a Mohd Shafi 6 éve
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Szerző: Leyla Izaguirre
Szerző: DIEGO TORRES PATASCOY
Szerző: PATRICIA ARARAT
Szerző: Mindomo Team
The extraction procedure are the same as outlined for the vitamin determination Common extract of the vitamin is concentrated and separated by HPLC.
The result expressed as μg niacin / g sample
Critical : toxicity of cyanogen bromide, the analysis must be carried out under fume hood.
niacin+cynogen bromide--> coloured compound with an intensity proportional to niacin concentration
1)Thiochrome is light sensitive 2)Thiamine is sensitive to heat especially at alkaline pH.
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.
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.
Digested with H2S04 and subsequently treated with a phosphatase preparation to free from natural ester and protein bond
The flourescent compound intensity proportional to the vitamin C content.
Dehydroascorbic acid + o-phenylenediamine-->fluorescent quinoxaline compound.
Ascorbic acid + o-phenylenediamine--> Dehydroascorbic acid
This method measures both ascorbic acid and dehydroascorbic acid.
L- dehydroascorbic acid determined by first converting it to L-ascorbic acid with a suitable reagent.
This method is not suitable for highly coloured products
At the endpoint, excess of unreduced dye is rose pink in acid solution lasting at least 10 sec.
Measures the decolourization of 2,6-dichorophenolindophenol dye by ascorbic acid.
L-ascorbic acid is oxidizes to dehydroascorbic acid by indicator dye.
The vitamin (L-ascorbic & -dehydroascorbic acid) is very susceptible to oxidative deterioration, which enhanced by: 1)High pH 2)Presence of ferric&cupric acid.
Conduct at low pH and with addition of chelating agent.
This method involve chromatographic separation and quantitative determination at 325 nm
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.
Measures unstable colour at A620nm that results from reaction between Vitamin A+antimony trichloride (SbCl3)
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
However, require high degree of analytical skill, time commitment, and costly equipment, thus not routinely used for analyses & labeling purpose.
Suitable for research, legislation, and labeling purpose.
provides most accurate estimate of fibre for wide range of foods
Total fibre = Soluble fibre + Insoluble fibre
Lignin in insoluble fraction is not hydrolyzed by acid but remains as insoluble complex which is removed by centrifugation, washed, dried & weighed.
Both fractions-->mixed with concentrated H2SO4-->hydrolyze cellulose&non-cellulose polysaccharides&mono- concentration are analyzed.
Soluble residue precipitated from solution by adding ethanol, removed by filtration, and then collected, washed and dried.
Insoluble residue is removed by centrifugation, filtration, washed with ethanol & acetone, dried.
Resultant solution contains mixture of soluble & insoluble fibre is separated by centrifugation.
Dry ground food-->suspend in 80% ethanol-->free sugars remove Lipids extracted with hexane. Starch digestion is completed by incubating with enzymes
Fibre = Monosaccharides + Lignin
Fibre fractions are hydrolyzed with H2SO4 and sugar content of the acid hydrolysates is determined. Lignin is determined gravimetrically.
Free sugars & lipids are extracted with ethanol & hexane. Starch is removed by enzymatic digestion and insoluble fibre is separated from soluble fibre.
Allow estimation of resistant starch.
Fibre = to the sum of all non-starch monosaccharide+lignin
Suitable method for determining fibre content in most foods (with low content of lignin).
Mass of fibre in original sample assumed to be equal to the total mono- present
Concentration of mono- is determined colourimetrically or chromatographycally.
Fibre is hydrolyzed using concetrated H2SO4 solution to break down starch into mono-.
Pure ethanol added-->ppt fibre, Separated from the digest by centrifugation-->washed-->dried.
Defatted food sample is heated in water--> Enzyme added
Greatly overestimate the fibre content with a high content of simple sugars
Suitable for routine fibre analyses for research, legislation and labeling purpose. The method can be used to determine fibre content in all foods.
Filtered fibre residues are washed with ethanol & acetone, oven dried, and weighed
One duplicate is incinerated to determine ash content
One duplicate is analyzed for protein determination
insoluble fibre is collected by filtration. Soluble fibre is precipitated by bringing the filtrate to 78% ethanol and collected by filtration.
Total fibre content – adding 95% ethanol to the solution. Solution-->filtered & fibre is collected.
Duplicates of dry, defatted food sample is enzymatically digested with: α-amylase, amyloglucosidase&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.
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.
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).
Digestible CHO, lipid, and protein-->selectively solubilized by chemical and/or enzymes. Indigestible materials--> collected by filtration Fibre residue-->quantitated gravimetrically.
Lignin
pectins
hemicellulose
cellulose
Oligosaccharides
found in pulses, onions, Jerusalem artichoke, garlic
Traditional soluble fiber
glucans in oats and barley, pentoses in rye
Resistant starch
found in whole grains, pulses, seeds
Traditional insoluble fiber
cellulose, hemicellulose, lignin in wheat and rice
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.
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
Do not use ether or acetone to clean off samples from prism because these solvents evaporate quickly and in that process change the temperature.
In practice, RI of CHO solution is usually measured at a boundary with quartz.
RI readings are normally expressed as % sugar wt./wt. or alternatively oBrix (g sucrose / 100 g of sample).
RI standard measurement are made specific at T (20oC) and wavelength
RI of a substance depends on :
Concentration Temperature (T) Wavelength of light
When electromagnetic radiation passes from one medium to another, it can change direction
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
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.
Concentration of an unknown sample is determined by measuring the angle of rotation and comparing it with a calibration curve
Concentration is determined from the specific optical rotation value, when no other optically active compounds are present and all other factors are held constant
CHO able rotate plane polarized light through an angle of rotation
Prior to analysis, sample solution must be clarified.
Angle of polarization proportional to the concentration of optically active molecules in solution
Plane polarized light passed through solution exhibiting optical activity, it rotated either to left (-) or right (+).
Asymmetric carbon atoms have the ability to rotate plane of polarization of polarized light
Require preparation of standard curve
The absorbance of the solution is determined at either 500 or 520nm against standard
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 reducing sugar when heated with alkaline copper tartrate reduce the copper, from cupric to cuprous state, thus cuprous oxide is formed
Same disadvantages as Lane-Eynon method
More reproducible and accurate
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.
The methods depends on the ability of reducing sugar to react with copper solution
Basic conditions (alkaline) are required to keep copper solution as copper hydroxide (Cu+).
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)
Amount of precipitate formed=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 – leads to the formation of a copper oxide precipitate
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
Determinations of reducing sugars in honey and other high-reducing sugar syrups
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.
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
Sources of error
4.Interferences from certain food constituents
3.Moisture adhering to walls
2.Atmospheric condition
1.Incomplete water extraction
Suitable for low moisture food that sensitives to decomposition or volatilization under vacuum or high temperature
5.Food with intermediate moisture levels
4.Food rich in reducing sugar & protein
3.Food with high volatile oils
2.Sugar rich food
1. Low moisture food
In KF volumetric titration
Volumetric titration applicable for moisture content below or equal 0.03%
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
KFR water equivalent must be determined first before amount of water in food sample can be determined
If moisture is inaccesible to reagent--> moisture extracted from food with appropriate solvent (ex: methanol) --> methanol extract titrated with KFR
KFR added directly as titrant if water in sample is accesible
KF reagent
Pyridine
Sulfur dioxide
Iodine
Methanol
Modified Karl Fischer Titration
C5H5n was added
Water remains + I2 --> Colourless solution Water used up--> Additional I2 observed as--> Dark red-brown (endpoint)
Reduction of iodine by S02 in the presence of water
Dean & Stark method
Advantages & Disadvantages of Distillation Method
Less accurate of reading than using weight measurement
Solubility of water in the distillation liquid
Incomplete evaporation of water
Some types of food are not applicable
Involve flammable solvents
Cheap equipment, easy to setup & operate
For food contain volatile oil
For low moisture food
Potential Sources Of Error With Distillation
Thermally labile component decomposed with production of water at elevated temperature used
Solution: Discontinue use of method Find alternative methods
Water droplets adhere to the inside of glassware
Solution: Use clean glassware
Formation of emulsions between water & solvent
Solution: Allow apparatus to cool after distillation completed & before reading the amount of moisture
Volume of water produced = total weight of food sample
Flask connected to condenser. Water vapour condensed & collected in graduated collection tube
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
Suitable for low moisture analysis for low moisture samples such as cheese, spices, oils
Reflux distillation
Better accuracy & precision than oven drying methods especially for low moisture sample
Use lower b.p solvent to reduce chemical reactions (distillations times increase)
During heating,water and immiscible solvents distills off together at a constant ratio at a lower temperature than the b.p of both components
Uses either solvent less dense than water or solvent more dense than water
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
Less thermal decomposition of some foods
Distilled water is condense--> Mixture that distills off collected in a collecting vessel--> Volume of water measured
Involves co-distilling water in food samples with high b.p solvent that immiscible in water
Practical consideration during moisture removal
Clumping & surface crust formation (Sand pan technique)
1.To prevent formation of surface crust 2.To disperse the sample
Add sand or other inert materials to prevent clumping of food
Sample pans
Sample--> Dried in oven--> store in dessicator -To ensure no residual moisture is attached to them
Handle pans with tongs because fingerprints can contribute to mass of a sample
Pan covers & lids- Control/prevent/spattering of sample
Aluminium pans- Cheap & have high thermal conductivity
Temperature control
Decompositon of other food component
Overestimation/Underestimation of true moisture content
Decomposition product : C02, C0, CH4, H20
Weight gain due to oxidation of unsaturated fatty acid
Use suitable time & temperature
Because heat sensitive component in food decomposed causing changes in mass leading to error of moisture content determination
Sample dimensions
High surface area, high rate of moisture removal
Moisture removal sometimes best achieved in 2 stage process
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
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
Advantage and disadvantage of oven drying method
Unsuitable for some food
Time consuming
Destructive
Many samples can be analyzed simultaneously
Officially approved for many applications
Relatively cheap
Precise
% Total solids (wt / wt) = wt of dry sample x 100 wt of wet sample
Thus, % Total solids = (100 - % Moisture)
% Moisture (wt / wt) = (wt of wet sample – wt of dry sample) x 100 ______________________________ wt of wet sample
Type of devices
Infrared drying
To obtain reproducible measurements we must
Thickness/dimensions of sample
Control distance between sample and IR lamp
Water molecules thermally excited
Heat penetrate intro sample being dried to evaporate water from sample
Microwave oven
Why samples placed at center & distributed evenly?
To avoid certain portion get burn while other area under processed
Water molecules absorbed microwave energy--> thermally excited--> evaporate
Weighed samples---> Placed for a specified time---> Power level & their dried mass is weighed Alternatively : Weighed samples---> Dried until constant mass reached
Vacuum oven
Air inlet & outlet : Carry out moisture to prevent accumulation of moisture within oven
Thermal energy : Applied directly via metallic shelf
Need dry air purge in addition to temperature & vacuum controls to operate within method definition
More complete removal of water and volatiles w/o decomposition
Dried under reduced pressure for a specified temperature & time.
Convection and forced draft oven
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
Thermal energy: Directly applied via shelf and air
Weighed samples---> placed in oven(specified time&temperature)--->mass determined or dried until constant mass achieved
Moisture content value obtained depend on
Type of sample
Type and condition of oven used
Time and temperature of drying
Thermal energy used to evaporate the moisture can be directly or indirect
-Heated under specified conditions until constant weighed achieved and calculate loss of moisture by loss of weight
Ways to overcome
Minimize headspace in sample container
Minimize exposure of sample to atmosphere during grinding
-Physically bound as a monolayer to food surface constituent
-Chemically bound -Does not freeze at low temperature -Reduced mobility of water
Held within food that are surrounded by physical barrier that prevents water from escaping
-Retains its physical form -Dispersing agent for colloids -Solvents for salts -Easily lost by evaporation
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.
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.
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.
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.
1)Small sample capacity. 2)Relatively expensive equipment.
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.
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. 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.
1.Sample is placed into a glass chamber, sealed and vacuum is applied.
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).
1. Minerals usually stay in solution. 2. Little / no loss from mineral volatilization 3. Rapid than dry ashing.
4. Solution cooled--> 50% of HCl is added and diluted with distilled, deionized water.
3. Boiling continue until solution become colourless or light in colour.
2. Sample solution--> heated(350oC)-->organic matter digested (leaving only mineral oxides in solution) & HNO3 is almost evaporated.
1. Oxidation of organic substances by strong acid (HNO3) and oxidizing agent, perchloric acid (HClO4).
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.
resistant to both acids & alkalies, inexpensive, but possible sources of contamination
Very inert and best crucible but expensive
Resistant to acid and halogen but not alkali in high temp.
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.
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.
4)The food sample is weighed before and after ashing to determine the concentration ash present.
% Ash (dry basis) = Mash x 100 Mdry
3)Most minerals are converted to oxides, sulfates, phosphates, chlorides or silicates.
2)Water & other volatile materials are vaporized organic substances--> burned in the presence of the O2 in air to CO2, H2O and N2.
1)Incineration at high temp. with muffle furnace (5250C or higher)
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.
high-temp. muffle furnace used temp. between 500 – 600oC.
to determine total ash and before an elemental analysis for individual minerals.
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
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
Used to estimate proteins in milk, wheat flour, soy products and meats
Dye bound = dye initial - dye free
Protein + excess dye--->protein-dye insoluble complex + unbound soluble dye
Unbound dye is inversely related to the protein content of the sample
The amount of unbound soluble dye is determined by measuring its absorbance
Proteins bind the dye =insoluble complex formed
Protein-containing sample+known excess amount of anionic dye in a buffered solution = protein positively charged
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
1)Very sensitive 2)Less affected by turbidity of the sample 3)More specific than most other methods 4)Relatively simple
Widely used in protein biochemistry
The absorbance of the solution is read at 650 nm
Freshly prepared Folin reagent added, mixed and incubated
Biuret reagent+diluted sample-->incubated at room temp. for 10 min.
Proteins to be analyzed diluted to an appropriate range
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)
Lowry method combines biuret reagent with another reagent (Folin-Ciocalteau phenol reagent) ; which reacts with tyrosine& trytophan residues in proteins
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
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
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
The absorbance of the mixture solution is read at 540 nm against blank reagent.
The mixture is allowed to stand at room temperature for 15 – 30 min.
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 colour intensity (absorbance) proportional to protein content of the sample
The absorbance read at 540 nm
Cupric ions (Cu2+) complexed with peptide bonds under alkaline conditions and produced a violet-purplish colour
Biuret method involves a reaction with peptide linkages
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
1)Applicable to all types of food 2)Relatively simple 3)Inexpensive 4)Accurate and good reproducibility – official method for crude protein content
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
Copper (II) sulfate
1)Act as catalyst 2)Convert organic nitrogen present to ammonium sulfate
Potassium sulfate
1)Use to increase boiling point of sulfuric acid 2)Accelerate digestion mixture to shorten the reaction
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
2. Total protein (g) per 100 g food sample = total nitrogen x factor for foodstuff analyzed
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)
A conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration
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
3)Distillation of diluted digest
The low pH of the solution in receiving flask converts the ammonia gas-->ammonium ion & simultaneously converts the boric acid-->borate ion
Ammonia gas liberated from solution-->moves out of the digestion flask into receiving flask (contains excess of boric acid)
2)Neutralization of diluted digest
Solution in digestion flask is made alkaline by addition of NaOH, which converts the ammonium sulfate into ammonia gas
digestion flask is connected to a receiving flask by a tube
1)Digestion--> heating with sulfuric acid+catalyst
converts nitrogen in the food-->ammonia Other organic matter--> CO2 & H2O
convert nitrogen to ammonium sulfate & complete oxidation
Result represents crude protein content
Borate anions are formed & titrated with standardized acid – converted to nitrogen in the sample
Total organic nitrogen converted to ammonium sulfate The digest neutralized with alkali--> distilled into boric acid solution
Proteins+other organic food component digested with H2S04+catalyst
Total protein content Amino acid composition Content of a particular protein in a mixture Nutritive value
To determine fat in milk
The percent fat is measured volumetrically and expressed as percent fat.
Then, strong hydrophilic nonionic polyoxyethylene detergent, sorbitan monolaurate added to separate fat from other food components.
Milk pipetted into a Babcock test bottle. An anionic detergent (dioctyl sodium phosphate) is added to liberate fat.
Detergent react with protein to form protein detergent complex to break up emulsions and release fat
Isoamyl alcohol improves fat separation, reduces effect of sulfuric acid & prevents charring of sugar
Simpler and faster than babcock
Wider application to a variety of dairy products
Directly read fromgraduated tube
Centrifuged-->incubated in 60-63 C water bath for 5 minutes
Amyl alcohol is added into mixture-->give clear homogenous fat column and the tube / butyrometer is carefully inverted.
H2SO4 added to milk in gerber tube-->digest CHO & protein-->release bound fat from milk & maintain fat in liquid state by generating heat
to determine essential oil in flavour extracts and fat in seafood
Does not recommend for chocolate or added sugar product due to charring of chocolate and sugars by sulphuric acid
Does not determine phospholipid in fat milk
Common method to determine fat content in milk.
The fat measured volumetrically but result expressed as %weight
Subsequent centrifugation and addition of hot water isolate fat for quantification in the graduated portion of test bottle
H2SO4 digest protein-->produce heat-->release bound fat from sample
Mixture shaken until homogeneous-->centrifuged-->submerged in water 63 C
H2SO4 + milk in babcock bottle
% fat = [(wt dish + fat) - (wt dish)] x 100 _____________________ wt of sample taken
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
To determine fat content in flour
Using HCL
fat-containing solvents (from repeated extraction) are pooled, solvent+fat-->evaporate-->fat-->weigh-->content determined.
Release of bound fat by alkaline digestion+ammonium hydroxide+addition of ethanol--> discontinuous extraction of fat using ethyl ether & pet-ether
Repeated 3 times
Weigh/measure test portion
brought to 20oC, Homogenous sample-->mixing and inverting the sample bottle/pouring back & forth between clean beakers.
Does not require removal of moisture
The extracted fat is dried to a constant weight
Fat is extracted with a mixture of ethyl ether & pet-ether in a Mojonnier flask.
Can determine cream, sweetened condensed milk products, ice-cream and other dairies product
% fat on dry weight basis = weight of fat in sample x 100 __________________ weight of dried sample
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
As the solvent passes through the sample, it extracts the lipids & carries them into the flask
Solvent build up in extraction chamber for 5-10 minutes--> surrounds sample-->siphons back to boiling flask
Avoid chanelling of the solvent
Provide soaking effects of the sample(better lipid extraction)
Flask heated-->solvent evaporates and moves up into condensor-->drip onto sample in extraction chamber
Thimble(sample) placed in an extraction chamber, which suspended above a flask containing the solvent & below the condenser
to increase the efficiency of lipid extraction from food
removes mainly non-polar lipids from sample
% fat on dry weight basis = weight of fat in sample (g) x 100 _____________________ weight of dried sample (g)
Weight of fat in sample = (beaker + extracted fat) – beaker
Incomplete extraction might occur
Faster & more efficient extraction method than Soxhlet extraction method
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
The solvent carries fat extracted as solvent drips through the sample, is collected back in the boiling flask
solvent from boiling flask continuously flows over the sample in ceramic thimble
Sample put in an extraction ceramic thimble and the solvent is added into the boiling flask
Less flammable than ethyl ether
More non-hygroscopic
Cheaper
Low boiling point fraction of petroleum
Forms peroxide
Hygroscopic
Generally expensive than other solvents
Better solvent for fat than pet-ether
Acid hydrolysis
Break both covalently & ionically bound lipids into easily extractable lipid forms
To digest non-polar solvents
Particle size reduction
sample + solvent are mixed in a high-speed comminuting device = better extraction
To reduce particle size and accelerate fat extraction
Lipid extraction efficiency from dried foods depends on particle size
Pre-drying sample
Importance
Helps fat free from the tissues of foods
Breaks fat-water emulsions – fat dissolve easily in organic solvent
Make a sample easier to grind - better lipid extraction
Vacuum oven drying at low temp – increase surface area for better lipid extraction
Type of analytical procedures used
Type and nature of lipids in food
Type of food
processing conditions depends on the total lipid content
shelf life of food product
development of low fat foods
standard of identity & nutritional labeling law
Contain general properties of lipid
Derived from neutral lipids or compound lipids
Sphingolipid
Compounds containing fatty acids, a nitrogen moiety, and phosphoryl group
Cerebrosides lipid
Compounds containing fatty acids, a CHO, and a nitrogen moiety
Phospholipid
glycerols esters of fatty acids, phosphoric acids & other groups containing nitrogen
Esters of fatty acid with alcohol
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)
Sufficient number of portion of the whole plant or part of the plant, or cuts of meat or fish flesh
Identical and representing the intrinsic properties with the bulk properties of the material.
