Introduction to Carbohydrates and their Qualitative Analysis

Experiment # 7: Introduction to Carbohydrates and their Qualitative Analysis
Abstract
Carbohydrates are defined as bio-molecules with a molecular formula of (CH2O)n, where the value of n > 3. Carbohydrates range from simple sugars to complex sugars and serves important functions in our body. Carbohydrates are the most abundant biological molecules found in any living organism. Estimation of carbohydrates has both prognostic and diagnostic value. Hence, carbohydrates are tested both qualitatively and quantitatively.
In the present laboratory experimentations, we evaluated the qualitative analysis of various carbohydrates as mentioned in our biochemistry manual. Such analysis was done to understand the chemical properties of each carbohydrate in relation to their molecular structure and the probable reactions they can undergo.
All the experimentations that we carried out with various qualitative reagents matched the expected results, except for Seliwanoff’s. Since, glucose is an aldose; it might have required more time to become dehydrated to respond to Seliwanoff’s test. Since, the experimentation was stopped early, such disparity between observed and expected results were noted. However, fructose responded to Seliwanoff’s in line with expected results.
Introduction to Carbohydrates and their Qualitative Analysis
Introduction
Carbohydrates are the most abundant biological molecules found in any living organism.

They are defined as compounds which contain carbon, hydrogen and oxygen. The ratios between these three atoms are usually in the form of 1:2:1. Carbohydrates are defined as compounds with the molecular formula of (CH2O)n, where the value of n should be greater than 3. Carbohydrates range from simple sugars to complex sugars (Bowen & Williams, 1957).
Simple sugars are termed monosaccharide and contain only one unit of simple sugar. When more than one units of single sugar are found in a carbohydrate molecule, they are referred to as polysaccharides. Simple sugars are designated as either aldohexoses or ketohexoses. Glucose is an example of aldohexose while fructose is an example of ketohexose. Aldohexoses bear a free aldehyde group in their molecule while ketohexoses contain a free ketone group in their molecule (Bowen & Williams, 1957).
Sugars made of two simple sugar units are called disaccharides. Sucrose is an example of disaccharide because it contains glucose and fructose. Starch, on the other hand, is a polysaccharide since it contains repeating units of simple sugars. Carbohydrates serve important functions in living systems. It is metabolized most readily for the production of energy through the generation of ATP. Further, carbohydrates also form the structural components of the living cell, like the cell membrane (Bowen & Williams, 1957).
Estimation of carbohydrates has both prognostic and diagnostic value. The common diagnostic importance lies in the quantitative estimation of glucose from blood samples for detection of diabetes mellitus. Apart from quantitative analysis, qualitative analysis is also done to detect the nature of carbohydrate present in a given sample. Often urinalysis involves qualitative testing because glucose is not present in the urine of a normal person and the expected results are negative for glucose. However, in the case of diabetes glucose appears in urine which can be detected by Benedict’s qualitative reagent. Various qualitative reagents are used in laboratory settings to specify the type of carbohydrate present in a given sample. The present article would evaluate various qualitative tests as per existing documentation in a manual of biochemistry (Bowen & Williams, 1957).
Materials and Methods
Materials
In this lab each group used the hotplate, large beaker, test tubes, 100 ml distilled water in the clean beaker for control tubes, ribose, glucose, maltose, sucrose, fructose, starch, anthrone reagent, iodine, Benedict’s reagent, Barfoed’s reagent and Seliwanoff’s reagent.
Procedure
The instructor assigned activities in revolving order then each groups wore gloves and goggles to start.
Iodine Test for Starch:
The group recorded the expected result in the data sheet, they marked two test tubs as S, G and W for starch, glucose and water. They added 2.0 ml of starch solution, glucose solution and distilled water into each tubes, respectively. They added 5 drops of freshly prepared I2/KI solution in each tube and they mixed it thoroughly. Then, they recorded the results and observation in the data sheet.
Benedict’s test for reducing carbohydrates:
The group recorded the expected result in the data sheet, they prepared a boiling water bath and marked 4 clean and dry test tubes as G, M, and W for glucose, maltose, sucrose and water respectively. They placed 5 drops of each sample to be tested into the respective test tubes. They added 30 drops of Benedict’s reagent to each sample and mixed it. Then, they placed the test tubes in boiling water at the same time for 5 minutes. After this had completed, they recorded all results and observation in the data sheet.
Barfoed’s test for reducing monosaccharide:
The group recorded the expected result in the data sheet, marked 6 clean and dry test tubes as R, G, M, S, F, and W for ribose, glucose, maltose, sucrose, fructose and water respectively. They placed 15 drops of each sample to be tested into the test tubes. They added 30 drops of Barfoed’s reagent to each sample and mixed it. Then, they placed the test tubes in boiling water at the same time for 5 minutes. After this had completed, they recorded all results and observation in the data sheet.
Seliwanoff’s test for ketoses:
The group recorded the expected result in the data sheet; they marked 3 clean and dry test tubes as G, F, and W for glucose, fructose and water respectively. They placed 2.0 ml of Seliwanoff’s reagent in each tube. They added 0.5 ml of glucose in tube labeled G, added 0.5 ml of fructose in tube labeled F and added 0.5 ml of water in tube labeled W. Then, they placed the test tubes in boiling water at the same time for 3 minutes. After this had completed, they recorded all results and observation in the data sheet.
Anthrone test:
The group recorded the expected result in the data sheet, marked 6 clean and dry test tubes as R, G, M, S, F, and W for ribose, glucose, maltose, sucrose, fructose and water. They added 0.25 ml of the respective carbohydrate solution to each sample and 0.25 ml water in the tube that is marked W. Also, they added 1.25 ml of Anthrone reagent to each tube and mixed it. Then, they placed the test tubes in boiling water at the same time for 15 minutes. After this had completed, they recorded all results and observation in the data sheet.
Finally, the groups cleaned the test tubes using test tube brush and soap and left all test tubes in the test tube rack upside down to dry. They disposed all test solution into the waste container and returned stockroom equipment and chemicals to the main station. Also, they cleaned their bench area and washed their hands with soap and warm water.
Results
Anthrone test:
Sample Expected
results (+/-) Observed
results (+/-) Observations
Ribose + + Dark green
Glucose + + Dark blue/green
Maltose + + Blue green
Sucrose + + Dark green
Fructose + + Dark blue/green
Water – – Dark yellow/ green
Table1: Anthrone test
Iodine test for starch:
Sample Expected
results (+/-) Observed
results (+/-) Observations
Starch + + Dark orange
Glucose – – Orange
Water – – Dark orange settling down at the bottom
Table2: Iodine test
Benedict’s test for reducing carbohydrates:
Sample Expected
results (+/-) Observed
results (+/-) Observations
Glucose + + Dirty brown/ red color
Maltose + + Dirty brown/ red color
Sucrose – – Stayed blue color
Water – – Stayed blue color
Table3: Benedict’s test
Barfoed’s test for reducing monosaccharides:
Sample Expected
results (+/-) Observed
results (+/-) Observations
Ribose + + 3rd to change
Glucose + + 2nd to change
Maltose + + 4th to change red/purple color
Sucrose – – Bright blue still
Fructose + + 1st to change
Water – – Bright blue still
Table4: Barfoed’s test
Seliwanoff’s test for ketoses:
Sample Expected
results (+/-) Observed
results (+/-) Observations
Glucose + – Clear, colorless
Fructose + + Turned peach first, then bright orange
Water – – Clear, colorless
Table5: Seliwanoff’s test
Discussion
From the above results, it is evident that our observed results matched with the expected results as described in our manual of biochemistry. The Anthrone test was a non-specific test for any sugars present in a sample. Since all our samples were either had a monosaccharide, disaccharide or polysaccharide, the anthrone test was positive. This means under acidic conditions all sugars gave a dark blue to green color (Bowen & Williams, 1957). Since water is not sugar it never responded to the anthrone test, which matched our expected results.
Since the iodine test is specific for starch, positive results were noted only with the starch sample. Iodine reacts with starch to form starch-iodide complex due to the distribution of electron charges between them. The formation of starch-iodide complex produces the characteristic color. Since, other samples did not contain starch the results were negative, which matched our expected results (Bowen & Williams, 1957).
Benedict’s solution is used to test for the presence of reducing sugars. Reducing sugars are defined as sugars that have a free aldehyde or ketone group. When reducing sugars are heated in the presence of strong alkali, they form enediols. Enediols reduce the cupric ions present in Benedict’s solution to cuprous ions. The red color is the color of cuprous oxide. Since, glucose and maltose had free aldehyde group and ketone group they gave positive results with Benedict’s solution (Simoni, Hill & Martha, 2002). Since, there are no free aldehyde group and ketone group in Sucrose, it never responded to Benedict’s, which again matched our expected results.
Barfoed’s test is specific for monosaccharide and disaccharide. The principle relies on the conversion of cupric acetate (present in Barfoed’s reagent) to the cuprous oxide that has a deep orange or red color. However, monosaccharides react faster than disaccharides with Barfoed’s reagent (Bowen & Williams, 1957). Fructose, Maltose and glucose are monosaccharides while sucrose is a disaccharide. This is the reason fructose, maltose and glucose responded to Barfoed’s earlier than sucrose. Such observations were consistent with expected results.
Seliwanoff’s test distinguishes aldoses from ketoses. The test is based on the principle that ketoses are dehydrated earlier than aldoses upon heating. The dehydrated ketoses react with resorcinol (present in Seliwanoff’s reagent) to produce the deep orange color. Since fructose is a ketose, it readily responded to Seliwanoff’s test. However, glucose never responded to the test in our experimentation. Thus, the observed results did not match the expected results with respect to glucose. Since, Glucose is an aldose; it might have required more time to become dehydrated to respond to Seliwanoff’s test. Since, the experimentation was stopped early such disparity between observed and expected results were noted (Bowen & Williams, 1957).
References
Simoni, R., Hill, R., & Martha, V. (2002). “Benedict’s Solution, a reagent for Measuring
Reducing Sugars: the Clinical Chemistry of Stanley R. Benedict.” J. Biol. Chem. 277 (16):
10–11
Bowen, G., & Williams, A. (1957). A Students’ Handbook of Organic Qualitative Analysis,
University of London Press