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Proximate analysis of Macro Nutrients
This document provides an overview of proximate analysis to determine macronutrients like carbohydrates, proteins, fats, and moisture content. It discusses the classification, isolation, and various quantitative analysis methods for each macronutrient. Proximate or Weende analysis partitions food compounds into moisture, ash, crude protein, crude lipid, crude fiber, and nitrogen-free extract. Common techniques described include Kjeldahl method for protein, Soxhlet extraction for fat, gravimetric methods for carbohydrates, and determining ash content.
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Proximate analysis of Macro Nutrients
- 1. PROXIMATE ANALYSIS OFMACRONUTRIENTS Presented by Y. Divya M.Pharm I-II Sem Pharmaceutical Analysis SPMVV
- 2. CONTENTS • Introduction a)Proximate Analysis •Analysis of Macronutrients a) Carbohydrates b) Proteins c) Fats • Determination of a)Ash Values b)Moisture Content c)Niterogen Content • Conclusion • References
- 3. INTRODUCTION Quantitative estimation ofMacronutrients 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.
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- 5. SCHEME OF PROXIMATEANALYSIS Sample Ground to pass seive #20 Dried in oven at 98-100 0C(Analytical Chemist) and 98-130 0C( Cereal Chemist) Air Dry Sample ASH Ether Extract KJELDHAL NITROGEN Residue Residue Residue Inorganic mineral matter acid digestion alkaline digestion CRUDE FIBER CRUDE PROTEIN ASH CRUDE FAT Solution Lipids Nitrogen Free Extract X 6.25
- 6. CARBOHYDRATES These are oneof 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
- 7. CLASSIFICATION Based on numberof sugar units in the total chain they are classified as: Monosaccharide: Single sugar unit Disaccharide: Two sugar unit Oligosaccharide: 3-10 sugar unit Polysaccharide: More than 10 units Chaining based on bridging on oxygen atoms: Glycoside bonds
- 8. Isolation Of Carbohydrates Usually avery 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
- 9. Food is dried under vacuum Ground to fine powder Defattedby solvent extraction Boil defatted sample with 80% alcoholic soln Mono & Oligo saccharides are soluble, but polysacchari des & 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
- 10. Since various othersmall 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
- 11. ANALYSIS OF CARBOHYDRATES ColoredReactions Chemical Reactions Titrimetric- lane Eynon Method Gravimetric- Munson & Walker Method Colorimetric- Anthrone Method Enzymatic Methods Physical Methods Polarimetry Refractive Index Density Infra Red Immuno Assay
- 12. Test Name ProcedureObservation 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
- 13. Test Name ProcedureObservation 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 test Iodine Test 0.5ml of fresh starch soln + 1 drop of I2 soln 0.5ml dextrin + 1 drop I2 soln Dark bule color Yellow-Negative Violet color Osazone Test 0.5g Phenyl hydrazine + 1 spoon Sodium acetate + 2ml glucose, heat on BWB for 45min Broomed shaped crystals (Glucose)
- 14. LANE EYNONMETHOD: Determines theconcentration 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:
- 15. 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
- 16. In 250ml ofErlenmeyer flask add 10ml of Felhing’s reagent Add 15ml sugar soln, boil for 15sec, till faint blue color appears Add 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 Value Factor can be obtained from literature METHOD:
- 17. GRAVIMETRIC METHOD Determines theconcentration 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.
- 18. ANTHRONE METHOD Colorimetric methodof 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
- 19. PROCEDURE: Take 3 testtubes Blank Standard Unknown Glucose (ml) 0.0 0.5 0.5 Distilled water (ml) 1.0 0.5 0.5 Anthrone (ml) 4.0 4.0 4.0 Mix well, place in BWB for 10 mins, cool to RT, Read Optical density Conc of Unknown = Abs of Unknown X Conc of Std Abs of Std
- 20. ANALYSIS OF POLYSACCHARIDES Starch isthe most common digestible polysaccharide found in foods Processed food samples are dried, ground. Dispersed in 80% ethanol Mono & Oligosaccharides are soluble Starch is insoluble and can be separated by filtering Sample Preparation
- 21. Gravimetrically Titrimetrically Physical Methods • By collecting,drying and weighing the precipitate • By determining the amount of iodine required to precipitate the starch • Density, Refractive Index, Polarimetry ANALYSIS METHOD FOR STARCH To starch add Enzyme Breakdown to Glucose Subject to analysis
- 22. ANALYSIS OF FIBERSDietaryFiber CellWall Polysaccharides Non Cell Wall Polysaccharides Lignin Cellulose – Principal structural component of all cell wall associated with Hemicellulose & Lignin These are Indigestable Carbohydrates but not derived from Cell wall of plants, Guar & Locust gum, agar, alginates etc Non carbohydrate polymer containing 40 aromatic sub units
- 23. METHOD OF ANALYSISFOR FIBERS Two 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
- 24. PROTEINS Proteins are large,complex, organic compounds composed mostly of amino acids linked with peptide bonds. Qualitative Analysis Quantitative Analysis a) Precipitation Reactions a) Kjeldhal Method b)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
- 25. PRECIPITATION REACTIONS Protein existin 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
- 26. Test Observation Inference Biuret2ml 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 heating Ninhydrin 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
- 27. QUANTITATIVE ANALYSIS Kjeldhal Method: Thisis 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.
- 28. The food sampleto be analyzed is weighed into a digestion flask (NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4 H3BO3 (boric acid) NH4 + + H2BO3 - (borate ion) H+ H3BO3 Digestion Neutralization Titration PROCEDURE:
- 29. This method isapplicable 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
- 30. A sample ofknown mass CO2, H2O and N2 Combustion (900 oC) Nitrogen Thermal conductivity detector The nitrogen content is then measured PROCEDURE:
- 31. These methods useeither 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 PRINCIPLES Direct measurement at 280nm Biuret Method Lowry Method
- 32. a)Direct Measurement At280nm: 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
- 33. The Lowry methodcombines 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 Proteins 750nm-for determination of low conc Proteins C) Lowry Method
- 34. AMINO ACID ANALYSIS Amino 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.
- 35. FATS Lipids can bedefined 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
- 36. CLASSIFICATION I. Simple CarboxylicEsters a) Fats or Glycerides- Acylglycerols b)Waxes II Complex Carboxylic Esters Glycerolipids Glycoglycerolipids Glycoglycerolipid Sulphates III. Complex Lipids (Amides) Sphingolipids & Glycosphingolipids IV.Precursor & Derived Lipids Acids (Phosphatidic & Bile acid) Alcohols (Sterols) Bases ( Sphinganines) V.Hydrocarbons (Straight & Branched) VI.Lipid Vitamins & Hormones
- 37. ANALYSIS OF FATS QualitativeAnalysis Quantitative Analysis Solubility Test Saponification Value Microscopic Properties Iodine Value Physical Test Hydroxyl Value Emulsion Formation Acid Value Sackowski’s Test Libermann Burchard Test Zak’s Reaction
- 38. QUALITATIVE ANALYSIS OFFATS Test 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 paper Emulsion 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)-Red Lower layer(H2SO4)-Yellow Libermann 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
- 39. QUANTITATIVE ANALYSIS OFFATS 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 mass Iodine 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.
- 40. Hydroxyl Value: It isexpressed as the mass of Potassium hydroxide in milligrams equivalent to the hydroxyl content of one gram of the chemical substance Acid Value: The mass of Potassium hydroxide in milligrams that is required to neutralize one gram of chemical substance
- 41. DETERMINATIONOF ASHVALUE Remnant of crudedrug 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
- 42. DETERMINATION OF TOTALASH Incinerate 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
- 43. DETERMINATION OF ACIDINSOLUBLE ASH Boilthe ash obtained in Total ash with 25ml 2M HCl for 5mins Collect the residue on ashless filter paper Wash with hot water & ignite Cool in dessicator and weigh. Calculate the % acid insoluble ash with reference to the air dried drug
- 44. DETERMINATION OF WATER SOLUBLEASH 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
- 45. MOISTURE CONTENT Moisture isexpected 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
- 46. AZEOTROPIC VOLUMETRIC METHOD TheAzeotropic 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
- 47. Procedure: Crude drug was weighedin flask Add toulene. Connect apparatus Receiving flask is filled with toulene Heated until no more water is distilled over Both toulene and water is distilled over As water is heavier than toulene, it sinks to bottom of receiving tube When the apparatus is cooled. Toulene and water is separated completely The volume of water distilled is given by % = 100 X N W W= wt in gm of material examined N= No of ml of water
- 48. KARL FISCHER TITRATION Mostextensively used chemical method for the determination of Total Moisture. It can be applicable to very small quantities of mositure The 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
- 49. Primary standardization ofreagentPlace36mlofdrymethanol intitrationvessel Add KFR & 150- 350gnm of sodium tartarate TitratewithKFR Determine endpoint using Pt electrode Determineaccuratevolume ofKFRrequired Calculate Water equivalent factor F = wt in mg of Sodium tartarate X 0.1566 ml of KFR
- 50. Test for sample %Water Content = Vol of KFR X Water Eq factor Wt of Sample
- 51. Determination of nitrogen Thetwo most commonly used methods for the estimation of nitrogen are: • Duma’s Method, • Kjeldhal Method
- 52. conclusion Finally it canbe 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.
- 53. References: 1.http://www.aquaculture.ugent.be/Education/coursematerial/online%20courses/ ATA/analysis/carb-mon.html 2. Hedge, JE 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#toc72531 4.http://csb.stanford.edu/class/public/readings/Molecular_Architecture_I_Lecture 2/Voet_and_Voet_BOOK_00_Chapter6_Protein_Structure.pdf 5.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.html 9.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.