PROXIMATE ANALYSIS OF MACRONUTRIENTS
Presented byY. DivyaM.Pharm I-II SemPharmaceutical AnalysisSPMVV
CONTENTS• Introduction
a)Proximate Analysis
• Analysis of Macronutrients
a) Carbohydratesb) Proteinsc) Fats
• Determination of a)Ash Values
b)Moisture Content
c)Niterogen Content
• Conclusion• References
INTRODUCTION Quantitative estimation of
Macronutrients is carried out by a method known as Proximate Analysis or Weende Analysis.
This method was developed in 1860 by Henneberg and Stohmann in Germany.
Proximate analysis partitioned the compounds in feed into 6 categories based on chemical properties of the compound.
Proximate Analysis
Moisture
Ash
Crude Protein
Crude Lipid
Crude Fiber
Nitrogen free extract
SCHEME OF PROXIMATE ANALYSIS Sample
Ground to pass seive #20Dried in oven at 98-100 0C(Analytical Chemist)
and 98-130 0C( Cereal Chemist)Air Dry Sample
ASH Ether Extract KJELDHAL NITROGEN
Residue
ResidueResidue
Inorganic mineral matter acid
digestion
alkaline digestion
CRUDE FIBER
CRUDE PROTEIN
ASH
CRUDE FAT
Solution Lipids
Nitrogen Free Extract
X 6.25
CARBOHYDRATES
These are one of the important components in many foods, Present as isolated molecules, Present associated (or) chemically
bound to other molecules.
If they are attached to proteins, they are known as Glycoproteins,When attached to lipids known as Glycolipids
CLASSIFICATION
Based on number of sugar units in the total chain they are classified as:Monosaccharide: Single sugar unitDisaccharide: Two sugar unitOligosaccharide: 3-10 sugar unitPolysaccharide: More than 10 unitsChaining based on bridging on oxygen atoms: Glycoside bonds
Isolation Of Carbohydrates
Usually a very little preparation is needed prior to analysis of carbohydrates
Aqueous solutions
Require only a little preparation
Carbohydrates bound to other components
Require isolation of carbohydrates from the rest of food prior to analysis
Like Fruit juices, Syrups, Honey
Nuts, Cereals, Fruits, Bread, vegetables
Food is dried under vacuumGround to fine powder Defatted by solvent extraction
Boil defatted sample with 80% alcoholic soln
Mono & Oligo saccharides are soluble, but polysaccharides & dietary fiber are not soluble
Filter and collect the two filterate fractions.
These two fractions are dried and weighed to determine their concentration
Procedure For Isolation Of Carbohydrates
Since various other small molecules like amino acids, organic acids, pigments, vitamins, minerals are also present in alcoholic extract, they are removed by following methods:
Treating the solution with Clarifying agents: Like heavy metal salts (Lead acetate), they form insoluble complexes that can be removed by filtration.
By passing it through one or more Ion Exchange Resins: Since many Mono & Oligosaccharides are polar non charges molecules they can be separated from charged molecules by passing through Ion exchange columns
ANALYSIS OF CARBOHYDRATES Colored Reactions Chemical Reactions
Titrimetric- lane Eynon MethodGravimetric- Munson & Walker
MethodColorimetric- Anthrone Method
Enzymatic Methods Physical Methods
PolarimetryRefractive IndexDensityInfra RedImmuno Assay
Test Name Procedure Observation Molisch Test 1ml test soln +
2drops of alpha napthol, mix and add Conc H2SO4 along sides of test tube
Purple ring appears at the junction of two
layers
Fehling’s Test 1ml of test soln + 1ml of Fehling’s reagent, heat on BWB for 5 mins.
Reddish Brown ppt
Benedict’s Test 1ml of test soln + 1ml of Benedict’s reagent, heat on BWB for 5 mins.
Reddish Brown ppt
Barfoed’s Test 1ml of test soln + 2ml of Barfoed’s reagent, heat on BWB for 2 mins.
Green, red or yellow ppt
COLOR REACTIONS OF CARBOHYDRATES
Test Name Procedure Observation
Seliwanoff Test 0.5ml of sample + 2ml of Seliwanoff’s reagent, heat on BWB for 2 mins
Red product
Hydrolysis Test 6ml of 1% Sucrose + 2 drops of Conc HCl, heat on BWB for 5 mins
Then test for Fehling &
Benedict and all other previous
testIodine Test 0.5ml of fresh
starch soln + 1 drop of I2 soln
0.5ml dextrin + 1 drop I2soln
Dark bule colorYellow-Negative
Violet color
Osazone Test 0.5g Phenyl hydrazine + 1 spoon Sodium acetate + 2ml glucose, heat on BWB for 45min
Broomed shaped crystals (Glucose)
LANE EYNON METHOD:Determines the concentration of
Reducing Sugars in a sample
The reducing sugars in the carbohydrate solution react with the copper sulfate present in the flask. Once all the copper sulfate in solution has reacted, any further addition of reducing sugars causes the indicator(Methylene Blue) to change from blue to white.
PRINCIPLE:
a
12.5g sample + 25 ml of 10% neutral lead soln + some qty of alumina cream. Make upto 250ml with lead acetate
Filter and collect filterate
100ml filterate + 10ml 10% soln of Potassium Oxalate, make upto 500 ml. shake well
Filter and collect filterate
Subject for analysis
SAMPLE PREPARATION
In 250ml of Erlenmeyer flask add 10ml of Felhing’s reagent
Add 15ml sugar soln, boil for 15sec, till faint blue color appearsAdd 2-5 drops of 1% aqueous soln of methylene blue, continue heating
Add sugar soln, until the reduction of dye.
Amount of Sugar = Factor X 100
Titer ValueFactor can be obtained from literature
METHOD:
GRAVIMETRIC METHODDetermines the concentration
of reducing sugars PRINCIPLE:Carbohydrates are oxidized in the presence
of heat and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which leads to the formation of a copper oxide precipitate. reducing sugar + Cu2+ + base oxidized sugar + CuO2
The amount of precipitate formed is directly related to the concentration of reducing sugars in the initial sample. The concentration of precipitate present can be determined gravimetrically or titrimetrically.
ANTHRONE METHODColorimetric method of determining the
concentration of the total sugars in a sample.
Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color.
The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. The solution is then allowed to cool and its absorbance is measured at 620 nm. This method determines both reducing and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid
PROCEDURE:Take 3 test tubesBlank Stand
ardUnknow
nGlucose
(ml)0.0 0.5 0.5
Distilled water (ml)
1.0 0.5 0.5
Anthrone (ml)
4.0 4.0 4.0Mix well, place in BWB for 10 mins, cool to RT,
Read Optical densityConc of Unknown = Abs of Unknown X Conc of Std
Abs of Std
ANALYSIS OF POLYSACCHARIDES
Starch is the most common digestible polysaccharide found in foods
Processed food samples are
dried, ground.Dispersed in 80% ethanol
Mono & Oligosaccharide
s are soluble
Starch is insoluble and
can be separated by
filtering
Sample Preparation
Gravimetrically
Titrimetrically
Physical Methods
• Density, Refractive Index, Polarimetry
ANALYSIS METHOD FOR STARCH
To starch add Enzyme
Breakdown to Glucose
Subject to analysis
ANALYSIS OF FIBERSDietary FiberCell Wall Polysacchari
desNon Cell
Wall Polysacchari
des
Lignin
Cellulose – Principal structural component of all
cell wall associated with Hemicellulose & LigninThese are Indigestable Carbohydrates but not
derived from Cell wall of plants, Guar & Locust gum,
agar, alginates etcNon carbohydrate polymer containing 40 aromatic sub
units
METHOD OF ANALYSIS FOR FIBERSTwo methods are available:
Gravimetrically: by weighing the mass of insoluble fiber fraction isolated from sample( Crude Fiber Method & Total, Insoluble and soluble Fiber Method)Chemically: by breaking down the fiber into its constituents monosaccharides
Remove Lipid
Add 1.25%H2SO4 & NaOH
Insoluble Residue
Remove Lipid
Add Enzyme &
95% alcohol
Insoluble Residue
Crude Fiber Method
Total, Insoluble and Soluble Fiber Method
PROTEINSProteins are large, complex, organic
compounds composed mostly of amino acids linked with
peptide bonds.Qualitative Analysis Quantitative Analysisa) Precipitation Reactions a) Kjeldhal Methodb)Color Reactions b) Enhanced Dumas
Method• Biuret Test c)UV Spectroscpic Method• Xanthoprotein Test d) Lowry Method• Millon Test• Ninhydrin Test• Hopkin’s Test• Aldehyde Test• Phenol Reagent Test
ANALYSIS OF PROTEINS
PRECIPITATION REACTIONS Protein exist in colloidal solution due to
hydration of polar groups (-COO, NH3
+, -OH) They can be precipitated by dehydration or
neutralization of polar groups. Test Observation Inference
By Salts 2ml Protein soln + equal vol of ammonium Sulphate
White ppt
Heavy Metal Salts
2ml of Protein soln + few drops of Lead acetate
White ppt
Alkaloidal Reagent
Sample + 1-2ml of Picric acid soln
Formation of ppt
Organic Solvents
Sample + 1ml alcohol, keep for 2mins
Formation of ppt
Heat Sample is heated over flame
Cloudy White ppt
Acids Sample + few drops 1% Acetic acid
White ppt
Test Observation Inference
Biuret 2ml sample + 10% NaOH, Mix, + 4-5 drops of 0.5% W/V Copper Sulphite Soln
Purplish Violet color
Xanthoprotein
2ml sample + 1ml Conc HNO3
heat for 2mins, cool + few drops of 40% W/V NaOH
Yellow color changes to
orange
Millon’s Test 2ml Sample + Millon’s Reagent
White ppt, turns to brick red on
heatingNinhydrin 2ml Sample + dil. Soln
of ninhydrin Violet color
Hopkin Cole’s Test
2ml Sample + few drops of HCHO + 2 drops HgSO4 ,mix + 2-4ml of HgSO4 along sides of test tube
Violet color ring at the junction of
two layers
Aldehyde Test
1ml Sample + Few ml PDAB in H2SO4
Mix and heat
Purple Color
QUANTITATIVE ANALYSISKjeldhal Method:
This is a more convenient and simpler method for the estimation of nitrogen and is largely used for the estimation of nitrogen in fertilizers, food stuffs, drugs, etc.PRINCIPLE:A known mass of the organic compound is digested (heated) with conc.H2SO4 in presence of potassium sulphate and a little copper sulphate or mercury in a long - necked flask called Kjeldahl’s flasks. Potassium sulphate raises the boiling point of and thus ensures complete reaction while copper sulphate or mercury catalyses the reaction.
The food sample to be analyzed is weighed into a digestion flask (NH4)2S
O4
+ 2 NaOH
2NH3 + 2H2O + Na2SO4 H3BO3 (boric acid)
NH4+ + H2BO3
- (borate ion) H+
H3BO3
Digestion NeutralizationTitration
PROCEDURE:
This method is applicable to all organic compounds containing nitrogen
Principle: A known mass of the organic substances is heated with excess of copper oxide in an atmosphere of CO2 .Carbon, hydrogen and sulphur (if present) are oxidised to CO2, H2O, SO2 while nitrogen gas is set free. Any oxide of nitrogen that may be formed is reduced back to free nitrogen by passing over a hot reduced copper gauze.
ENHANCED DUMAS METHOD
A sample of known mass
CO2, H2O and N2Combustion (900 oC)
Nitrogen
Thermal conductivity detector
The nitrogen content is then measured
PROCEDURE:
These methods use either the natural ability of proteins to absorb (or scatter) light in the UV-visible region of the electromagnetic spectrum, or they chemically or physically modify proteins to make them absorb (or scatter) light in this region
METHODS USING UV-VISIBLE SPECTROSCOPY
PRINCIPLESDirect measurement at 280nmBiuret MethodLowry Method
a)Direct Measurement At 280nm:Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm.
The Protein content of tryptophan and tyrosine remains fairly constant, and so the absorbance of protein solutions at 280nm can be used to determine their concentration
b)Biuret Method:When cupric ions (Cu2+) interact with peptide bonds under alkaline conditions a violet-purplish color is produced .And absorbance is read at 540 nm
The Lowry method combines the Biuret reagent with another reagent (the Folin-Ciocalteu phenol reagent) which reacts with tyrosine and tryptophan residues in proteins.
This gives a bluish color which can be read between 500 - 750 nm depending on the sensitivity required
500nm- for determination of high conc Proteins750nm-for determination of low conc Proteins
C) Lowry Method
AMINO ACID ANALYSISAmino acid analysis is used to
determine the amino acid composition of proteins.
A protein sample is first hydrolyzed (e.g. using a strong acid) to release the amino acids, which are then separated using chromatography, e.g., ion exchange, affinity or absorption chromatography.
FATS
Lipids can be defined as “Esters Of Fatty Acids” and are naturally occurring.
Lipids consist of numerous fat like chemical compounds that are insoluble in water but soluble in organic solvents.
Lipid compounds include Monoglycerides, Diglycerides, triglycerides, phosphatides, cerebrosides, sterols, terpenes, fatty alcohols, and fatty acids
CLASSIFICATIONI. Simple Carboxylic Esters
a) Fats or Glycerides- Acylglycerolsb)Waxes
II Complex Carboxylic EstersGlycerolipids
GlycoglycerolipidsGlycoglycerolipid SulphatesIII. Complex Lipids (Amides)
Sphingolipids & GlycosphingolipidsIV.Precursor & Derived Lipids
Acids (Phosphatidic & Bile acid)Alcohols (Sterols)
Bases ( Sphinganines)V.Hydrocarbons (Straight & Branched)
VI.Lipid Vitamins & Hormones
ANALYSIS OF FATSQualitative Analysis Quantitative Analysis
Solubility Test Saponification Value
Microscopic Properties Iodine Value
Physical Test Hydroxyl Value
Emulsion Formation Acid Value
Sackowski’s Test
Libermann Burchard TestZak’s Reaction
QUALITATIVE ANALYSIS OF FATSTest Observation Inference
Solubility Test a) Few drops of oil + 1-2ml Carotene.b)Sample + Chloroform/ Benzene
a) Formation of 2 layers (Insoluble)
b) Soluble
Microscopic Properties
Observed through Microscope
Rhombic shaped crystals
Physical Test Spot Sample on filter paper & Observe
Greasy spot penetrates filter
paperEmulsion Formation
Spot drop of oil on watch glass & 2-3 drops of water
Oil droplet is broken into fine droplets
Sackowwski’s Test
2ml organic soln + 2ml of Conc H2SO4 & boil for 3mins
Upper layer(Chloroform)-
RedLower layer(H2SO4)-
YellowLibermann Burchard
2ml of organic soln in chlororform + 5-6 drops acetic anhydride + 2 drops conc H2SO4
Rose to Bluish green color soln
Zak’s Reaction 2ml of organic soln in chlororform + Ferric chloride + Conc H2SO4
Red colored soln
QUANTITATIVE ANALYSIS OF FATS
Number of moles =
Saponification value:The number of milligrams of Potassium hydroxide required to saponify 1gm of fat under the conditions specified.No. of Moles = Mass of
oil Relative
atomic massIodine Value:
The mass of Iodine in gms that is consumed by 100gms o chemical substance.Used to determine “the amount of Unsaturation of fatty acids”.Higher the Iodine number, the more C=C bonds are present in the fat.
Hydroxyl Value:
It is expressed as the mass of Potassium hydroxide in milligrams equivalent to the hydroxyl content of one gram of the chemical substanceAcid Value:
The mass of Potassium hydroxide in milligrams that is required to neutralize one gram of chemical substance
DETERMINATION OF ASH VALUE
Remnant of crude drug after incineration
contains mostly inorganic salts and non-volatile inorganic
components known as “ASH” Ash value can be determined by: Total ash Acid insoluble ash Water soluble ash
DETERMINATION OF TOTAL ASHIncinerat
e 2-3gm of air dried
sample in silica crucible
At about 450oC,
until free from
carbon. Cool and weigh.
Collect the
residue &
incinerate it .
If carbon free ash content cannot be obtained
Then pass on to ashless filter paper
Later on filter paper
Calculate the %ash with reference to air dried
drug
DETERMINATION OF ACID INSOLUBLE ASH
Boil the ash obtained in Total ash with 25ml 2M HCl for 5mins
Collect the residue on ashless filter paper
Wash with hot water & igniteCool in dessicator and weigh.
Calculate the % acid insoluble ash with
reference to the air dried drug
DETERMINATION OF WATER SOLUBLE ASH
Boil the ash obtained in total ash with 25ml of water for 5mins
Collect insoluble matter on ashless
filter paper
Wash with hot water, ignite for 15min at a temp
450oC
Substract the wt of the insoluble
matter from the wt of the ash taken
Calculate the % of water soluble ash with reference to the air dried drug
MOISTURE CONTENT
Moisture is expected component of crude drug which must be eliminated as far as possible
Methods Available:
Loss on drying Azeotropic Volumetric
Method Karl Fischer Titration Colorimetric Method
AZEOTROPIC VOLUMETRIC METHOD
The Azeotropic Method gives a direct measurement of water (or) other volatile constituents present in crude drugs being examined when sample is distilled together within an immiscible solvent such as toluene to xylene, the water present in the sample is absorbed by the solvent. Dean Stark Apparatus
Procedure:Crude
drug was weighed in flask
Add toulene.Connect apparatu
s Receiving flask is filled with
toulene
Heated until no
more water is distilled
overBoth
toulene and
water is distilled
over
As water is heavier
than toulene,
it sinks to bottom of receiving
tubeWhen the apparatus is cooled. Toulene and
water is separated completely
The volume of water distilled
is given by% = 100 X N
WW= wt in gm of material examinedN= No of ml of water
KARL FISCHER TITRATION
Most extensively used chemical method for the determination of Total Moisture. It can be applicable to very small quantities of
mositureThe reagent contains solution of Iodine, Sulphurdioxide and Pyridine in dry Methanol constitutes KFR (Karl Fischer Reagent)
I2 + SO2 + 3C5H5N + H2O 2C5H5N + 2HI + C5H5N+SO3
C5H5N+SO3 + CH3OH C5H5N
OSO2CH3
H
Primary standardization of reagent
Place 36m
l of dry
methanol in titration ve
ssel
Add KFR & 150-350gnm of sodium tartarate
Titrate with KFR
Determine endpoint using Pt electrode
Determine accura
te volum
e of KFR require
d
Calculate Water equivalent factor
F = wt in mg of Sodium tartarate X 0.1566
ml of KFR
Test for sample
Add KFR
Titrate and determine end point
Calculate % Water content
Add KFR
Titrate and determine end point
Calculate % Water content
% Water Content = Vol of KFR X Water Eq factor
Wt of Sample
Determination of nitrogen
The two most commonly used methods for the estimation of nitrogen are:• Duma’s Method,• Kjeldhal Method
conclusion
Finally it can be concluded that, though the Proximate analysis and Dietary fiber analysis produce satisfactory results, still there is a need for development of few other advanced versatile methods,
Eg: Where in case of non-ruminants amino acid value is more important than total protein.
References:1.http://www.aquaculture.ugent.be/Education/coursematerial/online%20courses/ATA/analysis/carb-mon.html2. Hedge, J E and Hofreiter, B T (1962) In: Carbohydrate Chemistry 17 (Eds Whistler R L and Be Miller, J N) Academic Press New York.3.http://www.britannica.com/EBchecked/topic/479680/protein/72530/The-isolation-and-determination-of-proteins#toc725314.http://csb.stanford.edu/class/public/readings/Molecular_Architecture_I_Lecture2/Voet_and_Voet_BOOK_00_Chapter6_Protein_Structure.pdf5.http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html; 2003.6.http://quizlet.com/8801657/recreate_set/7.http://quizlet.com/8801729/color-reactions-of-proteins-flash-cards/8.http://people.umass.edu/~mcclemen/581Proteins.html9.http://www.sigmaaldrich.com/analytical-chromatography/analytical-reagents/amino-acid-analysis.html.10.O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.
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