EXPERIMENT 1: Qualitative Analysis of Carbohydrates

Carbohydrates

Most abundant biomolecules in nature

Carbohydrates in plants

Serves 2 purposes:

• Provides structural integrity in stems and roots in the form of cellulose

• Acts as a storage of glucose in the form of starch for the production of biochemical energy

Carbohydrates in humans

These molecules fulfill a more diverse set of functions such as:

• Providing energy for the cell through the oxidation of monosaccharides

• being part of the structural framework of nucleotides

• Acting as messengers in cell communication and recognition

Carbohydrates

• Polyhydroxylated aldehydes or ketones or molecules that yield polyhydroxylated aldehydes or ketone upon hydrolysis

• Can form glycosidic linkages with one another to form more structurally and biochemical complex carbohydrates such as monosaccharides, oligosaccharides, and polysaccharides

Monosaccharides

• Simplest type among the carbohydrates

• Can be classified based on the type of carbonyl group present (aldose or ketose) or based on the number of carbons present in its open chain structure (3C-triose, 4C-tetrose, 5C-pentose, 6C-hexose)

Carbonyl group (aldehyde or ketone) and hydroxyl group

• Functional groups found in carbohydrates

• Both functional groups are polar

Presence of hydroxyl groups

• Polar and allow carbohydrates to observe intermolecular hydrogwn bonding

• Gives carbohydrates high boiling and melting points and make them generally soluble in polar solvents such as water

Presence of different and multiple functional groups

Makes carbohydrates undergo different chemical reactions

Objectives of experiment 1

Classify carbohydrates based on the carbonyl functionality and based on the number of carbons, using qualitative chemical tests

Reagents

• Glucose

• Amylase

• Maltose

• Xylose

• Fructose

• Galactose

• Lactose

• Sucrose

• Molisch reagent

• Conc. Sulfuric acid

• Iodine solution

• Benedict's reagent

• Barfoed's reagent

• Seliwanoff's reagent

• Bial's orcinol reagent

Equipment

Water bath

To be borrowed by students from stockroom

• (20) test tubes (13 × 100 mm)

• (5) test tube holders

• (1) distilled water in wash bottles

Safety

• Wear PPE and lab goggles at all times

• Wash hands thoroughly with soap and water before leaving the laboratory

• Dispose gloves properly after performing the experiment

Preliminary procedures

• Label 10 test tubes with glucose, amylose, maltose, xylose, fructose, galactose, lactose, sucrose, unknown and blank (distilled water only)

• Place 15 drops of each sugar sample into their respective properly labelled test tubes

• Prepare boiling water bath setup

• Be sure to wash all the test tubes after each test before proceeding to the next

Molisch test

• Add 5 drops of Molisch reagent into each test tube

• Tilt the test tubes and allow 15 drops of concentrated H2SO4 to flow down

• Return the test tubes in the rack. Note your observations

Iodine test

• Add 5 drops of iodine solution into each test tube

• Return the test tubes in the rack. Note your observations

Benedict's test

• Add 25 drops of Benedict's reagent into each test tube

• Heat the test tubes in a boiling water bath for 2 minutes

• Remove the test tubes from the water bath and return them in the rack

• Note your observations

Barfoed's test

• Add 25 drops of Barfoed's reagent into each test tube

• Heat the test tubes in a boiling water bath for 5 minutes

• Record the time (in minutes and seconds) when the orange or red precipitate starts to appear

• Note: reducing monosaccharides will form orange or red precipitates faster (in less than 2 to 3 minutes) than reducing disaccharides (which may take up to 10 minutes)

Reducing monosaccharides

Will form orange or red precipitates faster in less than 2 to 3 minutes than reducing disaccharides which may take up to 10 minutes

Reducing disaccharides

Will form orange or red precipitate but may take up to 10 minutes

Seliwanoff’s test

• Add 25 drops of Seliwanoff’s reagent into each test tube

• Heat the test tubes in a boiling water bath until an observable change is observed

• Note the time when the colored product is observed.

Bial’s test

• Add 25 drops of Bial’s Orcinol reagent into each test tube

• Heat the test tubes in a boiling water bath and note your observations.

Excess sugar solution (waste generated)

Disposal container: sink

From Molisch test and Seliwanoff’s test (waste generated)

Disposal container: non-halogenated organic waste

From Iodine test (waste generated)

Disposal container: halogenated organic waste

From Benedict’s test, Barfoed’s test, and Bial’s test (waste geenrated)

Disposal container: metal-containing waste

Molisch test

• Test for:
  Carbohydrates (general test for all carbohydrates)

• Positive result:
  Formation of a violet/purple ring at the interface of the two layers

• Negative result:
  No violet ring formed

Iodine test

• Test for:
  Starch (polysaccharides, especially amylose)

• Positive result:
  Blue-black coloration

• Negative result:
  Solution remains yellow-brown (color of iodine)

Benedict’s test

• Test for:
  Reducing sugars (e.g., glucose, fructose, lactose, maltose)

• Positive result:
  Color change from blue to green, yellow, orange, or brick-red precipitate (depending on sugar concentration)

• Negative result:
  Solution remains blue

Barfoed’s test

• Test for:
  Monosaccharides (distinguishes monosaccharides from disaccharides)

• Positive result:
  Red precipitate (Cu₂O) formed within 1–2 minutes

• Negative result:
  No red precipitate within the given time (solution remains blue)

Seliwanoff’s test

• Test for:
  Ketoses (e.g., fructose) vs aldoses

• Positive result:
  Cherry-red color appears rapidly

• Negative result:
  No red color or only a faint pink color after prolonged heating

Bial’s test

• Test for:
  Pentoses

• Positive result:
  Blue-green color

• Negative result:
  No blue-green color (yellow or brown solution)