BIOL 1362 BIOCHEMISTRY I PRACTICAL

LABORATORY EXERCISE #2 CARBOHYDRATES

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Test #1 Acid Hydrolysis of Sucrose

Procedure: 1. Add 10 mL of the 0.1M sucrose solution to a boiling tube (large, thick-walled tube). Add 2 mL 0.1M HCl to the boiling tube. 2. 3. Heat in a boiling water bath for 10 min. Allow to cool to room temperature and then add NaOH to give a neutral or slightly alkaline solution (test the pH with pH paper). 4. Perform Benedicts and Seliwanoffs tests on the hydrolysate from step 3 and on the original sucrose solution. 5. Explain your observations.

Tips on the Use of a Burette Burettes are long cylindrical tubes of uniform diameter throughout the graduated length, terminating at the lower end in a glass or Teflon stopcock and a jet. When in use, a burette must be firmly supported on a stand. The burette should be soaped with the aid of a burette brush and rinsed with distilled water. Next, rinse the burette with a little of the solution to be used in the burette. With the stopcock in the closed position, add some solution (a maximum of 3mL) to the burette with the aid of a small funnel. Remove the funnel and roll the solution around in the burette (held almost horizontally) so that all sides are coated. Allow the solution to drain out through the jet. Repeat the rinse once. Clamp the burette vertically. Using the funnel, fill the burette to a little above the zero mark. Remove the funnel now.

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Discharge the solution through the jet until the lowest point of the liquid meniscus just touches the zero mark or some other mark which you can see comfortably (your eye should be at the same level as the meniscus to avoid errors due to parallax. A piece of white paper held behind the burette aids in reading the position of the meniscus. Inspect the jet to ensure that there are no air bubbles inside and that it is completely filled with solution. Now you are ready to start using the burette. First, RECORD the position of the meniscus this need NOT be zero. Place the fingers of the left hand behind the burette and the thumb in front, and hold the tap between thumb and the fore and middle fingers. (See diagram below) Any drop adhering to the side of the jet after liquid has been discharged should be added to the titration flask by washing it off with distilled water. During delivery of the solution, the flask should be rotated gently with the right hand to ensure proper mixing.

BIOL 1362 BIOCHEMISTRY I Test #2 Estimation of Ascorbic Acid

Ascorbic acid is a relatively potent reducing agent. The method used in this experiment involves the titration of ascorbic acid in the presence of an oxidation-reduction indicator 2,6-dichlorophenolindophenol, which is a combined oxidant and indicator. The extracts or samples to be tested are first made acidic with metaphosphoric acid to remove (precipitate) proteins and ferric ions.

Reagents: (these have been prepared for you)

1.

5% metaphosphoric acid Dichlorophenol-indophenol : preparation 30mg in 200mL water containing 45mg of NaHCO3. The solution was filtered into a dry dark bottle.

2.

3.

Standard Ascorbic acid solution 1mg/mL : preparation dissolve 100mg in 100mL of 5% metaphosphoric acid

Procedure:

A.

Standardization

(See Tips on the Use of a Burette)

Add the standard ascorbic acid solution to a burette.

Pipette 20mL of dichlorphenol-indophenol solution into an Erlenmeyer flask.

Titrate with the ascorbic acid until the last trace of pink colour disappears. The titration must be completed in 30 120 seconds because other reducing compounds slowly reduce the indophenol and so interfere with the titration.

Repeat the titration at least 3 times.

Calculate the mean S.D. ascorbic acid equivalence per 1mL of dye.

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B. Estimation of Ascorbic Acid in canned juice

1. Pour 10mL of canned orange juice into a measuring cylinder and acidify the canned juice with 1mL of glacial acetic acid.

Make up the volume to 125 mL with distilled water in a measuring cylinder.

Mix well (shake thoroughly & CAREFULLY)

Using a measuring cylinder, measure 60 mL of the preparation from step 4 and transfer to another. 250 mL conical flask or measuring cylinder

Add 50 mL of 5% metaphosphoric acid.

Make up the volume to 125mL by adding distilled water.

Transfer the solution to a burette.

Titrate at least three 5mL volumes of the dichlorophenol-indophenol solution.

Calculate the ascorbic acid content in mg per mL of canned orange juice

What volume of canned juice do you need to drink to provide the recommended daily allowance (RDA) of 75mg of vitamin C?

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Additional Discussion

1.

What are the major differences in chemical structure between starch and glycogen? How does cellulose differ from both of these?

2. 3.

Why was sucrose boiled in acid instead of alkali in test #2? How do your values for ascorbic acid content compare with published values for the ascorbic acid content of canned juice? Explain any differences.

4.

Show how vitamin C can be made from glucose and explain why it must be present in the diets of primates?