Proximate analysis of Macro Nutrients

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

Transcript of Proximate analysis of Macro Nutrients

  • PROXIMATE ANALYSIS OF MACRONUTRIENTS Presented by Y. Divya M.Pharm I-II Sem Pharmaceutical Analysis SPMVV
  • 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
  • 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 #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
  • 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 unit Disaccharide: Two sugar unit Oligosaccharide: 3-10 sugar unit Polysaccharide: More than 10 units Chaining 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 vacuum Ground to fine powder Defatted by 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
  • 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 Method Gravimetric- Munson & Walker Method Colorimetric- Anthrone Method Enzymatic Methods Physical Methods Polarimetry Refractive Index Density Infra Red Immuno 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 Fehlings Test 1ml of test soln + 1ml of Fehlings reagent, heat on BWB for 5 mins. Reddish Brown ppt Benedicts Test 1ml of test soln + 1ml of Benedicts reagent, heat on BWB for 5 mins. Reddish Brown ppt Barfoeds Test 1ml of test soln + 2ml of Barfoeds 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 Seliwanoffs 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)
  • LANE EYNONMETHOD: 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 Felhings 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:
  • GRAVIMETRIC METHOD Determines 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 METHOD Colorimetric 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 tubes 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
  • ANALYSIS OF POLYSACCHARIDES Starch is the 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
  • 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
  • ANALYSIS OF FIBERSDietaryFiber Cell Wall 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
  • METHOD OF ANALYSIS FOR 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
  • 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 Hopkins Test Aldehyde Test Phenol Reagent Test ANALYSIS OF PROTEINS