Tests for Carbohydrates

In this exercise, students explore a diversity of carbohydrate chemistry. In the first part, students perform four qualitative tests on eight sugars. The tests are Benedict's (reducing sugars), Barfoed's (monosaccharides), Seliwanoff's (ketoses), and Bial's (pentoses). The sugars we give them are glucose, fructose, lactose, maltose, ribose, sucrose, sorbose, and xylose (1% solutions). Structures for the first six are in most texts. The latter two are usually not mentioned and serve effectively as "unknowns."

The procedures for all the tests are identical: to 20 drops of the respective reagent, add 5 drops of the sugar, mix well, and place in a boiling water bath for 5-10 minutes. Results are tabulated as to color formation (clear vs. precipitate) and time required. The reagents used are Benedict's for reducing sugars, Barfoed's for monosaccharides, Seliwanoff's for ketoses, and Bial's for pentoses.

In the second part, students use these qualitative tests to determine the characteristics of sugars in four natural mixtures: corn syrup, honey, and acid hydrolysates of sawdust and dandelion root starch.

Corn syrup used to be glucose. Using a 2% solution of corn syrup in water, students got a variable ketose test. It turns out (read the label) it's a mixture of real corn syrup and high-fructose corn syrup. Students thus miss the point that corn starch is all glucose but gain the message about including fructose in corn syrup to increase its relative sweetness. Honey is made up as a 2% solution in water, and gives the expected test for ketose.

I made the wood and dandelion root extracts by adding 40 mL of 0.5 M HCl to 10 g sawdust (local sawmill) or 10 mL 1 M HCl to 4 g chopped dandelion roots (my yard) and incubating in sealed bottles in a 65 degree oven for three days. To each, I added 20 mL water, mixed well, and decanted into separate flasks. I then added 20 mL water to the sawdust and the roots and heated in a microwave oven for one minute, then vacuum filtered and pooled the filtrates. Adding 50% NaOH dropwise, I neutralized both extracts, then added 1 g Norit, mixed, and filtered through GF/C glass fiber filters. The filtrates were clear and pale yellow. The wood extract yielded pentose (xylose from hemicellulose), monosaccharide, and reducing sugar. Dandelion roots store sugar as inulin, a fructose polymer, and students' results were consistent with this, showing reducing sugar, monosaccharides, and ketose.

In the third part, students are presented with two bottles each containing white powder. One is starch, the other is cellulose. They look alike and of course share many chemical similarities. The students are asked to differentiate them on the basis of three tests: reaction with iodine, solubility in hot water, and digestion by amylase. The iodine test is of course classic. A small scoop of powder suspended in a few drops of water is treated with a few drops of an appropriate iodine/KI solution (we use 0.125% iodine/0.5% KI or a dilution of this).

For solubility in hot water and amylase digestion, students place a very small amount of each powder and 5 drops of water, in two separate tubes for each powder, four tubes all together. All four tubes are heated in a boiling water bath and solubility results are recorded. When the tubes are cool, students put one drop of a 2 mg/mL in water solution of pancreatic amylase (Sigma A3176 or equivalent) into one test tube containing each powder and let all four tubes stand at room temperature for 15 minutes. All four tubes are then tested directly with 20 drops of Benedict's solution as above. The results are definite; only starch disperses in the hot water and only starch gives an orange precipitate in only the tube treated with amylase. Cellulose is not affected by hot water and may give a small amount of precipitate (it looks as though the cellulose itself turns yellow).

Finally, students are asked (1) to identify which of the eight known sugars are monosaccharides, disaccharides, reducing sugars, ketoses, and pentoses, (2) to identify, based on their results, the molecular structures of sorbose and of xylose from groups of three structures,  (3) what they can conclude about what kinds of sugars are present in the four mixtures they tested, (4) to indicate a significant chemical difference between normal starch and dandelion root starch based on their results and what they know about starch, (5) to account for the ketose reaction in corn syrup, and (6)  to identify which of the white powders is starch and which is cellulose, based on all their results.

In previous years, we have included a study of fermentation. From my perspective, the only good reason for including fermentation is that it points up the reality of stereochemical differences between epimers like glucose and galactose. (It also adds a discriminating test for glucose and galactose if you want the students to identify single unknown sugars, which we used to do. I like better the approach above of having students characterize unknown sugars in natural mixtures.)

Several sugars were tested to see whether yeast could ferment them. The setup was quite simple. Students mixed equal volumes of a 2% suspension of yeast and a 1% solution of sugar. To see carbon dioxide formation, you need a way of trapping the gas.  One way to do this is to have the suspension in an inverted tube. It's a little tricky without real fermentation tubes, but not too difficult. Students filled small (10x75 mm) test tubes with the suspension, cover them temporarily with a small piece of paper towel, then invert them quickly and drop them upside-down into a larger test tube filled with water (over a sink to catch the spilled water). If done carefully, there should be no air bubbles in the inner tube. Another, simpler approach, is to draw the mixture into a small plastic syringe that can then be sealed. After incubation for an hour or two around 40 degrees, significant carbon dioxide is produced. We haven't tried detecting production of ethyl alcohol. Sucrose, glucose, and fructose are fermented, maltose is variable, and lactose, galactose, ribose, and xylose are not.


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Revised 8/31/06