San Pedro College  Biochemistry LaboratoryCustomize Laboratory Manual       (Activity 1-4)   Group 6- BMLS 2F Abundo, Simo...
ACTIVITY 1A & 1B                     SUBCELLULAR COMPONENTS OF THE LIVING CELL               QUALITATIVE TEST FOR THE CHEM...
Cont. sup 2                                                     Sediment 3                        Supernate 3             ...
Test for lipids                                                                Acrolein testSudan test   To 5 drops of the...
Biuret test- reduction of copper ions which then complexes with nitrogen atoms on      peptide bonds at high pH      Xanth...
Xanthoproteic test:    From left: sediment 1, sediment 2,    supernate 3, sediment 3    Sudan test:    From left: sediment...
Activity 2A                     Qualitative Test for Proteins/ Color Reaction TestI. OBJECTIVES       To detect proteins t...
III. RESULTS AND OBSERVATIONSQualitative test           Egg Albumin Solution                           Expected           ...
V. DOCUMENTATIONPreparation of Egg Albumin solutionTest results. From left: Sakaguchi test, Ninhydrin test, Xanthoproteic ...
ACTIVITY 2B                                PRECIPITATION TEST FOR PROTEINSI. OBJECTIVES        To observe the reactivity o...
E. Coagulation of Heat                                     Heat to Boiling 5ml of Albumin Solution                        ...
IV. ANALYSIS AND CONCLUSION   Guide Questions:1. Give the principles involved in each of the tests.    A. Precipitation re...
Note color change       Proteins will turn the solution into pink-purpleSchematic Diagram:                           To a ...
Precipitation by Alkaloidal reagent     From left: phospotungstic acid,     Picric acid, Tannic acid, TCA      Hopkin’s-Co...
Activity 3A                                            EnzymesI. OBJECTIVES       To be able to know the properties of an ...
Preparation of dilute solution of salivary amylase                                  Rinse the collector’s mouth           ...
III. RESULTS AND OBSERVATIONS                                  Result                            Expected ResultBiuret tes...
Enzymes are normally very specific, catalyzing at most only a few types of chemical reactions.The enzyme amylase acts on t...
Test for specificity of enzymeTEST TUBE 1 (cooked starch) TEST TUBE 2 (1% GLYCOGEN)                                 19
ACTIVITY 3B                                 FACTORS AFFECTING ENZYME ACTIONI. OBJECTIVES        To completely perform test...
III. RESULTS AND OBSERVATIONS Effect of Temperature:Temperature            15mins                30mins                45m...
IV. ANALYSIS AND CONCLUSION        Guide Question:1. Explain the mechanism of enzyme action at varying temperatures. Give ...
Activity 4                                               Nucleic Acid    I. OBJECTIVES             To be able to prepare n...
A.               Test for Purine1 ml solution + 1 ml       1 ml of water + 1      2N HCL                  ml 2N HCL       ...
III. RESULTS AND OBSERVATIONS    Test                          Experimental Result                 Expected Result    Test...
orcinol test for pentoses -Deoxyribose (contained in DNA) is converted       intohydroxylewulinic aldehyde when heated in ...
V. DOCUMENTATIONPreparation of nucleic acids (after the1st centrifugation)        The final test solutions (supernate)    ...
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Activity 1-4 Customozed Lab Manual

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Activity 1-4 Customozed Lab Manual

  1. 1. San Pedro College Biochemistry LaboratoryCustomize Laboratory Manual (Activity 1-4) Group 6- BMLS 2F Abundo, Simon Jonson Halog, John Anthony Canakan, Faraida R. Doplayna, ZipporahParagele, Kimberly Claire Tanseco, Karen 1
  2. 2. ACTIVITY 1A & 1B SUBCELLULAR COMPONENTS OF THE LIVING CELL QUALITATIVE TEST FOR THE CHEMICAL COMPONENT OF THE CELL I. OBJECTIVES: To determine the composition of organelles. To understand and appreciate the different biochemical systems through separating the subcellular components of a cell. To be able to perform different qualitative tests specific for carbohydrates, proteins, and lipids. To compare the relative amounts of carbohydrates, proteins, and lipids. II. SCHEMATIC DIAGRAM (1A): Wash off blood from chicken liver using a few drops of 5% TCA (trichloroacetic acid). Weigh the liver. Add 5 mL of suspending medium per gram of liver. Homogenize at low speed for 5-10 min. Centrifuge for 10 min. and decant. Sediment 1 Supernate 1Add 10 mL of 5% TCA and Centrifuge for 10mix. Test for carbohydrates, min. and decant.proteins, and lipids. Sediment 2 Supernate 2 Add 10 mL of 5% TCA and Centrifuge for 10 mix. Test for min. and decant. carbohydrates, proteins, and lipids. 2
  3. 3. Cont. sup 2 Sediment 3 Supernate 3 Add 10 mL of 5% TCA and Add 10 mL of 5% TCA and mix. Test for mix. Test for carbohydrates, proteins, carbohydrates, proteins, and lipids. and lipids.SCHEMATIC DIAGRAM (1B):Test for carbohydratesMolisch Test Benedict’s test To 5 drops of To 1 mL of Benedict’s sediment/supernate, add solution, add 2 drops of 5 drops Molisch reagent. the sediment/supernate. Layer with 1 mL of conc. Shake sulphuric acid. Perform Boil for 2 min. and cool. . control test using 1% ribose solution.Test for proteinsBiuret test Xanthoproteic test To 5 drops of the Place 1 mL of the sediment/supernate, add 5 sediment/supernate in a test drops of 10% NaOH and 1 tube. drop of .5% copper sulphate. Add 1 mL of conc. Nitric acid. Perform control test using 1% albumin. Heat and observe colour change. Cool and add 5 drops of ammonium hydroxide. 3
  4. 4. Test for lipids Acrolein testSudan test To 5 drops of the Place .5 g. Of sediment/supernate, potassium bisulphate add 5 drops of Sudan in a clean, dry test 4. tube. Perform control test Add a drop of the using 1% lecithin. sediment/supernate and heat. Note the odor produced.III.RESULTS AND OBSERVATION TESTS CONTROL EXPECTED SEDIMENT SEDIMENT SEDIMENT SEDIMENT RESULTS 1 2 3 4CARBOHYDRATES 1% RIBOSE MOLISCH PURPLE PURPLE - - - - BENEDICT’S BRICK - - - - REDPROTEINS 1% ALBUMIN BIURET VIOLET + - - - XANTHOPROTEIC YELLOW + - - -LIPIDS 1% LECITHIN SUDAN RED RED - - + - ACROLEIN ACRID + + - + ODORIV. ANALYSIS AND CONCLUSION Guide Questions: 1. Give the principle involved in each of the test used to determine the chemical components of the organelles. Molisch test- dehydration of the carbohydrate by sulphuric acid to produce an aldehyde Benedict’s test- reduction of copper ions by reducing sugars, which then can be detected by the formation of a brick red precipitate of cuprous oxide 4
  5. 5. Biuret test- reduction of copper ions which then complexes with nitrogen atoms on peptide bonds at high pH Xanthoproteic test- nitration of benzene ring present in the structure Sudan test- ability of fat cells to selectively absorb pigments in fat dyes such as Sudan IV Acrolein test- Whenever fat is heated in the presence of a dehydrating agent, fat molecules shed glycerol in the form of the unsaturated aldehyde- acrolein. 2. Explain the principle involved in the separation scheme for the subcellular components. The principle behind the separation scheme is differential centrifugation. Tissues or cells are first homogenized to release their internal contents. Larger particles settle faster than smaller one and this provides the basis for obtaining crude organelle fractions by differential centrifugation.V. DOCUMENTATION Molisch test: From left: sediment 1, sediment 2, supernate 3, sediment 3, control Benedict’s test From left: sediment 1, sediment 2, supernate 3, sediment 3 : Biuret test: From left: sediment 1, sediment 2, 5 supernate 3, sediment 3
  6. 6. Xanthoproteic test: From left: sediment 1, sediment 2, supernate 3, sediment 3 Sudan test: From left: sediment 1, sediment 2, supernate 3, sediment 3, control Acrolein test: From left: sediment 1, sediment 2, supernate 3, sediment 36
  7. 7. Activity 2A Qualitative Test for Proteins/ Color Reaction TestI. OBJECTIVES To detect proteins through the different color reaction tests. To know the significance of peptide bonds and amino acids in detecting proteins. To understand the principle behind every test.II. SCHEMATIC DIAGRAM 7
  8. 8. III. RESULTS AND OBSERVATIONSQualitative test Egg Albumin Solution Expected Results (+)Millon’s test Not performed Not performed Brick redXanthoproteic test + yellow yellowHopkins_cole test + VioletBiuret Test + violetNinhydrin test + Blue-violetSakaguchi test + redIV. ANALYSIS AND CONCLUSION Guide Questions: 1. Explain the principle involved in each of the test Millon’s Test- Millons reagent (Hg/HNO3) gives positive results with proteins containing the phenolic amino acid “tyrosine’’, the precipitate turns flesh to red color, if proteins containing tyrosine are present. Xanthoproteic Test- determined the amount of protein soluble in a solution using conc. nitric acid. The test gives a positive result in those proteins w/ amino acids carrying aromatic groups, esp. tyrosine. The proof is neutralized w/ an alkali, turning dark yellow. Hopkin’s Cole Test- There was a violet color produced at the point of contact. Tryptophan has an indole nucleus which is responsible for the violet ring found at the junction between the two layers. Biuret Test- Reduction of Cu2+ which then complexes with the N atoms on the peptide bonds at high pH. Ninhydrin Test- Amino Acid containing a free amino group and a free carboxylic acid group that react together with ninhydrin to produce colored products. Group on the alpha-carbon and can react with ninhydrin to produce blue purple product. Sakaguchi Test- In alkaline solution, protein containing Arginine gives red color with Alpha-Napthol and sodium hypochloride. 8
  9. 9. V. DOCUMENTATIONPreparation of Egg Albumin solutionTest results. From left: Sakaguchi test, Ninhydrin test, Xanthoproteic test, Hopkins –cole test,Biuret test 9
  10. 10. ACTIVITY 2B PRECIPITATION TEST FOR PROTEINSI. OBJECTIVES To observe the reactivity of reagents & solutions in each test To know the practical reactions in each test To Identify and observe the precipitate in each test which yield a positive result.II. II. SCHEMATIC DIAGRAM A. Precipitation Reaction by Concentrated Mineral and Organic Acids Place 1ml of Egg Albumin in each of the 4 test tubes Place 1ml of Egg Albumin in each of the 4 test tubes Add 5 drops of Add 5 drops of Conc. Add 5 drops of Nitric Add 5 drops Glacial Conc. Sulfuric HCl Acid HAc Acid Add 5 Precipitation by Metallic Salts B. drops of Conc. Place 1ml of Egg Albumin in each of the 5 test tubes Sulfuric Acid Place 1ml of Egg Albumin in each of the 4 test tubesAdd 1ml of Dil. Add 1ml Dil. Add 1ml Dil. Add 1ml Dil. Add 1ml Dil.Mercuric Acid Lead Acetate Copper Sulfate Ferric Chloride Barium Chloride C. Precipitation of Alkaloidal Reagents Place 1ml of Egg Albumin in each of the 4 test tubes Place 1ml of Egg Albumin in each of the 4 test tubes -Add 1ml of - Add 1ml of -Add 1ml of -Add 1ml of Picric Acid Sol’n TAC Acid Tannic Acid Phosphotungstic -Heat -Heat -Heat Acid -Heat D. Precipitation by Alcohol Place in each of the 3 test tubes 5ml of 95% Alcohol Place 1ml of Egg Albumin in each of the 4 test tubes -Add 2 drops -Add 2 drops -Add 3 drops of of Dil. HCl of 10% NaOH Egg Albumin -Add 3 drops of -Add 3 drops of Egg Albumin Egg Albumin 10
  11. 11. E. Coagulation of Heat Heat to Boiling 5ml of Albumin Solution Add 2 Drops of Acetic Acid F. Test for Denatured/Coagulated Proteins Perform the following tests; a) Milllon’s Test Suspend each precipitate Place 3ml of each test tube b) Xanthoproteic Test in 10 ml of Distilled Water c) Biuret Test d) Hopkins-Cole TestIII. Results and Observation:C. Precipitation by Alkaloidal Reagents Chemicals Results and ObservationsPicric Acid (+) Yellowish Sol’n/PrecipitateTrichloroacetic Acid (+) Whitish Sol’n/PrecipitateTannic Acid (+) Brownish Sol’n/PrecipitatePhosphotungstic Acid (+) Whitish Sol’n/PrecipitateD. Precipitation by Alcohol Results and Observations95% Ethyl Alcohol+Dil.HCl (-) Cloudy Solution/Precipitate95% Ethyl Alcohol+10%NaOH (-) Clear Solution/Precipitate95% Ethyl Alcohol (+) Whitish, Cloudy Sol’n/PrecipitateE. Coagulation by Heat Results and Observations5mL albumin sol’n + HAC (+) White Cloudy PrecipitateF. Test for Denatured/Coagulated ProteinsSuspended Millon’s Test Xanthoproteic Test Biuret Test Hopkins-Cole TestprecipitatePicric acid Not (+) (-) (-) performedTrichloroacetic (-) (-) (-)acidTannic acid (-) (+) (-)Phosphotunstic (-) (-) (-)acid 11
  12. 12. IV. ANALYSIS AND CONCLUSION Guide Questions:1. Give the principles involved in each of the tests. A. Precipitation reaction by concentrated mineral and organic acid Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect. As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. B. Precipitation by metallic salts Proteins are precipitated by salts of heavy metals, such as mercuric chloride, zinc sulfate,etc. in weak alkaline solutions, protein molecules carry negative charges and combine with positively charged metal ions to form insoluble salts which precipitate from the solution. The precipitated proteins aredenatured and this process is irreversible. C. Precipitation by alkaloidal reagents Alkaloidal reagents (e.g. tannic acid &trichloroacetatic acid) are high molecular weight anions. The negative charge of these anions counteracts the positive charge of the amino group in proteins giving a precipitate. D. Precipitation by Alcohol Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains. Where does the max. precipitation occur?3rd tube. E. Coagulation by Heat The result was coagulation of the albumin solution. Egg-white is faintly alkaline. Complete precipitation takes place only in faintly acid solution. The temperature at which coagulation takes place depends to a large extent on the amount of acid and of salts present F. Test for Denatured/Coagulated Proteins The term denaturation is used more frequently than coagulation by scientific investigators at the present time to denote certain changes in proteins. Definite characteristics of the proteins are changed when they are coagulated, among which is loss of solubility in water and dilute salt solutions. In some instances and under certain conditions the coagulation process may be reversible. Manner in which denaturation may be brought about. Coagulation of proteins may be brought about by a variety of processes. But in addition to heat the action of acids, alkalies, salts, alcohol, mechanical agitation, radiation, and ultra-sonic vibrations may denature the protein and convert it from a soluble into insoluble form weight anions. The negative charge of these anions counteracts the positive charge of the amino group in proteins giving a precipitate. Therefore, even a protein is denatured it will still give a positive result from the Qualitative of proteins.2-3. Using milk as test solution, how will you prove that milk is a protein? Show a schematicdiagram to arrive at your answer. Biuret solution is used to identify the presence of protein. Biuret reagent is a blue solution that, when it reacts with protein, will change color to pink-purple.Procedure: To a test tube, add 40 drops of milk solution Add 3 drops of Biuret reagent Shake gently to mix 12
  13. 13. Note color change Proteins will turn the solution into pink-purpleSchematic Diagram: To a test tube, add 40 drops of milk sol’n Add 3 drops of Biuret reagent (sodium hydroxide 10% & copper sulfate 0.5%) Shake gently to mix Change in color of sol’n to pink-purple confirms presence of proteinV. DOCUMENTATION Preparation of Egg Albumin solution Precipitation by Alcohol From left: NaOh, HCL, neutral 13
  14. 14. Precipitation by Alkaloidal reagent From left: phospotungstic acid, Picric acid, Tannic acid, TCA Hopkin’s-Cole Test From left: Tannic acid, TCA, Picric acid, Phosphotungstic acid Biuret Test From left: Picric acid, Tannic acid, TCA, Phosphotungstic acid Xanthroproteic Test From left: Picric acid, Phosphotungstic acid, TCA, tannic acid14
  15. 15. Activity 3A EnzymesI. OBJECTIVES To be able to know the properties of an enzyme To be able to test the presence of an enzyme To be able to test the specificity of an enzymeII. SCHEMATIC DIAGRAMPreparation of potato extract f Potato (Peeled) Grate/blendered (100ml of distilled H2O) Let it stand for 10 min and Stir Occasionally Strain (through cheese cloth) Filter (the extract through filter paper) Biuret test Test for catalase activity 5 ml of extract 1 ml of extract Mix 1 ml of 3% H2O2 Add 2 ml 10% NaOH soln Hold a splinter over the mouth of the tube mix. add 2-3 drops of Add 1 ml 0.5% Benzidine Copper sulfate 15
  16. 16. Preparation of dilute solution of salivary amylase Rinse the collector’s mouth several times with water Collect 1 ml of saliva Prepare 1:8 solution of saliva by adding 10 drops (0.25 ml) saliva with 20 ml water Use for testSpecificity of Enzyme Action Test tube 1 Test tube 2 2 ml 0.02 M phosphate buffer 2 ml 0.02 M phosphate buffer (pH 6.7) and 1 ml 0.9% (pH 6.7) and 1 ml 0.9% Sodium chloride sol’n Sodium chloride sol’n Add 1 ml cooked starch and 1 Add 1 ml 1% glycogen and 1 ml salivary amylase sol’n ml salivary amylase sol’n Stand for 15 minutes @ room temperature. Stir the mixture In each test tube place 1 drop of the solution in a spot plate Add 1 drop of Iodine sol’n. observed color produced. REPEAT AT 5 MINUTES 16 INTERVAL FOR 1 HOUR.
  17. 17. III. RESULTS AND OBSERVATIONS Result Expected ResultBiuret test + (violet) violetTest for Catalyse activity -splinter - Pooping sound -benzidine - Blue-green colorationTest for specificity of Enzyme actionTime (minutes) Test tube 1 Test tube 25 + green - No changes in10 + Darker green w/ - color observed purple coloration15 + Green with - darker purple coloration20 + Lesser green and - purple color25 + Presence of granule like subsatnce30 + Lesser green and - purple color35 + Lesser green and purple color40 + Much Lesser - green and purple color45 + green -50 + Lesser green - color55 + Almost orange -60 + Almost orange -IV. ANALYSIS AND CONCLUSION Guide Question: 1. Give the principle involved in Biuret test. Biuret test is based on the ability of Cu (II) ions to form a violet-colored chelate complex with peptide bonds (-CONH-groups) in alkaline conditions. 2. How will you proved the presence of catalase? Show chemical equation. A rapid appearance of sustained gas bubbles is an indication of the presence of a catalase.2 H2O2 → 2 H2O + O23. How will you prove the specific ation of salivary amylase? 17
  18. 18. Enzymes are normally very specific, catalyzing at most only a few types of chemical reactions.The enzyme amylase acts on the substrate starch catalyzing its break down into simple sugars.Amylase will not catalyze the breakdown of the complex sugar sucrose into simple sugars.Indicators can be used to test for the presence of these reactions like iodine solution.in theiodine test, amylose in the starch (straight chain) forms helices where iodine moleculesassemble and forming blue-black color. While amylopectin (branched portion) in glycogen formsmuch shorter helices and iodine are unable to assemble and only forming orange/yellow hue.V. DOCUMENTATION Preparation of potato extract Testing gas evolution by splinter Results for Buiret and catalase test Salivary amylase solution 18
  19. 19. Test for specificity of enzymeTEST TUBE 1 (cooked starch) TEST TUBE 2 (1% GLYCOGEN) 19
  20. 20. ACTIVITY 3B FACTORS AFFECTING ENZYME ACTIONI. OBJECTIVES To completely perform tests regarding the factors affecting enzyme action. To examine and explain the effect of temperature on enzyme. To determine the extent of digestion of enzyme through acid and base solutions with a controlled temperature. To verify the specific pH through the use of pH paper.To compare the obtained product with the standard solution.II. SCHEMATIC DIAGRAM Effect of Temperature: Prepare 3 test tubes each having 5ml. 1% cooked start soln +1ml saliva. Test tube # 1: Water bath in Test tube #2: Water bath in Test tube #3: Water bath in 40ºC. 60ºC. 10ºC. Take a drop after stirring and test Take a drop after stirring and test Take a drop after stirring and test with Iodinesoln. with Iodine soln. with Iodine soln. Perform Iodine test at 15mins interval for 1hr.Effect of pH: Prepare 4 test tubes containing equal amts of egg white. Measure height of coagulation for each test tube Coagulate in hot water bath.Test tube #1: 5ml 2% Test tube #2: 5ml 2% Test tube #3: 5ml Test tube #4: 5ml 2%pepsin + 10 drop 0.4% pepsin + 10 drop 2%pancreatin + 10 drop pancreatin + 10 dropHCl. 0.4% HCl. 0.4% Na2CO3. 0.4% Na2CO3. Determine pH of each sample Water bath test tubes @ 40ºCPerform Biuret test Perform Biuret test Perform Biuret test Perform Biuret testof 1ml supernatant of 1ml supernatant of 1ml supernatantdigestion ofof 1ml supernatant Measure each sampleliquid. liquid. liquid. liquid. 20
  21. 21. III. RESULTS AND OBSERVATIONS Effect of Temperature:Temperature 15mins 30mins 45mins 60mins Result Obtained Expected Color Expected Color Expected Color Expected Color Color Color Color ColorTest Tube #1 Powder Blue black Light Blue black Mossy Blue black Mossy Blue black 15min 30min 45min 60min@40 ºC Blue or Blue or Green or Green or - - +/- +/- something something something something dark in dark in dark in dark in color color color colorTest Tube #2 Dark Dark Dark Dark Orange or@ 60 ºC Blue Orange or Blue Orange or Blue; Orange or Blue; something something something traces something traces light in - - - - light in light in of light in of color color color yellow color yellow color colorTest Tube #3 Dark Blue black Blue Blue black Green Blue black Blue Blue black + + + +@ 10 ºC Blue or or or or something something something something dark in dark in dark in dark in color color color color Effect of pH: Test Tube Measurement Measure pH Color Expected Result (before ment color heating) (after heating) No.1 Pepsin + 31cm 30cm 6 Brown with traces Purple HCl of purple color + No. 2 Pepsin 31cm 29cm 9 Brown with traces Purple + Na2CO3 of purple color + No. 3 31cm 30cm 6 Purple Purple Pancreatin + + HCl No. 4 31cm 30cm 9 Purple Purple Pancreatin + + Na2CO3 No. 5 - - - - Purple + Controlled 21
  22. 22. IV. ANALYSIS AND CONCLUSION Guide Question:1. Explain the mechanism of enzyme action at varying temperatures. Give the principle involved.Being a protein, an enzyme is denatured at high temperature and becomes inactive. Initially asthe temperature increases, the rate of enzymatic reaction also increases but it has a limitation.When the temperature affects the molecular structure of the enzyme it gets inactivated.2. How does different pH affect enzyme action? Explain your results using this principle. DiscussIsoelectric pH of enzyme action.Enzymes are picky with pH levels, as they are with every thing else. They have an optimal levelat which they work the best, and anything above or below that level, their activity begins toslow down until they shut down all together.V. DOCUMENTATION After heating at controlled temperature (from Left to Right): Pancreatin (Na2CO3), Pancreatin(HCl), Pepsin (Na2CO3), Pepsin (HCl) Biuret Test Performed (from Left to Right): Pepsin (HCl), Pepsin (Na2CO3), 0.5% peptone standard, Pancreatin (Na2CO3), Pancreatin(HCl) Left side Spot plate (from top to bottom): 60ºC 15mins, 30mins, 45mins, 60mins. Right side Spot plate upper portion (from left to right):10ºC 15mins, 30mins, 45mins, 60mins. Right side Spot plate lower portion (from left to right): 40ºC 15mins, 30mins, 45mins, 60min. 22
  23. 23. Activity 4 Nucleic Acid I. OBJECTIVES To be able to prepare nucleic acid and perform the different tests for it. To understand the principle behind every test. To be able to chemical reactions behind every principle. II. SCHEMATIC DIAGRAM Preparation of Nucleic Acid Liver Homogenize with 1 volume of water 1 ml homogenate + 5% TCA (stir) Centrifuge (5 minutes) Residue/sediment + alcohol-ether mixture (3:10) Suspend with 7 ml TCA Heat at 90°C (15 mins) Cool and centrifuge Supernatant (make up a volume to 10 ml)Test for Purine Orcinol test for Pentose Orcinol test for Pentose Test for Phosphates 23
  24. 24. A. Test for Purine1 ml solution + 1 ml 1 ml of water + 1 2N HCL ml 2N HCL Place in water bath for 20 minutes 1 ml 2N acetate buffer 10 drops 10 % copper sulfate Heat in a water Place in boiling water bath for a few + 5 drops sat. sodium bath. Heat minutes. bisulfate. Mix. B. C.Orcinol test for Pentose Diphenylalanine test for Deoxyribose 0.5 ml sample + 1 ml water 1 ml sample + 1 ml diphenylalanine reagent 1 ml orcinol reagent Heat (5 minutes) in boiling Heat in water bath. water bath. Cool Cool D. Test for Phosphates I ml sample + 5 drops NHO3 5 drops Ammonium mmolybdenate soln. Heat 24
  25. 25. III. RESULTS AND OBSERVATIONS Test Experimental Result Expected Result Test for Purine Test tube 1 Test tube 2 White/tan flocculent ppt - - Orcinol test for Pentoses - green Diphenylalanine test for blue deoxyribose + Test for phosphates - yellowIV. ANALYSIS AND CONCLUSIONGuide questions:1. Discuss the different test used in the experiment test for purine - The NaHSO3 reduces Cu2+ to Cu+. Interaction of purines with Cu+ results in precipitation of insoluble yellow-white complex. Orcinol test for pentoses- the basis of this test relies on the conversion of pentose sugar, ribose, present in the RNA into furfural in the presence of hot acid. Furfural reacts with orcinol in the presence of ferric chloride to give a green color. The intensity of the color produced depends on the concentrations of HCL, ferric chloride and orcinol, as well as the duration of boiling. Diphenylamine test for deoxyribose- DNA contains deoxyribose. DNA can be identified chemically with the Dische diphenylamine test. The reaction between the Dische reagent and 2-deoxypentose results in the development of a blue color. The reaction depends on the conversion of the pentose to w-hydroxylaevulinic aldehyde which then reacts with diphenylamine to give a a blue colored complex. The intensity of the blue color is proportional to the concentration of DNA. Dische reagent does not react with the ribose sugar in RNA and does not form a blue-colored complex. Test for phosphates- In the test for presence of phosphates for DNA, a yellow precipitate is obtain. The ammonium molybdate solution reacted with the sample which yields yellow crystals, phosphoammoniummolybdate, which is a positive result .2. Show chemical equations. Give the principle. test for purine - The NaHSO3 reduces Cu2+ to Cu+. Interaction of purines with Cu+ results in precipitation of insoluble yellow-white complex. 25
  26. 26. orcinol test for pentoses -Deoxyribose (contained in DNA) is converted intohydroxylewulinic aldehyde when heated in concentrated H2SO4. This aldehyde reacts with diphenylamine forming blue colour complex. Diphenylamine test for deoxyribose- this reactions depends on the conversion of the pentose to w-hydroxylaevulinic aldehyde which then reacts with diphenylalamine to give a blue colored complex. test for phosphates- hydrolysis of pyrophosphate to phosphate forming a yellow precipitateHPO42-(aq) + 12MoO42-(aq) + 3NH4+(aq) + 23H3O+(aq)  (NH4)3[P(Mo3O10)4](yellow, s) 26
  27. 27. V. DOCUMENTATIONPreparation of nucleic acids (after the1st centrifugation) The final test solutions (supernate) Results: Test for Purine Result: Orcinol Test Result:Diphenylalamine Test Result: Test for phosphates 27

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