Proteins,Fats determination


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determination of proteins , fats and moisture in pharmaceutical preparation in pharmaceutical analysis department.

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Proteins,Fats determination

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  2. 2. PROTEINS Introduction IntroductionProteins are large, complex, organic compounds andare composed mostly of amino acids linked withpeptide bonds 2
  3. 3. Proteins differ from each other according to the type,number and sequence of amino acids that make up thepolypeptide backboneProteins are important constituents of foods for anumber of different reasonsThey are a major source of energy, as well as containingessential amino-acids oLeucine,oLysine, oIsoleucine andoTryptophan, oValineo Methionine, 3
  4. 4. •Structures of ProteinsPrimary structureSecondary structure Tertiary structure Quaternary structure 4
  5. 5. Primary structure•It is the linear sequence of amino acids joinedtogether by peptide bond.•It is simple and unfolded structure of polypeptidechains. 5
  6. 6. Secondary structure The primary structure of protein folds to formssecondary structure. It is regular, rigid and tubular Tertiary structure Two or more secondary structure combines toform a tertiary structure. It is a three dimensional folding structure bycompletes folding of the sheets and helices of asecondary structure. 6
  7. 7. Quaternary structureQuaternary structure refers to the wayindividual polypeptides combine to formcomplexes Quaternary structure 7
  8. 8. Amino acid An amino acid is a small organic moleculethat, as the name indicates, contains both anamino component and an acid componentNon-polar aliphatic R-groups 8
  9. 9. Aromatic R-groups Positively charged (= basic) R-groups Negatively charged (= acidic) R-groups 9
  10. 10. Qualitative analysis of Proteins Precipitation reactions Colour Reactions of ProteinsPrecipitation reactionsProtein exist in colloidal solution due tohydration of polar groups (-COO, NH3+, -OH)They can be precipitated by dehydration orneutralization of polar groups. Precipitation by saltsTo 2 ml of protein solution add equalvolume of saturated (NH4)2SO4 solutionWhite precipitation is formed 10
  11. 11. Precipitation by heavy metal saltsTo 2 ml of protein solution, add few drops ofHeavy Metals (lead acetate or mercuricnitrate) solution, results in white precipitationPrecipitation by alkaloidal reagentTo a few ml of sample solution add 1-2 ml ofpicric acid solution. Formation ofprecipitation indicates the presence ofproteins 11
  12. 12. Precipitation by organic solventsTo a few ml of sample solution, add 1 ml ofalcohol. Mix and keep aside for 2 min.Formation of white precipitation indicates thepresence of protein Precipitation by heatTake few ml of protein solution in a test tubeand heat over a flame. Cloudy whiteprecipitation is observed Precipitation by acidsTo 1 ml of protein solution in test tube, add fewdrops of 1% acetic acid, white precipitation isformed 12
  13. 13. Colour Reactions of ProteinsProteins give a number of colour reactionswith different chemical reagents due to thepresence of amino acid Biuret testThe Biuret test is a chemical test used fordetecting the presence of peptide bonds In the presence of peptides, a copper (II)ion forms violet-colored coordination in analkaline solution 13
  14. 14. To 2 ml of protein solution in a test tube add10% of alkaline (NaOH) solution. Mix and add 4-5drops of 0.5% w/v copper sulphite (CuSO4)solutionFormation of Purplish Violet Colour indicatesthe presentation of proteins 14
  15. 15. Xanthoproteic TestTo 2 ml of protein solution add 1 ml conc.HNO 3Heat the solution for about 2 minutes and coolunder tap waterA yellow colour is obtained due to the nitration ofaromatic ringAdd few drops of 40% w/v NaOH solution The yellow colour obtained initially changes toorange 15
  16. 16.  Millon’s TestWhen Millon’s reagent is added to a protein,a white precipitation is formed, which turnbrick red on heatingPhenols and phenolic compounds, whenmixed with Hg(NO3)2 in nitric acid and traces 16
  17. 17. Ninhydrin TestWhen protein is boiled with a dilute solutionof ninhydrin, a violet colour is produced Proteins Hydrolysis Amino acids Amino Acids + NinhydrinKeto acid + NH3 + CO2 + Hydrindantin NH3 + Ninhydrin Pink colour 17
  18. 18. Hopkin- Cole’s TestTo a few ml of protein solution in a test tubeadd few drops of formaldehyde solution(1:500) and 2 drops of HgSO4 (Oxidant)Mix thoroughly and add very gently 2-4 ml ofconc.HgSO4 along the sides of the test tubeThe formation of violet coloured ring at thejunction of the two layers is Observed 18
  19. 19. Aldehyde TestTo 1 ml of protein solution in test tube add fewml of PDAB in H2SO4.Mix the contents and heat if necessary.The formation of purple colour is observed Phenol’s reagent TestTo few ml of protein solution in a test tube add 1ml of NaOH solution (4% w/v) and 5 drops ofphenol’s reagent.The formation of blue coloured solutionObserved 19
  20. 20. Color Reactions of Proteins Test Composition of Reagent + Result (Color) Group Responsible ImportanceNinhydrin Triketohydrin Hydrate Blue or Purple Free amino and free Test for amino acid, peptides COOH in determining amino acidsBiuret NaOH + CuSO4 Violet Peptide linkages + Tripeptides up to proteinMillon’s Hg in HNO3 Red Hydroxyphenyl group + TryptophanXanthoproteic Conc. HNO3 Lemon yellow Benzene ring + Tyrosine, Phenyl alanine, TryptophanHopkins-Cole Glyoxylic acid and conc. Violet ring Indole group + Tryptophan H2SO4Liebermann Conc. HCl , sucrose Violet Indole group + TryptophanErlich’s Diazo Pb(OAc)2 Sullfanilic acid in Red orange – + Histidine and Tyrosine HCl + NH4OH lighter orangeSakaguchi 10% NaOH, ά naphtol, Intense red color Guanidine + Arginine alkaline hypobromiteAcree-Rosenheim HCHO conc. H2SO4 Violet ring Indole group +TryptophanReduced Sulfur KOH, Pb(OAc)2 Black ppt Sulfur + Cystine, Cystein and methionineBr water Br.H2O, amyl alcohol Pink Indole group + TryptophanMolisch ά naphtol in alcoholic Violet ring Carbohydrates Glycoprotein H2SO4Adamskiewez Glacial Acetic acid and Reddish violet Indole group + Tryptophan conc. H2SO4 ring at the junction 20
  21. 21. Quantitative Analysis of Proteins 21
  22. 22. Kjeldahl methodThe Kjeldahl method was developed in 1883by a brewer called Johann Kjeldahl A food is digested with a strong acid sothat it releases nitrogen which can bedetermined by a suitable titration technique.The amount of protein present is thencalculated from the nitrogen concentration ofthe food 22
  23. 23. Kjeldahl method Principles Digestion Neutralization TitrationThe food sample to be analyzed is weighed intoa digestion flask (NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4H3BO3 (boric acid) NH4+ + H2BO3- (borate ion) H+H3BO3 23
  24. 24. Enhanced Dumas method A sample of known mass Combustion (900 oC)CO2, H2O and N2 Nitrogen Thermal conductivity detector The nitrogen content is then measured 24
  25. 25. Methods using UV-visible spectroscopyThese methods use either the natural abilityof proteins to absorb (or scatter) light in theUV-visible region of the electromagneticspectrum, or they chemically or physicallymodify proteins to make them absorb (orscatter) light in this region Principles Direct measurement at 280nm Biuret Method Lowry Method Dye binding methods Turbimetric method 25
  26. 26. Direct measurement at 280nmTryptophan and tyrosine absorb ultravioletlight strongly at 280 nmThe tryptophan and tyrosine content ofmany proteins remains fairly constant, and sothe absorbance of protein solutions at 280nmcan be used to determine their concentration Biuret MethodA violet-purplish color is produced whencupric ions (Cu2+) interact with peptidebonds under alkaline conditions The absorbance is read at 540 nm 26
  27. 27. Lowry MethodThe Lowry method combines the Biuretreagent with another reagent (the Folin-Ciocalteu phenol reagent) which reactswith tyrosine and tryptophan residues inproteins. This gives a bluish color which can be readsomewhere between 500 - 750 nm depending onthe sensitivity required 27
  28. 28. Other Instrumental TechniquesMeasurement of Bulk Physical PropertiesMeasurement of Adsorption of RadiationMeasurement of Scattering of RadiationMethods Based on Different Solubility CharacteristicsSalting outIsoelectric PrecipitationSolvent Fractionation Ion Exchange Chromatography Affinity Chromatography Separation Due to Size DifferencesDialysisUltra-filtration Size Exclusion ChromatographyTwo Dimensional Electrophoresis 28
  29. 29. Amino Acid Analysis Amino acid analysis is used to determinethe amino acid composition of proteins.  A protein sample is first hydrolyzed(e.g. using a strong acid) to release the aminoacids, which are then separated usingchromatography, e.g., ion exchange, affinityor absorption chromatography. 29
  30. 30. FatsLipids can be defined as Esters of Fattyacids and are naturally occurring Lipids consist of numerous fatlike chemicalcompounds that are insoluble in water butsoluble in organic solventsLipid compounds include Monoglycerides,Diglycerides, triglycerides, phosphatides,cerebrosides, sterols, terpenes, fattyalcohols, and fatty acids 30
  31. 31. Types of fats CnH (2n+1) CO2HCnH(2n-1)CO2H 31
  32. 32. Classification ClassificationI. "Simple" Carboxylic esters A. Fats or glycerides (esters of fatty acids with glycerol e.g. acylglycerols) Monoglycerides Diglycerides Triglycerides B. WaxesII. Complex carboxylic esters •Glycerophospholipids •Glycoglycerolipids •Glycoglycerolipid sulfates 32
  33. 33. III. Complex lipids (containing amides) •Sphingolipids •Glycosphingolipids IV. Precursor and derived lipids •Acids (including phosphatidic acid and bile acids) •Alcohols (including sterols) •Bases (Sphinganines, etc.)V. Hydrocarbons •Straight-chain •Simple branched •PolyisoprenoidVI. Lipid vitamins and hormones 33
  34. 34. Qualitative Analysis of Fats1.Solubility test2.Microscopic Properties3.Physical test4.Emultion formation5.Sackowski’s test6.Libermann-Burchrd’s test7.Zak’s reaction 34
  35. 35. 1.Solubility testFew drops of oil in an test tube Few ml of oil sample 1-2 ml of carotene Chloroform BenzeneThe formation of two layers (Insoluble) Results in the soluble solution 35
  36. 36. 1. Microscopic PropertiesLipids appear in white shining chombic shapecrystals 2. Physical test•A little quantity of oil on a filter paper Few minutesThe greasy spot penetrating the filter paper 36
  37. 37. 4. Emultion formation A drop of oil on a watch glass Place carefully 2-3 drops of water over itOil droplet is broken into fine droplets,indicates the process of emulsification 37
  38. 38. 5. Sackowski’s test 2 ml of Organic solution (oil) in Chloroform 3 minutes 2 ml of conc.H2SO4Upper chloroform layer shows red colour andlower H2SO4 layer shows yellow colour 6. Libermann-Burchrd’s test2 ml of Organic solution (oil) in Chloroform(CHCL3) 5-6 drops of Acetic anhydride & 2 drops of conc.H2SO4Rose colour to Bluish Green coloured solution38
  39. 39. 7. Zak’s reaction2 ml of Organic solution in Chloroform (CHCL 3) FeCL3 in Acetic Acid conc. H2SO4 Red coloured solution Quantitative analysis of fats •Saponification value •Iodine value •Hydroxyl value •Acid value 39
  40. 40. Saponification value The number of milligrams of potassium hydroxide required to saponify 1gm of fat under the conditions specified Mass of oilNumber of moles = Relative atomic mass 40
  41. 41. Iodine valueThe mass of iodine in grams that isconsumed by 100 grams of achemical substanceUsed to determine the amount ofunsaturation in fatty acidsThe higher the iodine number, the more C=Cbonds are present in the fat 41
  42. 42. Hydroxyl valueIt is expressed as the mass ofpotassium hydroxide (KOH) inmilligrams equivalent to the hydroxylcontent of one gram of thechemical substance Acid valueThe mass of potassium hydroxide (KOH)in milligrams that is required toneutralize one gram ofchemical substance 42
  43. 43. Water / Moisture Determination Karl Fischer MethodThe Water Determination Test (Karl FischerMethod) is designed to determine watercontent in substances, utilizing thequantitative reaction of water with iodine andsulfur dioxide in the presence of a loweralcohol such as methanol and an organicbase such as pyridine, as shown in thefollowing formulaeH2O+I2+SO2 + 3 C5H5N 2(C5H5N +H) I- + C5H5N + SO3C5H5N + SO3 + CH3OH (C5H5N +H) O- SO2 +OCH3 43
  44. 44. 1.Volumetric titrationIodine required for reaction with water ispreviously dissolved in water determinationTS, and water content is determined bymeasuring the amount of iodine consumed asa result of reaction with water in a sample Volume(ml) of TS for Water determination consumed X f (mg/ml)Water = X 100 Weight of sample (mg) 44
  45. 45. 2. Coulometric Titration 2. Coulometric TitrationFirst, iodine is produced by electrolysis ofthe reagent containing iodide ion, and then,the water content in a sample is determinedby measuring the quantity of electricity whichis required for the electrolysis (i.e., for theproduction of iodine), based on thequantitative reaction of the generated iodinewith water. 45
  46. 46. References:1); 2003.4) Reagents9)O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Measurement withthe Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.10) Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal.Biochem. 163 (1987) 476-481.11) Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal.Biochem. 150 (1985) 76-85.12) Katan MB, Mensink RP, Zock PL. Trans fatty acids and their effect onlipoproteins in humans. Annu Rev Nutr 1995; 15:473-493.13) Firestone D (May-Jun 1994). "Determination of the iodine value of oils and fats:summary of collaborative study". J AOAC Int. 77 (3): 674–6. PMID 801221914)$FILE/B43.pdf 46
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  48. 48. THANKYOU