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The document provides an overview of Thin Layer Chromatography (TLC), including its principles, components, and applications in separating and analyzing mixtures. It details the preparation of TLC plates and chambers, the development process, and factors affecting retention and separation of molecules. Additionally, it includes practical steps for conducting TLC experiments and calculating retention factors (Rf).

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3 views

TLC-2022-Zoom (1)

The document provides an overview of Thin Layer Chromatography (TLC), including its principles, components, and applications in separating and analyzing mixtures. It details the preparation of TLC plates and chambers, the development process, and factors affecting retention and separation of molecules. Additionally, it includes practical steps for conducting TLC experiments and calculating retention factors (Rf).

Uploaded by

davidanani94
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Principles and Practice of Thin

Layer Chromatography
(TLC)
Learning Objectives

1. Gain basic understanding of thin layer chromatography (TLC)

2. Learn how to use TLC to separate and analyse mixtures


What is Chromatography?

Separation technique based on the different interactions of


compounds with a mobile phase and a stationary phase, as
the compounds travel through a supporting medium.
What are main the Components of Chromatography?

1. Mobile phase: a solvent that flows through the supporting medium

2. Stationary phase: a layer or coating on the supporting medium that


interacts with the analytes

3. Supporting medium: a solid surface on which the stationary phase is


bound or coated
Chromatographic Methods

Chromatography

1. Adsorption 2. Partition
compounds
partitioned between
two liquid phases

A. Thin Layer Chromatography B. Column Chromatography


What is TLC?

TLC is a simple, quick and inexpensive


method for separating and analyzing mixtures
based on the different interactions of
compounds with a mobile phase and a
stationary phase, as the compounds travel
through a supporting medium.
Uses of TLC

1. Separate mixtures into components

2. Purify compounds

3. Test purity of compounds

4. Monitor progress of chemical reactions


Types of Mixtures

1. Inorganic Mixtures
2. Organic Mixtures
3. Pigment/ Dye mixtures
4. Amino Acid mixtures
5. Lipid mixtures
6. Nucleotide Mixtures
7. Sugar Mixtures
Principle of TLC

TLC plate removed when


Mixture is TLC plate is Solvent moved solvent reaches almost top
spotted near placed a slowly up TLC of plate from the
the Bottom of shallow plate by capillary developing chamber. TLC
TLC plate solvent action plate is dried and separated
components are visualized

https://www.youtube.com/watch?v=iV1GfI_BbKE
TLC Chambers

Jar with a lid or Beaker with Chamber


a watch with a glass watch
Prepare TLC chamber

1. Pour solvent mixture to depth less


than 2 cm

2. Line chamber with filter paper to


saturate with the solvent vapour

3. Cover plate
Prepare TLC Plate

1. Measure 0.5cm from the bottom of TLC


Plate

2. Draw with pencil ORIGIN LINE across plate


at the 0.5cm

3. Mark name of samples to be spotted on the


plate, leaving enough space between the
samples
Spot TLC Plate

Step 1 Step 2 Step 3 Step 4 Step 5

Add few drops


of solvent to Swirl to dissolve Dip a microcarp in Make sure the Touch TLC plate with
sample mixture sample mixture sample mixture microcarp is filled filled microcarp to spot
to be analyzed
the plate
(if it is already
not in solution
Develop TLC Plate

1. Place TLC plate in developing


chamber

2. Allow the solvent to rise up the


plate to about 0.5cm below the
top of the plate

3. Remove the plate and mark the


solvent front

4. Allow the solvent to evaporate


Develop TLC Plate

1. Mark coloured spots by circling


lightly with a pencil before fading
away

2. Visualize spots not coloured under


UV lamp

UV is dangerous to the eyes and skin


Wear gloves and goggles
What is TLC-Retention Factor?
Distance travelled by a compound/spot divided by the distance travelled by the solvent
front
TLC-Retention Factor
TLC-Retention Factor
TLC-Retention Factor
1. Solvent system

2. Nature of Adsorbent Material

3. Thickness of Adsorbent Material

4. Amount of Material Spotted

5. Temperature

These factors are difficult to keep from one experiment to another,


hence Relative Rf is used
Factors Affecting Separation of Molecules
1.Polarity and size of the molecule
The More polar the molecule, the stronger attraction to stationary phase,
smaller Rf value

2. Polarity of the stationary phase

Electropositive character of the aluminum or silicon and the electronegative


oxygen create a polar stationary phase

3. Functional group
The more polar the functional group, the stronger it binds to the stationary phase
and the more slowly the molecules will move.

4. Remember like-dissolves-like
Separation of a mixture of molecules A & B

1. B molecules spend less time in the mobile phase

2. B molecules move through stationary phase


more slowly

3. B molecules do not move as far as A the same


amount of time

4. B is more polar than A

5. A less polar than B


Factors Affecting TLC-Retention

1. When comparing two different compounds


run under identical chromatographic
conditions, the compound with the larger
Rf is the less polar because it interacts less
strongly with the polar adsorbent on the
TLC plate

2. A compound with low polarity (non-polar)


will have a larger Rf value than a polar
compound
Application of Dyes to Plate F and G
Materials Required
1. Obtain two thin layer plates F and G from technologist
A. Brilliant Blue Dye
B. Blue-Red Dye 2. Draw line of origin 0.5cm from the bottom of on each plate
C. Yellow Dye
D. Light Blue Dye 3. Apply 1 ml of Brilliant Blue (A) towards the left of each plate on the line of
E. Mixture of A-D Dye origin
F. Aqueous Potassium acetate (10 x conc.)
G. Aqueous sodium Citrate-isopropanol 4. Rinse pipette and apply 1 ml of Blue–Red to the right of A on both plates
• 5 x5 cm thin layer, cellulose-based plate
• 5 μl glass capillary pipette 5. Apply I ml of C to the right of dye B on both plates
• 250 ml beakers
• Metric rulers 6. Apply I ml of D to the right of dye C on both plates
• 5 or 10 ml pipette
• Pipette pump 7. Apply I ml of E to the right of dye D on both plates

8. Let the samples spots dry for 5 minutes


Running TLC
1. Place the bottom edge of plate F into a beaker containing aqueous
potassium acetate (solvent F) ensuring solvent level is lower than sample
origin

2. Place plate G into a beaker containing sodium citrate-isopropanol (solvent G)

3. Allow plates to stand in solvent for about 12 minutes and should not reach
the top of the plate within this time

4. Remove plates and lay flat on a paper towel to dry

5. In millilitres, measure the distances from the sample origin to the centre of
each spot

6. Measure the distance of the solvent front from the origin

7. Calculate the Rf of samples A-D on both plate

Rf
https://www.youtube.com/watch?v=qdmKGs
kCyh8

https://www.youtube.com/watch?v=iV1GfI_B
bKE
Thank You

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