School of Chemical Science and Engineer

University of Experimental Technology Yachay Tech

Analytical Chemistry
Name: Jorge Ivn Crdenas Gamboa
Course:
Date: 07/09/2017
Title: Understanding chromatography using Thin Layer Chromatography (TLC)

1. Introduction
Thin layer chromatography and paper chromatography comprise planar chromatography. TLC
is the simplest of all the widely used chromatographic methods to perform. A suitable closed vessel
containing solvent and a coated plate are all that are required to carry out separations and
qualitative and semi qualitative analysis. With optimization of techniques and materials and the
use of available commercial instruments, highly efficient separations and accurate and precise
quantification can be achieved. Planar chromatography can also be used for preparative-scale
separations by employing specialized layers, apparatus and techniques.
Basic TLC is carried out as follows. A small aliquot of sample us placed near one end of the
stationary phase, a thin layer of sorbent, to form the initial zone. The sample is then dried. The end
of the stationary phase with the initial zona is placed into the mobile phase, usually a mixture of
two to four pure solvents, inside a closed chamber. If the layer and mobile phase were chosen
correctly, the components of the mixture migrate at different rates during movement of the mobile
phase through the stationary phase. This is termed development of the chromatogram. When the
mobile phase has moved and appropriate distance, the stationary phase is removed, the mobilse
phase is rapidly dried and the zones are detected in daylight or under ultraviolet (UV) light with
or without the application of a suitable visualization reagent.
Differential migration is the result of varying degrees of affinity of the mixture components for
the stationary and mobile phase. Various separation mechanisms are involved, the predominant
forces depending upon the extract properties of the two phases and the solutes. The interactions
involved in determining chromatographic retention and selectivity include hydrogen bonding,
electron-pair, donor/electron-pair acceptor, ion-ion, ion-dipole, and van der Waals interactions.
Among the latter are dipole-dipole, dipole-induced dipole and London forces interactions.
Sample collection, preservation, and purification are problems common to TLC and all other
chromatographic methods. For complex samples, the TLC development will usually nor
completely resolve the analyte from interferences unless a prior to TLC, to a derivative that is more
suitable for separation, detection, and/or quantification than the parent compound TLC can cope
with highly contaminated samples, and the entire chromatogram can be evaluated, reducing the
degree of cleanup required and saving time and expense. The presence of strongly adsorbed
impurities or even particles is of no concern, because the plate is used only once.
Compound identification in TLC is based initially on a comparison of values to authentic
references standards. values are generally not exactly reproducible from laboratory to
laboratory or even in different runs in the same laboratory, so they should be considered mainly as
guide to relative migration distance and sequences. Factors causing values to vary inclyde
dimensions and type of chamber, nature and sized of the layer, direction of the mobile-phase flow,
volume and composition of the mobile phase, equilibration conditions, humidity, and sample
preparations methods preceding TLC (Fried, 2003).
After a separation is complete, individual compounds appear as spots separated vertically. Each
spot has a retention factor ( ) which is equal to the distance migrated over the total distance
covered by the solvent. The formula is

=

The 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 value is less polar because it does not stick to the stationary phase as long as the polar
compound, which would have a lower value (Diaz, 2017).

2. Objectives
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.

3. Materials and Reagents

Knife /scissors/pencil/rule
TLC plates
Chromatography pipettes
Mortar
Chambers
Test tube (10ml)
Tweezers
Filter paper
Spatula
Beaker (100ml, 25ml)
UV light camera
Acetone
MgSO4
Methanol
 Dichloromethane
Carrot
Parsley
Pepper red
Spinach
Ethanol
Ethyl acetate
Hexane
Diethyl ether
4. Procedure
1. We took a quantity of every plant (spinach, parsley, pepper and carrot):
-3-4 leaves (spinach and parsley)- we cut with scissors
-Pepper skin and carrot skin
We deposited each vegetable in a different ortar and add and organic solvent in oder to
extract the pigments of each one. In the case of the spinach we left a small quantity of
MgSO4
2. We cut the plate with scissors (10cm high and 5cm wide, all depends of the quantity of
samples). We used a ruler and a pencil to lightly mark baselines on the silica side of the
plate (be careful not to remove any silica from the plate). We marked spots where you will
apply the samples.
3. We cut a sheet of filter paper to put inside the chamber (this will help Mobil phase). In the
selection of solvent or solvents taking in account eluotropic series where we found solvents
ordered by polarity. Once you got solvent or solvent mixture, with a quantity of 10ml
approx. Add it to the chamber paper filter we closed it and waited a couple of minutes until
the paper is soaked, then the chamber is ready to use.
4. We took the plate and spot the samples in marked places in baseline using TLC pipettes.
We applied samples different times, not only once!! And let dry the solvent in order to have
only compound in the spot. We placed the plate into the chamber as evenly as possible and
lean it against the side. Never allow the bulk solvent to rise above the line you drew. Allow
capillary action to draw the solvent up the plate until it is approximately 1 cm from the end.
Never allow the solvent to migrate all the way to the end of the plate.
5. We used a short-wave UV light and circle the components shown with a pencil.
6. We revised if our choice of solvent system based on the result of your initial TLC.
5. Results
To explain our results we have made 4 kinds of mixture to separate the solvents. To this we have
put attention in the table of polarity, taking two points in attention, since the most polarity like
water to the less polarity like pentane, we also have tried a mix of both, polar and non-polar. Our
objective were to find the best solvent to separate the mixture, it can be observed visually at the
moment to take retention factor due to in the best solvent we are going to have more division
because the pigments not only have one kind of pigments like carotenoids or chlorophyll, in the
next tables we are going to explain better this idea, demonstrating that TLC is the best method for
separating mixtures.
10 ml of Hexane
Distance for the Distance travelled by Retention factor (Rf)
solvent (maximum) component
Carrot 5,3 cm 0,25cm 0,047
Parsley 5,3 cm 0,5 cm 0,094
Red Pepper 5,3 cm 0,4 cm 0,0075
Spinach 5,3 cm 0,5 cm 0,094

5 ml of Hexane and 5ml of Methanol

Distance for the Distance travelled by Retention factor (Rf)
solvent (maximum) component
Carrot 4,8 cm -------- ----------
Parsley 4,8 cm 1,05 cm 0,218
Red Pepper 4,8 cm 0,05cm 2,1cm 0,010 0,44
1,5cm 2,6cm 0,31 0,54
Spinach 4,8 cm 1,05 0,218

5ml of Hexane and 5ml of ethyl acetate

Distance for the Distance travelled by Retention factor (Rf)
solvent (maximum) component
Carrot 5,95 cm 4,0cm 0,67
0,6cm 0,10
Parsley 5,95 cm 1,6cm 2,6cm 3,4cm 0,27 0,44 0,57
2cm 3cm 0,33 0,504
Red Pepper 5,95 cm 0,8cm 1,9cm 3,6cm 0,13 0,32 0,61
1,06cm 2,4cm 4cm 0,19 0,40 0,67
1,5cm 2,9cm 0,25 0,49
Spinach 5,95 cm 1,6cm 3,1cm 0,27 0,52
2,7cm 3,6cm 0,45 0,61
10 ml of ethyl acetate
Distance for the Distance travelled by Retention factor (Rf)
solvent (maximum) component
Carrot 5,8 cm ------- -------
Parsley 5,8 cm 2,1 cm 3,4 cm 0,36 0,59
3 cm 0,52
Red Pepper 5,8 cm 3,1cm 3,5 cm 0,53 0,60
Spinach 5,8 cm 1,5cm 2,8 cm 0,29 0,48
2,3cm 3,5 cm 0,4 0,60
As we know for every compound, it will have a Rf different, but theses samples are not just with
carotenoids and chlorophyll, according with the literature these compounds could have alfa
caroteno, beta caroteno, luteina and violaxtina in the samples like carrot and pepper but in the other
we can see chlorophyll a and chlorophyll b, feofitinas, xantofilas due to the great quantity of
pigments because not just this what I say before we note different colors for each compound
(Beley, s.f.).
The colors in all the samples were different for each one, for example in the sample a (carrot) the
colors are yellow-orange in the sample b (parsley) green soft and green dark, in the sample c(red
pepper) Orange dark and soft and in sample d (spinach) green soft and green, all these colors were
blurred or in different shades and it is due to the great quantity in pigments for each one of our
sample. To identify what pigment we are talking we should go to the literature and identify the Rf
because for each compound these values is specific, obviously, focusing on the same stationary
phase for each one. Finally, according to our results tables we could say that the best solvent is a
mix of hexane and ethyl acetate, because this fulfill with a special characteristic, both are as polar
as non-polar and this mixture will can dissolve more quantity of pigments, so, could identify not
just chlorophyll else other kinds of variation for each one.
Final Questions
1. Which of compound (pigment in your samples) is the most polar? And which of compound
is the most non polar? And why? (Find the compound structures on the open literature to
reason your answer)
According to the retention factor obtained (table 1,2,3,4) for the different displacements of the
pigments, the polarity seems to depend on the solvents used. For example, when using only hexane,
a non-polar solvent, the spinach retention factor is greater than the carrot retention factor. On the
other hand, when using a mixture of solvents whose polarity is much greater the result is that the
pigments present in the spinach are much more polar while the pigment present in the carrot is less
polar or non-polar. Through these explanation, we finally have decided that the most polar is the
chlorophyll b and the most non polar carotenoids, this is due to the literature who say about the
polarity of all the pigments shown in our chromatography paper.
2. Which eluents did you try? Which of different eluents allowed more separation of the
mixture? Why?
The best eluents in our case is showing in the table 3, a mixture of 5ml of Hexane and 5ml of ethyl
acetate. This can be explained by "equal dissolves the same" since using a non-polar solvent would
help the non-polar compound to adhere to the stationary phase, just as it happens with the use of
polar solvents with polar compounds, adhesion is much stronger while more polar is the solvent.
From this explanation at the moment when we use a eluents mostly non polar like hexane, the
chromatography paper only show few pigments, when a solvent mostly polar like ethyl acetate,
show a major quantity of pigments but are not enough, but a solution which contains polar and
non-polar characteristics separated more quantities of pigments, concluding that the mix of hexane
and ethyl acetate is the best eluents in our laboratory practice.
3. What is the Rf value in each case? (Compare for every compound)
Tables are shown before, in which we can see each sample, with the division between distance
travelled by component over distance travelled by solvent, showing different values Rf depending
of the eluent what we are using the solubility and de adhesion of the pigments to the
chromatography paper. The difference is in the solubility with respect to the eluent, in other words,
each compound will have a percent of polarity and depending of this, it will move around de
chromatography paper, seeing with a good eluent (in this case polar and non-polar) the quantity of
pigments in our samples, furthermore, for each one, we have a Rf factor, it means that we are
saying for an specific pigment, if the value of Rf between each compound is the same, it wants to
say that in the same compound we have the same pigment, and it has sense because these samples
share many pigments like I have said before chlorophyll and carotenoids the most representatives.
4. Check the answers above and discuss the following statement No matter what, in any
case Rf for a specific compound is the same
A compound's Rf value depends on interaction with stationary phase (TLC plate). In all of the
cases we work with the same stationary phase then, theoretically all of the Rf values for a specific
compound have to be the same. The value for each dissolvent is different because each one varies
on its adsorption, if we required a literature, in the case for the same stationary phase, and the same
samples we should get the same Rf value, on this way, each compound will have a specific value
of Rf as we can see on the analytical books as long as the conditions of the experiment are the
same in our case, we have varied some reagents because we learn about the best eluent at the
moment of work with pigments.

5. Explain what happen between stationary phase and pigments and draw it in order to
represent interactions or bonds.
Pigments and stationary phase are different interactions depending if the interactions are polar or
non-polar. First of all, as we have said before, the best stationary phase was a mixture between
polar and non-polar compounds and with this premise we could say: In polar pigments the
interactions are dipole-dipole, due to its character polar and in non-polar pigments, they have
shown interactions dipole-dipole instantaneous, dipole instantaneous-dipole induced and dipole-
dipole induced. All these interactions are given in the electronic cloud allowing to the pigments
move around the chromatography paper and theses interactions are shown here:

6. Conclusions
7. Attachments
Figure 1 Chromatography Paper for each simple

Figure 2 Best eluent, 5ml of Hexane and 5ml

of ethyl acetate

Figure 3 Chromatography paper shown in UV light

 8. Reference

Bibliography
Beley, S. (s.f.). eHow. Obtenido de http://www.ehowenespanol.com/pigmentos-vegetales-encontrados-
espinacas-info_192238/

Diaz, A. (2017). EXPERIMENT 1: UNDERSTANDING CROMATOGRAFY USING THIN LAYER

CHROMATOGRAPHY (TLC). Urcuqu: University Yachay Tech.

Fried, S. B. (2003). Handbook of Thin-Layer Chromatography. New York: Joseph Sherma and Bernand
Fried.