Biochemistry

Practical
Revision

Semester 1
2024/2025

1
 Carbohydrates

Carbohydrates are classified according to the degree of

complexity into:

1- Monosaccharides: simple sugars. Cannot be hydrolyzed into simpler

carbohydrate.
2- Oligosaccharides: On hydrolysis yield 2 to 10 monosaccharides units. The
most common form is disaccharide which consists of 2 monosaccharides.
3- Polysaccharides: On hydrolysis yield more than 10 monosaccharide units.

Monosaccharide:

• Monosaccharides are reducing sugars.

• All monosaccharides are reducing agents since they contain free aldehyde or ketone
group
Monosaccharides are the simplest sugars.
Monosaccharides are classified by two methods according to number of carbons or
according to carbonyl group.

Number of carbons: Carbonyl group:

• Trioses (3 carbons)
• Tetroses (4 carbons) • Aldoses
• Pentoses (5 carbons) • Ketoses
• Hexoses (6 carbons)

2
Disaccharides:

Each disaccharide molecule consists of two monosaccharide units joined by a

glycosidic bond.

Examples of Disaccharide:
1- Maltose (Malt sugar)
• Maltose is reducing sugar as it contains free aldehyde group
• Consists of 2 glucose units linked by 1,4 glycosidic bond

2- Lactose (Milk sugar)

• Lactose is reducing sugar as it contains free aldehyde group
• Consists of galactose and glucose units joined by 1,4 glucosidic bond

3- Sucrose (Cane sugar/ Table sugar)

• Sucrose is non-reducing sugar
• Consists of glucose and fructose units joined by 1,2 glucosidic bond

3
 Polysaccharides
• Polysaccharides are polymers of monosaccharide units joined by glycosidic bonds in
linear and branched manner.

Example of Polysaccharide
Starch
(Glucose units linked together by linear alpha 1,4 & branching alpha 1,6 glycosidic
bond).

Protein
Biological Importance of Protein:

• All enzymes are proteins.

• Hemoglobin is a protein.
• Proteins are important for growth.
• Antibodies are proteins.
• Some hormones are proteins as insulin.

Different Protein examples

(a) Albumin is simple protein: Albumin represents the highest concentration of plasma
proteins.
(b) Casein is conjugated protein: Casein represents up to 80% of the proteins in cow
milk.
(c) Gelatin is derived protein: derived from collagen after boiling. It is commonly used
as a gelling agent in food and Pharmaceuticals.

Normally no proteins are detected in urine. Detection of a protein as albumin in urine indicates
kidney disease.

4
 Urea & Uric Acid

Urea and uric acids are defined as non protein nitrogenous compounds (NPN)

NPN are:
q Urea
q Uric acid
q Creatine
q Creatinine
q Ammonia

Urea:
• It is one of the metabolic end products of protein.
• Urea is derived from ammonia (NH3)
• Ammonia is formed by removal of the amino group from amino acids
(deamination)
• Urea is only synthesized in liver and then transferred to blood and passes to the
kidneys to be excreted in urine.

Clinical value: assess both kidney and liver functions

1-kidney function: in kidney diseases the kidney fails to excrete urea so there is
an increase in blood urea and decrease of urea in urine.
2-Liver function: Liver is the only organ that converts toxic ammonia to non-toxic
urea so decrease in blood and urine urea level associated with increase ammonia
level indicates liver dysfunction

5
Uric Acid:

- It is one of the metabolic end products of purine

- Exogenous purines taken in diet: red meat, liver, sea-food

Clinical value

Gout:
is a metabolic disorder associated with increase in uric acids level in blood (> 7 mg/dl).

´ Excess uric acid in the blood may be precipitated as solid crystals (sodium urate) within joints.
´ If gout remains untreated, these urate crystals may settle in the joints and nearby tissues,
forming hard lumpy deposits called tophi.

q High levels of uric acid may also cause kidney stones or kidney failure.

6
 Practical test for Carbohydrates
A) Detection of carbohydrates
1) Molisch’s Test:

Principle:
Is a test for carbohydrates.
Observation:
Violet ring between 2 layers.
Result:
Positive result (violet ring) with Glucose, Fructose, Sucrose & Starch.
Negative result (No violet ring) with Albumin, Casein, Gelatin , Urea & Uric
acid.

B) Test for the reducing property of the sugars

2) Fehling’s test

Principle:
Aldehyde or ketone group in monosaccharides reduce the deep blue solution
of cupric hydroxide to a red precipitate of insoluble cuprous oxide.

Observation:
Reddish brown ppt.

Result:
Positive result (Reddish brown ppt) with Glucose& Fructose
Negative result (No Reddish brown ppt) with Sucrose & Starch

7
 C) Detection of ketose sugar (fructose or Sucrose):
3- Ketose test
Principle:
This test to detect ketohexoses.

Observation:
cherry red color

Result:
Positive result (Cherry red color) with Fructose & Sucrose.
Negative result (No Cherry red color) with Glucose& Starch

D) Test for presence of polysaccharides

(Starch)
4)Iodine test

Principle:
Iodine forms colored adsorption complexes with polysaccharides (Starch).

Observation:
Deep blue color.

Result:
Positive result (Deep blue color) with Starch
Negative result (No deep blue color) with Glucose, Fructose & Sucrose
1

8
Practical tests for Proteins:
A) Detection of Peptide bond
(Proteins)
1)Biuret Test:

Principle:

It is a general test for any substance contains at least 2 peptide bonds (Proteins).

Observation:
Violet color

Result:
Positive result (violet color) with Albumin, Casein and Gelatin
Negative result (No violet color) with Glucose, Fructose, Sucrose, Starch, Urea &
Uric acid.

B) Detection of Heat
Coagulable Proteins
(Albumin)
2)Heat Coagulation Test:

Principle
It is specific test for proteins that contains Albumin & globulin.
Egg white contains albumin, so it is heat coagulable

Observation:
White Coagulum

Result:
Positive result (white coagulum) with Albumin.
Negative result (No white Coagulum)) with Casein & Gelatin

9
C) Differentiation between
Casein & Gelatin
3) Reaction Test:

Principle
Casein is alkaline so it turns the red litmus paper into blue.
Egg white (Albumin) and gelatin are neutral.

Observation:
Red Litmus paper turns to Blue (Alkaline Protein)
Red litmus paper remains Red (Neutral Protein)

Result:
Red Litmus paper turns to Blue with Casein
Red litmus paper remains Red with Albumin & Gelatin

D) Detection of Tryptophan amino acid

4) Rosenheim / Tryptophan test

Principle
It is a general test for any proteins contains (indole ring) tryptophan amino acid.
Egg white (Albumin) and casein contain tryptophan amino acid.

Observation:
Violet Ring

Result:

Positive results (Violet Ring) with Albumin (Egg White) & Casein
Negative Results (No Violet Ring) with Gelatin.
10
 Practical tests for Urea & Uric acid:

A) Detection of carbohydrates
1) Molisch’s Test:

Principle:
Is a general test for carbohydrates.

Observation:
NO Violet (purple) ring.

Result:
Positive result (violet ring) with Glucose, Fructose, Sucrose & Starch Positive
Negative result (No violet ring) with Albumin, Casein, Gelatin, Urea & Uric acid.

Urea & Uric acid will give negative results as They are not Carbohydrates. They
are non-protein nitrogenous compounds.

B) Detection of Proteins
2) Biuret Test:

Principle:
It is a general test for any substance contains at least 2 peptide bonds
(Proteins).

Observation:
NO Violet Color.

Result:
Positive result (violet Color) with Albumin, Casein & Gelatin
Negative result (No violet Color) with Glucose, Fructose , Sucrose, Starch, Urea &
Uric acid.

Urea & Uric acid will give negative results as They are not Proteins. They are
non-protein nitrogenous compounds.

11
C) Differentiation between
Casein & Gelatin
3) Reaction Test:

Principle
Uric Acid is alkaline, so it turns the red litmus paper into blue.
Urea is neutral.

Observation:
Red Litmus paper turns to Blue (Alkaline Compund)
Red litmus paper remains Red (Neutral Compound)

Result:
Red Litmus paper turns to Blue with Uric Acid
Red Litmus paper remain Red with Urea

D) Detection of Urea
1) Na hypobromite Test :

Principle:
It is general test to detect presence of Urea.

Observation:
Marked effervescence

Result:
Positive result (Marked Effervescence) with Urea

12
E) Detection of Uric Acid
1) Follin Test:

Principle:
It is general test to detect presence of Uric acid (Partially reducing agent).

Observation:
Deep Blue Color

Result:
Positive result (Deep blue color) with Uric acid.

13
 Don’t Forget to
Write your report after identifying your
unknown

Write the physical properties of your unknown

Write your conclusion

(Mention the name of the unknown sample
and its chemical nature)

16
N.B: You will receive at the practical exams the Biochemical test steps &
General Scheme but without any colors (you should study the positive and
negative results for each test )

Example of the Scheme you will receive in Exam

22
 Unknown sample may be
Non-protein
Carbohydrates Protein nitrogenous
compounds
Glucose
Albumin
Urea

Fructose
Casein
Uric acid
Sucrose
Gelatin

Starch
 Violet color 1-Biuret test No Violet color
(Protein) (No Protein)

Violet Ring
2-Molisch test
2-Heat coagulation test Carbohydrate
(G,F,Suc,Starch) No Violet Ring
No Carbohydrate
No White (Urea or Uric acid)
White coagulum 3-Iodine test
coagulum (Casein or 3-Reaction test
No Deep blue color
(Albumin) Gelatin)
Deep blue color
(Glu,Fruc,suc)
(Starch) Red litmus paper Red litmus
3-Reaction test turns to blue paper litmus
3-Reaction test 4-Fehling (Uric acid) remain red
Red litmus paper
remain red 4-Fehling test test (Urea)
No reddish
Red LP Red LP brown ppt
turns to remain red Reddish No reddish brown 4-Follin 4-Na
ppt
blue brown ppt test hypobromite
(Gelatin) (Glu or Fru)
(Sucrose)
4-Rosenheim’s (Casein) 5-Ketose deep blue test
test test color Marked
Violet ring No Cherry 5-Ketose test effervescence
4- 4-
red color
Rosenhei Rosenhei
5-Ketose
m’s test m’s test Cherry red test
No Cherry red
Violet No Violet color Cherry red
color
ring ring (Fructose) color
(Glucose)
 Glucose Report
Test Observation Result

1-Biuret test No violet color Absence of Peptide bond

(Not Protein)
2- Molisch’s Deep violet ring between 2 layers Presence of Carbohydrates
test
No Deep Blue Color Absence of Polysaccharides
3- Iodine test
(Not Starch)
4- Fehling’s Reddish-brown ppt. Presence of reducing sugar
test

5- Ketose test No Cherry red color Absence of ketoses

Physical properties:
Color: colorless Odor: odorless Taste: sweet Appearance: clear Reaction: Neutral

Conclusion: Aldose-Monosaccharide-Carbohydrate
 Fructose Report
Test Observation Result

1-Biuret test No violet color Absence of Peptide bond

(Not Protein)
2- Molisch’s Deep violet ring between 2 layers Presence of Carbohydrates
test

No Deep Blue Color Absence of Polysaccharides

3- Iodine test (Not Starch)
4- Fehling’s Reddish-brown ppt. Presence of reducing sugar
test

5- Ketose test Cherry red color Presence of ketoses

Physical properties:
Color: colorless Odor: odorless Taste: sweet Appearance: clear Reaction: Neutral

Conclusion: Ketose -Monosaccharide-Carbohydrate

 Sucrose Report
Test Observation Result

1-Biuret test No violet color Absence of Peptide bond

(Not Protein)
2- Molisch’s Deep violet ring between 2 layers Presence of Carbohydrates
test

No Deep Blue Color Absence of Polysaccharides

3- Iodine test (Not Starch)
4- Fehling’s No Reddish-brown ppt. Absence of reducing sugar
test

5- Ketose test Cherry red color Presence of ketoses

Physical properties:
Color: colorless Odor: odorless Taste: sweet Appearance: clear Reaction: Neutral

Conclusion: Disaccharide-Carbohydrate
 Starch Report
Test Observation Result

1-Biuret test No violet color Absence of Peptide bond

(Not Protein)
2- Molisch’s Deep violet ring between 2 layers Presence of Carbohydrates
test

Deep Blue Color Presence of Polysaccharides

3- Iodine test (Starch)
4- Fehling’s No Reddish-brown ppt. Absence of reducing sugar
test

5- Ketose test No Cherry red color Absence of ketoses

Physical properties:
Color: whitish Odor: odorless Taste: tasteless Appearance: turbid Reaction: Neutral

Conclusion: Polysaccharide-Carbohydrate
 Albumin Report
Test Observation Result

1-Biuret test Violet color Presence of Peptide bond (Protein)

2-Heat coagulation White Coagulum formed Presence of heat coagulable protein

test (Albumin)

3- Reaction test Litmus paper Red Red Absence of alkaline protein

violet ring Presence of Tryptophan amino acid

4- Rosenheim test

Physical properties:
Color: whitish Odor: bad odor Taste: salty Appearance: turbid Reaction: Neutral

Conclusion: Simple protein

 Casein Report
Test Observation Result

1-Biuret test Violet color Presence of Peptide bond (Protein)

2-Heat coagulation No White Coagulum formed Absence of heat coagulable protein

test (Not Albumin)

3- Reaction test Litmus paper Red Blue Presence of alkaline protein (Casein)

violet ring Presence of Tryptophan amino acid

4- Rosenheim test

Physical properties:
Color: whitish yellowish Odor: bad odor Taste: bitter Appearance: turbid Reaction: Alkaline

Conclusion: Conjugated protein

 Gelatin Report
Test Observation Result

1-Biuret test Violet color Presence of Peptide bond (Protein)

2-Heat coagulation No White Coagulum formed Absence of heat coagulable protein

test (Not Albumin)

3- Reaction test Litmus paper Red Red Absence of alkaline protein

No violet ring Absence of Tryptophan amino acid

4- Rosenheim test

Physical properties:
Color: colorless Odor: bad odor Taste: tasteless Appearance: clear Reaction: Neutral

Conclusion: Derived protein

 Urea report
Test Observation Result

1-Biuret Test WriteNoyour

violet colorreport Absence of Peptide bond
(Not Protein)

2- Molish Test No violet ring between 2 layers Absence of Carbohydrates

3- Reaction test Litmus paper Red Red Absence of Alkaline

Compound

4- Sodium Marked effervescence Presence of Urea

hybobromite Test

Physical properties:
Color: colorless Odor: odorless Taste: tasteless Appearance: clear Reaction: Neutral

Conclusion: Metabolic endproduct of protein

 Uric acid report

Test Observation Result

1- Biuret Test No violet color Absence of Peptide bond

(Not Protein)
2- Molisch Test No violet ring between 2 layers Absence of Carbohydrates

3- Reaction test Presence of Alkaline

Litmus paper Red Blue Compound
Presence of partially
4- Follin’s Test Deep blue color reducing agent (Uric acid )

Physical properties:
Color: colorless Odor: bad odor Taste: bitter Appearance: clear Reaction: Alkaline

Conclusion: Metabolic endproduct of purines.