Post-Lab Discussion

Activity 1A

Subcellular Components of the Living Cell

Activity 1B

Qualitative Tests for the Chemical Composition of

Organelles

Tests for Carbohydrates

Molisch TestBenedict's Test

Molisch Test

Positive Result: PURPLE ring betweenw/ Ribose Sol'n acid and sediment

Benedict's Test

Positive Result: BRICK RED colorationw/ Ribose Sol'n

Tests for Proteins

Biuret TestXanthoproteic Test

Biuret Test

Positive Result: PURPLE colorationw/ Ribose Sol'n

Xanthoproteic Test

Positive Result: INTENSE YELLOW colorationw/ Ribose Sol'n

Tests for Lipids

Sudan TestAcrolein Test

Sudan Test

Positive Result: RED colorationw/ Ribose Sol'n

Acrolein Test

Positive Result: BITING ODORw/ Ribose Sol'n

Results

Analysis and Conclusion

Separation

Principle: Differential Centrifugation

Tissue or cells are first disrupted to release their internal contents (homogenate).

Larger particles sediment faster than smaller ones and this provides the basis for obtaining

crude organelle fractions by differential centrifugation.

Principle: Dehydration of the carbohydrate by sulfuric acid to produce an aldehyde

Reaction:

R-CHO + (conc.) H2SO4 → R-CHO (ex. furfural) + 3 H2O

R-CHO + 2 C10H8-OH (α-naphthol) → colored product

Molisch Test

Principle: Reduction of Cu2+ ions by reducing sugars, which then can be detected by the formation of a brick red precipitate of Cu2O (Cuprous oxide)

General Reaction:

R-CHO + 2Cu2+ + 4OH- →R-COOH + Cu2O + 2H2O

Benedict's Test

Principle: Reduction of Cu2+ which thencomplexes with the N atoms on thepeptide bonds, at high pH. [Amide bond is resposible for the colour.]

Reaction:

copper(II) ions + protein (peptide bonds) → biuret complex (PURPLE)

Biuret Test

Principle: Nitration of benzene ring present inthe structure

Reaction:

G-H + HNO3 →G-NO2 (YELLOW) + H2O

Xanthoproteic Test

Principle: Ability of fat cells to selectively absorb pigments in fat-dyes such as Sudan IV

Reaction: Absorption

Sudan Test

Principle: Whenever fat is heated in the presence of dehydrating agent, fat molecule sheds glycerol in the form of the unsaturated aldehyde - acrolein

Reaction:

Glycerol + KHSO4 → C3H4O (acrolein w/ biting odor)

Acrolein Test

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