PRACTICAL PROCEDURE OF PERFORMING TLC FOR THE

CONFIRMATION OF THE PRODUCT FORMATION IN AN

ORGANIC SYNTHESIS

Submitted By:
MEMOONA ARSHAD
Submitted To:
DR. MUHAMMAD AMIN
Registration No:
PCH072410001

MASTERS OF PHILOSOPHY
IN
ANALYTICAL CHEMISTRY

DEPARTMENT OF CHEMISTRY
THE UNIVERSITY OF LAHORE
SARGODHA CAMPUS
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 Table of Content
Sr. No. Title Page No.
1. Introduction 3

2. Principle 3

3. Materials 4

4. Components 4

5. Procedure 5

6. Data Collection 6

7. Sample Data 6

8. Calculations 6

9. Conclusion 6

10. Applications 6

11. Advantages of TLC 7

12. Disadvantages of TLC 7

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Introduction:
Thin Layer Chromatography is a kind of chromatography used to separate and isolate mixtures
that are non-volatile in nature. Just like other chromatography processes, this one consists of a
mobile phase and a stationary phase.
The latter one here is a thin layer of absorbent material, such as aluminium oxide, silica gel, or
cellulose. This layer is applied to plastic, glass, or aluminium foil sheets called an inert
substrate. The mobile phase in the TLC procedure is a solvent or a mixture of it.
If you want to learn more about the thin layer chromatography procedure, you have landed at
the right place. Here we will be discussing its principle, process, and applications in different
industries.
We will begin with the TLC principle.

Principle:
The separation principle of the TLC procedure is based on the given compound’s relative
affinity towards the mobile and the stationary phase. The process begins here by moving the
mobile phase over the stationary phase’s surface. During this movement, the higher affinity
compounds gain less speed as compared to the lower affinity compounds. This results in their
separation.
Once the procedure gets completed, different spots can be found on the stationary surface at
distinct levels, reflecting various elements of the mixture. Basically, the compounds that are
more attracted towards the stationary phase secure their position at lower levels while others
move towards the higher levels of the surface. So their spots can be seen accordingly.

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Materials:
- TLC plate (silica gel or alumina)
- Mobile phase (solvent mixture)
- Sample (crude product mixture)
- Standard (pure product, if available)
- UV lamp
- Chromatography chamber
- Capillary tube or TLC spotter
- Solvents (e.g., dichloromethane, chloroform, ethanol, acetone)
- TLC plate cutter or scissors
- Vials or tubes for sample and standard preparation
- Pipettes (10 μL and 100 μL)
- Timer
- Ruler or measuring device

Components:
A few components involved in the TLC procedure are as follows.
TLC Plates: These are used for applying the thin layer of stationary phase. They are inert or
stable in nature. The layer of stationary phase is kept even throughout these plates for better
analysis. Usually, ready-to-use plates are preferred by the people conducting experiments.
Mobile Phase: This comprises a solvent (or solvent mixture). The taken solvent needs to be
chemically inert, of the highest possible purity, and particulate-free. Only then can the TLC
spots be able to develop.
TLC Chamber: This is where the thin layer chromatography procedure takes place. It keeps the
dust particles away from the process and does not let the solvent evaporate. In order to
develop the spots appropriately, a uniform environment is maintained inside this chamber.
Filter Paper: This gets placed inside the chamber after being moistened with the mobile phase
solution. It ensures that the mobile phase rises uniformly throughout the TLC plate’s length.

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Procedure:
After collecting all these components, the process begins. Here are the steps followed in it:
1. Prepare the TLC plate:
- Cut the plate to size (about 5 cm x 10 cm)
- Wash the plate with ethanol or acetone in a chromatography chamber
- Allow the plate to dry completely
2. Prepare the sample and standard:
- Dissolve about 1-5 mg of the sample in 1-2 mL of solvent (e.g., dichloromethane)
- Dissolve about 1-5 mg of the standard in 1-2 mL of solvent (e.g., dichloromethane)
3. Spot the sample and standard:
- Use a capillary tube or TLC spotter to apply 1-5 μL of the sample solution to the TLC plate
- Apply the sample spot about 1 cm from the bottom edge of the plate
- Repeat for the standard solution
4. Develop the plate:
- Place the TLC plate in the chromatography chamber
- Add the mobile phase to the chamber (about 1-2 cm deep)
- Allow the plate to develop for 30-60 minutes
5. Visualize the plate:
- Remove the plate from the chamber
- Allow the plate to dry completely
- Visualize the separated components under UV light (254 nm or 365 nm)
This way, the TLC procedure gets completed. After analyzing the compound, it gets described
in its relative mobility’s terms, i.e., its Rf value is calculated. This value changes for each
compound, even under the same circumstances.
Usually, relative Rf comes into use here because keeping all the TLC factors constant may not
be possible. These aspects include adsorbent, temperature, adsorbent thickness, spotted
material’s amount, and solvent system. The formula used for Rf value calculation is:
Rf = (distance covered by the sample) / (distance covered by the solvent)

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Data Collection:
- Record the following data:
- Rf values (retention factor) for the sample and standard
- Distance traveled by the mobile phase
- Distance traveled by the sample and standard
- Color, intensity, and shape of the spots
- Take a photograph of the TLC plate under UV light

Sample Data:
| Compound | Rf Value | Distance Traveled (cm) | Color | Intensity | Shape |
| --- | --- | --- | --- | --- | --- |
| Sample | 0.5 | 3.5 | Yellow | Medium | Round |
| Standard | 0.5 | 3.5 | Yellow | Medium | Round |

Calculations:
- Rf value = (distance traveled by compound) / (distance traveled by mobile phase)
- Rf value = (3.5 cm) / (5 cm) = 0.5

Conclusion:
- The Rf values of the sample and standard match, indicating the presence of the product in
the sample.
- The color, intensity, and shape of the spots also match, further confirming the identity of the
product.

Applications:
Just understanding the principle and procedure of TLC is not enough. You also need to learn
about thin layer chromatography uses to see how and where it works in the real world. Some
standard TLC applications include:
 Being a separation process, TLC proves to be highly effective for separating
pharmaceutical formulations that consist of multiple components.
 The process can be used to examine a given product’s purity.
 Medicines like local anesthetics, analgesics, sedatives, hypnotics, anticonvulsant
tranquilizers, and steroids go through the TLC procedure for their qualitative testing.
 The cosmetic industry also uses TLC for checking the presence of preservatives in the
products.
 A given compound can be purified using TLC and then compared with a standard
sample.
 TLC also finds its use in Biochemical analysis. Here, it can be used for biochemical
metabolites’ separation from urine, blood plasma, serum, and body fluids.
 Just like the cosmetic industry, the food industry also utilizes TLC for the detection of
preservatives, artificial colours, and sweetening agents.
 A reaction’s progress can also get tracked with TLC to see whether it is complete or
not.
These were some typical thin layer chromatography applications that you can find at different
places. However, the procedure is not limited to these, and you can also see its use in a lot of
other industries.

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Benefits and Drawbacks:
Finally, let’s explore some of the benefits and drawbacks of using the thin layer
chromatography procedure.
Advantages of TLC
These include:
 The separated spots of TLC can be further visualized without any trouble.
 This chromatography process is cost-effective as compared to other methods.
 It can be used for a number of compounds, and it does not take much time because it
is quicker.
 The process is much more straightforward than other methods.
 TLC makes it simple to analyze any given compound’s purity standards.
 Several compounds can easily get isolated through TLC.
Disadvantages of TLC
The drawbacks of the process are:
 The TLC procedure can’t be used for lower detection limit experiments because it has a
high detection limit.
 The plates used in TLC do not possess a more extended stationary phase.
 Result reproduction is challenging in TLC.
 TLC is limited to qualitative analysis, and it can’t be used for quantitative analysis.
 The separation length is also restricted as compared to other chromatography
methods.
 The process here does not take place in a closed system. Therefore, aspects like
temperature and humidity can affect the results, making them inaccurate.