CHE 361

Chromatographic Technique
BSMRSTU
Dr. Bijan Mohon Chaki
Associate professor
Department of Chemistry
Begum Rokeya University, Rangpur
 TLC (Thin Layer Chromatography)

KEY CONCEPTS

 Introduction
 General Principle
 TLC Technique
 Applications
 Introduction
Chromatography
 Is the collective term for a set of laboratory techniques for the
separation of mixtures.
 Chromatography is a method by which a mixture is separated by
distributing its components between two phases. The stationary
phase remains fixed in place while the mobile phase carries the
components of the mixture through the medium being used
 The separation of materials is based on differential partitioning
[retardation] between the mobile and stationary phases.
 Introduction
Chromatography
The mobile phase (also known as solvent ) may be either liquid
or gas.
The stationary phase (also known as sorbent ) can be either a
solid or liquid, a liquid stationary phase is held stationary by a
solid (support or matrix)
The molecules in the mixture to be separated are the solutes
 Introduction

Types of Chromatography: Based on the mobile phase:

Gas Chromatography
Liquid Chromatography

Liquid Chromatography are also different types:

Thin layer Chromatography(TLC)

Paper Chromatography
Gel Filtration
Ion Exchange Chromatography
 Some factors that may affect separation

Some factors that may affect separation ( other than the chemical
nature of the substance)
1. Temperature: Since temperature can effect the solubility of
the solute in a given solvent. often a chromatography laboratory
has a fixed temperature for optimum results
2. Composition of the solvent : Since some compounds are
more soluble in one solvent than in the other the mixture of
solvents used affect separation of the compounds.
3. Impurity: Any ionized impurities in the support medium will
tend to bind or attract oppositely charged ions and will therefore
reduce the mobility of these solutes.
 Introduction

Thin Layer Chromatography (TLC) is an important

technique used for identification and separation of mixture
of chemical compounds into its individual components.

TLC is a form of liquid chromatography consisting of two

phases: A mobile phase (liquid) and A stationary phase
(solid).

Differences in the interactions between the solutes and

stationary and mobile phases enable separation.
 Principle
TLC technique involves the distribution of components of
a mixture to be separated between two phases.
The components of the mixture are partitioned between
an adsorbent(stationary phase), and a solvent (mobile
phase).
Different compounds will have different solubility and
adsorption to the two phases between which they are to be
partitioned.
In TLC separation of the individual substances is based on
their relative affinities towards stationary and mobile
phases.
 Principle

The stationary phase: is a thin layer of adsorbent

(usually silica gel or alumina) coated on a plate.
The mobile phase: is a developing liquid which flows
through the stationary phase, carrying the samples with
it.
Components with more affinity towards stationary phase
travels slower.
Components with less affinity towards stationary phase
travels faster.
 Principle
 Two common classes of TLC are:
-Normal Phase: Normal phase is the terminology used when the
stationary phase is polar for example silica gel, and the mobile
phase is an organic solvent or a mixture of organic solvents
which is less polar than the stationary phase.
-Reverse Phase: Reversed phase is the terminology used when
the stationary phase is a silica bonded with an organic substrate
such as a long chain aliphatic acid like C-18 and the mobile phase
is a mixture of water and organic solvent which is more polar
than the stationary phase.
 Mobile and Stationary Phase
 Adsorbents used as Stationary Phase:
-Inorganic: Silica Gel, Kieselguhr, Aluminium Silicate, Bentonite,
Silica gel-F (Fluorescing indicator added), Magnesium Silicate
(Florisil)
-Organic: Starch/Cellulose & its acetylates, Charcoal & activated
Charcoal, Dextran Gel, Polyamides.
 Solvents used as Mobile Phase:
-Petroleum ether, Benzene, CHCl3, CCl4, EA, Hexane etc.
Selection of Adsorbents and Solvents:
 Adsorbent should not adhere to glass plate.
 Solvents should be of high purity.
 Selected based on the nature of the compound to be
separated (polar or non polar.)
 Silica Gel
Silica gel is the most common adsorbent used in TLC
 Silica gel (SiO2) is solid, made synthetically from Sodium Silicate
 Silica gel is a porous, polymeric amorphous form and is composed of SiO2.
Due to its unique internal structure, silica gel is radically different to other
SiO2-based materials. It has a network of interconnected oxygen linkages
with many -hydroxyl groups extending from this matrix, pores and
channels.
 Si-O-Si-O-Si-O-Si-O-Si-O-Si-(OH)x
 They are very porous, have large surface area
that affect the separation characteristics
 The mode of separation is generally by adsorption or partition
 The more polar components will be adsorbed preferentially by the polar
layer, Hydrogen Bonding is the main force controlling adsorption between
the silica gel surface and the analyte functional groups
 Silica Gel
Silica gel is the most common adsorbent used in TLC
 It also comes with a fluorescing indicator added to it to make visualization
or detection of sample spots easier
 Silica gel is a porous, polymeric amorphous form and is composed of SiO2.
Due to its unique internal structure, silica gel is radically different to other
SiO2-based materials. It has a network of interconnected oxygen linkages
with many -hydroxyl groups extending from this matrix (at the surface of the
particle), pores and channels.
 Si-O-Si-O-Si-O-Si-O-Si-O-Si-(OH)x

 The surface of silica gel is highly polar, have large surface area that affect
the separation characteristics
 Silica Gel
Silica gel is the most common adsorbent used in TLC
 The polar functionality can bind analyte in two ways:
(i) Hydrogen Bonding-main force controlling adsorption between the silica gel
surface and the analyte functional groups
(ii) Through dipole-dipole interactions
More polar compounds will have greater interactions with the stationary
phase, so will move slower along it.
 Rf Value
Rf value indicates the position of migrated spots on
chromatogram.
In TLC the results are represented by Rf value which
represents the migration of solute relative to the solvent
front. The TLC plates can be used to calculate what is
called the retardation factor or retention factor the Rf
value. Rf value of an analyte is the ratio of the distance
travelled by the analyte/solute to that travelled by the
eluent front.

The Rf value is calculated as:-

Distance travelled by the solute
Rf Value =
Distance travelled by the solvent front
 TLC -Experiment
Prerequisites for TLC
Decide if you are going to do Normal or Reversed phase
chromatography
Prepare a plate or select a plate with the proper sorbent
material
Prepare the mobile phase
Mark the plate
Apply the sample
Develop the plate
Detect the analytes
 TLC -Experiment
During Performing the TLC Analysis
Types of Materials Needed
 Solvent bottles, 1 liter
 Small bottles, wide mouth 100 mL
 Graduated syringes, 1, 5 and 10 mL
 Pestle
 Graduated cylinders 25, 50 and 100 mL
 Volumetric glassware and pipettes
 Small sample vials 1.5 and 6 mL
 Micropipettes, 1, 2, 3, 4 and 5 microliters
 Pasteur pipettes and rubber bulb, assorted sizes
 Beakers, assorted small sizes
 DiSPO test tubes 3 to 10 mL sizes
 TLC -Experiment
Performing the TLC Analysis Procedures
 Preparation of sample
 Preparation of standards
 Preparation of developing solvent (mobile phase)
 Plate marking
 Spotting a plate
 Placing plate in development chamber
 Conditioning development chamber
 Development of plate
 Visualization and interpretation
 Estimation of concentration
 Calculations of Rf values
 TLC -Experiment
STEP 1:Preparation of Slurry
A plastic, glass or aluminum sheet is coated with a thin layer of
silica gel (adsorbent).
Plates must be dried, activated and stored in desiccator until
used.

STEP 2: Preparation of Tank

Solvent mixtures should be freshly prepared for analysis.
Solvent is poured down side of the tank (1.5cm depth).
Tank is covered with the glass lid and kept for saturation.

STEP 2: Application of sample spot

A very small amount of sample (solution) to be analyzed is
applied in a small spot with a capillary tube, ~1cm from the
bottom of the TLC plate.
 TLC -Technique

The TLC is developed in a

chamber which contains the
mobile phase (solvent).

When the mobile phase rises

up the plate up by capillary
action, the components
dissolve in the solvent and
move.
 TLC -Technique
 Individual components in the sample
move up at different rates.
 More polar analytes interact more
strongly with the stationary phase
move very slowly up.
 More nonpolar analytes interact less
strongly with the polar silica gel and
more strongly with the less polar
mobile phase move higher up.
 Once the solvent reaches the top
(below ~1-2 cm) of the TLC sheet the
plate is removed from the developing
chamber and position of solvent front
is marked.
 TLC -Technique
The solvent is allowed to
evaporate from the TLC sheet.
As the compound is colorless, it
can be visualized by suitable
methods.
o Unsaturation - Iodine vapors
o Amino acids –Ninhydrin reagent.
Also, manganese-activated zinc
silicate (fluorescent compound),
is added to the adsorbent that
allows the visualization of spots
under a black light (UV254 lamp).

Once visible, the Rf value of each spot can be determined.

TLC –Calculation of Rf Value
 Results of TLC –Rf Value
 Qualitative results of TLC
–expressed as fractions of 1.0
–can be expressed from Rf values (Ex: Rf x 100)
–no more than two decimal places

 Rf values can be used to aid in the identification of a substance by

comparison to standards.

 Comparison should be made only between spots on the same sheet,

run at the same time.
 Identical substances will have the same Rf value, whereas non-identical
compounds will differ in their Rf values.
 If Rƒ = 0 (Rƒ value of a solute is zero), the solute remains in the
stationary phase and thus it is immobile.
 If Rƒ value = 1 then the solute has no affinity for the stationary phase
and travels with the solvent front.
 Applications of TLC

TLC is used in qualitative and quantitative analysis to separate

organic compounds and to test the purity of compounds.

This technique is useful for separation of lipids, amino acids

and sugars etc.

It is useful in:

 Identification of components of a mixture.
 Following the course of a reaction,
 Analyzing fractions collected during purification,
 Analyzing the purity of a compound.
 Sample Questions of TLC

1) In Thin Layer Chromatography where silica gel is used as

stationary phase, the mobile phase is chloroform/methanol
(70/30).
a) If the mixture consists of 3 substances as R-COOH, R-OH
and RH, please write down which of these substances can be
A, which can be B, and which can be C and explain your
reasons.
b) As seen A and C substances are not separated from each
other (First TLC figure). What should we do to separate the
spots belonging to A and C substances to have the separation
seen in second figure.
c) If the same substances are analyzed by paper
chromatography, is there difference in the retention time of
analytes (A, B & C) Explain your reason(s).
 Sample Questions of TLC

Solvent front

Baseline or origin

A B C M A B C M

First TLC Second TLC

 Applications of TLC
2) Consider the following silica gel TLC plate of compounds , B, and C developed in hexanes.

Solvent front

Baseline or origin

A B C
a. Determine the Rf values of compounds A, B, and C run on a TLC plate using hexanes as the
solvent, if the spot finds at 0.40, 0.75 and 0.60 cm from the baseline and solvent travels 0.90
cm
b. Which compound A, B, or C is the most polar
c. What would you expect to happen to the Rf values, if you used acetone instead of hexanes as
the eluting solvent?
d. How would the Rf values change if eluted with hexanes using an alumina TLC plate
 Applications of TLC
Advantages of TLC
 Low cost
 Short analysis time
 Ease of sample preparation
 All spots can be visualized
 Sample cleanup is seldom necessary
 Adaptable to most pharmaceuticals
 Uses small quantities of solvents
 Requires minimal training
 Reliable and quick
 Minimal amount of equipment is needed
 Densitometers can be used to increase accuracy of spot
concentration
 Applications of TLC
Two Dimensional TLC and Preparative TLC (PTLC)
 Two dimensional means two
directional development of
analyte spots in a TLC. This
usually case for the mixture of
multiple components.
 First run the TLC as usual
very slowly as the analyte
contains complex mixture and
develop the spot
 After few hours the TLC is
turned into 90° clockwise and
the tank was filled up with a
different solvent system and
run.