Experiment # 4 PDF

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Experiment # 4 Properties of Carbohydrates BACKGROUND Carbohydrates have a carbon, hydrogen, and oxygen atom ratio that approximates that of CH20. The chemical structure is not similar to that of a hydrate. Chemically, carbohydrates are polyhydroxy aldehydes or ketones or substances that when hydrolysed, yield polyhydroxy aldehydes or ketones. Carbohydrates can be simple sugars or relatively complex substances such as starches and cellulose. The simplest carbohydrate is a monosaccharide. Monosaccharides cannot be hydrolysed to simpler carbohydrate molecules. Classification of carbohydrates is based on the number of monosaccharide units present in the molecule. A disaccharide is composed of 2 alike or different monosaccharide molecules. It yields these two monosaccharide molecules when hydrolysed. Oligosaccharides, on hydrolysis, yield 2 to 10 monosaccharide molecules. The monosaccharide molecules may be of only one kind, or they may be of two or more different kinds. Polysaccharides, when hydrolysed, yield many monosaccharide molecules, usually of only one kind. Monosaccharides are further classified according to the length of the carbon chain. Trioses have the chemical formula C3H603, tetroses C4H804, pentoses C5H10O5, and hexoses C6H1206. They can be further classified by the presence of the aldehyde or ketone group. If the carbohydrate contains an aldehyde, it is an aldose; if it contains a ketone, it is called a ketose. Usually, these are added as a prefix, e.g. aldohexose. The most common monosaccharides are glucose, galactose, and fructose. Glucose and galactose are aldohexoses; fructose is a ketohexose. The three most common disaccharides are sucrose (glucose and fructose), maltose (glucose and glucose), and lactose (galactose and glucose). Disaccharides can be hydrolysed to yield their respective monosaccharides by heating in a water solution containing a small amount of HCl or H2S04. The three most common polysaccharides, starch, glycogen, and cellulose, have the formula (C6H1005)x, where x ranges from about 200 to several thousand. All three are polymers of glucose. Many chemical tests have been devised to differentiate one carbohydrate from another. In this lab, a number of different chemical tests will be used to characterize monosaccharides, disaccharides and polysaccharides. This information will then be used to determine the composition of an unknown. The unknown can be a monosaccharide, disaccharide or a polysaccharide. Samples of commercially available polysaccharides such as cellulose will also be tested. A characterization scheme is shown in Figure 1 for this experiment. A discussion of each test is given below. Molisch's Test is a very general test for carbohydrates. The test is based on the formation of furfural, or hydroxyfurfural when a carbohydrate reacts with concentrated sulfuric acid. The furfural reacts with the Molisch reagent, (α-naphthol) to yield coloured condensation products. It will give a positive test for all carbohydrates.

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FIGURE 1 Molisch’s test

Starch, Sucrose, Galactose, Glucose, Fructose, Xylose, Arabinose, Lactose, Maltose

Non Carbohydrates

Benedict’s test (-) Nonreducing Clear Blue

(+) Reducing Brick-Red

Starch, sucrose

Galactose, glucose, fructose, xylose, arabinose, lactose, maltose

Iodine test (+)Purple

Starch

Barfoed’s test

(-) Brown/Orang.

Sucrose

(+) Brick- Red ppt (5 min)

(-) No reaction in 5 minutes (+) Brick- Red ppt in 20 min

Monosaccharide (Galactose, glucose, fructose, xylose, arabinose)

Disaccharide (Lactose, maltose)

Bial’s test (-) Brown/Green

(+) Blue

Hexoses (Galactose, glucose, fructose)

Pentoses (Xylose, arabinose)

Seliwanoff’s test (-) Colorless (1min) Aldohexoses (Galactose, glucose)

(+) Red (1 min) Ketohexoses (Fructose)

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Molisch's Test for arabinose, fructose, glucose, maltose and galactose (from left to right)

Benedict's and Barfoed's tests are both reduction tests. If carbohydrates have a reducing ability they have, or are able to form, a free aldehyde or ketone group in solution. In alkaline solution, copper(II) ions are then reduced to Cu20. All the common monosaccharides are reducing sugars. Some disaccharides and polysaccharides may initially be nonreducing but will show reducing properties after heating in an acidic solution because hydrolysis to their monosaccharides has taken place. Although very similar, results from the two tests will confirm the presence of monosaccharides and distinguish between reducing and nonreducing disaccharides. Benedict's Test shows the reduction of Cu2+ to Cu+, forming Cu20, which is a brick-red coloured precipitate. The colour in a positive Benedict's test may appear as green, yellow, orange, or red depending upon the amount of Cu20 suspended in the originally dark blue coloured reagent. The amount of Cu20 formed depends on the concentration of sugar in the solution. Reducing sugars, either mono- or disaccarides, give a positive test. This is a timed test as continued heating will hydrolyse disaccharides to monosaccharides which will then react with the Cu2+ to give a positive test. Barfoed's Test is used to distinguish between reducing mono- and disaccharides. This test is also a copper reduction reaction but differs from Benedict's in that the reagent is made in an acidic medium [copper(II) acetate and acetic acid]. A positive reaction within the stated time interval of 5 minutes indicates the presence of monosaccharides. Because this ion is weak, there is no positive reaction shown by reducing disaccharides unless they are heated and are hydrolysed by the acid present. A positive reaction after 20 minutes, that is, after hydrolysis has taken place, confirms the presence of the disaccharide. Characterization information is gained by recording observations after 5 minutes and again after 20 minutes.

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Benedict's Test for all nine tested sugars arabinose, fructose, glucose, maltose, galactose, lactose, starch, sucrose and xylose and distilled water (from left to right )

Barfoed's Test for galactose, glucose, fructose and xylose (from left to right) Page 4

Iodine reacts with starch, a polysaccaride of glucose, to form a deep blue/purple complex. When an acidified starch solution is boiled, it hydrolyses to yield oligomers, disaccharides and glucose itself. The iodine test can be used to follow the course of this hydrolysis. As both starch and sucrose will give a negative Benedict’s test, the iodine test can be used to distinguish sucrose from starch.

Iodine test for starch (left) and sucrose (right) Bial's Test is used to differentiate between pentoses and hexoses. Pentoses occur in both plants and animals. The pentoses ribose and deoxyribose are universally found in the nucleic acid portion of nucleoproteins of the cells. Bial's reagent contains orcinol (5-methylresorcinol) dissolved in concentrated HCl plus a small amount of FeCl3. When mixed with the reagent, the pentoses are converted to furfural, which reacts with the FeCl3 to yield a blue coloured compound.

Bial's Test for xylose, arabinose and glucose (from left to right) Page 5

Seliwanoff's Test distinguishes ketohexoses from aldohexoses and disaccharides. The only common ketohexose is fructose. The reaction between fructose and the reagent (resorcinol in dilute HCL) occurs within 1-2 minutes in boiling water. A reddish-coloured product is formed; this colour intensifies with further heating. Other carbohydrates will produce a faint red colour if heating is prolonged. The colour formation follows the transformation of glucose to fructose (by the catalytic action of HCl) or by the hydrolysis of sucrose.

Seliwanoff's Test for fructose, sucrose, glucose and galactose (from left to right) SAFETY PRECAUTIONS - Safety glasses, lab coats and gloves are required at all times. - Use concentrated acids/bases in the fume hood only. - Dispose of test solutions in the labelled containers provided. MATERIALS AND EQUIPMENTS: Test Tubes (~15 cm in height, 11 test tubes/test) Test Tube Rack Graduated Cylinders (10 mL) Hot Plate 500 mL Beaker Distilled water 2% solutions of arabinose, fructose, glucose, maltose, galactose, lactose, starch, sucrose and xylose An unknown carbohydrate solution for EACH group Tap water for water bath

Iodine solution (1% iodine in 2% potassium iodide) Concentrated Sulphuric Acid Benedict’s Reagent Barfoed’s Reagent Seliwanoff’s Reagent Bial’s Reagent Concentrated Hydrochloric Acid

10% Sodium Hydroxide

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Experiment # 4 PROCEDURE 1. Prepare a boiling water bath by placing approximately 200 ml of tap water in a 500 ml beaker on the hot plate. Keep enough water in the beaker through this experiment to heat the test tubes of solutions when required. 2. For each test, set up a test tube rack containing 11 clean test tubes; 9 test tubes for the 2% carbohydrate solutions; 1 test tube for the unknowns and 1 test tube for the distilled water blank (your negative control). Check each test for quantities to be measured out. 3. A droppers have been provided for each of the carbohydrate solutions. Please make sure there is no cross contamination between solutions. 4. When recording results, indicate whether a positive or negative colour change has occurred. For the conclusion, please be specific. For example, the Molisch test is a test for the presence of a carbohydrate. Your conclusion should then be either carbohydrates are present or no carbohydrates are present. Refer back to Figure 1 and your Prelab assignment to help you with your conclusions. a. Molisch Test (You will not perform the Molisch test in the lab) To a set of test tubes (step 2) add 10 drops of each solution. Add 4 drops of Molisch reagent to each tube and mix well. Be careful not to cross contaminate solutions. In the Fume Hood, tilt the test tube at an angle of about 45 degrees and very carefully and slowly pour 1mL of concentrated sulfuric acid from a 10 mL graduated cylinder down the side of the test tube so that the sulfuric acid forms a layer underneath the solution being tested. NOTE: It is very important that the lip of the graduated cylinder be touching the inner top of the test tube containing the carbohydrate and that the acid be poured slowly. Set the test tubes in the rack and observe for evidence of reaction at the interface of the two liquid layers. A purple coloured ring at the interface is a positive result. Record your results. Start performing the experiment # 4 from Benedict’s Test (b) b. Benedict's Test To a set of test tubes (step 2) add 10 drops of each solution. Add 1 mL of Benedict’s reagent to each test tube and mix well. Place all tubes in the boiling water bath for a maximum of 3 minutes. Observe the test tubes and record the results in the data sheets. As soon as a precipitate forms, indicating a positive result, the test tube can be removed from the water bath and results recorded. Note the appearance and colour of any precipitate. A colour change with no precipitate is a negative result.

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c. Barfoed's Test To a set of test tubes (step2) add 10 drops of each solution. Add 1 mL of Barfoed’s reagent to each test tube and mix well. Place the tubes in a beaker of boiling water. Note which samples give a precipitate in 5 minutes. Remove tubes with the precipitate from the water, allow the precipate to settle and record the results. Leave the remaining tubes (without the precipitate) in the boiling water for a total of 20 minutes. Again, record the results in this tubes after 20 min and make a conclusions on the data sheets. d. Iodine Test For this test, set up five test tubes. To each test tube, add 10 drops of glucose, starch, sucrose, distilled water and your unknown. Add 10 drops of iodine solution. A colour change to deep purple is considered to be a positive test.

e. Bial’s Test To a set of test tubes (step 2) add 10 drops of each solution. Add 1 mL of Bial’s reagent to each tube and mix well. Heat in a boiling water bath just until a blue colour develops. Observe and record the results in the data sheet. f. Seliwanoff's Test To a set of test tubes (step 2) add 10 drops of each solution. Add 1 mL of Seliwanoff's reagent to each tube and mix well. Heat the tubes in a boiling bath for maximum 3 min until a red color develops. Record the results. g. Hydrolysis Reactions (You will not perform this part in the lab but you should know the theory of the hydrolysis reactions on Starch, Sucrose and Maltose and Expected results for post-Hydrolysis Benedict reactions on Starch, Sucrose and Maltose. Perform the hydrolysis tests detailed below to determine if the test solutions are polymers and, if so, what are the constituent monomers or monosaccharides. Set up the following mixtures: a. 10 drops of starch solution + 5 drops of concentrated HCl b. 10 drops of sucrose solution + 5 drops of concentrated HCl c. 10 drops of maltose solution + 5 drops of concentrated HCl d. 10 drops of your unknown + 5 drops of concentrated HCl e. 10 drops of distilled water + 5 drops of concentrated HCl Heat the tubes in a boiling water bath for about 5 minutes. Cool the tubes under the stream of cold tab water. Neutralize the solutions with 10% NaOH (use red litmus paper). Withdraw 10 drops of the hydrolyzed starch and test with 10 drops of iodine as described in test (d). Note differences in colour from that observed in test (d). Record the results on the data sheet. Do not test the other solutions with iodine. Repeat the Benedict test using 10 drops of sample of each of the hydrolysed solutions and 1 mL of each test reagent, as described in procedure (b) above. Record your results on the data sheet. Include results from the first Benedict test in the Pre-Hydrolysis column. Page 8

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Iodine test for starch before and after the hydrolysis (from left to right)

Benedict test for starch after and before the hydrolysis (from left to right) Page 10

Experiment # 4 Table 1. Test Results Solution

Molisch

Conclusion

Benedict’s

arabinose

fructose

glucose

maltose

galactose

lactose

starch

sucrose

xylose

control (distilled water) unknown #---------Page 11

Conclusion

Experiment # 4Table 2. Test Results Solution

Barfoed’s (5 min.)

Barfoed’s (20 min.)

Conclusion

arabinose

Iodine

N/A

fructose

N/A

glucose

maltose

N/A

galactose

N/A

lactose

N/A

starch

sucrose

xylose

N/A

control (distilled water) unknown #----------

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Conclusion

Experiment # 4 Table 3. Test Results Solution

Bial’s

Conclusion

Seliwanoff

arabinose

fructose

glucose

maltose

galactose

lactose

starch

sucrose

xylose

control (distilled water) unknown # ______

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Conclusion

Experiment # 4 Table 4. Expected Test Results Solution

Iodine

Conclusion

Pre-Hydrolysis Benedict (from Table 1)

starch

sucrose N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

maltose

unknown # ______ control (distilled water)

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Conclusion

Expected Results for Post-Hydrolysis Benedict

Conclusion

POST-LAB QUESTIONS 1. You are given the following carbohydrates: arabinose, glucose, fructose, maltose, sucrose, and starch. Explain the results described below. (a) A carbohydrate solution (from the list above) gave a positive Molisch test and negative Benedict's, Barfoed's, Bial's, and Seliwanoff's tests. When treated with hydrochloric acid and boiled for several minutes, the solution showed positive Benedict's, Barfoed's, and Seliwanoff’s tests and a negative Bial's test. What carbohydrate was in the original solution? (b) A solution (from the list above) containing only one carbohydrate gave a Cu2O precipitate with Benedict's reagent. Which possible carbohydrates are present in the solution? (c) Another sample of the solution (from Q1b) failed to give a Cu2O precipitate with Barfoed's reagent. Which carbohydrate is present in the solution? 2. Describe how a solution of lactose would react toward these reagents. (a) Benedict's (b) Barfoed's (c) Seliwanoff s 3. What functional groups(s) are present in the reducing carbohydrates? 4. Which of the carbohydrates shown on the picture below is s reducing sugar and why? 5. Why is sucrose a non-reducing sugar? B

ow can you tell when the hydrolyses of starch is complete? Why does the test work this way? What is the monosaccharide that results at the end?

C

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