TEST FOR CARBOHYDRATES Activity 6 — Biochemistry LaboratoryNy i cries sno TEST FOR CARBOHYDRATES Carbohydrates are one of the main types of nutrients. They are the most important source of energy for your body. Your digestive system changes carbohydrates into glucose (blood sugar). Your body uses this sugar for energy for your cells, tissues and organs. It stores any extra sugar in your liver and muscles for when it is needed.A carbohydrate molecules is either a polyhydroxyaldehyde (aldose) or a polyhydroxyketone (ketose). This means it has an aldehyde or ketone and contains several hydroxyl groups per molecule. Plant produce glucose, the building block of starch and cellulose, through the process known as photosynthesis. 6CO, + 6H,O (light energy) ----- > C,H,.0, (glucose) + 60,Carbohydrates are divided into four major classes: Monosaccharides, Disaccharides, Oligosaccharides, and Polysaccharides. When a monosaccharide is treated with a concentrated acid solution, dehydration of the monosaccharide occurs. Disaccharides and polysaccharides are first hydrolyzed by the acid to their constituent monosaccharides. If the molecule is a pentose, the dehydrated product is a furfural while dehydration of a hexose yields hydroxymethylfurfural.Many i ae readily reduced alkaline solution of oxides of metals like copper, bismuth, mercury and silver, due to the presence of potentially free aldehyde or ketone group. Alkaline cupric hydroxide when heated is concerted into black cupric oxide. However, in the presence of a reducing sugar, it is reduced to yellow or red cuprous oxide. The variation in color is dependent upon the size of the particle. Fine precipitate is indicated by yellow color while coarse precipitate by red color. Uric acid and creatinine interfere with the test.Common Tests for Carbohydrates * Molisch’s Test —is a general test for carbohydrate. Compounds that are dehydrated by concentrated H,SO, to form furfural or hydroxymethylfurfural Will react with alpha napthol (in the Molisch’s reagent) to yielda purple condensation product. Although not a specific test for carbohydrates, a negative result is a good evidence of the absence of carbohydrates.Common Tests for Carbohydrates ¢ Benedict’s Test — isa specific test for reducing sugars or the presence of free or potential aldehydes. A positive test is the appearance of a red-orange precipitate Cu,O. However, the size of the particles may make it too orange or yellow or green if there is much deep blue cupric ion unreacted.Common Tests for Carbohydrates ¢ Barfoed’s Test —is atest used to distinguish between monosaccharides and disaccharides. A positive reaction may be only a little dark red precipitate in the bottom of the test tube. If this appears within 2-3 minutes, it indicates a monosaccharide, if it does not appear or show after 10 minutes, disaccharide is indicated.Common Tests for Carbohydrates + Seliwanoff’s Test ~— is a specific test for ketoses. The dehydration of ketohexoses with hot HCl occurs much faster than the dehydration of the eee ae aldohexoses. During the same time interval in which the dehydrated ketohexose reacts with the resorcinol (in the Seliwanoff's reagent) to form a bright red condensation product, the aldohexose yields only a pale pink coloration. This test is useful for distinguishing fructose (a ketohexose) from glucose, mannose and galactose.Common Tests for Carbohydrates + Bial’s Test — isa test for pentoses and nucleotides that contain the pentose sugar. It is based on the observation that furfural which is formed from a oe yields a blue-green compound when treated with orcinol in the presence of ferric ions (in the Bial’s reagent). It is not specific for pentoses since other compounds such as trioses, uronic acids and certain heptoses, will produce blue or green products. Hydroxymethylfurfural (formed from hexoses) will give a yellow-brown condensation product.Common Tests for Carbohydrates * Iodine Test for Starch — starch gives a blue color with iodineObjectives: * To perform test based on the production of furfural/furfural derivatives. * To perform test based on reducing property of sugars.Materials: Testtube Graduated Cylinder Medicine droppers Iron ring Iron stand Beaker Stirring rod Burner Wire gauze Test tube rack Testtube holder Molisch’s reagent Concentrated H2S04 Benedict's reagent Barfoed’sreagent Seliwanoff’s reagent Bial’s reagent Pentanol Dry yeast Starch solution odine in KI 1% solution of glucose, fructose, lactose, sucrose, galactose, Glycogen and riboseProcedureMOLISCH TEST Panay Aart || ciate |] actantene |] tentex | eccntoe [| Suen er aaa ee ee en Bee ee ome Dopintat ]| Opin aa oe fs | ca wevteyretprrs TY 1.¢ Add 3 drops of Molisch reagent to each sample and mix thoroughly ¥eytyF ++ ys ¥ 4. dincine the test tube ata 45°C angle and slowly adé 20 drops of concentrated sulfuric acd (H,S0,). Notice the formation of two layers. "Note: Do not point the Mouth ofthe test tube towards yourself or your classmates. Observe the color produced where the two layers meet syst + te FF YY 11 Record your cbservations. Remember that a purple colori postive result for menasaccharide,BENEDICT’S TEST Zatabelat | "Smears || “Seceace || "Soreacee Teeneace || "Saneacer || "Seneace || "Seneace |] “Seer EE) om |] ee mee caxen || sec thee || ovecen | ostaas20 Zborep im aaa opintot | Bopintet | [ Op into Bepimia |[ owpintar |[ oop inter | [ ove tet |” op inte "eucee con creroe || “Sa ee Sheseen || oes 20 folowing | souers_||_seuzers || setors an a 2.c Add 3 drops of Benedict's solution into each and mix by swirling + ttt +t ts ¥ 2d Put all the test tube at the same time into beaker full of boiling water. Note which solution reacts first.BARFOED’S TEST ib ily afi ELE a a a a a 2 a '3.¢ Add 3 mL of Barfoed’s reagent Mix and place at the same time in a boiling water bath up to 10 minutes. ¥¥ytyt +s Fe F‘SELIWANOFF’S TEST 4a Label all testtube ie ¥ FY of Pepzmte ]f om ¥ zt + i 4,¢ Add 3 ml of Seliwanoff’s reagent and place in a boiling water bath up t + + ts F ‘Add Record your observation.BIAL’S TEST croc TP? E0yF '5.c Add 3 mL of Bial’s reagent ¥yteyt &¢ s+ & + FY 5.4 Carefully, heat the mixture in a flame untd & bolls; then let i cool. the color is not east recognized, add Iml of pentanol and shake. ¥ + Fe ete tt Fy 5.e Record your observation.IODINE’S TEST ¥y¥ytyFt +e ee F ‘6a Label all testtube ¥Y Fy Fy Fy F '6.c Add 1-2 drops of lodine solution to each test tube containing samples. eeee Ey EES {iif none ofthe sample producedadeferert color compared withthe conrolsamples, edd 10 dropsof dled water to each esttube and another drop of edine ston. ¥yytyt+tFttt ‘0g-6kh Shake the test tube wellto ensure sufficient mixing of the sample and soWent. Record your observation. Remember that polysaccharides can combine with iodine to give able, red, violet or purple complex.RESULTSMoliesh’s Test Benedict's Test Barfoeds Test Bials Test Seliwanoff's test Fermentation Todine Test General test for Carbohydrates Test for reducing sugars Test to distinguish Mono and Disaccharides Test for pentoses Specific test for Ketoses Test for production of Carbon dioxide Presence of starch Purple/Violet ring Red Orange> Orange> yellow> green Dark red ppt- Monosaccharides No reaction- Possible for disaccharides Blue-green compound Red-Brown color means: hexoses Bright red condensation Formation of bubbles Deep blue or violet colorGlucose Brick: an Pp ing ne ell Beene s e : Sucrose oe ial a a Cherr solution ey caer Rea + + es = 7 Fructose Brick rea a coer eset Purple Ring wihingmis. Rei brow color : m Galactos + + ae 2 Lactose a te : i a Purple Ring Rib + + e ss is i: ose Za ree pra (ere Purple Ring ana rs Starch + 3 - 2 a Dae nie Purple Ring rs Glycogen + = 7 - a Red Brown Se Purple Ring Dis. H20 E ColorlessNy i cries sno Molisch’s Test vis a general test for carbohydrate. Compounds that are dehydrated by concentrated H,SO, . yyield a purple condensation product. 7 Although not a specific test for carbohydrates, a negative result is a good evidence of the absence of carbohydrates.Ny i cries sno Benedict’s Test > a specific test for reducing sugars or the presence of free or potential aldehydes. > A positive test is the appearance of a red-orange precipitate Cu,O. However, the size of the particles may make it too orange or yellow or green if there is much deep blue cupric ion unreacted.Barfoed’s Test 7a test used to distinguish between monosaccharides and disaccharides. ~(+) a little dark red precipitate in the bottom of the test tube. If this appears within 2-3 minutes, it indicates a monosaccharide, if it does not appear or show after 10 minutes, disaccharide is indicated. rylose glucose fructose cs fructose lactose asec)Seliwanoff’s Test ya specific test for ketoses. ~ Form a bright red condensation product, the aldohexose yields only a pale pink coloration. This test is useful for . distinguishing fructose (a ketohexose) ke from glucose, mannose and galactose.Ny i cries sno Bial’s Test ~a test for pentoses and nucleotides that contain the pentose sugar. 7 It is not specific for pentoses since other compounds such as trioses, uronic acids and certain heptoses, will produce blue or green products. ~ Hydroxymethylfurfural (formed from hexoses) will give a yellow-brown condensation product.lodine Test for Starch ~ starch gives a blue color with iodine » give blue color due to amylose which forms a deep- blue complex , which dissociates into heating. Amylopectin solutions are colored blue-violet or purple.Guide QuestionsWhat are the product formed when each of the following carbohydrates is hydrolyzed? a. Sucrose H rai kev Hq SC. o eo HOH Sucroseb. Lactose CH,OH 0. CH,OH HOH ‘CH2OH OH ects OH O. OH 0. OH OO OH OH + OH OH OH 10 on OH OH OH OH Lactose D-Galactose D-Glucosec. Maltose cu,OH cuO4 cu,04 Qe Ky & maltose wader anesGijon non —o—KAgut HO Starch \ HoH ‘cH, HoH cH,0H 4 Hyon Hoo WW 4 4 4 4 9 NH o NLL Nt a OH ion On OH { orvese CH,OH 4 ,H oH_y HO a OH GlucoseWhy can’t human digest cellulose? ¢ Human doesn’t have enzyme cellulase that breakdown cellulose and cellobiose. ¢ Grazing animals (horses, cows, sheep, cattle, etc.) lack the enzymes, however, their intestinal tracts contain bacteria that produce Cellulase; termites also contain the same microorganisms, which enable termites to use wood as their source of foodDa EE Fructose: Glucose ur — om ovcthern, Explain the asi \f ute difference in the Glyceraldehyde Fructose-6-P ‘ise Sf ves ~=metabolism of Fructose-1,6-diP fructose and Moen etonenP glucose in the th ae human body. ‘AestyiCo-a y ‘ Oz Fatty acidsCC Glucose is absorbed and transported qu) directly to the body cells to fuel their Fain Givens metabolism, and to eventually form sap 4 water and carbon dioxide through the tricarboxylic acid (TCA) cycle. It does Glucose-6-P. not undergo any hepatic uptake, and in Glucdse-1-P a state of excessive energy intake, i another pathway is used to store it in Fructogs-64 the form of glycogen, and a third is to Glycogen be converted to fatty acids and Fructose-1,6-diP deposited in fat tissue in the form of triglycerides. When the excessive ingestion of energy becomes chronic, both muscle and fat Dr-hydrony. «+ Glycereldehyde-3-P =» Di-hycrony- acetone-P acetone-P i! cells become insulin resistant and Pyruvate glucose uptake at the periphery becomes less, leading to increased insulin secretion as the cells demand fostvicos more glucose supply. This leads finally to < ., y \ the development of type 2 co, Fatty acids diabetes mellitus.G@tuT2) quuT) orp —_Fiuctose Glucose are — | cress) It Fructose has a low glycemic index (Gl) ADP of only 23, compared to glucose (and neAMP Fryctose-1 Glucose-6-P. used as the standard) which has a Gl of " \t Glucose-1-P 100. Its ingestion is followed by rapid Glyceriidehyde ——-—Fructoge-6-P | absorption which leads to a small ATP It Glycogen _PestPrandial rise in blood glucose. trate However, the liver is the main site of Fructose-1,6-diP fructose metabolism, where fructose is converted to fructose-1-phosphate, Dithydroxy- => Giyceraldshyde-2°? =~ Di-hydroxy- thereby proving that it is not subject to acatone-P phophofructokinase regulation, which is the main rate-limiting step in the metabolism of glucose. In other words, fructose can end up as glucose, fatty acids or as lactate. acetone-PWhich of the following carbohydrate test is the appropriate test to detect the presence of sugar in urine? Explain why. Perform the test two hours after a meal. In the initial stages of the disease, a diabetic does not lose sugar in his urine, when on empty stomach. Hence if the benedict's test is performed in the fasting state, it is possible to miss the diagnosis of the disease. Even if sugar is detected in the urine by benedict's test, the diagnosis of diabetes should be confirmed by blood-analysis.-End of Slide-