EXPERIMENT 15

[carbo.lab]

OBJECTIVES:

1. To define CARBOHYDRATE and to name some simple carbohydrates.

2. To be able to perform qualitative tests on carbohydrates.

3. To be able to identify an unknown carbohydrate by performing tests on it.

DISCUSSION:

Carbohydrates are "hydrates of carbon". They contain only Carbon, Hydrogen and Oxygen. These 3 elements are bonded in different ways. The alcohol (-OH) group is usually found in carbohydrates, as well as an aldehyde group (-CHO) or a ketone group (-CO-).

The most important simple carbohydrate is GLUCOSE. The molecular formula is C₆H₁₂O₆. There are 5 -OH groups and one -CHO group, usually arranged in a ring form. FRUCTOSE is another simple carbohydrate with the same molecular formula, but a slightly different structure. It is much sweeter than glucose. Glucose and fructose are "simple sugars". They are called "monosaccharides", or "hexoses". "Hex-" means six carbon atoms and "-ose" means a sugar. It is difficult to show these structures on paper, especially with only a typewriter, so we will have models of them in the laboratory.

SUCROSE is a double sugar, or "disaccharide". It is the common table sugar we use and is usually obtained from sugar cane. It is formed from one glucose unit and one fructose unit and has the formula C₁₂H₂₂O₁₁. LACTOSE is found in milk and is commonly called "milk sugar". It is also a disaccharide and has the same molecular formula as sucrose, but a slightly different structure.

STARCH and CELLULOSE are POLYSACCHARIDES. They are formed from large numbers of glucose units. They differ from each other only in the way the O atoms link the glucose units together. Cellulose is relatively unreactive; it is not digestible by humans.

You will perform three tests (plus one demonstration) on these 5 compounds and the same tests on an UNKNOWN. By comparing the results, you will be able to identify the Unknown. As you do each test, note whether the result is positive or negative. The tests are discussed below with the Experimental Procedure.

MATERIALS:

5 % aqueous solutions of: fructose, sucrose, cellulose, starch, lactose

Reagents: Benedict's Reagent, Seliwanoff Reagent, Iodine Reagent

EXPERIMENTAL PROCEDURE:

Obtain 5 mL of an Unknown solution from the Instructor. It is one of the five Known sample solutions. This will be used for three different tests, so divide it into 4 portions. Don't waste it. Be sure you record its sample number or letter. The Instructor will tell you the test results for the Fermentation test.

Each test is done for each KNOWN plus your UNKNOWN. Set up 6 clean test tubes. Add one of the solutions to be tested to each tube, then add the reagent.

1. FERMENTATION. (demonstration)

Yeast reacts with fructose, sucrose and starch to form CO₂ and ethyl alcohol. Cellulose and lactose resist fermentation. A positive test will show the formation of gas. The Instructor will demonstrate this with the 5 known samples.

a. Yeast suspension is put in a large test tube, then 20-30 mL of the sample is added. A small test tube is put UPSIDE DOWN into the larger test tube. The whole is inverted to let the air out of the small tube. Return to the upright position and remove the cork.

b. The large tube is put in a warm water bath (37^oC) for several hours. If bubbles collect in the small tube, that is a POSITIVE reaction and indicates fructose, sucrose or starch.

2. BENEDICT'S TEST. Carbohydrates containing free carbonyl groups (-CO-) will react with Benedict's Solution. A positive test will show a red, orange, or greenish color. If there is no reaction, the solution will remain blue. Among our samples, only fructose and lactose have positive tests. Cellulose and starch will give weak positive tests only if they are heated a long time.

a. Set up a 250 mL beaker half full of water and boil it. Keep this hot. It is our "water bath.

b. Add about 10 drops of the test solution to the test tube.

c. Add about 2 mL of Benedict's Solution to each test tube and mix well. Heat the mixture in the hot water bath for 5 minutes.

d. If the color changes, that is a POSITIVE test.

3. SELIWANOFF TEST. This contains a dye named resorcinol. Ketoses (fructose) and aldoses (sucrose) react with the reagent and a positive test is when a red color is formed. DANGER: the reagent is strongly acid. If you get any on your hands, wash them immediately. If any is spilled, add bicarbonate solution to neutralize and then wash it up.

a. Add about 10 drops of the test solution to the test tube.

b. Add 5 mL of Seliwanoff Reagent to each test tube. Mix well. Heat the mixture in the hot water bath for 5 minutes.

c. If a red color develops, it is a ketohexose (ie: fructose).

4. IODINE TEST. Starch forms a dark blue or black color when it reacts with iodine (I₂). Cellulose and the simple sugars give no reaction. Starch exists as "grains". These do not react easily with iodine. However, when the starch is heated, the grains open up (something like popcorn) and then the starch is very reactive. [That is also why foods containing starch, such as potatoes or rice, are more easily digestible when they are cooked than when they are raw.]

a. Place 1 mL of the test solution in a test tube and add 2-3 drops of Iodine Solution.

b. A blue or black color is a positive test for starch.

EXP # 15 - LABORATORY REPORT Name ______________ Sec ____

For each test, record whether it is positive (+) or negative (-). Give a brief description of a + test.

For example: Fructose / fermentation: + because a gas formed.

UNKNOWN number = ______. It is _______________________.

REVIEW QUESTIONS

1. What is a HEXOSE?

2. ETHANAL, or ethyl aldehyde, has the molecular formula C₂H₄O.

Write its structural formula.

3. What is a DISACCHARIDE?

4. Sucrose is heated. It becomes black and steam escapes. Complete and balance the equation:

C₁₂H₂₂O₁₁ ==> C +