The objective of this experiment was to ascertain the reactions of carbohydrates in three tests: Benedict’s test, Barfoed’s test, and Iodine test. Carbohydrates have a vital role in living organisms and primarily contribute to energy production. They are made up of carbon, oxygen, and hydrogen with a ratio of 1:2:1. Carbohydrates consist of three main components: monosaccharides, disaccharides, and polysaccharides. Monosaccharides are individual CH2O chains while disaccharides form through dehydration reaction between two units. Despite their differences, both monosaccharides and disaccharides belong to the category of sugars.
Polysaccharides are formed by connecting three or more monosaccharide units. Carbohydrates are produced through the combination of carbon dioxide and water molecules, and they contain two functional groups: hydroxyl groups and carbonyl groups. Within the realm of carbohydrates, a sugar that possesses an aldehyde group is known as a reducing sugar, while one that does not have the aldehyde group is categorized as a non-reducing sugar.
The Benedict’s test is utilized for determining if sugars are reducing or non-reducing. It involves the addition of a solution called Benedict’s reagent, which contains Cu2+ ions, to an alkaline solution with sodium citrate present to maintain dissolved cupric ions. Under these alkaline conditions, ketoses undergo an isomeric transformation into aldoses. This transformation leads to the reduction of the blue Cu2+ ion and formation of cuprous oxide (Cu2O), resulting in the production of a brick red-orange precipitate. Glucose serves as an example of a reducing sugar, while sucrose acts as an example of a non-reducing sugar.
Carbohydrates are composed of monosaccharides, which are the smallest molecules within this category. Monosaccharides are classified based on their carbon count, such as pentose with five carbons and hexose with six carbons. These single sugar units typically contain three to seven carbon atoms. Within these molecules, each carbon atom is linked to a hydroxyl group except for one that forms a carbonyl group through bonding with an oxygen atom. An aldehyde is designated when the carbonyl group exists at the end of the chain, while it is referred to as a ketone if it appears elsewhere in the molecule.
The Barfoed’s test is a useful method for identifying different monosaccharides, such as ribose, deoxyribose, glucose, fructose, and galatose. It relies on the fact that monosaccharides capable of reduction produce Cu2O more quickly than disaccharides. The test solution contains cupric ions in an acidic environment which allows for oxidation of monosaccharides without affecting disaccharides. By carefully controlling the duration of heating, only monosaccharides are reduced while disaccharides remain unreactive. A positive result is indicated by the formation of a red Cu2O precipitate. Ketoses do not undergo isomerization when exposed to this specific reagent.The Barfoed test is used to differentiate between monosaccharides and non-monosaccharides. In contrast, the Iodine test helps distinguish non-polysaccharides. If it is not a non-polysaccharide, then the Iodine test can further determine whether it is a branched or unbranched polysaccharide. The Iodine test is especially effective in identifying starch, which produces a distinct deep blue-black color complex when combined with iodine. Starch consists of ?-amylose and amylopectin, both being helix saccharide polymers. When iodine enters the amylose coil, it forms a large complex polysaccharide, resulting in the formation of the blue-black hue.
Simpler oligosaccharides and monosaccharides do not form this complex with iodine.
Methods/Procedures: For the Benedict test, we had to reheat our water since we had difficulty with the burner and that was an extra procedure. Also, instead of using a cylinder to measure the appropriate solution, we tried to put all the test tubes next to each other and match up the first measured test tube with the appropriate liquid. For the other tests, we followed the same procedures as mentioned on pg 14 of “Experiments in Biology” by Linda R.
All samples, except for Sucrose, Glycogen, Starch, and Distilled water, displayed reducing properties in the Benedict test. Barfoed’s test confirmed that Fructose, Glucose, Galactose, and Equal are monosaccharides. The Iodine test indicated that sweet and low, glycogen, and starch are polysaccharides. Distilled water was classified as a non-reducing sugar and does not fall into either the monosaccharide or polysaccharide category. Glucose was determined to be both a reducing sugar and a monosaccharide.
Fructose, Glucose, and Galactose exhibit similar results as they are both reducing sugars and monosaccharides. However, Maltose, despite being a reducing sugar, does not fall into the categories of monosaccharides or polysaccharides. Surprisingly, Lactose yields identical outcomes to Maltose. In contrast, Sucrose is considered a non-reducing sugar that does not fit into the classifications of polysaccharides or monosaccharides. Glycogen, another non-reducing sugar, acts as a branched polysaccharide. Starch, which also lacks reducing properties, functions as an unbranched polysaccharide. Lastly, the unknown substance known as “Sweet and Low” was determined to be both a reducing sugar and a branched polysaccharide.
The second Unknown B is also a reducing sugar, but it is a monosaccharide. During the Benedict test, the sample started as a blue solution and ended with a red precipitate, indicating that it was reducing. In the Barfoed test, the initial blue solution also had precipitate in some samples, indicating that they were monosaccharides. Starch and sucrose, for example, remained blue and showed a positive test. However, fructose, glucose, and maltose formed a red precipitate, indicating a negative test.
The presence of disaccharide is indicated by the blue solution remaining after heating, while the formation of a brick red precipitate indicates the presence of monosaccharide. In the Iodine test, mostly yellow results indicated non-polysaccharides, but there were some samples that showed a rust or blue-black color, indicating either branched or unbranched polysaccharides. Glycogen and sweet and low were identified as branched polysaccharides based on their rusty color at the end, while starch was identified as an unbranched polysaccharide due to its blue-black color change.
Using the Iodine and Barfoed test, we were able to identify the possible disaccharide samples. When the carbohydrate solution is heated with Benedict’s reagent, a red brick precipitate forms, indicating a reducing sample. This precipitate is formed from CuO in the Benedict’s solution, as monosaccharides in the sample reduce Cu2SO4 to CuO. Any monosaccharide that can undergo this reaction is classified as a reducing sugar.
The Benedict’s test showed that distilled water, which had a blue color, is non-reducing. Additionally, the absence of a precipitate in the Barfoed’s test and the yellow color observed when reacting with iodine indicate that distilled water is also non-monosaccharides and non-polysaccharide. Based on this, we can conclude that distilled water is likely a disaccharide. On the other hand, glucose, which turned red during the Benedict’s test and formed a precipitate in the Barfoed’s test, can be classified as a reducing monosaccharide. Therefore, we can infer that it will test negative for being a polysaccharide in the iodine test.
Both Fructose and Glucose are reducing monosaccharides, and they both exhibit a red color after the Benedict’s test. Galactose shows the exact same reaction as Fructose and Glucose. On the other hand, Maltose differs from the rest as it is a disaccharide – evidenced by negative results in both the Barfoed and Iodine tests. Despite this, it still qualifies as a reducing sugar since it turns orange in the first test. Lactose produces the same results as Maltose, which can possibly be attributed to their similar grouping. In contrast, Sucrose is a non-reducing disaccharide sugar; both the Barfoed and Iodine tests yield negative results and the Benedict test indicates a blue solution.
Distinct from other substances, glycogen was identified as a non-reducing and branched polysaccharide. This distinction was evident through the negative result of the Barfoed’s test and the rusty coloration produced by the Iodine test. Similarly, starch also exhibited non-reducing properties and was a polysaccharide but lacked branching, as indicated by the blue-black solution. Unknown A, referred to as Sweet and Low, demonstrated expected characteristics of being both a reducing sugar and a branched polysaccharide, as observed during testing with a rusty coloration. Therefore, it is confirmed that the outcome of the second test was negative.
Unknown B was both equal and a reducing sugar, but it was a monosaccharide because of the precipitate. To summarize, using all three tests was helpful in confirming results from the other tests and determining which samples were likely disaccharides. For instance, although Glycogen did not show a reducing reaction in the iodine test, it did indicate the presence of sugar. Therefore, I believe that employing multiple tests can validate or invalidate a hypothesis and provide a more precise answer rather than making assumptions.
