Date of submission :

Objective

What is Qualitative test for Carbohydrates?............................................... 1

Why Qualitative test for Carbohydrates ?................................................... 1

Different types of Qualitative tests for Carbohydrates ............................... 1

-Molisch’s Test..................................................................................... 2

-Benedict’s Test..................................................................................... 3

-Fehling’s Test...................................................................................... 4

-Barfoed’s Test..................................................................................... 5

-Seliwanoff’s Test................................................................................. 6

-Iodine Test.......................................................................................... 7

-Bial’s Test........................................................................................... 8

What is Genomic DNA Extraction?........................................................... 9

Why Genomic DNA Extraction?............................................................... 9

Genomic DNA Extraction for Eukaryotes................................................ 10

Genomic DNA Extraction for Prokaryotes.............................................. 12

Others..................................................................................................... 14
Qualitative Tests for Carbohydrate

Qualitative tests for carbohydrates are chemical tests used to detect the presence
and type of carbohydrates in a sample. These tests identify carbohydrates based on
their chemical properties, such as the ability to reduce certain compounds or react
with specific reagents.

Carbohydrates can be classified into monosaccharides, disaccharides, and

polysaccharides, and each group can be detected using different qualitative tests.
These tests are often used in laboratories to determine the nature of an unknown
carbohydrate sample.

Why Qualitative Tests for Carbohydrate?

• Identification of Carbohydrates: Helps determine the type of carbohydrate (simple

sugar or complex sugar).
• Differentiation: Distinguishes between reducing and non-reducing sugars, aldoses
and ketoses, and mono- and disaccharides.
• Diagnostic Use: Used in clinical settings (e.g., detecting glucose in urine).
• Educational Purposes: Fundamental for understanding carbohydrate chemistry in
laboratories.

Examples of Qualitative Tests for Carbohydrate

• Molisch’s Test: Detects all types of carbohydrates.

• Benedict’s Test: Detects reducing sugars (e.g., glucose, fructose).
• Fehling’s Test: Another test for reducing sugars.
• Barfoed’s Test: Differentiates monosaccharides from disaccharides.
• Seliwanoff’s Test: Distinguishes ketoses from aldoses.
• Iodine Test: Detects polysaccharides like starch.
• Bial’s Test: Detects pentoses (5-carbon sugars).

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Molisch’s Test
Purpose: To detect the presence of all types of carbohydrates (monosaccharides,
disaccharides, and polysaccharides).
Principle: Under acidic conditions, carbohydrates are dehydrated to form furfural or
hydroxymethylfurfural. These compounds react with α-naphthol to produce a violet-
colored ring.
Requirement: Test tubes,Molisch’s reagent (α-naphthol in ethanol), Concentrated sulfuric
acid (H₂SO₄), Carbohydrate solution.
Reagents:
Molisch’s reagent (α-naphthol in ethanol)
Concentrated sulfuric acid (H₂SO₄)
Procedure:
1. Add 2-3 drops of Molisch’s reagent to 2 mL of the carbohydrate solution.
2. Carefully add 1 mL of concentrated sulfuric acid along the side of the test tube to form a
layer.

Fructose Glucose Sucrose Starch

Result:
A violet ring appears at the interface, indicating the presence of carbohydrates.

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Benedict’s Test

Purpose: To detect the presence of reducing sugars (e.g., glucose, fructose, lactose).
Requirement: Test tubes, Benedict’s reagent, Carbohydrate solution, Water bath or
Bunsen burner
Principle: Reducing sugars have a free aldehyde or keto group that can reduce
copper(II) ions (Cu²⁺) in Benedict’s reagent to copper(I) oxide (Cu₂O), forming a
colored precipitate.
Reagents:
Benedict’s reagent: Copper sulfate (CuSO₄), sodium carbonate (Na₂CO₃), and
sodium citrate.
Procedure:
1. Add 2 ml of Benedict’s reagent to 1 ml of the carbohydrate solution.
2. Heat the mixture in a boiling water bath for 3-5 minutes.
3. Observe any color change or precipitate formation.

Result:
Blue precipitate = No trace of reducing sugar.
Green precipitate = Low concentration of reducing sugar.
Yellow/Orange precipitate = Moderate concentration of reducing sugar.
Red precipitate = High concentration of reducing sugar.

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Fehling’s Test

Purpose: To detect the presence of reducing sugars.

Requirement: Test tubes, Fehling’s solution A and B, Carbohydrate solution, Water
bath or Bunsen burner.
Principle: Reducing sugars reduce copper(II) ions (Cu²⁺) in Fehling’s solution to
copper(I) oxide (Cu₂O), producing a red precipitate.
Reagents:
Fehling’s Solution A: Copper sulfate (CuSO₄)
Fehling’s Solution B: Potassium sodium tartrate (Rochelle salt) and sodium
hydroxide (NaOH)
Procedure:
1. Mix equal volumes of Fehling’s solution A and B.
2. Add 2 ml of this mixture to 1 ml of the carbohydrate solution.
3. Heat the solution in a boiling water bath for 3-5 minutes.

Result:
A red or orange precipitate indicates the presence of reducing sugars.

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Barfoed’s Test

Purpose: To differentiate between monosaccharides and disaccharides.

Requirement: Test tubes, Barfoed’s reagent, Carbohydrate solution, Water bath
Principle: Monosaccharides reduce copper(II) acetate in Barfoed’s reagent to
copper(I) oxide, forming a red precipitate. This reaction happens faster for
monosaccharides than for disaccharides.
Reagents:
Barfoed’s reagent (copper acetate in acetic acid)
Procedure:
1. Add 1 mL of Barfoed’s reagent to 1 mL of the carbohydrate solution.
2. Heat in a boiling water bath for 2-3 minutes.

Result:
Red precipitate within 2-3 minutes indicates monosaccharides. Delayed red
precipitate indicates disaccharides.

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Seliwanoff’s Test

Purpose: To differentiate between aldoses and ketoses.

Requirement: Test tubes, Seliwanoff’s reagent, Carbohydrate solution, Water bath
Principle: Ketoses (e.g., fructose) are dehydrated more rapidly than aldoses,
forming furfural derivatives that react with resorcinol to produce a red color.
Reagents:
Seliwanoff’s reagent - Resorcinol in dilute hydrochloric acid (HCl)
Procedure:
1. Add 2 ml of Seliwanoff’s reagent to 1 ml of the carbohydrate solution.
2. Heat the mixture in a boiling water bath for 2-3 minutes.

Fructose Sucrose Aldose control DW

Deep red Cherry red Faint red No change

Result:
A red color indicates the presence of ketoses. Aldoses give a slower or no reaction.

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Iodine Test

Purpose: To detect the presence of starch (a polysaccharide).

Requirement: Test tubes, Iodine solution, Carbohydrate solution
Principle: Starch reacts with iodine to form a blue-black complex due to the helical
structure of amylose.
Reagents:
Iodine solution - Iodine in potassium iodide (KI) solution
Procedure:
1. Add a few drops of iodine solution to 2 ml of the carbohydrate solution.
2. Gently mix the solution.

Glucose Sucrose Starch control DW

Result:
A blue-black color indicates the presence of starch.

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Bial's Test

Purpose: To detect the presence of pentoses (5-carbon sugars).

Requirement: Test tubes, Bial’s reagent, Carbohydrate solution, Water bath.
Principle: Pentoses are dehydrated by hydrochloric acid to form furfural, which
reacts with orcinol and ferric chloride to produce a green or blue color.
Reagents:
Bial’s reagent - Orcinol, ferric chloride (FeCl₃), and hydrochloric acid (HCl)
Procedure:
1. Add 2 ml of Bial’s reagent to 1 ml of the carbohydrate solution.
2. Heat the mixture in a boiling water bath for 2-3 minutes.

Pentose

Result:
A green or blue color indicates the presence of pentoses.

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Genomic DNA Extraction

Genomic DNA extraction is the process of isolating pure DNA from cells or
tissues for various applications such as PCR, sequencing, and genetic analysis. The
protocol varies depending on the type of cell (eukaryotic or prokaryotic) due to
differences in cell structure and DNA organization.

Why Genomic DNA Extraction?

Genomic DNA extraction is performed for various scientific, medical, and

industrial purposes because it provides the foundation for studying the genetic
material of organisms. The main reasons why genomic DNA extraction is done are-
1. Research and Genetic Studies
2. Medical Applications
3. Forensic Science
4. Agricultural Biotechnology
5. Industrial Applications
6. Educational and Training Purposes

General Steps for Genomic DNA Extraction

Steps in Genomic DNA Extraction

Sample Collection: Obtain the biological material (e.g., blood, tissue, bacteria
culture).
Cell Lysis: Disrupt the cell membrane (and nuclear membrane for eukaryotes)
to release DNA.
Protein and RNA Removal: Add enzymes like Proteinase K to digest proteins.
Treat with RNase to degrade RNA.
DNA Extraction: Use methods like phenol-chloroform or silica-based columns
to separate DNA from other cellular components.
DNA Precipitation: Add alcohol (ethanol or isopropanol) to precipitate DNA.
DNA Washing: Rinse with ethanol to remove impurities.
Resuspension: Dissolve the DNA in a buffer (e.g., TE buffer) for storage.

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Genomic DNA Extraction for Eukaryotes

Eukaryotic genomic DNA extraction involves isolating high-quality DNA from

cells that have a complex structure, including a nucleus where the DNA is stored.
This process is essential for genetic analysis, sequencing, and various molecular
biology applications.

Steps in Eukaryotic Genomic DNA Extraction

• Sample Collection
-Collect eukaryotic cells from tissues, blood, or cell cultures.
-Ensure samples are fresh or preserved appropriately.

Tissue Sample Buccal Swab Saliva Sample Blood Sample

• Cell Lysis
-Disrupt the cell and nuclear membranes to release DNA:
-Lysis Buffer: Contains detergent (e.g., SDS) to break lipid bilayers.
-Proteinase K: Added to degrade proteins, including histones.

Figure : Eukaryotic cell Lysis

• RNA Removal
-Add RNase to degrade RNA, which may contaminate the DNA.

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• DNA Extraction
-Separate DNA from proteins and other cellular debris using one of these
methods:
1. Organic Extraction: Use phenol-chloroform to denature proteins and extract
DNA.
2. Silica-Based Methods: DNA binds to silica membranes in spin-column kits for
purification.
3. Magnetic Beads: DNA binds to magnetic beads, making it easy to isolate.

• DNA Precipitation
-Add alcohol (ethanol or isopropanol) and salt (e.g., sodium acetate) to
precipitate DNA.
-Centrifuge to pellet the DNA.

Figure : DNA Precipitation with Alcohol

• Washing and Resuspension

-Wash the DNA pellet with 70% ethanol to remove impurities.
-Dry the pellet and dissolve it in TE buffer or distilled water.

Figure : DNA Purification

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Genomic DNA Extraction for Prokaryotes

The process of genomic DNA extraction from prokaryotes is designed to isolate

DNA from cells with a simpler structure compared to eukaryotes. Prokaryotic cells,
such as bacteria, have a cell wall and lack a nucleus, making the protocol distinct in
certain steps.

Steps in Prokaryotic Genomic DNA Extraction

• Sample Collection
-Obtain bacterial cells from a culture or environmental sample.
-Pellet the cells via centrifugation to concentrate them.

Figure : Environmental Sample Figure : Laboratory Sample

• Cell Lysis
-Disrupt the cell wall and plasma membrane to release DNA:
➤Gram-Negative Bacteria: Use detergents (e.g., SDS) directly.
➤Gram-Positive Bacteria: Pre-treat with lysozyme or enzymes to degrade the
thick peptidoglycan cell wall.

Figure : Prokaryotic Cell Lysis

• Protein Digestion
-Add Proteinase K to degrade proteins and free the DNA.
-Incubate at an appropriate temperature (e.g., 55–60°C).
• RNA Removal
-Treat the lysate with RNase to remove contaminating RNA.
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• DNA Extraction
-Separate the DNA from proteins and debris using one of these methods:
1. Phenol-Chloroform Extraction: Add phenol and chloroform to the lysate,
centrifuge to separate layers, and collect the aqueous DNA-containing phase.
2. Spin-Column Methods: Use silica membrane-based columns where DNA
binds and impurities are washed away.
3. Magnetic Beads: Bind DNA to magnetic beads and wash impurities away.

Figure : Protein removal

• DNA Precipitation
-Add alcohol (e.g., ethanol or isopropanol) and a salt (e.g., sodium acetate) to
precipitate DNA.
-Centrifuge to pellet the DNA.

• Washing and Resuspension

-Wash the DNA pellet with 70% ethanol to remove residual contaminants.
-Air-dry and dissolve the DNA in TE buffer or distilled water.

Figure : DNA Extraction from Gram Positive Bacteria

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Contribution

2301010 - Utsho Saha

Study on Genomeic DNA Extraction from Eukaryotes.
Merge Information on the Topic
Relevant Figures collection.
2301011 - Md. Eachin Mia
Study on Genomeic DNA Extraction from Prokaryotes.
Merge Information on the Topic
Relevant Figures collection.
2301012 - Rafia Mahmud
Study on Qualitative Tests for Carbohydrates.
Merge Information & Relevant Figures collection on the topic.
Arranging the Assignment together

References

Textbooks:
Biochemistry by Lubert Stryer – Detailed information on carbohydrate structure and tests.
Practical Biochemistry: Principles and Techniques by Keith Wilson and John Walker – Comprehensive
procedures for biochemical assays.
Biochemistry Laboratory: Modern Theory and Techniques by Rodney Boyer – Laboratory protocols for
carbohydrate tests.
Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press.
Green, M. R., & Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press.
Ausubel, F. M., et al. (2002). Short Protocols in Molecular Biology. Wiley.
Laboratory Manuals:
Vogel's Textbook of Practical Organic Chemistry by Brian S. Furniss et al. – Procedures for qualitative
analysis of carbohydrates.
Wilson, K. (2001). "Preparation of Genomic DNA from Bacteria." Current Protocols in Molecular
Biology.
2. Pitcher, D. G., et al. (1989). "Rapid Extraction of Bacterial Genomic DNA with Guanidium
Thiocyanate." Letters in Applied Microbiology.
A Review on Qualitative and Quantitative Analysis of Carbohydrates.
Laboratory Activities to Introduce Carbohydrate Qualitative Analysis to College Students.
Online Educational Resources:
Chem LibreTexts – "Qualitative Tests for Carbohydrates"
NCBI Bookshelf – Comprehensive carbohydrate classification and tests.
QIAGEN: Prokaryotic and Eukaryotic DNA Extraction Protocols.
Thermo Fisher Scientific: DNA Extraction Protocols.
New England Biolabs (NEB): Prokaryotic and Eukaryotic DNA Extraction.
Isolation of genomic DNA

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