carbohydrates experiment in biochemistry lab report

Qualitative Analysis of Carbohydrate

The qualitative analysis of carbohydrate is the significant test for detecting and classifying carbohydrates, depending on the colour change followed by chemical reactions . Carbohydrate is an organic biomolecule that has a chemical formula C m (H 2 O) n . The carbohydrate consists of three atoms, viz. carbon , hydrogen and oxygen as the main constituent, where the ratio between the hydrogen and the oxygen is 2:1.

Carbohydrates act as the major energy source for all living organisms, and they are primarily of three kinds, namely mono ,  oligo and polysaccharides . Monosaccharides include simple crystalline sugars that are readily soluble in water. Oligosaccharides include low molecular weight sugars and exist as a polymer of monosaccharides covalently linked through glycosidic linkages.

Polysaccharides include high molecular weight sugars and exist either as a polymer of the same monosaccharides (homopolysaccharides) or different monomers (heteropolysaccharides). In this context, we will discuss the different chemical tests for the qualitative analysis of carbohydrates.

Content: Qualitative Analysis of Carbohydrate

Meaning of qualitative analysis of carbohydrate, molisch’s test, benedict’s test, fehling’s test, barfoed’s test, bial’s test, seliwanoff’s test, iodine test.

The qualitative analysis of carbohydrates is detected based on the reagent’s utilisation  and the reaction between the test sample and reagent. The reaction of the test sample with the chemical reagent gives a significant colour , through which we can detect the presence or absence of carbohydrates.

Video: Qualitative Analysis of Carbohydrate

Methods for Qualitative Analysis of Carbohydrate

We can determine the presence of carbohydrates in the test sample by employing the following chemical tests:

It is the most common method for the detection of carbohydrates. Molisch’s test makes use of Molisch’s solution (contains α-naphthol in 95% alcohol) and concentrated H 2 SO 4 .

Principle : Molisch’s test detects the carbohydrate presence, which principle is based upon the dehydration reaction . The carbohydrates in the sample get dehydrated into aldehyde by the addition of concentrated H 2 SO 4 .

The aldehyde formed is either furfural (produced by the dehydration of pentoses or pentosans) or hydroxymethylfurfural (produced by the dehydration of hexoses or hexosans). Then, the α-naphthol in the molisch reagent reacts with the aldehyde and develops a purple condensation product.

Procedure :

• Take a sample and add water to make 2 ml of solution.
• Add 2 drops of Molisch’s reagent.
• Then, pour 5 ml of concentrated H 2 SO 4 from the side of the inclined test tube.
• Observe the colour change in the tube.

Result interpretation :

• Positive result: Violet ring appears at the junction of two layers, i.e. between the sugar and acid.
• Negative result: Green or brown colour appears.

Application : It detects the presence of all carbohydrates.

It is also used for the analysis of carbohydrates as reducing sugars. Reducing sugar consists of a free aldehyde or ketone group. Benedict’s test makes use of Benedict’s solution as a chemical reagent, which contains copper sulphate, sodium citrate, and sodium carbonate with a pH of 10.5.

Principle : The reducing sugar will reduce into the enediols by reacting with an alkaline reagent, i.e. Benedict’s solution. The reducing sugar gives a green to brick-red colour precipitate depending upon the sugar concentration. The colour change is due to the reduction   reaction of copper (II) to copper (I) in the solution that develops a red-coloured precipitate.

The resulting precipitate is water-insoluble. The sodium carbonate provides the alkaline condition or maintains the alkalinity in the redox reaction solution. Sodium citrate binds with the copper- II ions to prevent the reduction into copper I during storage.

• Prepare 1 ml of a solution by adding a test sample and water.
• Add 2 ml of Benedict’s solution to the test tube.
• Heat the solution in the water bath for 3 minutes.
• Observe the test tube for any colour changes.

• Positive result: Green to brick-red precipitate forms.
• Negative result: Colour remains unchanged.

Application : It detects the presence of reducing carbohydrates.

This method is also useful for the analysis of reducing sugars. It makes use of Fehling’s solutions A and B  as chemical reagents. Fehling’s solution A contains copper sulphate pentahydrate in 1 L of distilled water. Fehling’s solution B contains potassium sodium tartrate and sodium hydroxide in 1 L of distilled water.

Principle : In Fehling’s test, a reduction reaction occurs between the aldehyde or keto groups of the reducing sugar and the alkaline cupric hydroxide that later reduces into cuprous oxide. This cuprous oxide gives a brick-red coloured precipitate in the solution.

• Add 2 ml of the test sample to a test tube.
• Then, add Fehling’s A and B solutions in equal proportion to the above sample.
• After that, place the test tube in a hot water bath for 3 minutes.
• At last, observe the colour change in a test tube.

• Positive result: Brick-red precipitate forms.

This method is also used for the analysis of monosaccharide-reducing sugars. Barfoed’s test makes use of Barfoed’s solution , which contains copper acetate in the dilute acetic acid with a pH of 4.6.

Principle : In Barfoed’s test, the reducing monosaccharide is oxidized by the copper ion in the solution to form a carboxylic acid and copper (I) oxide, which results in the formation of a red-coloured precipitate.

• Add 1 ml of the test sample to a test tube.
• Then add 2 ml of Barfoed’s solution to the test tube.
• Place the tube in a water bath for boiling for up to 1 min.

• Positive result: Gives red-coloured precipitate.

Application : It determines the presence of reducing monosaccharides like glucose.

It is most commonly used for the detection of pentose sugars or pentoses. By this method, we can also differentiate pentoses from hexoses by means of colour change. Bial’s test makes use of Bial’s solution, which contains orcinol, hydrochloric acid and ferric chloride.

Principle : Here, the pentoses react with the Bial’s reagent and convert it into furfural derivatives due to dehydration by HCl. Then, orcinol and furfural condense in ferric ion presence and develop a green-coloured compound.

• Take 2 ml of the test solution in the test tube.
• Then, add 5 ml of Bial’s reagent.
• Heat the solution in a water bath for 1 min.
• Cool and observe the solution for any colour change.

• Positive result: Gives green-coloured precipitate.
• Negative result: The solution remains unchanged.

Application : Used for the detection of the pentose monosaccharides like ribose.

It is most commonly used for the detection of Keto-sugars or Ketoses. By this method, we can also differentiate Ketoses from Aldoses by means of colour change. Seliwanoff’s test uses Seliwanoff’s reagent that contains 0.5 g of resorcinol per litre of 10% HCl.

Principle : In Seliwanoff’s test, ketoses react with the HCl of Seliwanoff’s reagent and yield furfural derivatives due to dehydration . Then, resorcinol and furfural react to give a deep red colour to the solution.

• Take 1 ml of test solution in the test tube.
• Then, add 3 ml of Seliwanoff’s reagent.
• Cool and observe the solution for the colour change.

• Positive result: Gives a deep red colour to the solution.

Application : Used for the detection of fructose, sucrose etc.

It is widely used for the detection of starch in the solution. In an iodine test, iodine acts as an “ Indicator ”.

Principle : In the Iodine test, an iodine solution reacts with the starch (contains α-amylose and amylopectin polymers). This reaction between starch and iodine results in a starch-iodine complex , which gives a blue-black colour to the solution.

• Then, add 2 drops of iodine solution.
• Observe the solution for the colour change.

• Positive result: Gives blue-black colour to the solution.
• Negative result: Gives a brown-yellow colour to the solution.

Application : It detects polysaccharides like starch.

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Experiment 6: Qualitative test of carbohydrates

Related Papers

magendira mani vinayagam

miran mardin

Jean Diane Rose Pilapil

This experiment aims to introduce you with the identification of unknown carbohydrates. The test samples were 1% Dextrin, 1% Galactose, 1% Glucose, 1% Lactose, 1% Sucrose, 1% Maltose and 1% Starch. The test solutions in the Molisch test were treated with Molisch reagent and concentrated sulfuric acid. In the Orcinol test, test solutions were added with Bial’s reagent and was heated in a flame. For the Seliwanoff’s test, the test solutions were added with Seliwanoff’s reagent and was heated in a water bath. And for the Barfoed’s and Benedict’s tests, the test solutions were added with the reagent. Heated in water bath. In Barfoed’s test, the time was recorded when precipitate forms. Thus, the postive result for Molisch test is purple liquid layer. In the Orcinol’s test the positive result for pentoses is blue or green color and hexoses is yellow or brown color. For Seliwanoff’s test, the postive result is red product. Lastly, for Barfoed’s and Benedicts test were the presence of brick red precipitate.

DonNy MildEw

emmanuel auguis

georgia obligado

Adrian Javier

Post Experiment Questions: 1) Enumerate at least five (5) qualitative tests used for detection of a carbohydrate in an unknown sample a. Molisch Test b. Benedict's Test c. Barfoed's Test d. Osazone Test e. Seliwanoff's Test 2) What are the reagents used and what are the components of each of these reagents enumerated in number 1 a. Molisch Reagent and Sulfuric Acid o Components: Molisch Reagent-A solution of ∝-naphthol in ethanol. b. Benedict's Reagent o Components: A mixture of Sodium carbonate, Sodium citrate and Copper (II) sulfate pentahydrate. c. Barfoed's Reagent o Components: 0.33 molar solution of neutral Copper acetate in 1% Acetic acid solution. d. Phenylhydrazine Mixture o Components: 0.5 g of Phenylhydrazine hydrochloride, 0.1 gram of Sodium acetate and 10 drops of Glacial Acetic acid. e. Seliwanoff Reagent o Components: Resorcinol and Concentrated Hydrochloric acid 3) Discuss the principle underlying each of the qualitative test enumerated in No. 1. What will positive results show? a. Molisch's Test o Carbohydrates undergo dehydration when heated with concentrated H2SO4 to form furfural derivatives. Furfural derivatives obtained are condensed with alpha-naphthol to give colored compounds hence the presence of carbohydrate is confirmed. The positive result yielded will be a purple ring. b. Benedict's Test o Reduction-reaction is carried out in a weak alkaline medium through the presence of sodium carbonate. CuSO4.7H2O in an alkaline citrate solution is the reagent that would participate in the reduction process. The copper in the Benedict's reagent is reduced to Copper (II) Oxide, which then precipitates. The reducing sugar would be the reducing agent, giving out electrons in the copper-containing reagent and allowing precipitation to occur. c. Barfoed's Test o The reaction is based on the reduction of cupric acetate by reducing monosaccharides and reducing disaccharides. The reaction with disaccharides is slower because disaccharides have to get hydrolyzed first and then react with the reagent cupric acetate to produce cuprous oxide. The positive result will be a brick-red precipitate. d. Osazone Test o Osazone formation is an indication of the presence of reducing sugars. The reaction between phenylhydrazine and the carbonyl group of the sugar form phenylhydrazone,

Volume 7, Issue 2

Kestutis Bendinskas

There is a multitude of chemical and biochemical detection methods for sugars. Which ones would be most practical in an undergraduate laboratory setting? How to best detect non-reducing disaccharides? How to make such lab fun for students to perform? After trying several spectrophotometric methods, it was found that chemical detection by dinitrosalicylic acid and biochemical detection by hexokinase/glucose-6-phosphate dehydrogenase reagent are most appropriate. Sucrose, a non-reducing disaccharide was digested chemically with hydrochloric acid and biochemically with invertase. It was concluded that chemical detection and biochemical detection compliment each other. Chemical digestion method was preferred over the digestion by invertase. These methods were applied for testing the validity of sugar ingredients printed on drink labels as well as the measurement of sugar levels in ripening bananas at two different conditions. The comprehensive comparison of these methods and the detecti...

nalluri mallikarjuna rao

DR. Prithviraj Bhandare

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Carbohydrate Research

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Qualitative Identification of Carbohydrates: Lab Report

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This laboratory experiment aimed to identify a carbohydrate substance through a series of qualitative tests. Various chemical tests, including Benedict's Test, Bial's Test, Barfoerd's Test, Seliwanoff's Test, Glucose Oxidase Test, and Starch Iodine Test, were conducted to determine the identity of an unknown carbohydrate sample. The results of these tests led to the conclusion that the unknown carbohydrate was ribose, a reducing sugar, pentose, and monosaccharide.

Introduction

Qualitative identification of substances is a fundamental aspect of chemistry. In organic chemistry, physical constants such as melting points have traditionally been used to identify unknown compounds.

On the other hand, inorganic substances can be identified through various methods such as the precipitation of a solid, flame tests, or the formation of colored substances. Modern techniques like chromatography and spectroscopy have also become essential tools in qualitative analysis.

In the field of biochemistry, identifying specific compounds can be challenging because many tests rely on the reactions of functional groups. Consequently, these tests may yield positive results for multiple compounds.

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Therefore, to determine the presence of a particular compound, several tests often need to be performed to narrow down the possibilities through a process of elimination.

Carbohydrate chemistry provides an excellent opportunity to explore qualitative testing. In this experiment, we aimed to identify simple carbohydrates using a set of tests commonly employed for this purpose. By conducting these tests with various carbohydrates, we sought to identify the unknown carbohydrate sample.

Materials and Methods

The following tests were performed to identify the unknown carbohydrate:

Each test was conducted following standard procedures, and the results were recorded for analysis.

Data Analysis

The results of the tests conducted on the unknown carbohydrate are presented in the following table:

Based on the test results, we can conclude that the unknown carbohydrate is a reducing sugar, a pentose, and a monosaccharide.

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This conclusion is drawn from the positive reactions in the Benedict's Test, Bial's Test, and Barfoerd's Test, which are specific to these carbohydrate characteristics. Other tests, such as Seliwanoff's Test, Glucose Oxidase Test, and Starch Iodine Test, yielded negative results, indicating that the unknown carbohydrate does not possess the characteristics associated with these tests.

The carbohydrate identified in this experiment is ribose. Ribose was the only carbohydrate among the ten tested that exhibited positive reactions in the Benedict's Test, Bial's Test, and Barfoerd's Test. These three tests collectively confirm the presence of a reducing sugar, a pentose, and a monosaccharide. The unknown carbohydrate matched ribose more closely in color, particularly in Barfoerd's Test, where both showed a dark blue precipitate.

It is worth noting that there was some initial uncertainty between identifying the unknown as either ribose or arabinose, as both carbohydrates tested positive in the crucial tests. However, the greenish tint observed with arabinose distinguished it from the unknown, which displayed a color more akin to ribose's dark blue.

In conclusion, the qualitative identification of carbohydrates through a series of chemical tests led to the determination that the unknown carbohydrate in this experiment is ribose. The positive results in the Benedict's Test, Bial's Test, and Barfoerd's Test provided strong evidence for the presence of a reducing sugar, a pentose, and a monosaccharide. This identification enhances our understanding of qualitative testing in carbohydrate chemistry.

Recommendations

Further experiments could involve a wider range of carbohydrates to expand the scope of qualitative testing and improve accuracy in identifying unknown samples. Additionally, exploring more advanced spectroscopic techniques may offer enhanced capabilities for carbohydrate identification.

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• Chemistry Practicals
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• Tests Of Carbohydrates

Tests of Carbohydrates

Carbohydrates are the most abundant and diverse class of organic compounds occurring in nature. It played a key role in the establishment and evolution of life on earth by creating a direct link between the sun and chemical energy.

Table of Contents

• Materials Required
• Apparatus Setup
• Observations and Inference
• Results and Discussions
• Precautions

Frequently Asked Questions on Tests of Carbohydrates

To study some simple tests of carbohydrates in the given sample.

The word carbohydrate is formed from the words carbon and hydrogen. Carbohydrates are combinations of the chemical elements carbon and hydrogen plus oxygen. In the natural world, carbohydrates are the most common chemical compound used for food.

The following are the tests to identify the presence of carbohydrates.

• Molisch’s test
• Fehling’s test
• Benedict’s test
• Tollen’s test
• Iodine test

(a) Molisch’s Test:

Molisch’s test is a general test for carbohydrates. This test is given by almost all of the carbohydrates. In this test, concentrated sulfuric acid converts the given carbohydrate into furfural or its derivatives, which react with α-naphthol to form a purple coloured product.

The chemical reaction is given below.

(b) Fehling’s Test:

This test is given by reducing sugars. To the aqueous solution of carbohydrate fehling’s solution is added and heated in water bath. The formation of red precipitate confirms the presence of reducing sugars. The copper ions present in fehling’s solution in +3 state is reduced to +2 oxidation state and in alkaline medium it is precipitated as red cuprous oxide .

Note: The appearance of red precipitate confirms the presence of carbohydrates.

(c) Benedict’s Test:

This test is given by reducing sugars. in an alkaline medium, sodium carbonate converts glucose to enediol and this enediol reduces cupric to cuprous forming cuprous hydroxide. This solution is kept in sodium citrate and on boiling, red precipitate of cuprous oxide is formed.

(d) Tollen’s Test:

This test is given by reducing sugars. Carbohydrates react with Tollens reagent and forms a silver mirror on the inner walls of the test tube. This confirms the presence of reducing sugars. Silver ions are reduced to metallic silver.

(e) Iodine Test:

This test is only given by starch. Starch reacts with iodine solution forms complex blue colour solution. On heating the blue colour disappears and on cooling the blue colour reappears.

Materials Required:

• Molisch’s reagent
• Fehling’s reagent
• Benedict’s reagent
• Tollen’s reagent
• Iodine solution
• Concentrated sulfuric acid
• Sodium hydroxide solution
• Test tube holder
• Test tube stand
• Bunsen burner

Apparatus Setup:

Preparation of Reagents:

• Molisch’s reagent – It is prepared by adding α-naphthol in 10% alcoholic solution.
• Fehling’s reagent – It is a solution of 1ml each of Fehling’s A and Fehling’s B
• Fehling’s solution A – Dissolve copper sulfate in distilled water and add a few drops of sulfuric acid.
• Fehling’s solution B – Dissolve sodium potassium tartrate and sodium hydroxide in 150ml of distilled water.
• Benedict’s reagent – To a solution of sodium citrate 0.25g of anhydrous sodium carbonate in distilled water is added and copper sulfate solution is added to it. Make the total volume up to 125ml by adding distilled water.
• Tollen’s reagent – Add sodium hydroxide solution to the silver nitrate solution. Then add ammonium hydroxide solution dropwise till the precipitate dissolves. The clear solution is called Tollens reagent.
• Iodine solution – Iodine solution is obtained by dissolving iodine in potassium iodide solution.
• Take 2ml of the given sample solution in a clean test tube.
• Add 2-3 drops of Molisch reagent slowly.
• Now add concentrated sulfuric acid along the sides of the test tube.
• The acid layer forms a layer at the bottom.
• Note the junction of the two layers.
• If there is a formation of the violet ring then the presence of carbohydrate is confirmed.
• Take 2ml of given sample solution in a clean test tube.
• Add 2 ml of Fehling’s solution A and Fehling’s solution B to it.
• Keep the solution in a boiling water bath for about 10 minutes.
• If there is the formation of red precipitate then the presence of carbohydrate is confirmed.
• Take the given sample solution to be tested in a clean test tube.
• Add 5ml of Benedict’s reagent to it.
• Boil the solution for about 2 minutes.
• Cool the solution and observe the solution.
• If there is formation of green, red or yellow precipitate then there is presence of reducing sugars.
• Take the given sample solution in a clean test tube.
• Add 2-3ml of tollens reagent to it.
• Keep the test tube in a boiling water bath for 10 minutes.
• If there is the appearance of shiny silver mirror confirms the presence of reducing sugars.
• Take the sample solution to be tested in a clean test tube.
• Add 2-3 drops of iodine solution.
• Observe the change in colour.
• If there is the appearance of a blue colour then the presence of starch is confirmed.

Observations and Inference:

Results and Discussions:

The given organic compound is a _________ (reducing sugar/starch/carbohydrate) compound.

Precautions:

• Handle the acids like concentrated sulfuric acid with care.
• Always use droppers to take reagents from the reagent bottles.
• While heating the reaction mixture do it carefully.

Also, Check ⇒ Structure & Properties of Maltose

What are carbohydrates?

Carbohydrates are polyhydroxy aldehydes, ketones or compounds that are transformed into one of a large group of organic compounds that occur in foods and living tissues, including sugars, starch and cellulose.

What is Fehling’s solution?

Fehling’s solution is prepared by mixing two solutions Fehling’s A and Fehling’s B. Fehling’s A contains copper sulfate solution whereas Fehling’s B contains potassium hydroxide and potassium sodium tartrate.

If a compound gives a positive test for iodine test, what is the nature of the compound?

If a chemical compound gives positive iodine test then the given organic compound is starch.

What are the most important constituents of food?

Carbohydrates, proteins and fats are the most important constituents of food.

What are reducing and non-reducing sugars?

Non-reducing sugars do not have an OH group attached to the anomeric carbon so other compounds cannot be reduced. Glucose is a reducing sugar. A disaccharide may be a reducing sugar or non-reducing sugar. Maltose is a reducing sugar, while sucrose is a non-reducing sugar.

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