Form or structure of a molecule plays a significant role in the function of that molecule. Since we are interested in the function of molecules, it helps to study their structure. One of the major classes of organic compounds found in cells is carbohydrates.
These carbohydrates are made of carbon, hydrogen, and oxygen in a ratio of 1:2:1, respectively, with a general formula of X(CH2O)n. When the carbohydrate consists of one unit of sugar, X=1, it is called a monosaccharide. If it consists of two units, X=2, the carbohydrate is called a disaccharide. Carbohydrates made up of more than two units, X>2, are called polysaccharides. Carbohydrates can also be branched or unbranched depending on the type of linkage.
Those with alpha 1:4 linkages are linear or unbranched, while those with alpha 1:6 linkages are branched. Carbohydrates are necessary biomolecules because they play a role in energy metabolism as a source of potential chemical energy. Additionally, they are important building blocks for other biomolecules. The word carbohydrate is very general, so in order to understand these molecules more precisely, we need to be able to identify more specific classifications. Our experiments try to accomplish this using three common bioassay tests.
The first, the Benedict test, will test various compounds for reducing sugars. All six-carbon hexose sugars are reducing carbohydrates, as are most disaccharides. Sucrose is the exception. Most polysaccharides are not reducing. Secondly, we have the Barfoed test, which is designed to test for monosaccharides. The third and final Iodine test is used to test for polysaccharides that are either branched or unbranched.
By combining these tests, we were able to make accurate predictions about the carbohydrate contents of a given sample. Now, let’s take a closer look at how these bioassays work. The Benedict and the Barfoed tests are based on the reaction of cupric ions with aldehyde or ketone groups. In the presence of a reactive group, the blue cupric ions are reduced to red cuprous ions. The Benedict test is a basic solution and upon heating turns green, yellow, orange, or brick red, which indicates a positive reaction. The final color is dependent on the number of reactive sites available; green indicates few sites, yellow more, and red denotes many sites.
The Barfoed solution is acidic and only free aldehyde or ketone groups of monosaccharides can reduce the blue ions to red ions. The color change to red will occur immediately. The lack of a change indicates only that the solution is not a monosaccharide. The iodine test is used for polysaccharides. Iodine combines with any existing alpha helices. The more coiled the sample, the darker the iodine will turn. The color change can range from deep black-blue with a sample of many coils to a rust-red violet with fewer coils and more branchings. When there are no coils, there is no color change. Mono- and disaccharides give negative results.
In summary, this lab attempts to investigate several different samples by means of a series of tests, and based on the combined results of all three tests, we can attempt to understand the carbohydrate composition of unknown samples.
We hope to be able to predict the results of three bioassays for an unknown solution if given its saccharide type and reducing property. We should also be able to predict the saccharide type and reducing capability of an unknown solution if given the results of the three bioassays.
Materials and Methods
Like any other experiment, this experiment needs some specific materials, including a beaker, graduated cylinder, hot plate, 11 test tubes, test tube holder, wax pencil, liquid soap, and test tube brush. Additionally, we used the Barfoed reagent, Benedict reagent, and iodine reagent.
Our eleven samples were distilled water (control), glucose, fructose, maltose, lactose, sucrose, glycogen, starch, potato soup, and dilute honey. First, we marked our test tubes with the wax pencil to keep track of the substances, then we placed the eleven samples in the corresponding tubes. The first test that we performed was the Benedict test, followed by the Barfoed test, and ending with the iodine test. When needed, the samples were heated, and our results were immediately recorded in the following tables. In all three cases, distilled water was used as a control.
*The details of the materials and the methods can be obtained from the lab manual: Experiments in Biology, From Chemistry to Sex by Linda Van Thiel, page 13.
The actual results of the Benedict test are as follows: distilled water remained blue, glucose turned a dark green, fructose turned blue-green, galactose was red, maltose was slightly red, lactose was blue-green on the top of the test tube and red on the bottom, sucrose, glycogen, starch, and potato soup were all negative (blue). Finally, the dilute honey sample was dark orange.
The actual results of the Barfoed test are as follows: distilled water formed no precipitate, glucose, fructose, and galactose formed a red precipitate, maltose, lactose, sucrose, glycogen, starch, and potato soup did not form a precipitate, dilute honey formed a precipitate.
The actual results of the iodine test are as follows: distilled water, glucose, fructose, maltose, lactose, and sucrose all remained yellow or negative. Glycogen turned a rust color, starch was black-blue, potato soup was rust-colored, and the final sample, dilute honey, remained yellow.
Combining the three tests, we have the overall results as follows: for our control distilled water, we can conclude that it is non-reducing, non-monosaccharide, and non-polysaccharide. Glucose, fructose, and galactose were all reducing, monosaccharides, non-polysaccharides. Maltose and lactose were both reducing, non-monosaccharides, non-polysaccharides. Sucrose was non-reducing, non-monosaccharide, non-polysaccharide. Glycogen was a non-reducing, non-monosaccharide, and a branched polysaccharide. Starch was a non-reducing, non-monosaccharide, and an unbranched coiled polysaccharide. Potato soup was non-reducing, non-monosaccharide, and a branched polysaccharide. Dilute honey was reducing, monosaccharide, and a non-polysaccharide.
Let’s continue the discussion of this lab with a closer look at our monosaccharides. Based on our results, we can conclude that glucose, fructose, galactose, and dilute honey are the monosaccharides since they all formed a precipitate in the Barfoed test. The sample of dilute honey was of the greatest interest to me since we did not know prior to the test whether it was a monosaccharide or not. I suspected that it was reducing since the honey was diluted. I do not believe we could dilute a non-reducing carbohydrate since it will not dissolve.
Based on the precipitate formation of dilute honey in the Barfoed test, it can be concluded that it is comprised of monosaccharides. Looking at our results, I can reasonably conclude that the disaccharide samples are maltose, lactose, and sucrose since they all were negative for both the Barfoed and iodine tests. If we also look at the probable disaccharides, we see that none of our tests used were designed to specifically test positively for them. Since we know that disaccharides are comprised of two monosaccharides by way of a dehydration reaction, we could test for disaccharides by adding water to the possible disaccharide samples and maybe heat them so they will undergo a hydrolysis process, then run them through the Barfoed test again. If the sample which was negative in the Barfoed test before adding water was positive after adding water, then we could conclude that the original sample was a disaccharide.
Our tested samples that we believe to be polysaccharides are glycogen, starch, and potato soup since they all had some color change in the presence of Lugol’s iodine. Polysaccharides can be further classified by their overall structure, in particular, whether they are highly branched, highly coiled and unbranched, or both slightly coiled and branched. We learned that the starches can be coiled profusely or coiled with no branches. The iodine test will result in a different degree of color change based on the amount of coiling present.
Namely, a highly coiled carbohydrate will turn a dark blue-black color. The particular highly coiled polysaccharide that we discussed in class is amylose, which is an unbranched storage starch found in plants. Since our starch sample turned black, it may be comprised of amylose starch. The potato soup sample did not turn as dark, a color indicating to me that the starch in this sample probably consisted of smaller starch units called dextrin.
Dextrin has very short terminal ends that coil only slightly so the color change would not be so dramatic as in the presence of highly coiled starch like amylose. The potato soup was made from dehydrated buds. This dehydration process of the fresh potatoes does cause structural changes in the starches. A fresh potato sample, I predict, would turn dark black since its starches would be intact.
Glycogen turned a rust color as we should expect since we know that glycogen is a slightly coiled polysaccharide. I did predict prior to the experiment that the color change in the presence of iodine would be different for starch and glycogen since they have different coiling characteristics. The data, in my opinion, did not conflict with our expected results. These tests, when used together, allow us to make predictions about unknown samples with confidence. I believe that the data provide sufficient information to better understand carbohydrates and how we can more precisely describe carbohydrates.