Activity No. 4
CHROMATOGRAPHY
Casiño, Jeanne Pearl C.
Bachelor of Science in Chemistry
Ms. Nova Fe E. Anorico, RCh, MSc
April 2021
INTRODUCTION
1.1 Rationale of the Experiment
Chromatography is a technique used to separate components in a mixture and can be
used to partially identify the components. It works in a way were each liquid undergoes
adsorption in a slightly different way and spends more or less time in either the solid or the
liquid phase. One of the liquids might spend much longer in the solid phase than in the
liquid, so it would travel more slowly over the solid; another one might spend less time in
the solid and more in the liquid, so it would go a bit faster (Woodford, 2020). In this
experiment, we used two different types of chromatography in separating the organic
components, the thin layer chromatography and column chromatography. Thin layer
chromatography is a technique used to isolate non-volatile mixtures, the process involved
the use of the most known adsorbent, the silica gel. The mixture was placed in a plate and
is emerged in a bath of solvent up until below the mark. The components started to separate
and were then gradually moving upward, from the mobile phase to stationary phase with
different velocities. To calculate the retention factor, the following formula was used:
Rf = Distance Travelled by Sample
Distance Travelled by Solvent
Column chromatography on the other hand, is another solid-liquid technique in which
the two phases are a solid (stationary phase) and a liquid (moving phase). The theory of
column chromatography is analogous to that of thin-layer chromatography. The sample is
dissolved in a small quantity of solvent (the eluent) and applied to the top of the column
(Welleslly, n.d.). During the laboratory instead of the solution rising up, it flows down
through the column filled with the adsorbent. The column was loaded with mixture, sand,
solution and solvent. When everything is already in tacked and settled, the data was the
collected through a numerous labeled flask by opening the stopcock. Along with it, the
component started to separate differentiating which is which with colors of red, blue and
such. Just like the tlc, this method establishes its own equilibrium between the solute
adsorbed on the silica gel and the eluting solvent that is flowing down through the column.
Objectives of the Experiment
The main objectives of this experiment is to separate organic compound through
chromatography. The experiment also addressed the following:
1. To separate the components using thin layer chromatography.
2. To separate the product using column chromatography.
METHODOLOGY
1. Experimental Set-Up
Part A. Separating Components using Thin Layer Chromatography
Figure 1. Thin Layer Chromatography Experiment
1.1 Preparation of Materials and Chemicals
In the thin layer chromatography experiment, a specific materials and chemicals were
prepared. The experiment required the following: tlc plate, solution with components, solvent,
pencil, uv light, solvent, developing chamber, beaker cover, capillary tube and flask.
1.2 Procedure in Separating the Compound using TLC
To perform the experiment, a line was drawn near at the bottom of the tlc plate with the use
of a pencil. A capillary tube was then inserted in a flask containing the component, to suck some
of the solution through a capillary action. The content in the tube was disposed right unto the line
in the tlc plate by pressing the end of the tube three times.
The spotted tlc plate was put inside the beaker but was reassured that the line was above the
solvent to avoid washing out the component. For the solvent not to evaporate the beaker was then
covered. The solvent began to rise from the mobile phase to the stationary phase of the plate,
wherein the component is also dragged upward. The plate was then removed when the solvent
almost reaches the top of the plate. With the use of a pencil, the place where the solvent stop was
mark with a line. The plate was then leave for a moment to dry.
Since the component isn’t visible enough an ultraviolet light inside a special box was used to
trace and analyzed the data.
1.3 Schematic Diagram
Figure 2. Schematic Diagram for Separating the Components using TLC
TLC Plate and the Sample Compound
- mark a line near at the bottom of
the plate
- Insert a capillary tube in a flask to suck
some of the components in the solution
- Deposit the content in the tube to the
mark of the plate
Developing Chamber
-
Put the plate inside the chamber
Add a solvent and make sure
It will not reach the mark
Close the beaker with something
to avoid evaporation
Mobile Phase to Stationary Phase
-
-
Watch the solvent rise from
MP to SP
Notice the component being drag
upward and separates
Remove the plate as soon as
it reaches nearly at the top and mark the
spot
Use a uv light carefully to gather the
data needed
End of Experiment
PART B. Separating the Compound using Column Chromatography
Figure 3. Column Chromatography Experiment
1.4 Preparation of Materials and Chemicals
In the column chromatography experiment, a specific materials and chemicals were
prepared. The experiment required the following: Liquid funnel, glass column, eluent, sand,
adsorbent, mixture, flask, iron stand, iron lamp, cotton, stopcock, rotary evaporator, tlc and pipette.
1.5 Setting-Up the Compound and the Column
A cotton was placed at the bottom of the column using a wire. The column was then clamp
in an iron stand. A layer of sand was added inside together with the solvent filling one-third of the
column. After that, an amount of silica gel powder was poured into the beaker containing the
solvent, inside the fume hood to avoid inhaling the toxic. The mixture was then stirred until its
completely slurry. To get in to the column a spatula and solvent was used to push the slurry inside
and gently patted to settle. Another set of sand was added onto the column along with the solvent
to drip.
1.6 Loading the Column with Mixture
The mixture was dissolve in the smallest amount as possible for it to be pipetted onto the
column. It was then drip directly into the sand without touching the sides of the column. Finally,
small amount of solvent was drain to load the mixtures onto the stationary phase.
1.7 Adding the Solvent
A solvent was added to the column through a pipette by squirting it to the side, until it
reaches several inches above the sand.
1.8 Collecting Fractions
The solvent was dripped out of the column and was collected through a series of labeled
flask. It was noticed that the components began to separate as they move through the column. To
separate properly an optical flow rate was applied. To complete the process, an amount of eluent
was gradually added onto the column until all the data needed were collected.
1.9 Analyzing Fraction
A 5-10 vertical lines were drawn in the plate, and was numbered according to its
corresponding fractions. Each line has a spot for each fractions and the plates were then
developed. All the fractions were then combine into a rotary evaporator to evaporate the solvent
away.
2.0 Schematic Diagram
Figure 2. Schematic Diagram for Separating the Compound using
Column Chromatography
Setting-Up
-
Place a cotton at the bottom
Clamp the column
Add sand and solvent
Mix solvent and silica powder
Put the slurry in the column using
Spatula and settle
Loading Column with Mixture and Solvent
-
Dissolve the mixture into the smallest
amount and drip into the column using
pipette
- Drain the solvent until mixture reaches
SP
- Add solvent through the side
Collecting and Analyzing Fraction
-
Drip the solvent
Collect the data needed
Analyze
End of Experiment
RESULTS AND ANALYSIS
The following data shown in Table 1.1 and Table 1.2 are obtained from the results,
after completely separating the components using two different types of chromatography. The
thin layer chromatography and column chromatography.
Table 1.1 Results in Separating the Components Using Thin Layer Chromatography
Result
Identification
Rf values
Separated
Non-polar
0.91
Separated
Polar
0.45
Solution and
Solvent
Table 1.1 shows the result in separating the components using tlc. The solution containing
the components was placed in a plate containing silica gel and put in a solvent bath. With this, the
eluting power of the solvent will increase making those polar and non-polar substance dissolved
and eluted. In this case, you will see that both components are completely separated but one is a
polar and the other is non-polar. Looking at the retention factor non-polar seems to travel a farther
distance than the polar, that is because polar molecules have stronger bonds which is ideal for
adsorbent. It will spend most of its time in the stationary phase that slower its movement, while as
of the less polar it was carried out quickly up unto the mobile phase (Chromatography, 2007).
Table 1.2 Results in Separating the Product using Column Chromatography
Result
Entire Sample
Product Separated
Table 1.2 shows the result in separating the product using column chromatography.
The column was filled with the entire sample according to their respective layers, wherein the
product must sit in between the sand and the slurry also known as the stationary phase. The solvent
level was lowered, in order for the separating process to run smoothly. Solvent plays a vital role
in separating the product, because of its polarity. When it passes through the column it affects the
relative rates in which the mixture moves through( ). So as for the entire sample after it was settled
inside the column and ready for the process, they are being collected through series of flasks, and
an amount of solvent was gradually being added for those polar molecules will compete the polar
solvent. In analyzing if the product was successfully separated the entire data was run through a
rotary evaporator. Rotary evaporator is a device used to remove solvents from the sample by
evaporation (“Rotary evaporator”, 2020). The solvent began evaporating until the product was
completely isolated.
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