
the fat contain was read directly from the garduated tube
the tube is centrifuged and incubated in waterbath at 60-63 degree celcius for 5 min
the amyl alcohol is added into mixture to give a clear homogenous fat column and the tube is carefully inverted
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
Babcock Method
Principle
the fat is measured volumetrically,but the result is expressed as a% fat by weight
subsequent centrifugation and addition of hot water isolate fat for quantification in the graduated portion of test bottle
sulphuric acid:digest protein,generates heat and release the bound fat from the sample
the mixture then shaken until homogenous,centrifuged,and submerged into water at 63 degree celcius
H2SO4 is added into a known amount of milk in Babcock bottle
Mojonnier Method
function of reagents
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
ammonia:neutralize sample and dissolve protein
alcohol:precipitates protein;prevent possible gel formation
promptly weigh/measure the test portion
homogenous sample is prepared by mixing and and inverting the sample bottle or by pouring back&forth between cean beakers
sample is brought to 20 degree celcius
does not require prior removal moisture from sample
the extracted fat is dried into a constant weight and expressed as a percent of fat by weight
fat is extracted with a mixture of ethyl-ether&pet-ether in a Mojonnier flask
to determine fat in cream,sweetened condense milk products,ice-cream,frozen desserts and other dairy based products
for analysis of fat in milk
Soxhlet Method
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
extraction is carried out 4hrs or until 16 hrs
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
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
avoid channeling of the solvent
provide soaking effect to the sample(provide more complete extraction of fat from the sample)
the flask is heated,the solvent evaporates,move up into the condenser(converted into a liquid&drips back into the extraction chamber containing sample)
thimble(containing sample) is placed in an extraction chamber,which suspended above a flask containing the solvent&below the condenser
sample is ground into small particles&placed in a porous thimble
at low temperature(40-50 degree celcius overnight or 95-100 degree celcius for 5 hrs)
high moisture food > 10% requires pre dryingsample under vacuum <100 mm Hg
commonly used method to increase the efficiency of lipid extraction from food
removes mainly non-polar lipids from sample
Goldfisch Method
the solvent may preferentially take certain routes through the sample
channeling of solvent may occur(inefficient/incomplete extraction)
more efficient than Soxhlet extraction method
faster
procedure
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
solvent:hexane,pet-ether,diethyl ether
allow the solvent from a boiling flask to continuously flows over the sample that held in a ceramic thimble
sample required removal of moisture(vacuum or dried) and ground to small particle size prior to analysis
sample is put in an extraction ceric thimble,and the solvent is added into the boiling flask
Chapter 2: Moisture
Chemical Method
Karl-Fischer Titration
if the sample containning low levels of moisture,colourimetric is ideal procedure
applicable for sample with moisture content 0.03% or more
the end point colour is dark red brown
excess I2 that cannot react with water can be determined visually
iodine and SO2 in the appropiate form are added to the sample in a closed chamber protected from athmospheric moisture
before amount of water found in a food sample can be determined,a KFR water equivalent(KFReq) must be determined
then methanol extract is titrated with KFR
if it inaccessible to the reagent,the moisture the extracted from the food with appropiate solvent such as methanol
in KFR volumetric reaction,the reagent is added directly as a titrant if the water in the sample accessible
std reagent for general work using methanolic solution: 1 iodine:3 SO2: 10 pyridine
the reagent for Karl-Fischer titration consist of iodine,pyridine,sulphur dioxide and methanol
the above reaction has been modified by adding solvent;C5H5N
the volume of iodine required to titrated the water can be related to moisture content
once the water has been used up,addtional iodine observed as dark red-brown as an end point
any water remains in the sample react with iodine&produce colourless solution
based on the fundamental reaction involving reduction of iodine by sulphur dioxide in the presence of water
Distillation method
Dean and Stark Method
distillation of water soluble component
incomplete evaporation of water(underestimation of moisture content)
solubility of water in the distillation liquid
reading the meniscus of receiving tube to determine the volume of water may be less accurate than using weight measurement
not applicable for some type of food
used of flammable solvents
relatively time consuming
destructive
Advantages
equipment cheap,east to set up&operate
applied for food containning volatile olls such as herbs or spices
suitable for application for food with low moisture content
volume of water produced by distillation read from a tube and serves as a function of the total weight of food sample
the water vapour then condensed&collected in graduated collection tube
the flask containing the sample&the organic solvent is attached to a condenser
safe to use
less dense than water
have a higher boiling point than water
insoluble with water
a known weight of food is placed in a flask with oganic solvent;eg xylene or toluene
Oven Drying Method
Disadvantages
time consuming
Unsuitable for some type of food
Destructive
Advantage
many sample can be analyzed simultaneously
officially approved for many applications
easy to use
relatively cheap
precise
Principles
the moisture content value obtained is highly influenced by:
type of sample
type of oven used and condition of oven
infrared(IR)drying
microwave oven
vacuum oven
convection and force draft oven
time and temperature of drying
the thermal energy used to evaporate the water from a food sample can be provided directly or indirectly
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
Chapter 1 Introduction
Evaluation of analytical data
Standard reference/internal control sample
Standard curves
Coefficient of variation
Standar deviation
%Relative error
Repeatability(reproducibility)
reproducibility: test results are obtained with the same method on identical test items in different lab,with different operators using different equipment
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
precision under repeatability conditions
Precision
how close replicate measures are indicates reproducibility
Accuracy
effect of other substances in the analyzed product
innacuracy inherent in the procedure
how close experiment measure is to the correct/true value of an analyzed
Sample preparation
analysis of sample should be performed 3 times(triplicate)
an ideal sample of analysis should be identical/representing the intrinsic properties with the bulk properties of material
Sampling
analysis
sample preparation
sampling
Food Analysis
Sample Preservation
sample characteristic
mirobial growth
using chemical preservative
drying
freezing
low temperature
heat treatment
unsaturated lipid component
frozen storage
use antioxidant to retard lipid oxidation
under nitrogen
light sensitive sample
placed in opaque container
wrapped in aluminium foil
should store in the condition that prevent degradation
change in food composition
action of mirobrobes
enzymic action
evaporation
to prevent changes in food composition and degradation
analytical method:use of official method in analysis
depends on proper selection and preparation of food sample&appropiatecalculations and enterpretationof data
analysis of chemical composition and physical properties of food
a part of quality management programme
monitor food composition to ensure quality and safety of food supply
