Carbohydrates

                                        Corresponding Chapters from Reference Books                                          

• Carbohydrates and Glycobiology                                    - By Lehninger
• Sugars and Polysaccharides                                             - By Voet & Voet 
• Carbohydrates                                                                   - By Stryer                           

Fehling’s Test

                    Principle                  

                                 Fehling's test differentiates between aldehydes and ketones. Aldehydes can be oxidized by Cu²⁺ in the presence of a strong base to form carbonic acids. Ketones cannot be oxidized by this reaction. When the Cu²⁺ oxidizes the aldehydes it is reduced to Cu⁺, and forms the compound Cu₂O, which is a reddish precipitate. That is how you know you have an aldehyde.

                            Fehling's reagent is usually formed by mixing CuSO₄, sodium citrate, and sodium carbonate. The carbonte is used to raise the pH, but doing so would normally form Cu(OH)₂, a precipitate. That is where the sodium citrate comes it---the citrate complexes the Cu2⁺ ions and prevents them from precipitating.

              Fehling's solution               

                    Fehling's solution is a chemical test used to differentiate between water-soluble aldehyde and ketone functional groups, and as a test for monosaccharides. The test was developed by German chemist Hermann von Fehling in 1849.

                    Fehling's solution is always prepared fresh in the laboratory. It is made initially as two separate solutions, known as Fehling's A and Fehling's B. Fehling's A is a blue aqueous solution of copper(II) sulfate, while Fehling's B is a clear solution of aqueous potassium sodium tartrate (also known as Rochelle salt) and a strong alkali (commonly sodium hydroxide).

                    Equal volumes of the two mixtures are mixed together to get the final Fehling's solution, which is a deep blue colour. In this final mixture, aqueous tartrate ions from the dissolved Rochelle salt chelate to Cu²⁺ (aq) ions from the dissolved copper(II) sulfate, as bidentate ligands giving the bistartratocuprate(II)^4- complex as shown below. The tartarate ions, by complexing copper prevent the formation of Cu(OH)₂ from the reaction of CuSO₄.2H₂O and NaOH present in the solution.

Procedure:

- To 1 mL of Fehling’s solution A (aqueous solution of CuSO4) add 1 mL of Fehling solution B (solution of potassium tartrate).

- Add 2 mL of the sugar solution, mix well and boil.

•  Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.

Questions:

• Write the reaction(s) involved in Fehling’s Test.
• What is the function of tartrate?
• Some disaccharides such as maltose are reducing agents, whereas others, such as sucrose are not. Explain briefly by incluiding the structures of the sugars.

Carbohydrates as Reducing Sugars

                                                                             Primary Concept                                                   

                                                A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. The enolization of sugars under alkaline conditions is an important consideration in reduction tests. The ability of a sugar to reduce alkaline test reagents depends on the availability of an aldehyde or keto group for reduction reactions. A number of sugars especially disaccharides or polysaccharides have glycosidic linkages which involve bonding a carbohydrate (sugar) molecule to another one, and hence there is no reducing group on the sugar; like in the case of sucrose, glycogen, starch and dextrin. In the case
of reducing sugars, the presence of alkali causes extensive enolization especially at high pH and temperature. This leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH. These sugars, therefore, become potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so fort. Most commonly used tests for detection of reducing sugars are Fehling’s Test, Benedict’s Test and Barfoed’s Test.

1. Fehling’s Test
2. Barfoed’s Test
3. Seliwanoff’s Test
4. Bial’s Test

Fehling’s Test

 

Fehling’s Solution (deep blue colored) is used to determine the presence of reducing sugars and aldehydes. Perform this test with fructose, glucose, maltose and sucrose.

Procedure:

- To 1 mL of Fehling’s solution A (aqueous solution of CuSO4) add 1 mL of Fehling solution B (solution of potassium tartrate).
- Add 2 mL of the sugar solution, mix well and boil.

•  Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.

Questions:

•  Write the reaction(s) involved in Fehling’s Test.
•  What is the function of tartrate?
•  Some disaccharides such as maltose are reducing agents, whereas others, such as sucrose are not. Explain briefly by incluiding the structures of the sugars.

Barfoed’s Test

Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so that is can only be reduced by monosaccharides but not less powerful reducing sugars. Disaccharides may also react with this reagent, but the reaction is much slower when compared to monosaccharides. Perform this test with glucose, maltose and sucrose.

Procedure:

- To 1-2 mL of Barfoed’s reagent, add an equal volume of sugar solution.
- Boil for 5 min. in a water bath and allow to stand.

• You will observe a brick-red cuprous oxide precipitate if reduction has taken place.

Questions:

• Write the reaction(s) involved in the Barfoed’s Test.
• When you test starch with Barfoed’s reagent, what would be the answer, positive or negative? Explain your answer by giving reasons and structures.

Seliwanoff’s Test

Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose and if it contains an aldehyde group, it is an aldose. This test is based on the fact that, when heated, ketoses are more rapidly dehydrated than aldoses. Perform this test with glucose, fructose, maltose and sucrose.

Procedure:

- Heat 1 mL of sugar solution with 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10% HCl) in boiling water.

• In less than 30 seconds, a red color must appear for ketoses.
• Upon prolonged heating, glucose will also give an appreciable color.

Questions:

• Write the reaction(s) involved in Seliwanoff’s Test.
• What is the funtion of resorcinol?
• What is the aim of using a strong acid?
• What is the result of testing sucrose with Seliwanoff’s reagent? Explain your answers by giving reasons and structures.

Bial’s Test

Bial’s Test is to determine the presence of pentoses (5C sugars). The components of this reagent are resorcinol, HCl, and ferric chloride. In this test, the pentose is dehydrated to form furfural and the solution turns bluish and a precipitate may form. Perform this test with ribose and glucose.

Procedure:

- To 5 mL of Bial’s reagent, add 2-3 drops of sugar solution and boil.

• Upon boiling, note the green-blue color formed.

Questions:

• Write the reaction(s) involved in Bial’s Test.
• Is it possible to distinguish DNA and RNA structures by using Bial’s Test?
• The boiling step is common for each test for the reducing sugars. Why boiling is necessary for the reduction to take place?

Solubility Tests

- Apply this test to all carbohydrates provided.
- Observe the solubility of the carbohydrates both in water and ethanol.
 DO NOT depend on your solubility observations during identification of your unknown compound.

Molisch’s Test

                                                                             Primary Concept                                                             
                                               Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde (either furfural or a derivative), which then condenses with the phenolic structure resulting in a red or purple colored compound.


Procedure:

- Apply this test two different carbohydrate solutions of your own choice, preferably to one monosaccharide and one polysaccharide.

- Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of Molisch’s reagent (10% α-naphthol in ethanol).

- Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a layer at the bottom of the tube.

- Observe the color at the interface between two layers and compare your result with a control test.

 A brown color due to charring must be ignored and the test should be repeated with a more dilute sugar solution.

Questions:
 Write the reaction step(s) involved in this test?
 Give an example of a protein structure that would give positive test with Molisch’s Reagent.

Burdwan University BT 1st Year

BT - 101 Chemistry of Biomolecules

1. Carbohydrates
2. Amino-acids Peptides and Proteins
3. Lipids
4. Nucleic-acids
5. Enzymes
6. Synthesis and Breakdown of Carbohydrates
7. Plant and animal Hormones

BT - 102 Genetics

1. Mendelian Inheritance
2. Genetic Changes
3. Bacterial and Viral Genetic Systems
4. Human Genetic
5. Chromosomal Abnormalities
6. Population Genetics

BT - 103 Microbiology

1. Microbial World
2. Microbial Growth
3. Microbial Metabolic Diversity
4. Soil Microorganism and Nutrient cycling
5. Plant Microbe Interactions
6. Microbial Infection and Disease
7. Chemotherapy and Antibiotics

BT - 104 Cell Biology

1. Cellular Basis of Life
2. Cellular Information
3. The Cell Surface
4. Energy Transduction
5. Endo-membrane System
6. The Cytoskeleton
7. Cell Reproduction
8. Tools and Techniques

BT - 105 Mathematics

1. Differential Calculus
2. Integral Calculus
3. Statistics and Probability

BT - 106 Communicative English

1. Communication and communicative activities the notions of encoder and decoder and the message and the medium.
2. Concise grammatical structures and key vocabulary for general as well as specific purpose accuracy and appropriateness in the use of English.
3. English speech sound and sound combinations.
4. Elements of spoken English.
5. Topic of discourse, mode of discourse and style of discourse with special reference to scientific discourse.
6. Writing notes, reports, proceedings etc.
7. Expanding and summarizing
8. Narrating and describing.
9. Tutorial for each topic.

BT - 107 Practical Biochemistry

BT - 108 Practical Microbiology

Quantitative Analysis of Carbohydrates

                                         Primary Concept                                                                                                                    

                         A carbohydrate is an organic compound with the general formula Cm(H2O)n, that is, consists only of carbon, hydrogen and oxygen, with the last two in the 2:1 atom ratio. Carbohydrates make up the bulk of organic substances on earth and perform numerous roles in living things.
The carbohydrates (saccharides) are divided into four chemical groups: monosaccharides, disaccharides, oligosaccharides and polysaccharides. Polysaccharides serve for the storage of energy (e.g., starch in plants and glycogen in animals) and as structural components (e.g., cellulose in plants and chitin in arthropods). Structural polysaccharides are frequently found in combination wit proteins (glycoproteins or mucoproteins) or lipids (lipopolysaccharides). The 5-carbon monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD and NAD) and the backbone of the genetic molecule known as RNA. The related deoxyribose is a component of DNA. Saccharides and their derivatives include many other important biomolecules that play key roles in the immune system, fertilization, preventing pathogenesis, blood clotting and development.
This experiment aims to introduce you with the identification of unknown carbohydrates. To gain maximum benefit, observations should be related, as far as possible, to the structure of the substances examined.

Some important points:-

1. Most of the tests and reactions described are not quantitative and volumes are approximate, despite these facts some tests do not work if quantities greatly in excess of those stated are used.

2. DO NOT place your pipettes in reagent bottles as this leads to contamination.

3. In most tests, it is important to apply a control test using water instead of the solution under examination. If you are in doubt about the result of a test, perform the reaction with a suitable known compound.

4. In this experiment, sugar samples are given in their solid state. To perform each procedure, you should prepare your own sugar solution by taking very small amounts of solid sugars.

5. When you need to boil your sample in a test tube, prepare a hot water in a large beaker and put your test tube inside the beaker. DO NOT forget to put boiling chips in the beaker.

TESTS ON CARBOHYDRATES

1. Molisch’s Test
2. Solubility Tests
3. Carbohydrates as Reducing Sugars
   • 3A. Fehling’s Test
   • 3B. Barfoed’s Test
   • 3C. Seliwanoff’s Test
   • 3D. Bial’s Test
4. Action of Alkali on Sugars
5. The Inversion of Sucrose
6. Iodine Test

Specification of Carbohydrate:-