Lab.1. Thin - layer chromatography

Lab.1. Thin - layer chromatography

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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

F. Rouessac, A. Rouessac, Chemical Analysis, Modern Instrumentation Methods and Techniques,

6th ed., 2004, Chapters, 1, 3, 5.

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 aluminium (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

http://www.youtube.com/watch?v=e99nsCAsJrw&feature=player_detailpage

http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html


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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

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.

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

conventional chamber (in cuboid vessel)

Mobile

phase

Station

ary

phase

Valve

Mobile

phase

Chromatographic

plate

Chromatographic

chamber

A B


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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.

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.

Solvent front

Start line


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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. Aluminium oxide

Aluminium oxide (Al2O3) is another adsorbent, which is often used as stationary phase in

laboratory practice. TLC aluminium oxide plates usually comprise neutral or basic aluminium

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 aluminium oxide plates, while neutral compounds can be successfully separated

with neutral aluminium 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.


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Fig. 4. Fragment of cellulose macromolecule

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

Phase

Chromatographic

Mechanism Typical Application

Silica Gel Adsorption steroids, amino acids, alcohols, hydrocarbons,

lipids, aflatoxin, bile acids, vitamins, alkaloids

Silica Gel RP reversed phase fatty acids, vitamins, steroids, hormones,

carotenoids

Cellulose,

kieselguhr partition

carbohydrates, sugars, alcohols, amino acids,

carboxylic acids, fatty acids

Aluminium

oxide adsorption

amines, alcohols, steroids, lipids, aflatoxins, bile

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)


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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.

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 φA 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

constant

Dipole

moment

[D]

Ability to

hydrogen bond

formation

Polarity

(P’)

Elution strength

Aluminium Silica

hexane 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/ H-donor 3.9 0.82 0.60

ethanol 24.55 1.69 H-acceptor/ H-donor 8.8 0.88 0.69

methanol 33 1.70 H-acceptor/ H-donor 5.1 0.95 0.70

Source Wikipedia


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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).


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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. 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.

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

increase of solute migration distances

Inrease o enhance of solute retardation factor, RF


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1 2 M 3 4

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 each

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)

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

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

(distance of solvent front migration)

B

Start line

A Solvent front

a4 a3

a2

a1

b


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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 are 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).


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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)


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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, e.g. 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 7 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.


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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, RF, of solutes in

systems with silica gel and different solvents, is the elution strength

Solute

Hexane

= 0.00

Toluene

= 0.22

Acetone

= 0.56

The solvent front

migration distance,

start – finish (b)

Colour of the

dye

Migration

distance

(a)

RF Migration

distance

(a)

RF Migration

distance

(a)

RF

Dye 1

Colour …….

Dye 2

Colour…….

Dye 3

Colour …….

Dye 4

Colour ……

Mixture

Formula to use:

RF = a/b, RF – retardation factor


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Answer the questions:

Which solvent is characterized by the highest elution strength?

Arrange the eluotropic series for solvents/eluents used.

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

Solute

Hexane

= 0.00

Toluene

= 0.22

Acetone

= 0.53

Separation factor Separation

factor

Separation

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?


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Indicate the chromatographic system, which facilitates good separation of all investigated

mixture components.

PART II.

ELUOTROPIC SERIES OF SOLVENTS IN TLC SYSTEMS WITH ALUMINIUM OXIDE

Use the procedure from PART I, steps 1- 6, for aluminium oxide plates.

Table.3. Migration distance (mm) and retardation factor, RF, values of solutes in systems with

aluminium oxide and different solvents; is the elution strength

Solute Hexane

= 0.00

Toluene

= 0.29

Acetone

= 0.56

The distance of

solvent front

migration, start –

finish (b)

Colour of the

dye

Migration

distance

(a)

RF Migration

distance

(a)

RF Migration

distance

(a)

RF

Dye 1

Colour …….

Dye 2

Colour…….

Dye 3

Colour …….

Dye 4

Colour ……

Mixture


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Which solvent has the highest elution strength? Arrange the eluotropic series of solvents for

chromatographic systems with aluminium oxide.

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 aluminium oxide and

solvents/eluents specified

Formulas to use:

Solute

Hexane

= 0.00

Toluene

= 0.29

Acetone

= 0.56

Separation factor

(α)

Separation factor

(α)

Separation

factor (α)

Dye 1/ Dye 2

Dye 2/ Dye 3

Dye 3/ Dye 4


For which solvent the separation factor shows the highest values?


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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 aluminium oxide. Fill

in Table 5 with appropriate data.

Table.5. The values of retardation factor, RF, obtained for the systems with silica gel and

aluminium oxide

Solute

Hexane Toluene Acetone

Silica

gel

Aluminium

oxide

Silica

gel

Aluminium

oxide

Silica

gel

Aluminium

oxide

Dye 1

Colour

…….

Dye 2

Colour…….

Dye 3

Colour

…….

Dye 4

Colour ……

Mixture


Have you obtained the same results for the systems with silica gel and aluminium oxide? If

not, then try to explain the difference/s?


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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).

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.


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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)

Colour of the

dye

Migration

distance (a)

RF Migration

distance

(a)

RF Migration

distance

(a)

RF

Dye 1

Colour …….

Dye 2

Colour…….

Dye 3

Colour …….

Dye 4

Colour ……

Colour of the

dye


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

Lab.1. Thin - layer chromatography

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