EXPERIMENT 1.

CARBOHYDRATES AND RELATED COMPOUNDS

Carbohydrates
- make up the bulk of organic substances on earth

- class of natural compounds made up of carbon, hydrogen, and oxygen (H:O = 2:1)

- contain either an aldehyde or a ketone group and many hydroxyl groups

- aldehyde = polyhydroxy aldehyde/aldose; ketone = polyhydroxy ketone/ketose

- aldose = potentially-free aldehyde; ketose = potentially-free ketone

- presence of aldoses or ketoses in a chemical structure of a carbohydrate is

responsible for the positive results in various reactions

- broken down to form energy, releasing water and carbon dioxide

* Excess carbohydrates = stored in the liver as glycogen or converted into fats

* Plants create carbohydrates via photosynthesis (use of solar energy)

* Monosaccharide - single carbohydrate molecule containing 3-7 carbons

- ex. Glucose and Fructose

* Disaccharide - two monosaccharides linked together

- ex. Sucrose and Lactose

* Polysaccharide - many saccharides linked together

- ex. Starch, Pectin, Glycogen, Cellulose

Importance of Carbohydrates

• Important source of carbon compounds and chemical energy

> Immediate use (glucose, fructose)

> Storage in the form of polysaccharides (starch, glycogen)

• Structural function (cellulose, chitin)

* Structural Polysaccharides - frequently found in combination with proteins (glycoproteins/

mucoproteins) or lipids (lipopolysaccharides)

Subgroups of Carbohydrates
1. Monosaccharides - can be written as Fischer projections

2. Disaccharides - (or sometimes oligosaccharides)

3. Polysaccharides
* Fischer Projections - used to indicate the stereochemistry of each chiral carbon in a .. . ...
..... molecule

- drawn vertically, with the carbonyl carbon at the top

- horizontal bonds point out of the page (toward you)

- vertical bonds point into the page (away from you)

Representations of Chain Molecules

A. Conventional Fischer Projection

B. “Line Structure” Variation of Fischer Projection - carbons and hydrogens are not shown

C. “Zigzag” Style - preferred by organic chemists

* Most monosaccharides exist in a cyclic form

- the aldehyde or ketone group reacts with one of the -OH groups on the other
end of the same molecule to form a cyclic hemiacetal

PROCEDURE

A. Physical Properties
Physical Solubility in Solubility in
Carbohydrate Classification Taste
Appearance Water 95% Ethanol

Glucose Monosaccharide Sweet White powdery Soluble Insoluble

Galactose Monosaccharide Sweet White powdery Soluble Insoluble

Yellowish-white
Fructose Monosaccharide Sweet Soluble Insoluble
crystalline

Insoluble

Lactose Disaccharide Sweet White powdery Soluble

Sucrose Disaccharide Sweet White crystalline Soluble Insoluble

White
Gum Arabic Polysaccharide Tasteless amorphous Soluble Insoluble
powder

White
Starch Polysaccharide Tasteless amorphous Soluble Insoluble
powder

White
Glycogen Polysaccharide Tasteless amorphous Soluble Insoluble
powder

White
Inulin Polysaccharide Tasteless amorphous Soluble Insoluble
powder

① Which of the carbohydrates taste the sweetest? Monosaccharides

② Which of the carbohydrates taste the least sweet? Polysaccharide

③ Which among the monosaccharide and disaccharide taste the least sweet?
Monosaccharide = galactose; disaccharide = lactose

④ What component of sugar is responsible for the taste? Hydroxyl (-OH) group

⑤ In which reagent were the sugars consistently insoluble? Support your answer.
Ethanol. Alcohol molecules have only 1 polar area and a larger non-polar area,
making it a bad dissolver of polar substances like sugars

B. General Test for Carbohydrates

1. Molisch Test

- qualitative test for determining the presence of carbohydrates in a solution

- positive for all kinds of carbohydrates in free form or combined form

- (+) = appearance of reddish violet/purple colored ring at the junction of the two
liquids

> Molisch Reagent = 1% solution of alpha-naphthol in alcohol

* Test involves the test tube tilted at about 30-45 degrees and added with conc. H2SO4

 2. Anthrone Test

- for both qualitative and quantitative analysis of carbohydrates

- same principle as methyl furfurals (giving condensation products with anthrone

that are bluish green in color)

> Anthrone Reagent = anthrone + conc. H2SO4

Carbohydrate Solution Color Reaction with Molisch Color Reaction with Anthrone

Glucose Purple ring Bluish green

Ribose Purple ring Bluish green

Lactose Purple ring Bluish green

Sucrose Purple ring Bluish green

Gum Arabic Purple ring Bluish green

Starch Purple ring Bluish green

Water Yellowish sol’n Cloudy white

① What is the composition of the Molisch reagent? 1% alpha-naphthol and alcohol

② What is responsible in the formation of the colored ring? Furfural derivatives

C. Chemical Tests for Carbohydrates

1. Reduction

* Reducing Sugar - one that can be oxidized

- molecule contains a free anomeric carbon (since it is the open-chain form of

the aldehyde that can react and be oxidized)

a. Fehling’s and Benedict’s Reagent

- blue colored reagents

> Fehling’s Reagent = Cu2+ + Na2CO3 + potassium tartrate

- contain Cu2+ ions in an aqueous basic solution

- (+) = reducing agent/sugar is present = Cu2+ is reduced to Cu = formation of

Cu2O (red ppt) (may need to be heated for precipitate to form)

* Benedict’s Test - qualitative test; color depends upon amount of reducing sugar
. present in the mixture (produces Cu2O)

> Benedict’s Reagent = copper + sodium acetate + acetic acid

> blue solution = none

> green/yellow ppt = traces of reducing sugar

> orange red ppt = moderate

> brick red ppt = large amount of reducing sugar

 b. Barfoed’s Test

- Barfoed’s reagent is much milder than Fehling’s reagent

> Barfoed’s Reagent = CuSO4 · 5H2O + NaOH

- distinguishes a reducing monosaccharide from a reducing disaccharide

- different types of sugars react at different rates

- Reducing monosaccharides = react quickly

- Reducing disaccharides = react very slowly or not at all

- (+) = presence of reducing monosaccharides = Cu2O (dark red precipitate)

c. Tollen’s Reagent

> Tollen’s Reagent (colorless) = silver nitrate (AgNO3) and ammonia

- turns Ag+ to Ag

- (+) silver mirror

d. Nylander’s Reagent
> Nylander’s Reagent = bismuth nitrate (Bi(NO3)3), potassium sodium
tartrate, potassium hydroxide (KOH)

- turns Bi to metallic Bi (black ppt)

3+

e. Picric Acid
- (O₂N)₃C₆H₂OH / 2,4,6-trinitrophenol
> Picric Acid Reagent = picric acid and Na2CO3
- (+) = formation of picramic acid (C6H5N3O5) (mahogany red)

Tests Glucose Fructose Maltose Sucrose

Fehling’s Brick red ppt Brick red ppt Brick red ppt Blue sol’n

Benedict’s Brick red ppt Brick red ppt Brick red ppt Blue sol’n

Brick red ppt in Brick red ppt in

Barfoed’s Blue sol’n Blue sol’n
blue sol’n blue sol’n

Tollen’s Silver mirror Silver mirror Silver mirror Colorless sol’n

Nylander’s Black ppt Black ppt Black ppt Colorless sol’n

Picric Acid Mahogany red ppt Mahogany red ppt Mahogany red ppt Yellow sol’n

① Which of the unknowns is not a reducing sugar? Sucrose

② Support your answer. The test results on every test came out negative

③ What test differentiates a monosaccharide from a disaccharide? Barfoed’s Test

④ Classify the sugars

 Reducing Non Reducing

Monosaccharide Polysaccharide
Disaccharide Disaccharide

Glucose Maltose Sucrose

Fructose

2. Color Tests of Saccharides (Dehydration)

* Dehydration - action of strong acids

- occurs when aldopentoses and aldohexoses are heated with strong acids

- dehydrated carbohydrates form furfural derivatives (for pentoses) and

hydroxymethylfurfural derivatives (for hexoses) which will condense with various
phenolic substances (alpha naphthol, resorcinol, orcinol) to give a colored
complex

Tests

a. Seliwanoff’s Test (Resorcinol-HCl Test)

> Seliwanoff’s Reagent = resorcinol and non-oxidizing acid (HCl)

- for presence of a ketose

- (+) = ketose becomes dehydrated = cherry red complex (not a precipitate)

- timing for heating is important

- overheating will give a false positive result

b. Bial’s Test (Orcinol-HCl Test)

> Bial’s Reagent = orcinol and HCl
- determine the presence of pentoses (5C sugars)

- when pentoses are heated with conc. HCl, furfural is formed

- furfural condenses into orcinol in the presence of ferric ion, turning into
condensed furfurals (blue-green)

* Hexoses generally react to form green, red, or brown products

c. Mucic Acid Test (Oxidation of Sugar to Sugar Acids)

- highly specific for galactose

- independently present in solutions or obtained by hydrolysis of lactose

- galactose is converted into saccharic acid on heating with HNO3 (strong

oxidizing agent)

- saccharic acid is insoluble in cold water

- formation of saccharic acid (white crystals?) = presence of galactose

- mucic acid/galactaric acid (a saccharic acid) is formed from galactose due to

the oxidation of both aldehyde and primary alcoholic group at C1 & C6

 * Mucic acid is also known as 1,6-dicarboxylic acid (which is an aldaric acid)

* Aldaric acid - products of oxidation of both the aldehyde and the primary alcohol
groups of the sugars

Carbohydrates Seliwanoff’s Bial’s Test Mucic Acid

Glucose No color change Green

Fructose Cherry red complex Dark green

Galactose No color change Green White crystals

Lactose No color change Greenish yellow

Maltose No color change Greenish yellow

Sucrose Cherry red complex Green

Ribose No color change Blue-green

① Classify the sugars as aldose and ketose:

Aldose Ketose

Glucose Fructose

Fructose Sucrose

Galactose

Lactose

Maltose

Ribose

① What test differentiates an aldose from a ketose sugar? Seliwanoff’s test

② What test differentiates pentosans from ketosans? Bial’s test

③ What test is specific to keto sugars? Seliwanoff’s test

④ Which of the sugars tested is a pentosan? Support your answer. Ribose. It came out
positive with the Bial’s test, which is used for determining the presence of pentoses

⑤ Which of the sugars tested aldaric? Glucose and galactose (maltose?)

 3. Tests to Determine the Presence of Potentially-Free Aldehyde or Ketone Group

a. Moore’s Test
- action of strong alkalis

- includes the addition of strong NaOH (40% NaOH)

- (+) = yellow to yellowish brown?

Time Glucose Fructose Sucrose

After 1 minute Reddish brown sol’n Golden yellow sol’n Colorless

After 3 minutes Reddish brown sol’n Golden yellow sol’n Colorless

After 5 minutes Reddish brown sol’n Golden yellow sol’n Colorless

Final results Reddish brown sol’n Golden yellow sol’n Colorless

① What is the color of the final product formed? Glucose = brown; fructose =
golden yellow; sucrose = colorless

② What is the name of the final product formed? Caramel

b. Phenylhydrazine Reaction/Osazone Formation

- includes the addtion of phenylhydrazine reagent, saturated NaCl solution, and
glacial acetic acid

- (+) = osazone (bright yellow crystals)

Carbohydrate Glucose Fructose Lactose Sucrose

Results Yellow crystals Yellow crystals Yellow crystals Yellow solid

① Which of the sugars has a potentially (a) free aldehyde and (b) free ketone
group? (a) glucose and lactose; (b) fructose

② Support your answer. Fructose, glucose, and lactose formed yellow crystals

③ What are the tests to determine potentially free aldehyde and ketone groups?
Moore’s test and osazone formation

NOTE!

* (+) Fehling’s, Benedict’s, Tollen’s, Nylander’s, Picric acid = reducing sugar

* (+) Barfoed’s = reducing monosaccharide

* Ex. (+) Fehling’s, (-) Barfoed’s = reducing disaccharide

* Reducing sugar = has a free aldehyde/ketone group