Year2 Bioclab 1 - TLC

Makenzie Bailey
620147606
September 7, 2022
Thin layer chromatography
Principle of Thin layer chromatography
Thin layer chromatography is a separation technique used to separate mixtures based on differences in
polarity. Separation of mixtures depends on the ability of compounds to move based on their affinity
to either the mobile phase or stationary phase. The mobile phase is the solvent, and the stationary
phase is a thin layer of absorbent for example: silica gel or aluminium oxide. The principle behind
thin layer chromatography is that the mobile phase will move over the stationary phase by capillary
action so that compounds that have a higher attraction for the stationary phase will move slower than
those that are attracted to the mobile phase so that a separation can be observed. Polar compounds are
absorbed to the stationary phase and will therefore travel less distance than non-polar compounds
which have are weakly absorbed to the stationary phase.
Components of Thin layer chromatography
o Stationary phase - Selection
The stationary phase is a TLC plate which is a thin layer of silica gel or alumina spread over a glass,
aluminium, or plastic surface. It was important to consider the different properties of the samples
when selecting a TLC plate such as size and reactivity. For example, silica gel is acidic and if used
with a basic sample will result in poor separation.
Preparation of plate
Using two pencils to prop a ruler above the plate was used to draw a straight line at the baseline of the
plate. This is done to ensure that the layer was not damaged. Marks were then made equal distance
from each other on the baseline for the samples and label each mark with the name of each sample.
o Mobile phase
Non-polar compounds in the sample moved with the mobile phase up a TLC plate. This is because the
non-polar compounds will make weak interactions with the polar molecules in stationary phase and
therefore move further up the plate.
o Chamber saturation
The TLC chamber was saturated to maintain equal humidity. This was done by firstly pouring
the selected solvent below the baseline. Then, placed two filter papers against the walls of the
tank and gently tipping the tank to moisten them. A lid was then placed over the tank to
ensure that the solvent does not evaporate and to maintain equal pressure within the tank.
o Sample
To prepare the samples, a solvent in which the sample would be soluble was added. Then use
a clean capillary tube to apply drops of the sample to the baseline. Allowing each spot to dry
between applications. The plate was then placed in the TLC tank and the lid shut. Once the
spots are developed, the plate was removed and observed under a UV light making sure to
draw a line indicating how far the solvent travelled.
The Retention Factor (Rf) is used to identify compounds. The Rf value is equal to the distance
travelled by the compound divided by the distance travelled by the solvent.
Solvent front
TLC Plate
Origin/Baseline
THIN LAYER CHROMATOGRAM
Rf= Distance travelled by the sample / distance travelled by the solvent

Rf =
Problems that arise with thin layer chromatography
1. The eluent is above the spotted sample – this will totally dissolve the sample before the eluent
can interact with the stationary phase resulting in the plate not developing as it should.
2. Leaving the TLC plate in the chamber too long – this will cause the solvent to surpass the top
of the plate. This will affect the Retention factor value as there will be no way to accurately
measure the distance travelled by the solvent.
3. Sample is too concentrated- this is called streaking; this is when the sample travels up the
stationary phase as streak instead of as separate spots.
4. Not enough solvent – this will affect the retention factor since not enough solvent is present to
travel up the late.
5. Sample spot is too large – this could cause overlapping with other samples of similar Rf
values.
Advantages of TLC
1. Separation is done in a short time

2. Components of mixtures are easily separated and easy to recover
3. Requires less sophisticated instruments
Disadvantages of TLC
1. Only applicable for soluble mixtures

2. Results are difficult to reproduce
3. Since TLC plate length is limited, separation only takes place up to a certain length.
Uses and application of thin layer chromatography
1. Used to follow the progress of chemical reactions

2. Separation of colours, sweeting agents, preservatives in the food industry
3. Used to identify drugs, inks, DNA samples in forensics
Different types of chromatography
1. Thin layer chromatography

2. Gas-liquid chromatography
3. Liquid- liquid chromatography
4. Liquid- solid chromatography
5. Paper chromatography
6. Supercritical fluid chromatography
7. High performance liquid chromatography
References
Chemistry LibreTexts. (2019, December 6). Thin Layer Chromatography. Chemistry
LibreTexts.
https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments
/General_Lab_Techniques/Thin_Layer_Chromatography
Dave, P. (2019, March 1). Thin Layer Chromatography (TLC). Www.youtube.com.
https://youtu.be/I4u_1ST7Ezk
Nichols, L. (2017, October 20). 2.3E: Step-by-Step Procedures for Thin Layer
Chromatography. Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab
_Techniques_(Nichols)/02%3A_Chromatography/
2.03%3A_Thin_Layer_Chromatography_(TLC)/2.3E%3A_Step-by-
Step_Procedures_for_Thin_Layer_Chromatography
Thin Layer Chromatography. (2019, February 21). BYJUS; Byju’s.
https://byjus.com/chemistry/thin-layer-chromatography/
Thin Layer Chromatography (TLC)-Animation- Chromatographie sur Couche Mince (CCM).
(2020, January 17). Www.youtube.com; Biology with Animations.
https://youtu.be/rMGQavOMAmc

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Makenzie Bailey

Thin layer chromatography

Principle of Thin layer chromatography

Components of Thin layer chromatography

o Stationary phase - Selection

THIN LAYER CHROMATOGRAM

Rf= Distance travelled by the sample / distance travelled by the solvent

Problems that arise with thin layer chromatography

1. Separation is done in a short time

1. Only applicable for soluble mixtures

Uses and application of thin layer chromatography

1. Used to follow the progress of chemical reactions

Different types of chromatography

1. Thin layer chromatography

Chemistry LibreTexts. (2019, December 6). Thin Layer Chromatography. Chemistry

Dave, P. (2019, March 1). Thin Layer Chromatography (TLC). Www.youtube.com.

Chromatography. Chemistry LibreTexts.

Thin Layer Chromatography. (2019, February 21). BYJUS; Byju’s.

Thin Layer Chromatography (TLC)-Animation- Chromatographie sur Couche Mince (CCM).

(2020, January 17). Www.youtube.com; Biology with Animations.

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