Introduction to chromatography

1. Introduction to
Chromatography

2. Syllabus Contents
 Adsorption and partition column chromatography
 Thin layer chromatography
 Paper chromatography
 Electrophoresis

3. Adsorption and
Partition column
chromatography
METHODOLOGY,
ADVANTAGES,
DISADVANTAGES
AND
APPLICATIONS.

4. Chromatography
 Group of methods used as a way of separating mixtures into individual
components.
 Basic set up requires two phases,
 Interest
Stationary Phase
Mobile Phase
Solute/ Mixture

5. HISTORY
 1903 - Mikhail Tswett, Estonia
 The first report of a chromatographic application for the separation of mixtures in
chemistry
 Setup: glass column - buret
 Solute mixture: pigments of plants
 Stationary Phase: starch
 Mobile Phase: solvent
 Separation: Separate colour bands on the column……. hence chromatography
 Removed the bands separately and extracted the individual pigments off the starch

6. Classification of Chromatography
Chromatography
Adsorption
Ex: Column
Partition
Ex: HPLC
Mechanism of
separation

7. Classification of Chromatography
Chromatography
Solid phase
Solid-liquid
Ex-Column
Solid-gas
Ex-GSC
Liquid phase
Liquid-liquid
Ex- HPLC
Liquid –gas
Ex- GLC
Based on phases

8. Classification of Chromatography
Chromatography
2- D/ Planner
Paper
Thin Layer
3-D/Column
Packed
Open tubular
Shape of
Chromatographic Bed

9. Adsorption Chromatography
Solute
Adsorbs on SP
MP added
Solute remains
adsorbed on SP
Solute retains
on SP
Solute
Adsorbs on SP
MP added
Solute desorbs
to MP
Solute elutes
with MP
 Choice of Solute

10. Partition Chromatography
Solute
Partitions on SP
MP added
Solute remains
in SP
Solute retains
on SP
Solute
Partitions to SP
MP added
Solute
Partitions to MP
Solute elutes
with MP
 Choice of Solute

11. Adsorption & Partition Chromatography
 Choice of Solute
Solute
Adsorbs on SP
MP added
Solute desorbs
to MP
Solute elutes
with MP
Solute
Partitions to SP
MP added
Solute
Partitions to MP
Solute elutes
with MP

12. Terminology
 The analyte is the substance to be separated during
chromatography.
 A chromatogram is the visual output of the chromatograph.
 The eluate is the mobile phase leaving the column.
 The eluent is the solvent that carries the analyte
 The detector refers to the instrument used for qualitative and
quantitative detection of analytes after separation.

13. Adsorption
Column
Chromatography
A typical Adsorption
Chromatographic
column is illustrated

14. Theory
 The distribution of a solute between the mobile and stationary phases in
chromatography is described by κ , the partition coefficient, defined by:
 Κ = Cs / Cm …………..(1)
where
 Cs: Concentration of solute in the stationary phase
 Cm: Concentration of the solute in the mobile phase.
 The mobile phase serves to carry the sample molecules through the
chromatographic column.

15. Theory
 During the sample molecules transportation through the column, each
analyte is retained according to that compound's characteristic affinity for
the stationary phase.
 The time that passes between the sample injection and peak maximum is
called the retention time.
 The area underneath each peak is proportional to the amount of co
responding analyte in solution.

16. Retention Time
 The retention time, tR , is given in seconds by:
 tR = tS + tM …………………….(2)
 where
 tS is the time the analyte spends in the stationary phase and
 tM is the time spent in the mobile phase.
 tM is often referred to as the dead, or void time, as all components spend
tM in the mobile phase.

17. Adsorption chromatograohy
 Adsorption chromatography is one of the oldest types of
chromatography.
 It utilizes a mobile liquid or gaseous phase that is
adsorbed onto the surface of a stationary solid phase
 The equilibriation between the mobile and stationary
phase accounts for the separation of different solutes.
 Adsorption chromatography is process of separation of components
in a mixture introduced into chromatography system based on the
relative difference in adsorption of components to stationary phase
present in chromatography column

18. Partition chromatography
 This form of chromatography is based on a thin film formed on the
surface of a solid support by a liquid stationary phase
 Solute equilibrates between the mobile phase and
the stationary liquid.
 Chromatography in which separation is based mainly on differences
between the solubility of the sample components in the stationary phase
or on differences between the solubility of the components in the mobile
and stationary phases