University of Research and Technological Investigation YACHAY TECH

School of Chemical Sciences and Engineering Department of Chemistry

Chemical Analysis II - Understanding Chromatography Using Thin Layer Chromatography (TLC)

Michelle Beatriz Chicaiza Lema (0502998867)

Urcuqu, September 15 2017
Introduction
The technique chromatography is used to
separate mixtures of substances into their
components. All forms of chromatography
work on the same principle that consist of
stationary phase and a mobile phase. The
mobile phase flows through the stationary
phase and carries the components of the
mixture with it. Different components travel
at different rates.
Thin layer chromatography (TLC) is done
with a layer of silica gel or alumina coated
onto a piece of glass, metal or rigid plastic.
TLC is an analytical tool widely used because
of its simplicity, relative low cost, high
sensitivity, and speed of separation.
One of the components of the TLC is
silica gel (alumina) which is the stationary
phase that is highly polar (standard) or non-
polar (reverse phase). The stationary phase
for TLC also often contains a substance
which fluoresces in UV. The mobile phase is
a suitable liquid solvent or mixture of
solvents.
Applying a sample in solution to the plate,
then "run" the plate by allowing a solvent (or
combination of solvents) to move up the plate
by capillary action. Depending on the
polarity of the components of the mixture,
different compounds will travel different
distances up the plate. More polar
compounds will "stick" to the polar silica gel
and travel short distances on the plate, while
non-polar substances will diffuse into the
solvent and travel large distances on the plate.
After a separation is complete, individual
compounds appear as spots separated
vertically. Each spot has a retention factor
(Rf) which formula is:
=
The Rf value can be used to identify
compounds due to their uniqueness to each
compound. When comparing two different
compounds under the same conditions, the
compound with the larger Rf value is less
polar because it does not stick to the
stationary phase as long as the polar
compound, which would have a lower Rf
value.
Objective
To remove pigments from plants.
The student will be able to separate
and identify compounds (pigments)
present in different plant sample by
TLC.
The student will identify same
compounds of different samples.
The most suitable mobile phase for a
good chromatographic separation will
be found
Materials and reagents
Knife /scissors/pencil/rule
TLC plates
Chromatography pipettes
 Mortar capillary action to draw the solvent up the
Chambers plate until it is approximately 1 cm from the
Test tube (10ml) end. Never allow the solvent to migrate all the
Tweezers way to the end of the plate. Remove the plate
Filter paper and immediately draw a pencil line across the
Spatula solvent front.
Beaker (100ml, 25ml) 5. Visualizing: Use a short-wave UV light and
UV light camera circle the components shown with a pencil
Acetone (C3H6O) 6. Reviewing and changing: revise your choice
Magnesium sulfate (MgSO4) of solvent system based on the results of your
Methanol (CH3OH) initial TLC.
Dichloromethane (CH2Cl2) Results and calculations
Ethanol (C2H6O)
Ethyl acetate(C4H8O2) In the TLC the samples corresponds to:
Hexane(C6H14) A: carrots
Diethyl ether((C2H5)2O)
B: parsley
C: Pepper
Procedures
D: Spinach
1. Preparation of samples: For first TLC:
Take a quantity of every plant spinach, Solvent used: hexane 10 mL
parsley, pepper and carrot:
Deposit each vegetable in a different mortar 0,7
= = 0,14; without color in TLC
and add an organic solvent in order to extract 5
the pigments of each one. 0,7
= = 0,14; without color in TLC
*Spinach case: we are going to crush spinach 5
0,8
leave with a small quantity of acetone MgSO, = = 0,16; without color in TLC
and the Parsley with acetone 5
2. Prepare TLC plates: 0,8
= = 0,16; without color in TLC
Cut the plate and use a ruler and a pencil to 5
lightly mark baselines on the silica side of the
plate. Mark spots where you will apply the For second TLC:
samples Solvent used: Ethyl acetate 10mL
3. First of all, cut a sheet of filter paper to put
inside the chamber. Poured 10 ml of the 3,5
= = 0,73;
solvent mixture on the chamber with paper 4,8
filter close it and wait a couple of minutes 4,2
= = 0,88;
until the paper is soaked. 4,8
4. Spotting and developing: take the plate and 4,6
spot the samples in marked places in baseline = = 0,96;
4,8
using capillaries. Place the plate into the 4,2
chamber as evenly as possible and lean it = = 0,88;
4,8
against the side. Never allow the bulk solvent
to rise above the line you drew. Allow
 For third TLC: compound. We should take into account that like
Solvent used: methanol 10mL dissolve like. Moreover, in the result of TLC
4,7 shows that are two kinds of Chlorophyll, the
= = 0,73;
5,5 green color correspond to Chlorophyll a and
4 bluish green color correspond to Chlorophyll b.
1 = = 0,73;
5,5 In the other hand, some samples contains acetone
4,6 or other solvent that produce that it acquire
2 = = 0,84;
5,5 certain polarity. And it influences in the result of
3,7 the Rf of the TLC. Finally, in the TLC samples
= = 0,67;
5,5 were observed some enlarged spots of pigments
3,9 and it is due the sample of pepper was very
= = 0,71;
5,5 concentrated at preparation time.
4,6
= = 0,84 ; Conclusion
5,5
For fourth TLC: Thin Layer Chromatography is an
Solvent used: 2,1Ml methanol, 2,9Ml easy technical that allows to separate
ethyl acetate, 5 mL hexane compounds in a sample. In the case of
4,9
= = 0,94; experimental process the pigments of
5,2 plants.
3,3
1 = = 0,63; The principal mechanism of
5,2
chromatography is based on the
3,7
2 = = 0,71; stationary phase (adsorption based of
5,2
4 intermolecular forces) and the mobile
3 = = 0,77; phase (solubility of eluent).
5,2
4,9 The factors like concentration of the
4 = = 0,94; sample can influence the distance of
5,2
4,1 spot can move in mobile phase.
1 = = 0,79; The polarity of the solvent show us
5,2
5 more clearly to differentiate the
2 = = 0,96
5,2 polarity of compound that are present
4 in the molecule.
= = 0,77;
5,2 The values of Rf are mainly
4,2 depending of the conditions like: kind
2 = = 0,81;
5,2 of adsorbent, eluent, plate.

Final Questions
Observations: the adsorption is produced in the
Which of compound (pigment in your samples) is
stationary phase where the silica gel acts with
the most polar? And which of compound is the
polar groups of the compounds that are studied.
most non polar? And why?
Also, the solubility is present in the solvent that
had chosen depending of the polarity of
 The most polar pigment in the samples is the and not act as a catalyst in reactions of
chlorophyll, because it moves a large distance decomposition. The adsorbent interacts with the
thought the plate. The polarity is determined by substances by dipole-dipole interaction or by
the number and nature of the functional groups hydrogen bond if present as indicate in the next
present in the compound. The more polar solutes figure:
will be more retained since they are more
strongly adsorbed to the active centers of the
stationary phase, in this case in the silica gel.
While non-polar ones will elute more easily. In
the experiment the carotene is the most non polar
pigment.
Which eluents did you try? Which of different
eluents allowed more separation of the mixture?
Why?
We used for the first plate hexane, then
ethyl acetate, methanol and finally a mixture of
methanol and hexane and ethyl acetate. Figure 1 the next figure represents the structure of
the silica gel and their intermolecular forces
The order of elution of a compound is increased
In the case of chlorophyll a polar
by increasing the polarity of the mobile phase or
molecule has hydrogens that allows to
eluent. In the case of hexane the spots are very
form a hydrogen bond with the silica gel.
closer to the baseline. When we used ethyl
acetate the spots are dispersed. But, when we
used methanol the sports are very differentiable,
especially for chlorophyll. Also, the order of
elution of a compound is increased by increasing
the polarity of the mobile phase or eluent.
This may be a single solvent or two miscible
of different polarity. The following eluents are in
increasing order of eluting strength: hexane <
ethyl acetate < methanol.
Explain what happen between stationary phase
and pigments and draw it in order to represent
interactions or bonds.
The two most used adsorbents in
stationary phase are silica gel (SiO2) and alumina
(Al2O3), both of polar character. The adsorbent Figure 2 the next figure represents the structure of
chlorophyll a and b.
must be inert with the substances to be analyzed