Carbohydrates or sugars are an important source of energy for living organisms. The structure of carbohydrates contain both an alcohol and carbonyl functional group. Monosaccharides consist of a single sugar molecule. These can be represented either in a linear form or in the hemiacetal form. The transformation from one form into the other is shown below for the monosaccharide glucose.

In glucose formation of the hemiacetal occurs because the –OH group on the 5^th carbon can twist around allowing the oxygen’s electrons (negative in nature) to react with the positively polarized carbon in the carbonyl group forming a bond and creating a ring. Two ring forms are possible, a and b. The a form arises when the OH on carbon 1 is trans (on the opposite side of the ring) from carbon 6. The b form arises when the OH on carbon 1 is cis (on the same side of the ring) from carbon 6. Carbon 1 is very reactive and has the ability to interconvert from the a form- to the linear chain - to the b form. The carbon that is the carbonyl carbon in the linear form (in the case of glucose carbon 1) is given the term the anomeric carbon. Since it is so reactive, it is easily oxidized by other molecules. If carbon 1 is oxidized, then it is reducing the molecule it is reacting with. If a sugar has a free anomeric carbon then we can also call it a reducing sugar because of its ability to reduce other molecules.

A disaccharide is a molecule containing two monosaccharides linked through a glycosidic linkage (this is defined as an oxygen atom linking two monosaccharides). Glycosidic links between monosaccharides form during condensation (dehydration) reactions. Sucrose, a disaccharide, is shown below. The monosaccharides in sucrose are glucose and fructose. These are linked by an a(1à 2) glycosidic linkage. It is considered a because the first monosaccharide is the a-anomer, and the (1à 2) refers to the carbons linked on the glucose and fructose respectively.

a-D-glucose fructose

Notice that the fructose and the glucose are joined at the anomeric carbon of glucose. Because the anomeric (very reactive) carbons are joined and not able to react to oxidize anything, sucrose is considered a non-reducing sugar.

Two other common dissacharides are maltose, one of the products of starch digestion, and lactose, a sugar found in mammalian milk.

Polysaccharides that contain a number of monosaccharides linked by glycosidic linkages also exist. Glucose linked a(1à 4) is found in the plant storage polysaccharide, starch. Glucose linked b(1à 4) is found in the structural polysaccharide, cellulose. Glycosidic links like the ones found in di- and polysaccharides can be broken during hydrolysis reactions. These usually require enzymes for efficient hydrolysis. Humans possess enzymes that can break down the a(1à 4) glycosidic linkages in starch. During digestion, starch is broken down into maltose (disaccharides of a(1à 4) linked glucose) in the saliva by the enzyme a-amylase. Humans do not possess an enzyme that can hydrolyze the b(1à 4) link found in cellulose. Some organisms like cows and termites contain bacteria that possess such enzymes. These enzymes are called cellulases. This also means that humans can not use cellulose as a source of glucose.

This lab uses the technique gel filtration chromatography to separate a solution of glucose and starch. Separation by gel filtration relies on the following theory: molecules are separated on the basis of molecular weight, size, and shape. A liquid chromatography column (glass or plastic tube) is packed with porous gel beads (usually crosslinked polysaccharides called dextrans). The pore size is chosen so that small molecules can enter the beads, but the large ones can’t. The small molecules must pass through a larger column volume than the large ones that go around the gel beads in the column. The larger molecules will exit first.

Figure 1. A gel filtration column with two different size molecules applied. The larger molecules exit the column first.

In the first part of this experiment, glucose (smaller molecule) present in a piece of candy is isolated from the other ingredients in the candy. Since glucose is a reducing sugar, the presence of glucose can be tested by having glucose reduce another molecule. The Benedict’s test reduces copper (II) to copper (I) using a reducing sugars if present. A positive test (reducing sugar present) will result in a color change from pale blue to dark green to red brick when the solution is heated.

In the second part of the experiment, you will verify that the enzyme found in saliva, a-amylase is able to hydrolyze (break glycosidic linkages) starch. You will incubate a starch solution with the amylase and then separate the unhydrolyzed starch from the sugar using gel filtration. Maltose is also a reducing sugar and should give a positive Benedict’s test.

1. Take one smartie and cut it in half.

2. Place half of the smartie in the mortar and pestle, and crush the smartie.

3. Add the crushed smartie to 100 mL of distilled water and dissolve.

1. Mount the PD-10 gel column vertically, using a ring stand and clamp.

2. Number a set of test tubes from 1 to 10. Put 1ml of water into another unlabeled test tube. You will use this tube as a guide to determine when you have collected 1mL in each of the fraction test tubes (1 thru 10). (Note: mark this height on the test tubes you labeled 1 to 10.) Place the test tubes in a test tube rack.

3. Remove the top from the column. Place a small beaker under the column. Remove the stopper from the column tip and allow the eluant (out-flowing liquid) to drip out of the column until the flow stops.

4. Add 1 to 2 mL of the 0.9% aqueous NaCl to the top of the column to flush the column. Leave column open until flow stops.

5. In a separate test tube put 1.0 ml of the 0.2% glucose solution that you prepared.

6. Add just 1mL of the solution to the top of the gel column and continue to collect eluant in the same beaker as step #3. Discard all eluant washings.

7. When the eluant flow has stopped, cap the outlet spout.

8. Add 0.9% aqueous NaCl solution to fill the top of the column and lower the gel column so that the spout is slightly above test tube 1.

9. Remove the outlet cap and collect 1mL of eluant into each test tube 1 through 10. The column will need to be refilled with 0.9% aqueous NaCl during the fraction collection.

10. Cap the outlet spout after 1mL of eluant in test tube 10 has been collected. Make sure the space above the gel column is approximately half full of NaCl solution before continuing.

1. For the glucose test: Place 1mL of Benedict’s solution into each of the original test tubes, 1 to 10.

2. Gently heat these test tubes in a warm water bath on a hot plate for 10 minutes. (Heat the water bath to 80°C.) A reducing sugar will produce a red, green, or yellow precipitate.

3. Place waste from the Benedict’s tests in the aqueous waste container. All other waste can be disposed of down the drain.

4. Clean all test tubes to be used in the next procedure.

 

1. Place 1mL of 0.5% aqueous starch solution into a test tube and add approximately an equal volume of 1.0 % amylase solution. Incubate the mixture in a warm water bath (50°C) for 10 to 15 minutes.

2. Remove the test tube from the water bath and allow it to cool to room temperature.

3. Remove the end cap from the column and flush the column with 10 mL of the 0.9% NaCl solution. This will wash out any residual material. Again, collect this in a small beaker.

4. Add 1.0 mL of the hydrolyzed starch to the top of the gel column and continue to collect eluant in the beaker. Discard all eluant washings.

5. Repeat steps 7 to 10 as described in the isolation of glucose, using the hydrolyzed starch sample.

6. Perform the Benedict’s test for the presence of maltose on the eluted samples.

7. Before returning the plastic column, rinse the column with 5 mls of distilled water to flush out the NaCl. Thank you.

 

Fraction #	Part A. Glucose in Candy	Part B. Maltose from Starch
1
2
3
4
5
6
7
8
9
10

QUESTIONS

Answer the following questions using complete sentences in well organized paragraphs.

Which test tubes showed the presence of a reducing sugar for each experiment performed?

Why doesn’t starch give a (+) positive glucose test?

Below is the structure for the disaccharide lactose. The monosaccharides in lactose are galactose (left) and glucose (right).

                   Is lactose a reducing sugar? How do you know?

                   Identify the glycosidic linkage in lactose.