Experiment 4: Properties of Carbohydrates - Comprehensive Notes

Experiment 4: Properties of Carbohydrates

Intended Learning Outcomes

• Describe the physical characteristics of carbohydrates (solubility, taste, physical appearance).
• Perform general and classification tests for carbohydrate identification.

Materials

• 1% solutions of: ribose, glucose, fructose, lactose, sucrose, starch, inulin, glycogen, cellulose.
• Molisch reagent.
• Concentrated sulfuric acid (H2SO4).
• Benedict's reagent.
• Saturated picric acid.
• 10% Na2CO3 solution.
• 30% NaOH solution.
• Barfoed's reagent.
• Seliwanoff's Reagent.
• Bial's reagent.
• I2 in KI solution

Introduction to Carbohydrates

• The term "carbohydrate" originates from the general molecular formula $C<em>r(H</em>2O)$, once considered hydrates of carbon.
• This view is inaccurate, but the term persists.
• Monosaccharides are the simplest carbohydrates.
  • Polyhydroxy aldehydes (aldoses, general structure A).
  • Polyhydroxy ketones (ketoses, general structure B).

Monosaccharides Classification

• Monosaccharides typically consist of 3-6 carbons.
  • Trioses: 3 carbons.
  • Tetroses: 4 carbons.
  • Pentoses: 5 carbons.
  • Hexoses: 6 carbons.
• Examples: ribose, glucose, fructose.

Structures of Monosaccharides

• Glucose: An aldose with the formula $C<em>6H</em>{12}O_6$
• Fructose: A ketose with the formula $C<em>6H</em>{12}O_6$
• Ribose: An aldose with the formula $C<em>5H</em>{10}O_5$

Cyclic Forms of Monosaccharides

• In solution, monosaccharides exist in cyclic forms due to the reaction between the aldehyde/ketone group and an -OH group on the same molecule, forming a cyclic hemiacetal.
• Anomers: α-D-glucose and β-D-glucose are anomers.
• Equilibrium exists in solution between cyclic and open-chain forms, allowing interconversion of α and β forms.

Disaccharides

• Two monosaccharides combine with the loss of one water molecule to form a disaccharide.
• The bond connecting monosaccharide units is a glycosidic bond.
• Examples: sucrose (table sugar), lactose (milk sugar).

Polysaccharides

• Polysaccharides consist of many linked monosaccharides.
• Examples: starch, pectin, glycogen, cellulose.

Qualitative Chemical Tests for Carbohydrates

• Used to classify molecules according to structural type and identify structural features.
• Experiments involve analyzing known solutions and an unknown carbohydrate solution.

I. Physical Characteristics of Carbohydrates

• Pre-lab: Complete a table assessing taste, physical appearance, and solubility in water and ethanol for various carbohydrates.

II. General Test for the Presence of Carbohydrates: Molisch's Test

• Discovered by Hans Molisch.
• Involves adding Molisch's reagent to the analyte, followed by concentrated sulfuric acid ($H<em>2SO</em>4$).
• Formation of a purple or purplish-red ring at the contact point indicates the presence of carbohydrates.
• Almost all carbohydrates give a positive reaction (exceptions: tetroses and trioses).

Procedure for Molisch's Test

1. Add 2 mL of ribose, glucose, fructose, lactose, sucrose, starch, and water to separate test tubes.
2. Add 1 mL of Molisch's reagent to each tube and mix.
3. Slowly pour concentrated $H<em>2SO</em>4$ down the side of each tilted test tube (45°). Do not shake.
4. Observe for the formation of a purple or purplish-red ring. Record results.

III. Classification Tests for Carbohydrates

• Classify carbohydrates according to structural types.
• Determine functional groups in carbohydrate molecules.

Test for Reducing Sugar

• Reducing sugar: oxidized by a weak oxidizing agent in basic aqueous solutions.
• Non-reducing sugar: not oxidized.
• All aldoses are reducing sugars due to the aldehyde functional group.
• Ketoses, though containing a ketone, show reducing properties due to keto-enol tautomerization in basic aqueous solutions, converting them to aldoses.
• All monosaccharides are reducing.
• Disaccharides can be reducing or non-reducing depending on whether they contain a free anomeric carbon.
  • Free anomeric carbon allows the ring to open and expose the aldehyde group.
• All polysaccharides are non-reducing.
• Weak oxidizing agents used: Benedict's reagent, Fehling's reagent, Tollen's reagent, Nylander's reagent, picric acid with sodium carbonate solution (all in basic conditions, pH > 7).

1. Benedict's Test

• Cupric ion ($Cu^{2+}$) oxidizes aliphatic aldehydes, including α-hydroxyaldehydes like aldoses.
• Positive test: formation of a brick red precipitate of cuprous oxide ($Cu_2O$).
• Color varies (red, orange, green) depending on sugar concentration.
• Used to quantitatively estimate sugar amount and detect glucose in urine.

Procedure for Benedict's Test

1. Add 2 mL of each carbohydrate solution (ribose, glucose, fructose, lactose, sucrose, starch, water) to separate test tubes.
2. Add 3 mL of Benedict's reagent to each tube and mix.
3. Heat in a boiling water bath for 2 minutes.
4. Observe for yellow to brick red precipitate. Record results.

2. Picric Acid Test

• Saturated picric acid solution (yellow) is used as an oxidizing agent in an alkaline environment (10% $Na<em>2CO</em>3$).
• Positive result: mahogany red colored solution due to the reduction of picric acid to picramic acid.

Reaction Example

• Glucose reacts with picric acid under alkaline conditions:

$CHO - (CHOH)<em>4 - CH</em>2OH + Picric Acid \rightarrow Gluconic Acid + Picramic Acid$

Procedure for Picric Acid Test

1. Add 2 mL of each carbohydrate solution to separate test tubes.
2. Add 2 mL of saturated picric acid solution, followed by 0.5 mL of 10% $Na<em>2CO</em>3$ solution. Mix well.
3. Heat in a boiling water bath for 2 minutes.
4. Observe for mahogany red coloration. Record results.

3. Moore's Test

• Reducing sugars have free carbonyl groups that undergo aldol condensation in a basic environment.
• Product for reducing sugars: caramel with a brown color and characteristic odor, intensified by acidification with diluted sulfuric acid.

Procedure for Moore's Test

1. Add 1 mL of each carbohydrate solution to separate test tubes.
2. Add 1 mL of 30% NaOH solution and heat on a direct flame for 1-2 minutes.
3. Observe for yellowish-brown color with characteristic caramel odor. Record results.

Barfoed's Test for Monosaccharides

• Distinguishes between monosaccharides and disaccharides based on reducing properties of sugars.
• Oxidizing agent: cupric ion ($Cu^{2+}$).
• Positive test for monosaccharides: brick red precipitate of $Cu_2O$ within 2-3 minutes.
• Disaccharides require longer time (10 minutes) to form the precipitate.

Procedure for Barfoed's Test

1. Add 2 mL of each carbohydrate solution to separate test tubes.
2. Add 3 mL of Barfoed's reagent to each tube and mix.
3. Heat in a boiling water bath for 5 minutes.
4. Observe for brick red precipitate against a dark background. Record results.

Seliwanoff's Test for Ketoses

• Distinguishes aldoses from ketoses.
• Seliwanoff's reagent dehydrates ketoses rapidly to form furfural derivatives, which condense with resorcinol to give a cherry red complex.
• Positive test: cherry red solution after 2 minutes.
• Responded by keto-containing carbohydrates (monosaccharides or disaccharides).

Procedure for Seliwanoff's Test

1. Add 2 mL of each carbohydrate solution to separate test tubes.
2. Add 3 mL of Seliwanoff's reagent to each tube and mix.
3. Heat in a boiling water bath for 2 minutes.
4. Observe for cherry red solution. Record results.

Bial's Test for Pentoses

• Distinguishes pentoses from hexoses based on color development.
• Pentoses dehydrate and react with orcinol and ferric ion to generate a bluish-green solution.
• Hexoses yield different products, giving muddy-brown, yellow, or gray solutions.

Procedure for Bial's Test

1. Add 2 mL of each carbohydrate solution to separate test tubes.
2. Add 2 mL of Bial's reagent to each tube and mix.
3. Heat in a boiling water bath for 2 minutes.
4. Observe for bluish-green color. Record results.

Iodine Test for Polysaccharides

• Specific test for polysaccharides, which have highly coiled structures like starch.
• Iodine forms a coordinate complex with the helically coiled polysaccharide chain due to adsorption.
• Color depends on the length of the linear chain available for complex formation.
• Color change of $I_2$ in KI solution (brown) indicates the presence of polysaccharides.
• Different colors are produced for different polysaccharides.

Procedure for Iodine Test

1. Add 2 mL of each carbohydrate solution to separate test tubes.
2. Add 1 drop of $I_2$ in KI solution to each tube and mix.
3. Observe for color changes. Record results.
4. Repeat steps 1-3 with glycogen, inulin, and cellulose solutions. Record results.

Tables for Recording Results

• Table 2: General and Classification Tests of Carbohydrates (Molisch's, Benedict's, Picric Acid, Moore's, Barfoed's, Seliwanoff's, Bial's, Iodine).
• Table 3: Iodine Test for Polysaccharides (Starch, Glycogen, Inulin, Cellulose).