Carbohydrates—Sugars Carbohydrates are polyhydroxy aldehydes, polyhydroxy ketones, or compounds which can be hydrolyzed to them. Carbohydrates include sugars, starches, and cellulose. In this experiment, different reactions are used to distinguish different classes of sugars, and also, glucose pentaacetate, a derivative of glucose, is synthesized. Sugars are water-soluble carbohydrates. A sugar which cannot be hydrolytically converted into simpler carbohydrates is called a monosaccharide. A sugar, which can be hydrolyzed into two equivalents of monosaccharide, is called a disaccharide. Depending on the functional group it bears, a monosaccharide can be classified as an aldose, if it contains an aldehyde group, or a ketose, if it contains a keto group. Depending on the number or carbons it has, a monosaccharide can also be classified as: e.g., a triose (three carbons), a tetrose (four carbons), a pentose (five carbons), a hexose (six carbons), etc. For example arabinose, bearing an aldehyde terminating group on a five carbon chain, is an aldopentose, and fructose, containing a keto group on a chain of six carbons, is a ketohexose. CHO CH 2 OH HO OH O H OH OH HO H H OH CH 2 OH CH 2 OH D-Arabinose D-Fructose Disaccharides are made up of two monosaccharide units bound through glycoside linkage to each other. Sucrose, table sugar, is a disaccharide which, on hydrolysis, gives two different monosaccharides: glucose and fructose. HOH2 C Sucrose O HO OH OH O OH HOH2 C CH 2OH O OH Test to distinguish monosaccharides from disaccharides—Barfoed’s test The reagent is cupric acetate in acetic solution. Monosaccharides react with this reagent within 5 minutes to give a red precipitate, cuprous oxide. Disaccharides, however, take a longer time to react since the aldehyde function is masked as an acetal. R O C H + 2 Cu2+ blue R O C OH + Cu 2O red ppt Test to distinguish ketoses from aldoses-Seliwanoff’s test Under acidic conditions, ketoses dehydrate very rapidly to give furfural which then reacts with resorcinol (1,3-dihydroxybenzene) to give colored product (Equation 1). CH 2 OH O R H OH H H OH H OH H HO O C H 2 OH H OH OH R H+ H -H 2 O H HO O CH O H OH H H+ O R CHO -2 H 2 O CH 2 OH R = H ; fu rfu ra l R = C H 2 OH ; 5 -h yd ro xy me thy lfu rfu ral R = H ; k e to p en to se R = C H 2 OH ; k eto h ex os e HO O R CHO OH resorcinol red colored product However, under the same conditions, the formation of furfural from aldoses is slow, presumably since a β-elimination is required in the dehydration to furfural (Equation 2). CHO CH O H C OH H C OH H C OH -H 2 O H C OH C O CH 2 H C ! -El im H C OH CH O C H 2 OH H C OH H C OH H C OH H C OH CH 2 OH CH 2 OH R H O H HO CHO H+ OH R O CH O -2 H 2 O R = H ; a ld o pe n to s e R = C H 2 OH ; al d oh e xo se Test to distinguish pentoses from hexoses-Bial’s test The reagent for the Bial’s test is a solution of orcinol 5-methyl-1, 3-benzenediol (5methylresorcinol) and ferric chloride in concentrated hydrochloric acid. As shown in equations (1) and (2), under acidic conditions, pentoses undergo dehydration to give oxo-2, 4-cyclopentadiene-2carboxaldehyde (furfural) while hexoses give oxo-2, 4-cyclopentadiene-5-hydromethyl-2-carboxaldehyde (5-hydroxymethylfurfural). Furfural reacts with orcinol in the presence of ferric chloride to give a blue-togreen color. 5-Hydroxymethylfurfural reacts with orcinol in the presence of ferric chloride to give a brown-to-grey color. Test to distinguish reducing sugars from non-reducing sugars-Benedict’s test or Fehling’s test Aldehydes and α-hydroxy ketones reduce Benedict’s solution (an alkaline solution of cupric ion complexed with citrate ion), or Fehling’s solution (an alkaline solution or cupric ion complexed with tartrate ion). R O C H + 2 Cu2+ blue R O C OH + Cu 2O red ppt Sugars containing these groups are known as reducing sugars. All monosaccharides are reducing sugars. Disaccharides containing a free hemi-acetal group are also mild reducing agents. Most disaccharides are reducing sugars (an important exception is sucrose). O OH H OH 2 C Fre e He m i-A ce tal O HO Su c ros e OH O OH C H 2 OH OH O H OH 2 C OH Acetylation of glucose-preparation of glucose pentaacetate In solution, sugars exist in more than one form. Often, the open straight chain form and a cyclic hemiacetal form (six-membered ring or five-membered ring) are the major isomers present. In forming the cyclic isomer, the carbonyl carbon becomes a chiral center. As a result, two stereoisomeric configurations (anomers) are formed, the α-anomer and the β-anomer. The anomer in which the -OH group at C-1 is below the ring (down) in the Haworth projection, is called α. The anomer in which the OH group at C-1 is above the ring (up) in the Haworth projection is called β. 6 CH 2 OH 1 CHO H HO H H 5 OH H OH OH 6 CH 2 OH 4 O OH OH 3 1 2 OH Haworth Projection of !-D-glucopyranose OH 6 CH 2 OH 5 4 D-Glucose O OH OH OH 3 1 Haworth Projection of "-D-glucopyranose 2 OH Glucose pentaacetate can be prepared by acetylation of glucose with acetic anhydride. In the presence of an acidic catalyst (for example, zinc chloride), the preferred product is the α-anomer. In the presence of a basic catalyst (such as sodium acetate), the β-anomer predominates. 1 CHO H OH HO H H H OH OH 6 CH 2 OH D-Glucose 6 CH2 OAc (CH 3 CO)2 O Catalyst 5 4 AcO O OAc 3 OAc 1 2 OAc H 3C O C O O C CH3 Acetic Anhydride (Ac 2 O) O C CH 3 Ac Experimental Procedure: (Timing: one 3 h period, do part I while heating the reaction mixture in part II) Put on your safety goggles I. Test for Sugars: Prepare a beaker of boiling water for this experiment. Perform the following tests for the following 1% sugar solutions: arabinose; fructose; glucose; lactose; maltose; sucrose, and an unknown obtained from your instructor. A. Barfoed’s Test Place 1 mL of Barfoed’s reagent1 and 10 drops of each 1% sugar solutions in separate, labeled test tubes. Heat the tubes in boiling water for 5 min. Remove the tubes and observe the results. The presence of a red precipitate (cuprous oxide) indicates a positive test for monosaccharides (then do tests B, D, E for these sugars). Disaccharides give negative test (then do tests B and C for these sugars). B. Benedict’s Test Place 1 ml of 1% sugar solutions in labeled test tubes and add 5 ml of Benedict’s reagent4. Heat the tubes in boiling water for several min. A red precipitate is a positive test for reducing sugar. C. Hydrolysis of a Disaccharide Place 10 ml of the above 1% disaccharide solution in a large test tube. Add 1 mL of 3 N HCl, and heat the solution in boiling water for 10 min. Carefully neutralize the solution with 3 N NaOH to give the hydrolysis products (presumably monosaccharides). Then use the product of this step to do tests D and E. D. Seliwanoff’s Test Place 10 drops of 1% sugar solutions in labeled test tubes and add 2 mL of Seliwanoff’s reagent2. Heat the tubes in boiling water for 2 min. Ketohexoses give red solutions and ketopentoses give bluegreen solutions. Aldoses provide no color. E. Bial’s Test Place 10 drops of 1% sugar solutions in labeled test tubes. Add 2 mL of Bial’s reagent3 and heat the tubes in boiling water until a color appears. Pentoses give a blue-to-green color solution. Hexoses give a brown-to-grey color solution. If the color is not distinct, add 4 mL water and 1 mL of 1-butanol. Shake the tube vigorously and observe the result. The colored product should be in the 1-butonal layer. F. Unknown Sugar Sample Depending on the results for the known sugars and comparing them to the results for the unknown, determine which sugar the unknown is. Flow Chart Form of Directions: Perform Test A (Barfoed’s) on all sugars If sugar tests positive (red precipitate) perform the following: Perform Tests B (Benedict’s), D (Seliwanoff’s), E (Bial’s) If sugar tests negative follow these steps: Perform Test B (Benedict’s) on original 1% sugar solution. Do Step C (Hydrolisis of a Disaccharide) and use product for the following: Perform Test D (Seliwanoff’s). Perform Test E (Bial’s). Results from Part I: Sugar solution A. Barfoed’s test B. Benedict’s test C. Bial’s test D. Seliwanoff’s Conclusion test (e.g. aldopentose) 1. 2. 3. 4. 5. 6. 7. Note 1 2 3 4 Barfoed’s reagent: Mix 33.3 g of cupric acetate with 5 mL of acetic acid, and then dilute to 500 mL. Seliwanoff’s reagent: Dissolve 0.25 g of resorcinol in 500 mL of 6 N hydrochloric acid (1 volume of concentrated HCl and 1 volume of distilled water). Bial’s reagent: Dissolve 1.5 g of orcinol in 500 mL of concentrated hydrochloric acid, and then add 2 mL of 10% aqueous ferric chloride. Benedict’s reagent: Dissolve 88 g of hydrated sodium citrate and 50 g of anhydrous sodium carbonate in 400 mL of distilled water with heating. Add 8.8 g of cupric sulfate crystals dissolved in 50 mL of distilled water. Dilute the mixture to 500 mL. The solution should be clear, if not filter it. II. Acetylation of Glucose (do part 1 or 2) CAUTION: Acetic anhydride is a lachrymator and should be measured under the hood on a bicarbonate tray. Cap the bottle immediately after use. All glassware should be dry. 1. Preparation of α-Glucose Pentaacetate In a porcelain mortar, grind a small amount of anhydrous ZnCl2.5 Place 0.5 g of the powdered zinc chloride in a 100 mL round-bottomed flask. Add 14 mL (15 g, 0.15 mol) of acetic anhydride. Attach an air-cooled condenser to the flask, and heat gently. Slowly add 2.7 g (0.015 mol) of powdered anhydrous glucose. Since the reaction is vigorous, do not add all the glucose in one portion. Remove the condenser before addition, and replace the condenser and swirl the reaction mixture between additions. After all the glucose is added, put the condenser in place, and heat the mixture for one hour. Then, slowly pour the reaction mixture, with vigorous stirring, into a beaker containing 150 mL of crushed ice. Stir until all the ice has melted, and the oil which separates solidifies. Collect the product, α-glucose pentaacetate, by suction filtration. The product can be recrystallized form about 15 ml of methanol. (α-glucose pentaacetate has mp 100° C) 2. Preparation of β-Glucose Pentaacetate In a porcelain mortar, grind and mix 2.7 g (0.015 mol) of anhydrous glucose and 1.5 g of sodium acetate. Transfer the mixture to a 100 mL round-bottomed flask, and add 14 mL (15 g, 0.15 mol) of acetic anhydride. Attach a condenser to it, and heat the mixture on a steam bath for one hour. Swirl the reaction mixture frequently during the heating period. Then, slowly pour the mixture, with vigorous stirring, into a beaker containing 150 mL of crushed ice. Stir until all the ice has melted. Collect the product with suction filtration. The product, β-glucose pentaacetate, can be recrystallized from about 15 mL of methanol. (βglucose pentaacetate mp 134° C). Note: Zinc chloride reacts with moisture in the atmosphere. Minimize its exposure to atmosphere by carrying this out quickly. Questions for Report: 1. How useful are the following reagents in classifying an unknown sugar? Barfoed’s reagent; Seliwanoff’s reagent; Bial’s reagent; Benedict’s reagent. 2. What is a reducing sugar? 3. Define and illustrate: a. Disaccharide b. Monosaccharide c. Ketohexose d. Anomer 4. What is acetylation? Name two acetylating reagents. 5. In the preparation of glucose pentaacetate, the warm reaction mixture was poured, with stirring, into 125 ml of ice in a beaker. What is the purpose of this step?