Proximate analysis of Macro Nutrients

54
PROXIMATE ANALYSIS OF MACRONUTRIENTS Presented by Y. Divya M.Pharm I-II Sem Pharmaceutical Analysis SPMVV

description

Analysis of Carbohydrates, Proteins, Fats, Ash content, Moisture content

Transcript of Proximate analysis of Macro Nutrients

Page 1: Proximate analysis of Macro Nutrients

PROXIMATE ANALYSIS OF MACRONUTRIENTS

Presented byY. DivyaM.Pharm I-II SemPharmaceutical AnalysisSPMVV

Page 2: Proximate analysis of Macro Nutrients

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

Page 3: Proximate analysis of Macro Nutrients

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.

Page 4: Proximate analysis of Macro Nutrients

Proximate Analysis

Moisture

Ash

Crude Protein

Crude Lipid

Crude Fiber

Nitrogen free extract

Page 5: Proximate analysis of Macro Nutrients

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

Page 6: Proximate analysis of Macro Nutrients

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

Page 7: Proximate analysis of Macro Nutrients

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

Page 8: Proximate analysis of Macro Nutrients

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

Page 9: Proximate analysis of Macro Nutrients

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

Page 10: Proximate analysis of Macro Nutrients

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

Page 11: Proximate analysis of Macro Nutrients

ANALYSIS OF CARBOHYDRATES Colored Reactions Chemical Reactions

Titrimetric- lane Eynon MethodGravimetric- Munson & Walker

MethodColorimetric- Anthrone Method

Enzymatic Methods Physical Methods

PolarimetryRefractive IndexDensityInfra RedImmuno Assay

Page 12: Proximate analysis of Macro Nutrients

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

Page 13: Proximate analysis of Macro Nutrients

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)

Page 14: Proximate analysis of Macro Nutrients

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:

Page 15: Proximate analysis of Macro Nutrients

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

Page 16: Proximate analysis of Macro Nutrients

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:

Page 17: Proximate analysis of Macro Nutrients

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.

Page 18: Proximate analysis of Macro Nutrients

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

Page 19: Proximate analysis of Macro Nutrients

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

Page 20: Proximate analysis of Macro Nutrients

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

Page 21: Proximate analysis of Macro Nutrients

Gravimetrically

Titrimetrically

Physical Methods

• Density, Refractive Index, Polarimetry

ANALYSIS METHOD FOR STARCH

To starch add Enzyme

Breakdown to Glucose

Subject to analysis

Page 22: Proximate analysis of Macro Nutrients

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

Page 23: Proximate analysis of Macro Nutrients

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

Page 24: Proximate analysis of Macro Nutrients

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

Page 25: Proximate analysis of Macro Nutrients

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

Page 26: Proximate analysis of Macro Nutrients

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

Page 27: Proximate analysis of Macro Nutrients

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.

Page 28: Proximate analysis of Macro Nutrients

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:

Page 29: Proximate analysis of Macro Nutrients

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

Page 30: Proximate analysis of Macro Nutrients

A sample of known mass

CO2, H2O and N2Combustion (900 oC)

Nitrogen

Thermal conductivity detector

The nitrogen content is then measured

PROCEDURE:

Page 31: Proximate analysis of Macro Nutrients

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

Page 32: Proximate analysis of Macro Nutrients

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

Page 33: Proximate analysis of Macro Nutrients

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

Page 34: Proximate analysis of Macro Nutrients

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.

Page 35: Proximate analysis of Macro Nutrients

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

Page 36: Proximate analysis of Macro Nutrients

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

Page 37: Proximate analysis of Macro Nutrients

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

Page 38: Proximate analysis of Macro Nutrients

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

Page 39: Proximate analysis of Macro Nutrients

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.

Page 40: Proximate analysis of Macro Nutrients

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

Page 41: Proximate analysis of Macro Nutrients

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

Page 42: Proximate analysis of Macro Nutrients

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

Page 43: Proximate analysis of Macro Nutrients

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

Page 44: Proximate analysis of Macro Nutrients

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

Page 45: Proximate analysis of Macro Nutrients

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

Page 46: Proximate analysis of Macro Nutrients

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

Page 47: Proximate analysis of Macro Nutrients

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

Page 48: Proximate analysis of Macro Nutrients

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

Page 49: Proximate analysis of Macro Nutrients

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

Page 50: Proximate analysis of Macro Nutrients

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

Page 51: Proximate analysis of Macro Nutrients

Determination of nitrogen

The two most commonly used methods for the estimation of nitrogen are:• Duma’s Method,• Kjeldhal Method

Page 52: Proximate analysis of Macro Nutrients

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.

Page 53: Proximate analysis of Macro Nutrients

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.

Page 54: Proximate analysis of Macro Nutrients

THANK

“U”…

..