Lab.2.

Thin layer chromatography

Key words:

Separation techniques, compounds and their physicochemical properties (molecular

volume/size, polarity, molecular interactions), mobile phase, stationary phase, liquid
chromatography, thin layer chromatography, column chromatography, retardation factor,
elution, chromatogram development, qualitative and quantitative analysis with
chromatography techniques, eluotropic series, elution strength.

Literature:

D.A. Skoog, F.J. Holler, T.A. Nieman: Principles of Instrumental Analysis; 637 - 718
Search on www pages “Thin-layer chromatography principles”
For example: MIT Digital Lab Techniques Manual you find on
http://www.youtube.com/watch?v=e99nsCAsJrw&feature=player_detailpage
Basic equipment for modern thin layer chromatography:
www.camag.com/downloads/free/brochures/CAMAG-basic-equipment-08.pdf
other examples:
en.wikipedia.org/wiki/Thin_layer_chromatography
www.chemguide.co.uk/analysis/chromatography/thinlayer.html
www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/th
in_layer_chrom.html

Theoretical background

Chromatography is the separation technique in which separated solutes are distributed

between two phases: stationary and mobile. The first phase can pose a layer of
sorbent/adsorbent (0.1 to 0.25 mm in thickness) fixed to a carrier plate made of glass, plastic
or aluminum (used in technique named as thin-layer chromatography, TLC) or placed inside
of a steel tube as a column bed (used in a technique named as high-performance liquid
chromatography, HPLC, or generally in column liquid chromatography, LC). The second
phase, mentioned above, constitute liquid or gas phase. Various organic (e.g. methanol,
hexane, acetone) and inorganic (e.g. water) solvents or their mixtures (e.g. acetone and
 Lab.2. Thin layer chromatography

hexane, methanol and water) can be used as the mobile phases. So each chromatographic
system consists of:
a) stationary phase,
b) mobile phase,
c) mixture of components to be separated.
A solution of the component mixture is usually introduced into the chromatographic system
by injection (in HPLC or classical column chromatography in entrance to the column) or by
spotting/application onto start line (in TLC). In column chromatography the mobile phase is
pumped through the adsorbent bed or its flow is caused by gravitation as it is demonstrated in
Fig 1A. In thin layer chromatography mobile phase is driven into movement by capillary
forces (solvent wets adsorbent layer on the chromatographic plate by capillary forces) as it is
demonstrated in Fig 1B. Under such circumstances mixture components migrate along the
stationary phase (adsorbent) according to the direction of flow of the mobile phase.

Mobile
phase

Station
A B
ary
phase

Chromatographic
plate
Valve

Chromatographic
chamber

Mobile
phase

Fig. 1. (A) Classical column chromatography, (B) chromatogram development in

conventional chamber (in cuboid vessel)

Migration velocities of mixture components are slower from that o the mobile phase. It is
because of time, which separated molecules spend in the stationary phase. Arrangement of
solute zones on the chromatographic plate after chromatogram development is demonstrated
in Fig. 2.
 Lab.2. Thin layer chromatography

Solvent front

Start line

Fig. 2. Thin layer chromatogram of dyes, 1 and 10 – dye mixture, 2 – 9 single dyes
The time the separated molecules spend in the stationary phase depends on their interactions
with stationary and mobile phases. It means the mixture components can be separated in the
chromatographic system if they demonstrate different migration distances, i.e. if they show
different energy of molecular interactions with components of the chromatographic system.
Following molecular interactions of solutes with elements of stationary and mobile phases can
take place in any chromatographic system: hydrogen bond, dipole – dipole, dipole – induced
dipole, ion – dipole, instantaneous dipole – induced dipole (London dispersion forces), ion –
ion.

The stationary phase

1. Silica gel
Silica gel is composed of silicon dioxide (silica). The silicon atoms are bonded via oxygen
atoms in a giant covalent structure. However, at the surface of the silica gel -OH groups are
attached to the silicon atoms. So, on the surface of silica gel Si-O-H groups are present
instead of Si-O-Si ones. This makes silica surface very polar.
Fig. 3 shows the model of a small part of the silica surface.
 Lab.2. Thin layer chromatography

Fig.3. A simplified model of silica gel surface

There are also silica based adsorbents, which are non-polar, i.e. chemically modified silica.
Modified silica gel is formed by chemical reaction of its surface with e.g.
trichlorooctadecylsilane or other reagents. Thus the surface polarity decreases and then its
hydrophobicity increases.

2. Aluminum oxide
Aluminum oxide (Al2O3) is another adsorbent, which is often used as stationary phase in
laboratory practice. TLC aluminum oxide plates usually comprise neutral or basic aluminum
oxide. These kinds of plates provide distinct separation features with regard to a pH range of
the mobile phase used. Under aqueous conditions basic compounds can be well separated
with basic aluminum oxide plates, while neutral compounds can be successfully separated
with neutral aluminum oxide ones.

3. Cellulose
Cellulose is the next adsorbent used as a stationary phase in chromatography systems,
especially in TLC. Macromolecules consisting of D-glucose units coupled -glycosidically at
positions 1 and 4 by oxygen atoms stand for this adsorbent. A section of a cellulose chain is
shown in Fig. 4.

Fig. 4. Fragment of cellulose macromolecule

 Lab.2. Thin layer chromatography

There are two kinds of cellulose layers used in TLC, native cellulose (400 -500 units per
chain) and micro-crystalline cellulose that is prepared by the partial hydrolysis of regenerated
cellulose and comprises between 40 and 200 units per chain.
Similarly to the silica gel, cellulose surface can be modified by esterification (e.g.
acetylation).

Table.1. TLC stationary phases (adsorbents), mechanism of separation and examples of

compounds separated with TLC
Stationary Chromatographic
Typical Application
Phase Mechanism

steroids, amino acids, alcohols, hydrocarbons,

Silica Gel Adsorption
lipids, aflaxtoxin, bile acids, vitamins, alkaloids

fatty acids, vitamins, steroids, hormones,

Silica Gel RP reversed phase
carotenoids

Cellulose, carbohydrates, sugars, alcohols, amino acids,

partition
kieselguhr carboxylic acids, fatty acids

Aluminum amines, alcohols, steroids, lipids, aflatoxins, bile

adsorption
oxide acids, vitamins, alkaloids

Solvents

As it has been mentioned above, the choice of the mobile phase for chromatographic
separation is dependent on interactions between mixture components in question with
stationary phase. If polar interactions are involved in this process then solvents of dispersive
character to molecular interaction (like hexane) in mixture with polar ones (e.g. ethyl acetate)
are chosen as mobile phase solution. Analogously, if dispersive interactions predominate
between adsorbent surface and solutes then solvents of polar properties (methanol or
acetonitrile) in mixture with water are preferred.
 Lab.2. Thin layer chromatography

The strength of solvent to elute solute molecules from the adsorbent surface (stationary phase)
is characterized by polarity index (P’), which ranges from 0 (for non-polar solvent, e.g.
pentane) to 10.2 (very polar one, water). When the mobile phase is a mixture of two solvents
A and B then its polarity index, P’AB, is calculated according the following formula:

P’AB = φAP’A + φBP’B (1)

Where P’A and P’B are the polarity indexes of pure solvents A and B, respectively, and φ A and
φB are the molar fractions of A or B in the mobile phase, respectively.
The polarity of a solvent can be evaluated by examining its dielectric constant (ε), dipole
moment (δ) and ability to hydrogen bond formation.

Table.2. Properties of solvents applied in liquid chromatography

Solvent Dielectric Dipole Ability to Polarity Elution strength
constant moment hydrogen (P’)
Alumina Silica
[D] bond
formation
hexene 1.88 0.00 not form 0.1 0.01 0.00
toluene 2.38 0.36 not form 2.4 0.29 0.22
chloroform 4.81 1.04 H-donor 4.1 0.40 0.26
dichloromethane 9.1 1.60 H-donor 3.1 0.42 0.30
tetrahydrofuran 7.5 1.75 H-acceptor 4.0 0.45 0.53
ethyl acetate 6.02 1.78 H-acceptor 4.4 0.58 0.48
acetone 21 2.88 H-acceptor 5.1 0.56 0.53
acetonitrile 37.5 3.92 H-acceptor 5.8 0.65 0.52
2-propanol 18 1.66 H-acceptor/ 3.9 0.82 0.60
H-donor
ethanol 24.55 1.69 H-acceptor/ 8.8 0.88 0.69
H-donor
methanol 33 1.70 H-acceptor/ 5.1 0.95 0.70
H-donor
Source Wikipedia
 Lab.2. Thin layer chromatography

Eluotropic series of solvents

Solvents are arranged in a series according to increase of their elution strength in a
chromatographic system with given stationary phase. Each adsorbent (stationary phase)
possess its own eluotropic series of solvents.

Mechanisms of chromatographic separation

Several mechanisms are involved in solute separation in chromatographic system. The most
often applied mechanisms of chromatographic separation are presented in Fig. 5.

Fig. 5. The mechanisms of chromatographic solute separation often applied in laboratory

practice

Adsorption mechanism of chromatographic separation is very often used for solute separation.
Migration of solute in chromatographic system in which adsorption mechanism is involved
depends on:
1. molecular interactions of solute with stationary phase,
2. molecular interactions of solute with solvent (eluent, mobile phase components).
 Lab.2. Thin layer chromatography

If polar solutes are strongly bonded (adsorbed) to polar stationary phase then relatively polar
(strong) solvent as the mobile phase has to be applied to elution of such solutes. If the solvent
is too “weak” then migration of the solutes is small, the solutes show short migration
distances. It can be said their retention is strong. Usually under such circumstances mixture
components are not well resolved.

If the mixture components are nonpolar their molecular interactions (e.g. dipole – induced
dipole or/and London dispersion forces) with polar adsorbent are weak. The solutes are then
weakly attracted by polar stationary phase (show weak affinity with the stationary phase), and
can be easily eluted from the chromatographic system. It can be said their retention is small.
Generally speaking, if stationary phase is more polar than mobile phase then chromatographic
system is named as normal phase system. Analogously, if mobile phase is more polar than
stationary phase then chromatographic system is named as reversed phase system.

Possible interactions of various solute molecules with silica gel stationary phase are
presented in Fig. 6.

increase of solute migration distances

enhance
Inrease oof solute retardation factor, RF

Fig.6. Influence of various functional groups in solute molecule on its migration distance and
retardation factor. The coloured, dashed lines indicate hydrogen bonds between solute
molecule and silica stationary phase
 Lab.2. Thin layer chromatography

A shape of the separated molecule also influences on its bonding with the stationary phase
surface. Flat molecules can be more strongly retained by the adsorbent surface than branched
ones. The solute molecules with multiple polar groups are in position to more strongly interact
with the surface of polar stationary phase than the solute molecule with lower number of
polar groups. However, due to steric hindrance all polar groups of the solute molecule cannot
take part in molecular interactions with the adsorbent surface. In such case prediction of
solute retention is more complicated.
In adsorption chromatography solute elution is based on displacement of its molecules from
stationary phase surface by solvent molecules. It is because solvent molecules show ability
to interacts with the stationary phase. It means in any chromatographic system adsorption of
the solute molecules is not permanent state. Affinity of solute with the mobile phase
components (such as solubility) also influences on its retention. Stronger molecular
interactions of the solutes and mobile phase components lead to decrease of solute retention,
the solutes are then easily eluted from any chromatographic system (TLC and HPLC).

Retention and separation parameters

Retardation factor, RF, is a characteristic parameter of investigated solute/s in a given
chromatographic system. It corresponds to relative migration of solute/s in comparison to
solvent migration. RF values range from 0.0 to 1.0. Definition of RF is presented by the
equation 2 and in Fig. 7.

(2)

A B
Solvent front

b
a1
a2
a3 a4
Start line
1 2 M 3 4 4

Fig. 7. (A) Solutes applied on the start line of the chromatographic plate and (B)
chromatographic plate after chromatogram development; a1, a2, a3 and a4 – the migration
 Lab.2. Thin layer chromatography

distances of the solute zones 1, 2, 3, 4, respectively; b - the mobile phase migration distance
(distance of solvent front migration)

Substance showing RF value of 0.4 spends 2/5 of the experiment (chromatogram

development) time in the mobile phase and 3/5 of the experiment time in the stationary phase.
The solute with RF values of 0.6 spends 3/5 of the chromatogram development time in the
mobile phase and 2/5 of the chromatogram development time in the stationary phase. It means
the first solute migrated shorter distance in comparison with the second one. The difference of
the RF values is equal to 0.2. The solute zones on chromatographic plate migrated different
distances, and then their separations is possible..

The retardation factor can be converted into retention factor, k, with the following equation

(3)
This factor is a measure of retention of solutes in column chromatography systems. It
expresses how many times longer a solute spends in the stationary phase in comparison to that
in the mobile phase.
The separation factor, α, is another chromatographic parameter. It determines separation
selectivity of two solutes in a given chromatographic system. Its value can be equal to or
higher than 1.0. It is calculated with the following equation:

(4)
If is equal to 1.0 then two solutes cannot be separated. Then one should search another
chromatographic system, which enables to obtain higher separation factor than 1.0.

Application of chromatography
The main application of chromatographic processes involves:
1. resolution of mixtures into their components,
2. purification of substances (including technical products) from their contamination,
3. determination of homogeneity of chemical substances,
4. comparison of substances suspected of being identical,
5. quantitative separation of one or more constituents from complex mixture
6. concentration of materials from dilute solutions (plant extracts).
 Lab.2. Thin layer chromatography

EXPERIMENTAL PART
HORIZONTAL DS CHAMBERS (www.chromdes.com)

In standard version (DS-II) the Horizontal DS Chamber for TLC consists of a flat PTFE plate (4) with
five rectangular depressions: two containers/reservoirs (2) of eluent and a central tray with three
troughs (7) and the chromatographic plate (3). The chamber is covered with a large cover plate (1).

Principle of action

Development of chromatogram is started by shifting the plates (1) to the chromatographic plate (3)
which brings a narrow zone of the absorbent layer on the plate (3) into contact with the eluent from
one or two sides. Fig. 8 shows the situation before chromatogram development and Fig. 9 during
chromatogram development. The eluent in containers/reservoirs (2) is covered with the glass plates (1)
so that a vertical meniscus of the eluent is formed. Because the bottom of the containers/reservoirs (2)
is slightly slanted, the meniscus moves in the direction of the chromatographic plate (3) during the
development process, to the complete absorption of the eluent by the adsorbent layer.

1 3 6 5 1

Fig. 8

4 2 8 7 8 2

1 3 6 5 1

Fig. 9

4 2 8 7 8 2

1 – cover plate of eluent reservoirs, 2 – eluent reservoirs, 3 – chromatographic plate, 4 – PTFE plate, 5
– large cover plate, 6 – cover plates of troughs, 7 – troughs for vapour saturation, 8 – eluent (blue area)
 Lab.2. Thin layer chromatography

PROCEDURE
Draw slightly marked start lines (use a soft pencil!) on a 5 x 10 cm chromatographic
plates (glass carrier plates with thin layer of adsorbent, ca. 0.2 mm in thickness, eg. silica gel,
aluminium oxide) about 1 cm from its bottom edge (5 cm in length, see Figs. 7A and 7B).

PART I
ELUOTROPIC SERIES OF SOLVENTS IN SYSTEM WITH SILICA GEL

Brief description:
Step 1
Apply side by side about 5 μL of sample solutions [mixture + several single dye solutions]
onto the star line of the chromatographic plate using spotting capillary tubes. Fill the
capillary by dipping it in the dye solution then gently touch the tip of the capillary tube to the
adsorbent layer and make the spot (the smaller the spot the better results). Clean the capillary
tube with acetone. Repeat the application procedure with the remaining solutes investigated.
Remember that each solute requires clean capillary tube for sample application.
NOTE: The spots applied should be placed on start line, which is 1.0 cm apart from lower
edge of the chromatographic plate (see Figures 5 A, B), and the neighbouring spots on the
start line should be approximately 1 cm apart.
Step 2
Add 2 mL of solvent (hexane, acetone, ethyl acetate or toluene) to the reservoirs of the
chromatographic horizontal DS chambers (one solvent to one chamber). Then insert the
chromatographic plate with spots applied on it into the chromatographic chamber. Start to
develop chromatograms in each chamber.

Step 3
When the solvent front approaches to the end (finish line) of the chromatographic plate, then
remove the wet plate from the chamber. Place the plate in a laboratory hood to complete
evaporation of solvent.
Step 4
Measure distances travelled by the solute zones from the start line (origin) to the middle of
the spot for all compounds. Record the obtained data in Table 1.
 Lab.2. Thin layer chromatography

Step 5
Calculate retardation factor, RF, of investigated solutes and use them to fill Table 1.
Table.1. The values of migration distance (mm) and retardation factor, R F, of solutes in
systems with silica gel and different solvents, is the elution strength

Hexane Toluene Acetone

Solute
= 0.00 = 0.22 = 0.56

The solvent
front
migration
distance, start
– finish (b)

Migration RF Migration RF Migration RF

distance distance distance

(a) (a) (a)

Dye 1

Dye 2

Dye 3

Dye 4

Mixture

Formula to use:
RF = a/b, RF – retardation factor
Answer the questions:

Which solvent is characterized by the highest elution strength?

Arrange the eluotropic series for solvents/eluents used.

 Lab.2. Thin layer chromatography

What components comprise the investigated sample mixture?

Step 6

Apply the data from Table 1 for calculation of the data in Table 2.

Table 2. The values of separation factor, α, of solutes chromatographed in systems with silica
gel and different solvents, is the elution strength

Hexane Toluene Acetone

Solute
= 0.00 = 0.22 = 0.53

Separation factor Separation Separation

factor factor

Dye 1/ Dye 2

Dye 2/ Dye 3

Dye 3/ Dye 4

Formulas to use:

Place for calculations:

Answer the question:

Indicate chromatographic system, which is characterized by the highest values of separation

factor?

Indicate the chromatographic system, which facilitates good separation of all investigated
mixture components.
 Lab.2. Thin layer chromatography

PART II.
ELUOTROPIC SERIES OF SOLVENTS IN TLC SYSTEMS WITH ALUMINUM OXIDE
Use the procedure from PART I, steps 1- 6, for aluminum oxide plates.

Table.3. Migration distance (mm) and retardation factor, RF, values of solutes in systems with
aluminum oxide and different solvents; is the elution strength

Hexane Toluene Acetone

Solute
= 0.00 = 0.29 = 0.56

The distance of
solvent front
migration, start
– finish (b)

Migration RF Migration RF Migration RF

distance distance distance

(a) (a) (a)

Dye 1

Dye 2

Dye 3

Dye 4

Mixture

Answer the questions:

Which solvent has the highest elution strength? Arrange the eluotropic series of solvents for
chromatographic systems with aluminum oxide.
 Lab.2. Thin layer chromatography

Step 7

Apply the data from Table 3 for calculation of the data in Table 4.

Table 4. Separation factor, α, values of solutes in systems with aluminum oxide and
solvents/eluents specified

Formulas to use:

Hexane Toluene Acetone

Solute
= 0.00 = 0.29 = 0.56

Separation factor Separation factor Separation

(α) (α) factor (α)

Dye 1/ Dye 2

Dye 2/ Dye 3

Dye 3/ Dye 4

Answer the question:

For which solvent the separation factor shows the highest values?

PART III
COMPARISON OF ELUTION STRENGTH OF SOLVENTS IN SYSTEMS WITH SILICA
AND ALUMINA

Step 8
Comparison of the results obtained for the systems with silica gel and aluminum oxide. Fill
in Table 5 with appropriate data.
 Lab.2. Thin layer chromatography

Table.5. The values of retardation factor, RF, obtained for the systems with silica gel and
aluminum oxide
Hexane Toluene Acetone

Solute Silica Aluminum Silica Aluminum Silica Aluminum

gel gel gel
oxide oxide oxide

Dye 1

Dye 2

Dye 3

Dye 4

Mixture

Answer the question:

Have you obtained the same results for the systems with silica gel and aluminum oxide ? If
not then try to explain the difference/s?

PART IV
ELUTION STRENGTH OF MIXED SOLVENTS IN SYSTEMS WITH
SILICA GEL
Step 9
Pour 2 mL portion of the eluent solution (5%, 10%, 40% v/v, acetone in hexane) into the
shallow reservoir of single chromatographic chamber (one solution into one chromatographic
chamber).
Step 10
Put a piece of blotting paper on the chamber bottom.
Step 11
Pour the solvent on the blotting paper (approximately 0,5 mL of solvent).
 Lab.2. Thin layer chromatography

Step 12
Insert the chromatographic plate with applied samples (spots) into the chromatographic
chamber. The adsorbent layer should be placed face down in the chromatographic chamber.
Cover the chromatographic chamber with the glass cover plate.
Step 13
Equilibrate chamber atmosphere with solvent vapours for 15 min.
Step 14
Start chromatogram development. When the solvent front reaches the finish line, remove the
wet chromatographic plate from the chamber. Place the plate in a laboratory hood, to dry the
adsorbent layer of the chromatographic plate.
Step 15
Measure the migration distances of solute zones (distance from the start/origin to the middle
of solute zone for all compounds) and record the obtained values in Table 6.
Step 16
Calculate retardation factor, RF, values of the solutes.

Table 6. Migration distance (mm) and retardation factor values of investigated solutes in the
systems with silica gel and acetone + hexane
Eluent 5% acetone in hexane 10 % acetone in hexane 40% acetone in hexane

The migration
distance of solvent
front (start –
finish) (b)

Migration RF Migration RF Migration RF

distance (a) distance distance
(a) (a)

Dye 1

Dye 2

Dye 3

Dye 4

Mixture
 Lab.2. Thin layer chromatography

Answer the questions:

Does composition of the mobile phase influence on migration distance of solute zone/s?

Arrange the solvent mixtures/solutions in respect of their elution strength in silica gel system