FDSC 450 United Arab Emirates University TLC and Column Chromatography Lab Report Lab report Writing: Abstract: last thing to write (A short paragraph desc

FDSC 450 United Arab Emirates University TLC and Column Chromatography Lab Report Lab report Writing: Abstract: last thing to write (A short paragraph describing the aims, results and outcomes of the experiment or investigation. One to two sentences for each of these sections should be sufficient. Do not use references within the abstract section. The complete abstract should be no more than 200 words. Generally the information can be presented in five or six well chosen sentences.) Introduction:TLC and Column Chromatography, Artificial food pigments (Dyes) and different polarities. Materials and Methods: as per the Manuals. You can summarize and illustrate. Results and Discussion: use data from the figure 7 in ppt.. Don’t forget to write the results before adding graphs and tables. Conclusion References in the write format, at least 5, most of them coming from papers, 10 % you can use educational content: e.g.: https://www.flinnsci.com/api/library/Download/dfbc96d40dcd4b218eb533be51d670a9using the manuals and ppt. take care of plagiarism percentage. THIN-LAYER AND COLUMN
CHROMATOGRAPHY
FDSC450 – Dr. Camila da Rosa
Chromatography is a laboratory technique used to
separate a mixture of compounds. The mixture is dissolved
in a fluid called the mobile phase, which carries it through a
structure holding another material called the stationary
phase. The various constituents of the mixture travel at
different speeds, causing the separation of different
compounds so they can be analysed separately.
Thin-Layer Chromatography: TLC
It provides a rapid separation of compounds with different polarities, and thereby gives an indication of the
number and nature of the components of a mixture.
TLC can also be used to identify compounds by comparison with known samples, to check the purity of a
compound, or to monitor the progress of a reaction, an extraction, or a purification procedure.
TLC is normally done on a small glass or plastic plate coated with a thin layer of a solid — the most
common are silica (SiO2) or alumina (Al2O3). This is the stationary phase.
The mobile phase is an organic solvent or solvent mixture.
Figure 1. TLC plates.
What is the Principle of TLC?
1. The sample mixture is applied near the bottom of the
plate as a small spot, then placed in a jar containing a
few mL of solvent.
2. The solvent climbs up the plate by capillary action,
carrying the sample mixture along with it.
3. Each compound in the mixture moves at a different rate,
depending on its solubility in the mobile phase and the
strength of its absorption to the stationary phase.
4. When the solvent gets near the top of the plate, it is
allowed to evaporate, leaving behind the components
of the mixture at various distances from the point of
origin.
Figure 2. Scheme of the Thin-Layer Chromatography Principle.
5. The ratio of the distance a compound moves to the
distance the solvent moves is the Rf value (retention
factor). This value is characteristic of the compound, the
solvent, and the stationary phase.

Column chromatography
Column chromatography is one of the most useful methods for the separation
and purification of both solids and liquids. This is a solid – liquid technique in
which the stationary phase is a solid and the mobile phase is a liquid.
The sample is carried down a column of silica or alumina by solvent, and the
separate components of the mixture are captured as they elutes from (exit)
the column. This can be done by allowing the solvent to flow under the force
of gravity, but this is slow.
This technique utilizes a stationary phase, which is packed in a column, and a mobile phase that passes
through the column. This technique exploits the differences in polarity between compounds, allowing the
molecules to be facilely separated.
The two most
common
stationary phases
for column
chromatography
are silica gel
(SiO2) and
alumina (Al2O3),
Figure 3. The proper setup for a column
chromatography experiment prior to the addition of
the sample.
The solvent(s)
chosen for the
mobile phase
are dependent
on the polarity
of the
molecules
being purified.
Figure 4. As the compounds traverse through the stationary
phase they begin to separate. In this experiment the TPP
(dark purple-reddish band) travels through the column
slightly faster than the fluorenone (yellow band).
Figure 5. As the compounds elute off the column they are
collected in test tubes. The compounds being separated in
this experiment are colored, so they can be visually identified.
The Stationary and Mobile phases
Silica and alumina are relatively polar stationary phases. Both have OH groups on their surfaces that
interact strongly with polar compounds.
A
B
Figure 6. Chemical structures of silica (A) and alumina (B).
Such compounds are adsorbed strongly and therefore move along the plate slowly, while non-polar
compounds are absorbed only weakly and are therefore carried along the plate more quickly.
Solvent polarity also affects how fast compounds travel. Polar compounds are carried along quickly by
polar solvents, but move slowly or not at all with non-polar solvents.
Because non-polar compounds don’t adhere strongly to the silica, they tend to move more quickly in
most solvents.
The table below lists several common chromatographic solvents in order of increasing dielectric constant, ?,
which is a measure of bulk polarity. Since a solvent’s chromatographic “eluting power” (ability to move
compounds) is roughly related to its polarity, this is an approximate eluotropic series.
Table 1. Dieletric constant of different solvents
Caution must be taken to ensure that the
solvent system chosen for the column
chromatography has the appropriate
polarity to create separation between the
compounds in the mixture. The solvent
choice is crucial to successful separation
using column chromatography. To identify
an optimal solvent system, a series of thin
layer chromatography (TLC) experiments
should be conducted prior to performing
the column chromatography experiment.

Food Dyes:
Food dyes can be separated and identified by chromatography techniques.
Pretend that this plate has 10 cm
Dye 6: 5cm
Dye11: 3cm
Dye10: 2cm
Dye 25: 1cm
Dye 23: 8 cm
Dye 17: 0.5 cm
Calculate the Rf.
Figure 7: TLC plate for food dyes.
How to calculate the Rf?
Figure 8: Demonstration on how to obtain the Rf values.
Lab report Writing:
Abstract: last thing to write
Introduction:
TLC and Column Chromatography
Artificial food pigments (Dyes) and different polarities.
Materials and Methods as per the Manuals. You can summarize and illustrate.
Results and Discussion use data from the figure 7. Don’t forget to write the results before adding
graphs and tables.
Conclusion
References in the write format, at least 5, most of them coming from papers, 10 % you can use
educational content: e.g.:
https://www.flinnsci.com/api/library/Download/dfbc96d40dcd4b218eb533be51d670a9
FDSC 450 – Food Analysis Lab Manual
TLC – Thin Layer Chromatography for different food pigments
•
It is routinely used by researchers in the field of phytochemicals,
biochemistry, and so forth, to identify the components in a compound
mixture, like alkaloids, phospholipids, and amino acids.
Principle
Similar to other chromatographic methods, thin layer chromatography is also
based on the principle of separation.
1. The separation depends on the relative affinity of compounds towards
stationary and the mobile phase.
2. The compounds under the influence of the mobile phase (driven by capillary
action) travel over the surface of the stationary phase. During this
movement, the compounds with higher affinity to stationary phase travel
slowly while the others travel faster. Thus, the separation of components in
the mixture is achieved.
3. Once separation occurs, the individual components are visualized as spots
at a different level of travel on the plate. Their nature or character are
identified using suitable detection techniques.
TLC system components consist of
1. TLC plates, preferably ready-made with a stationary phase: These are
stable and chemically inert plates, where a thin layer of stationary phase is
applied on its whole surface layer. The stationary phase on the plates is of
uniform thickness and is in fine particle size.
2. TLC chamber. This is used for the development of the TLC plate. The
chamber maintains a stable environment inside for proper development of
spots. It also prevents the evaporation of solvents and keeps the process
dust-free.
3. Mobile phase. This comprises of a solvent or solvent mixture. The mobile
phase used should be particulate-free and of the highest purity for proper
development of TLC spots. The solvents recommended are chemically inert
with the sample, a stationary phase.
4. A filter paper. This is moistened in the mobile phase, to be placed inside
the chamber. This helps develop a uniform rise in a mobile phase over the
length of the stationary phase.
Materials
? Capillary tubes (or syringes) (to apply samples to plates)
? Developing tank, with lid
? Food pigments
? Pencil
? Thin-layer chromatography plates: aluminium sheets silica gel 60 F254, 0.2 mm
thick coating, 20 × 20 cm.
? Solvent: Chlorofom/Methanol (3:1).
Procedure
2. With a pencil, make a thin mark 2 cm from the bottom of the plate to apply the
sample spots.
3. Make marks with a pencil to divide the plate into 4 “lanes” of equal width.
4. Use capillary tubes. The application should be done as a streak across the
center of the lane origin.
5. Below the origin line, write the identity of the sample/standard in each lane.
6. Allow spots to dry.
7. Apply more sample to the same spot.
8. Write your name and the solvent used in the top right corner of the plate.
9. Write the name or numbers of the sample in the bottom of the lines.
10. Add a thin layer of solvent (Chloroform/Methanol – 3:1) in the chamber.
11. Place the plates with the sample line facing the mobile phase, such that the
sample spots are above the level of mobile phase (but not immersed in the solvent)
for development (figure 1).
12. Close the lid and observe the mobile phase travels over the stationary phase.
13. Calculate the retention factor (Rf) of all dots on the plate. The Rf is defined as
the ratio of the distance travelled by the solute to the distance travelled by the
solvent front (figure 2).
Figure 1: TLC system components.
Figure 2: Demonstration on how to calculate the retention factor (Rf)
FDSC 450 – Food Analysis Lab Manual
Column Chromatography for different food pigments
Column chromatography is one of the most useful techniques for purifying
compounds. This technique utilizes a stationary phase, which is packed in a
column, and a mobile phase that passes through the column. This technique
exploits the differences in polarity between compounds, allowing the molecules to
be facilely separated. The two most common stationary phases for column
chromatography are silica gel (SiO2) and alumina (Al2O3), with the most
commonly used mobile phases being organic solvents. The solvent(s) chosen for
the mobile phase are dependent on the polarity of the molecules being purified.
Typically, more polar compounds require more polar solvents in order to facilitate
the passage of the molecules through the stationary phase. Once the purification
process has been completed the solvent can be removed from the collected
fractions using a rotary evaporator to yield the isolated material.
Principles
The sample mixture is placed on the top of the column and absorbed onto the top
of the stationary phase. Subsequently, the mobile phase is applied to the column
and used to elute the mixture through the stationary phase. Column
chromatography exploits a molecule’s polarity to separate the compounds. The
difference in polarity leads to variances in the rate at which the molecules travel
through the column, which effectively separates the compounds from one another.
The mobile phase is collected in small fractions in test tubes as it elutes off the
column, thus allowing for the isolation and purification of the compounds. Lastly,
the solvent is removed using a rotary evaporator to yield the isolated compound(s).
Column chromatography’s versatility and convenience has made it one of the most
widely used techniques for purifying compounds. Unlike recrystallization (another
commonly
used
purification
technique) compounds purified with
column
chromatography do not have to be solid. Column chromatography is also capable
of isolating a number of compounds from a mixture. Another advantage of column
chromatograph is that very little needs to be known about the compound’s physical
properties in order to use this purification method, making this technique very
valuable when synthesizing or isolating novel compounds, in which little is known
about the compound(s).
Solvent
The rate at which a compound traverses through the column is highly dependent
on the mobile phase being utilized. Typically, the more polar the solvent the faster
the compounds will pass through the column. Polar solvents have a greater affinity
for the solid phase, limiting the interactions between the compound(s) and the solid
phase, allowing the compounds to elute more rapidly. Caution must be taken to
ensure that the solvent system chosen for the column chromatography has the
appropriate polarity to create separation between the compounds in the mixture.
The solvent choice is crucial to successful separation using column
chromatography. To identify an optimal solvent system, a series of thin layer
chromatography (TLC) experiments should be conducted prior to performing the
column chromatography experiment. In some case it may be necessary to use a
binary solvent system.
Selecting a Solvent System
1. Identify a solvent system that produces a retardation factor (Rf ) between
0.2–0.3 for the desired compound on a TLC plate.
2. Start experimenting different solvents such as chloroform and methanol as
the mobile phase for the TLC experiment.
If the Rf is greater than 0.3, try less polar solvent, such as hexane. If the Rf is less
than 0.2, try adding a small amount of a polar solvent such as methanol.
The optimal solvent system may require a mixture of two solvents.
Be careful not use more than 10% methanol as the mobile phase for a silica
column.
Procedure
1. Silica Gel Slurry
Pour the silica gel into an Erlenmeyer flask. The weight of the packing material
should be roughly 50x that of the sample being separated. If the compounds being
separated have very similar Rf values, then it may require using a larger amount
of silica per sample, which is the case in this example.
Place 10 g of silica in the Erlenmeyer flask, since 50 mg of sample (45 mg of
fluorenone and 5 mg of tetraphenylporphyrin) are being isolated.
Add the solvent system to the Erlenmeyer flask containing the silica gel. Add
enough solvent to ensure that all of the silica gel is well solvated. The silica will not
dissolve, but the mixture will be visually noticeable when solvated. Once the
solvent has been added swirl the Erlenmeyer flask to ensure that all of the silica is
well solvated.
2. Preparation of the Column
a. Select the appropriately sized column. Typically, the column should be filled
about half way with silica gel slurry. The larger the sample being purified, the larger
the column required.
b. Plug the bottom of the column with a piece of glass wool. Using a long rod, make
sure the wool is firmly lodged in the bottom of the column just above the stopcock.
c. Once the wool is firmly in place, apply a thin layer of sand over the glass wool.
Note: If the column is equipped with a glass frit above the stopcock, this step
should be omitted.
d. Clamp the column in the vertical position to a ring stand.
e. Using a funnel, gently pour the prepared slurry of silica gel into the column. You
may need to add additional solvent to transfer the slurry from the Erlenmeyer flask
to the column. Using a pipette, wash down any silica gel that sticks to the sides of
the column.
f. As the silica gel is settling in the column, gently tap the sides of the column to
ensure that the silica gel packs tightly and excludes any air bubbles.
g. Open the stopcock and allow solvent to drain into a clean Erlenmeyer flask until
just before the silica gel and the solvent front meet. The silica gel should never go
dry until the procedure is complete.
h. Place a thin layer of sand on top of the silica gel (Figure 1). Using a pipette,
wash down any sand that may have stuck to the sides of the column.
i. Drain any additional solvent until the sand is dry, but not down to the silica gel
layer.
3. Adding the Sample to the Column
j. Dissolve the sample in the smallest amount of solvent possible (using the same
solvent that was used to make the silica gel slurry).
k. Using a pipette, gently add the sample to the top of the column.
l. Once the sample has been applied to the top of the column, open the stopcock
and allow the solvent to drain through the sand layer but not the silica gel layer.
Use a very small amount of solvent to wash down any sample that may have clung
to the sides of the column. Drain this additional solvent through the sand layer as
well.
4. Eluting the Sample through the Column
m. Using a pipette, very gently add 4–5 mL of solvent in such a manner that does
not disturb the sand layer.
n. Place a funnel at the top of the column and very slowly and gently fill the
remainder of the column with solvent.
o. Open the stopcock and allow the solvent to drain through the column.
p. Begin collecting the mobile phase as it drains from the column into test tubes.
q. Test tubes should be placed in a test tube rack in a sequential manner.
r. Add additional solvent to the top of the column as needed until all the desired
compounds have eluted from the column.
5. Recovering the Constituents
s. If the compounds are colored, then they can be visually identified. However, if
the compounds are colorless, they will have to be identified using ulta-visible (UV)
light (if the compounds contain conjugation) or with the appropriate stain. The
purity of the compounds can be verified using thin layer chromatography.
t. Identify the test tubes that contain the desired compound(s).
w. Merge all of the fractions that contain the desired isolated compound(s) into a
pre-weighed round-bottom (RB) flask. Do this for each compound being isolated.
v. Evaporate the solvent by placing the RB flask on the rotary evaporator.
x. Once all of the solvent has been removed, weigh the RB with the dried product
and subtract the initial weight of the RB to obtain a yield.

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