NCCU Chemistry Lab Objectives Question

Using robust details and ample evidence, create a reflection paragraph that describes 4 learning objectives you met while performing this experiment. View the learning objectives from the lab manual provided and select four to focus your writing on. Make this a well-constructed paragraph by including an introduction and conclusion sentences, avoiding bulleted lists.a 250-word count goal.

Virtual Lab Manual
Thin Layer Chromatography:
Separate a mixture and monitor
the progress of a reaction
Interactions are key to understanding the chemistry behind this important lab technique. In
this simulation you will learn the principles of Thin Layer Chromatography (TLC), diving into
how polarity affects the separation of compounds. Experiment with different mobile phases
to achieve the correct solvent ratio, monitor the progress of a reaction to determine when it
has gone to completion, and analyze your results by calculating the correct Rf value.
Explore the interactions involved in Thin Layer Chromatography
Dr. One has been experimenting. In the Lab you can observe the separation of pigments in a
spinach leaf, but how did they achieve this? Welcome to the world of Thin Layer
Chromatography! Using a solvent, a stationary phase, a sample, and some time, you will be
able to separate the components of a mixture. Learn about the interactions by taking a visit
to the world’s largest TLC plate (awaiting official certification), and zoom into the stationary
phase to observe how TLC separates a sample.
Determine the mobile phase through trial and error
After you have explored the interactions, it is time to determine the mobile phase solvent
ratio for your own TLC plate. Trial combinations of different solvents to separate compounds
with different polarities, until you have found the perfect mobile phase to separate the
molecules of interest. The joys of a virtual lab means you get instant feedback, and don’t
have to wait for several TLC plates to develop.
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Monitor the progress of a reaction via TLC and analyse the results
Once you have calculated the correct solvent ratio, it’s time to get to work. Assemble and run
your own TLC experiment to monitor the progress of a reaction. Select the appropriate
apparatus, experiment with how to handle and place the plate, and correctly spot the sample
onto the stationary phase. Will you be able to successfully remove the plate from the
chamber at the right time? After you have run the experiment, move onto analyzing your TLC
plate under UV light, determining when the reaction has gone to completion. The virtual lab
saves time again, with rapid results! Then you will cap it all off by measuring the distance the
spots have moved to calculate the Rf value. Will you be able to successfully analyze your
results and figure out at what time the reaction completed?
Learning Objectives
At the end of this simulation, you will be able to…
● Assemble, run, and analyze your own TLC experiment
● Understand the key principles of Thin Layer Chromatography and how the mobile and
stationary phases interact with a sample to enable separation
● Monitor the progress of a reaction via TLC
● Quantify and interpret the spots on a complete TLC plate
● Measure migration distances and use them to determine the Rf values of different
compounds of interest
Techniques in Lab
Thin Layer Chromatography (TLC)
Thin Layer Chromatography
Thin Layer Chromatography (TLC) is an analytical technique that separates components
within a mixture. It can be used to determine the quantity and identity of different
compounds in a reaction mixture and monitor the progress of a reaction. All forms of
chromatography work upon the same principle.
TLC Procedure
The procedure of performing a TLC experiment is outlined in detail below:
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Figure 1: TLC Assembly Diagram
After determining your solvent system, add around 1cm of solvent to a beaker.
2. Add a filter paper to the beaker – where the bottom of the filter paper is submerged
in solvent and reaches to the top of the beaker.
3. Cover the beaker with a lid or watch glass. This ensures the volatile solvent does not
evaporate from the beaker.
4. Mark a straight line, 1.5 cm from the bottom of the TLC plate in pencil. Add a mark
and label under the baseline where each sample will be spotted onto the plate making sure not to touch the surface of the silica plate or press too hard with the
5. Using a capillary tube, spot a small amount of the sample in a concentrated area on
the TLC plate at the correct marking. Let the spot dry and spot again 2-3 times, to
ensure a sufficient amount of sample is present. Repeat this process for each sample.
6. Place the TLC plate carefully into the developing chamber with forceps ensuring the
baseline is not submerged into the solvent. If this is the case, you have added too
much solvent.
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7. Cover the TLC chamber and allow the mobile phase to be drawn up the TLC plate
through capillary action.
8. Remove the TLC plate from the chamber, when the solvent front is approximately 1 cm
from the top of the TLC plate. Allowing the TLC plate to dry before proceeding to
Choosing the correct Solvent System
The choice of a good solvent system for the mobile phase is highly dependent on the nature
of the compound that is analysed. Often a few trial TLC plates, in different ratios of polar and
non-polar solvent systems, are run to achieve a desired R f range of 0.3 – 0.7. In addition to
effectively separating the samples of interest, a good solvent system moves all compounds
from the baseline but does not migrate compounds to the solvent front. Although some
inferences can be made in relation to the polarity of the samples with the polarity of the
solvent, the particular ratio and choice of solvent system can be only truly determined
experimentally. It is important to bear in mind that even small additions of polar solvent can
result in large changes in separation.
The most polar solvent system will have a greater attraction to the stationary phase than the
samples. Therefore, the samples will be carried all the way to the solvent front. Whereas, the
least polar solvent system will have no interaction with the stationary phase, leaving the
compounds at the baseline.
TLC Visualization Methods
The most common method of visualization for the TLC of organic compounds is using UV
light. The surface of the TLC plate is coated with a compound that fluoresces under UV-C
light at 254 nm causing the plate to appear green. Most organic compounds, with functional
groups, will quench this fluorescence and appear as dark spots on the TLC plate. However,
this requires the sample to have functional groups that will absorb the UV light. Conjugated
systems, and aromatic compounds will often quench this fluorescence – appearing visible
under UV.
However, certain compounds will not be visible UV light, such as simple alcohols and
aldehydes therefore chemical staining methods or dips may be required. This involves
exposing the TLC plate with a reactive compound. The chemical stain will react with the
compounds on the plate, but not the mobile phase producing the visualisation. Unlike UV,
this method of visualisation is destructive and the samples cannot be recovered. Common
staining compounds include iodine, permanganate and phosphomolybdic acid (PMA).
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Retention Factor
The retention factor or Rf value of a compound in TLC is a measure of how far a sample has
moved up the plate in relation to the solvent front. It is a ratio and can be calculated as:
Figure 2: Rf Value calculation procedure
A TLC experiment should aim to have an Rf value in the range of 0.3 – 0.7. Rf values are
specific to a solvent system and the sample, therefore if the experiment was replicated a
database of Rf values can be used to identify the different compounds on the plate, for
specific compounds and solvent systems. The value is most often used to distinguish
between spots in a Lab report.
NOTE: The Rf value is not to be confused with the retention factor k’, also known as the
capacity factor, used in HPLC.
Separation Principles
Intermolecular forces are fundamental to the separation of compounds via TLC. We have
three components at play here, the stationary phase, the mobile phase, and the sample. The
balance between the interactions of these components causes separation.
The stationary phase consists of a 3D silica gel network of repeating silicon oxygen chains,
with OH groups creating a polar surface, as shown in figure 3.
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Figure 3: TLC interaction diagram
This interacts with the sample, which is often a mixture of compounds with differing
polarities. Compounds with polar functional groups will have a stronger interaction with the
TLC plate than non-polar compounds. As they will interact with the surface via a range of
intermolecular forces including, dipole-dipole interactions, hydrogen bonding (functional
group dependant), and van der waals. Whereas, non-polar compounds will only interact via
weak van der waals forces. Polar compounds will be adsorbed more strongly onto the TLC
plate and will not move as far, creating a separation.
The final component in this interaction puzzle is the mobile phase, this is the one you have
the most control over. The choice of mobile phase will be dependent on the samples you are
separating. The solvent itself has an affinity for the mobile phase, and the samples are
essentially competing with the binding sites of the plate with the solvent. The mobile phase
pushes the samples up the plate. If the solvent has too weak an interaction with the mobile
phase, the samples will remain at the baseline. Whereas, if it has too strong an interaction, it
will displace the samples all the way up the top of the plate. The balance of the interaction
is finding a solvent with a similar affinity to the stationary phase than the samples, causing
different compounds to move at different rates.
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