REVISED P02

Icha – Imad – Adrian

QC by Ler
 Introduction
A. Carbohydrates
Carbohydrates perform numerous roles in the living things this means saccharides
and their derivatives play key roles in immune system, fertilization, preventing
pathogenesis, blood clotting, and development.
❖ Monosaccharide
The 5-carbon monosaccharide ribose is an important component of:
➢ Coenzymes (ATP, FAD, NAD)
➢ The backbone of RNA
❖ Polisaccharide
Serves as the storage of energy (ex: starch and glycogen)

B. Proteins
Proteins have an important role in the:
❖ Lifespan of human life
❖ Life Quality of human life

C. Lipids
Important function of lipid in living organisms:
❖ Cell membrane structure
➢ Barrier of the cell
➢ Controls membrane fluidity
➢ Controls the flow of material (going in and out the cell)
❖ Energy storage
example: fat stored in adipose tissue
❖ Lipid hormones
example: steroids and eicosanoids – mediate communication between cells
❖ Signal Transduction
Function in the transmission of information in cells
❖ Lipid Vitamins
Required for metabolism (usually as coenzymes)

2 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism

 Carbohydrates

1. Molisch Test
❖ General test of carbohydrates
❖ Reagent: alpha-naphthol 95% alcohol (Molisch Reagant)
❖ This reaction depends on the formation of furfural and its derivative
❖ If the result is positive carbohydrates = a violet ring form = carbohydrate is present

This test is carried out by using solution of Monosaccharide (could be
glucose, fructose, galactose), Disaccharides (could be galactose, maltose),
and Polysaccharides (amylum).

Steps:
1. 2 ml of solution that is going to be tested to test tube
2. add 3 drop of Molisch reagent to (1) – then mix
3. add 2 ml of concentrated sulfuric acid (ADD SLOWLY in 90o, down the
side of the tube – and DON’T MIX)
4. If carbohydrate is present, a violet ring will appear in the middle of both
solution

1) Benedict’s Test
❖ To identify reducing sugar
Reducing sugar: sugar that is capable to act as a reducing agent
because it has free aldehydes or ketones. ALL monosaccharide are
reducing sugar.
❖ Reagent: Cupric Sulphate, Sodium Carbonate, and Sodium Citrate
❖ Depends on: the reduction of alkaline cooper solution by reducing
sugar which leads to the forming colored precipitate:
➢ Green: found traces of reducing sugar
➢ Yellow: moderate amount of reducing sugar
➢ Red: large amount of reducing sugar
➢ Blue/no change of color: no reducing sugar is found

Steps:
1. 1 mL of Benedict’s solution to test tube
2. Add 2 mL of solution to be tested (glucose/fructose, lactose/maltose, and
sucrose.)
3. Mix and keep in a boiling water bath for 3 minutes
4. Allow to cool slowly
5. Observe the color change and whether a precipitate has formed

3 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism


2) Modified Barfoed’s Test
❖ To differentiate monosaccharide and disaccharides or to detect
presence of monosaccharide in disaccharides.
❖ Reagent: Cupric acetate dissolved in water + lactic acid
❖ If monosaccharide is present, the solution will turn Dark Blue.



Steps:
1. 1 mL Barfoed’s Solution + 1 mL (5 drops of solution to be tested + 15 drops water) in test
tube
solution to be tested = lactose, sucrose, glucose, and water as a “blank”
2. Heat in boiling water bath for 3 minutes
3. Place in cold water to cool for 5 minutes
4. Add 1 mL of Phosphomolydic reagent and mix
5. If the solution turns dark blue, means that it’s a monosaccharide
If the solution turns light blue, means monosaccharide are absent in the disaccharide

3) Seliwanoff Test
❖ To differentiate between aldose and ketose
❖ The reaction depend on the formation of 4-hydroxy-
methyl-furfural and its reaction with resorcinol (1,3
dihydroxy-benzene) to form a red-colored compound.
❖ No ketone – No difference
= No difference = red solution

Ketone is present – red solution

Steps:
1. 2 mL of Seliwanoff’s reagent + 2 mL solution to be tested (glucose,
fructose, sucrose)
2. heat in hot water bath exactly for 60 seconds
3. observe the change of color

4 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism



Protein
1) Biuret Test
(is the most common protein test)
❖ To determine peptide bond in a protein
❖ Protein dissolve cooper hydroxide to form colored complexes
❖ Violet color indicates the presence of protein

Steps:
1. 1 mL of albumin solution + 1 mL sodium hydroxide
2. add 2 drops of cooper sulfate solution to (1)
3. shake and observe violet color
4. repeat with the other solution



2) Millon’s Reaction
❖ to determine the monohydroxy-benzene group in a sample
❖ the reaction depends on the presence of monohydroxy-benzene
derivative (such as tyrosine and phenol)
❖ the reaction does not take place satisfactorily in the presence of
CI- or NH4+ (its not useful in urine analysis)

Steps:
1. 1 mL of albumin solution in test tube
2. add 2 drops of Millon’s reagent to (1)
3. mix well – a white precipitation forms
4. heat carefully for 5 minutes
5. observe, if red or pinkish white precipitate forms then it’s positive (monohydroxy-
benzene group is present)
The result could be:
➢ No precipitate (clear solution, tidak ada endapan) = Gelatin
➢ Pink or pinkish white precipitate (tidak larut) = Albumin
➢ Brick red precipitate = Casein

5 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism

 3) Action of Alcohol Upon Protein
❖ To know the reversibility of protein

Note:
❖ Since protein can from precipitate with alcohol, by adding water some protein will
become clear again – this are the characteristic of reversibility of protein.

Steps:
1. 2 mL of Albumin solution + 2 mL alcohol
2. Mix well
3. Add 3 mL water then mix again
4. Note the result
❖ Result:
➢ Transparent and clear solution (reversible denaturation) =
Gelation
➢ Solution contain colloid/white precipitate (irreversible
denaturation) = Casein
➢ Cloudy and murky solution (irreversible denaturation) =
Albumin

6 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism

 Lipid

B. Lipid
1) Hubl’s Test
❖ To determine degree of unsaturation of lipid or
fatty acid
❖ Lipids could undergo an addition reaction
❖ To determine double bond, samples are reacted
with Hubl’s solution.

Note:
❖ The decreasing intensity of the violet color
(which is the color of Hubl’s solution) denotes
the the presence of double bonds.
❖ That is because Hubl’s main task is to break
double bonds. If the solution have double bond, Hubl’s will work to break the
double bond, that is why the color intensity of Hubl itself will decrease.
THIS BASICALLY MEAAANSSSS
➢ If you drop Hubl in and the color intensity decrease = double bond is present
If you drop Hubl in and the color stays violet = no double bond

Steps:
1. add 2 drops of Hubl’s iodine solution into 2 ml of X
X = butter / oil / chloroform

Results of this experiment will be as shown in the table bellow
X Margarine Oil Chloroform
Color after Hubl is added Cream Clear yellow Pink

Color intensity of the Hubl Color intensity Color intensity Color Intensity
color fades fades doesn’t fade
Presence of double bonds Double bonds Double bonds No Double Bonds

7 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism

 E. POST TEST NOTES - (ini penting bgt guys seriusan)

Unknown Sample

+ NaOH + CuSO4
Blue Purple
(Carbohydrates) (Protein)

Molisch Test Millon Test

(+) (-)
Violet ring no purple ring (-) No Precipitate, Clear (+) Pink (+) Brick red
solution Precipitate Precipitate

Not
Barfoed’s Test
carbohydrates Gelatin Albumin Casein

(-) Light Blue = (+) Dark Blue =

Disaccharides Monosaccharides
Add water + Alcohol

Benedict Benedict
Test Test

Irreversible
Reversible
Denaturation
Denaturation Irreversible
Not Brick red? Brick red? Must be
Denaturation
= Sucrose = Maltose or brick red (no precipitation –
(white precipitate –
Lactose tidak larut, ada
gaada endapan)
endapan putih

Seliwanoff test

No color = Red Solution =

Aldose = no Ketose = Ketone is
ketone present

Glucose or Fructose or
Galactose Sucrose

8 BBS MODULE I Practical 02—Carbohydrate, Protein, and Lipid of Living Organism