Chapter 2

Basic Chromatography

1
 Chromatography
 Chromatography is a physical method of separation
in which the components to be separated are
distributed between two phases (KD/P =
Distribution/partition constant)
 one of which is stationary (stationary phase) while
the other (the mobile phase) moves through it in a
definite direction.
 The chromatographic process occurs due to
differences in the distribution constant of the
individual sample components.

KD of Cpd A = [A]S / [A]M

KD = Distribution constant of compound A
[A]S = concentration of compound A in stationary phase
2
[A]M = concentration of compound A in mobile phase
 Purpose of Chromatography
• Analytical :
 A very thin layer of stationary phase coated on
supporting materials
 For qualitative and quantitative analysis
 Deals with analytes in micrograms quantities
 Chemical profile (composition) of a sample
 To check the purity of the compound
 for screening purpose
eg. TLC; HPLC; GC
• Preparative :
 Relatively thicker layer of stationary phase coated on
supporting materials
 Deals with analytes in milligrams or large quantities
 vital for both isolation and purification purpose
 purify and collect one or more components of a sample
eg. PTLC; Prep. HPLC; Prep GC
3
Classification according to the force of
separation:
1- Adsorption chromatography.
2- Partition chromatography.
3- Gel filtration chromatography.
4- Ion exchange chromatography.
5- Affinity chromatography.

4
 Adsorption Chromatography
 One of the oldest types of
chromatography around
It involves
a stationary solid phase
a liquid or gaseous mobile phase
Not used as widely as partition
chromatography
 used mainly in TLC & CC packed
with silica gel/alumina
Separation is based on
adsorption strength 5
 Adsorption versus Absorption:

 In absorption one substance penetrate in to

the bulk of another substance.

 Adsorption is a surface phenomenon where

interaction takes place only on the surface of
one substance.

6
 Adsorption Chromatography
 Adsorbents:
It is a solid stationary material where it is either
coated or packed on solid supporting materials
The most common are Silica gel & Alumina in which
the interactions with solute molecules is due to OH
groups present on their surface.

7
Normal Silica gel phase

8
9
10
Adsorption strength of Solutes
 The adsorption strength of compounds increases
with increasing polarity of functional groups, as
shown below:

 As stationary phase SiO2 is polar

-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONHR, -CO2H.
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)

Olefins < Ethers < Esters < Lactones < Aldehydes < Amines <Phenols < Acids.

11
 Thin layer chromatography (TLC)
 TLC: a thin layer of solid stationary phase (silica or
alumina) is coated on solid supporting materials (on
glass, plastic or aluminum plates).

 TLC is a method for identifying substances and testing

the purity of compounds.

 TLC is a useful technique because it is relatively quick

and requires small quantities of material.

 TLC is an analytical method for screening of cpds

12
 Thin layer chromatography (TLC)

 Separations in TLC involve distributing a mixture of two or more

substances between a stationary phase and a mobile phase.

 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 travels up the stationary phase,
carrying the samples with it.

 Components of the samples will separate on the stationary

phase according to
how much they adsorb on the stationary phase versus
how much they dissolve in the mobile phase.
Adsorption strength
13
THIN LAYER CHROMATOGRAPHY
Individual components move up at different rates,
depending on intermolecular forces between the
component and the silica gel stationary phase and
the component and the mobile phase.

 The stationary phase is SiO2 and is very “polar”.

 It is capable of strong dipole-dipole and H-bond donating and
accepting interactions with the “analytes” (the components being
analyzed).
 More polar analytes interact more strongly with the stationary phase
in move very slowly up the TLC plate.
 By comparison, the mobile phase is relatively nonpolar and is
capable of interacting with analytes by stronger London forces,
 More nonpolar analytes interact less strongly with the polar silica
gel and more strongly with the less polar mobile phase and move
higher up the TLC plate.
14
TLC

15
 Interpreting the Data
 The Rf (retention factor) value for each spot should be calculated
 Rf = Distance travelled by compound
Distance travelled by the solvent front (mobile phase).
 For two closely migrating components, optimum resolutions are usually
obtained when the Rf’s of both compounds are between 0.2 and 0.5

 It is characteristic for any given compound on the same stationary

phase using the same mobile phase for development of the plates.

 Hence, known Rf values can be compared to those of unknown

substances to aid in their identifications.

16
17
 Rf values often depend on
 the type of stationary phase and
 the solvent used in the TLC experiment.
 Temperature (to small extent)

 the most effective way to identify a compound is to spot

known substances – authentic - next to unknown substances
on the same plate.)

 In addition, the purity of a sample may be estimated from the

chromatogram.

 An impure sample will often develop as two or more spots,

while a pure sample will show only one spot
18
 Identifying the Spots (visualization)
Three techniques involved
1. If the spots can be seen (visible),
outline them with a pencil.

2. If no spots are obvious, the most

common visualization technique is
to hold the plate under a UV lamp.

3. Derivatization by spraying with

appropriate reagents (chemical
treatment)

19
THIN LAYER CHROMATOGRAPHY

Once the solvent is within ~1-2 cm of the

top of the TLC sheet, the TLC is removed
from the developing chamber and the
farthest extent of the solvent (the solvent
front) is marked with a pencil.
The solvent is allowed to evaporate from
the TLC sheet in the hood.
The spots are visualized using a UV lamp.

 A fluorescent compound, usually Manganese-

activated Zinc Silicate, is added to the adsorbent
that allows the visualization of spots under a
blacklight (UV254).
The adsorbent layer will fluoresce light green by
itself, but spots of analyte quench this
fluorescence and appear as a dark spot. 20
 THIN LAYER CHROMATOGRAPHY - Visualization

• As the compounds being separated may

be colorless, several methods exist to
visualize the spots:
• Visualization of spots under a UV254 lamp.
The adsorbent layer will thus fluoresce
light green by itself, but spots of analyte
quench this fluorescence.
• Iodine vapors are a general unspecific
color.
• Specific color reagents exist into which
Chromatogram of 10 essential oils,
Stained with vanillin reagent. the TLC plate is dipped or which are
sprayed onto the plate.

• Once visible, the Rf value of each spot

can be determined 21
 Visualizing Agents
Alkaloids: Dragendorff’s reagent

Cardiac glycosides: Antimony trichloride

Sugar: Aniline phthalate

Amino acids: Ninhydrin

22
 Resolution
 The separation between two
analytes on a chromatogram can be
expressed as the resolution, Rs and
can be determined using the
following equation:

Rs = (distance between center of spots)

(average diameter of
spots)

In TLC, if the Rs value is greater than

1.0, the analytes are considered toRs > 1
be resolved. Rs < 1
23
Solvent Properties and Elution Strengths

24
Elution Strength of Mixed Solvents
The elution strength of the mixture is assumed to be the weighted average of the
elution strengths of the components:

eonet = eoA (mole % A) + eoB (mole % B)

where: mole % A = (moles A) / (moles A + moles B)

Thus, to determine the eonet of a solvent mixture, the molar ratio of the solvents must
first be calculated.
For example, the eonet of a solvent mixture prepared from 1.0 mL of ethyl acetate plus
9.0 mL of hexanes is calculated as shown below:

eonet = eoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +

eohexane [(moles hexane)/(moles EtOAc+moles hexane)]

where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]

thus: eonet = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)

(0.659g/mL)/86.18g/mole)]}
{(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)}

and eonet = 0.067

25
 Resolution defines as the separation b/n two
spots (compounds)

 How to improve resolution (Rs < 1)

 Multiple chromatography
 Two dimensional chromatography

26
 Multiple chromatography
 Multiple development: the chromatogram is
repeatedly developed in the same direction and
thus the complete resolution of two or more
substances which have Rf values close together
can be obtained.

 As the mobile phase one can use either

 the same solvent system or
 different solvent systems.

27
Two-dimensional chromatography:
 This technique is applicable when large numbers of
substances are to be separated on a single chromatogram.
 Development in a direction perpendicular to the first, and
with a solvent system different from that used initially is
often necessary.
 The sample is applied on one corner of a square piece
of chromatogram and after development with the
first solvent, the chromatogram is dried , rotated 90o
and developed in the second direction.
 Usually, different types of solvents systems are used in
each direction. It is essential that the first solvent be
completely volatile.

28
29
30
 TLC Problems: Troubleshooting
Problem Causes Solution
• Over migration Developer too polar Reduce polarity
• Under migration Developer too non-polar Increase polarity
• Distorted solvent front Developer not equilibrated Equilibrate
• Distorted spots Wrong adsorbent Change plates
• Distorted spots Spotted too much Change concentration
• No separation Wrong developer Change developer
• No separation Wrong adsorbent Change plate type
• Tailing Spot overloading Reduce concentration
• Tailing Component is basic Increase basicity
• Tailing Component is acidic Increase acidity
• Tailing/no separation Decomposition Developer/plate

31
 PTLC Chromatography

PTLC: preparative thin layer chromatography

• PTLC:–
 Preparative technique
 Adsorption chromatographic technique
 Preparative scale (1 mm thickness of stationary
phase coated on a glass plate with 20 cm X 20 cm
in size)
 Larger amount (milligrams or larger quantity can
be separated)
 Deals with isolation of compound
32
 Column Chromatography (CC)
 CC is an example of Adsorption Chromatographic
methods in which:
The stationary phase is packed into a column.
The mobile phase is a moving liquid or gas.

Two types of Column chromatography

Stationary phase is held in a narrow tube through which
the mobile phase is forced
 under pressure (Flash column chromatography) or
 under the effect of gravity (Normal column
chromatography)
33
Column Chromatography

34
 Packing & operating the column
1- Packing
 The selection of the method of packing
depends mainly on the density of the
solid stationary phase.
 Two techniques used are
1. Dry packing method &
2. Wet (Slurry) method.
In all cases avoid inclusion of air bubbles

35
 2- Sample Application

1. Apply evenly & in a concentrated solution to

the top of the column which is protected
from disturbance (e.g. add glass wool or
filter paper).
2. Adsorption of the mixture on silica gel is also
appropriate when the sample has problem
of solubility

36
3. Elution techniques

37
 4- Detection
 On-column detection for colored or
fluorescent compounds directly after
developing the chromatogram.

 Monitoring of eluted fractions (TLC or PC).

 Using special detectors connected to the

column such as refractive index, UV
detectors, etc…

38
Factors affecting solutes separation in CC ( Factors affecting column
efficiency)

39
Applications in separation of natural products
 Alumina: sterols, dyestuffs, vitamins, esters,
alkaloids & inorganic compounds.
 Not used for compounds containing phenolic
or carboxylic groups

 Silica gel: sterols, phenolic & amino acids.

 Carbon: peptides, carbohydrates & amino acids.

 Calcium carbonate: carotenoids & xanthophylls.

40