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I.
I. Introduction

II.
II. Classification of chromatographic methods

III.
III. Principle of chromatography

IV.
IV. High performance liquid chromatography (HPLC)

V.
V. Gas chromatography (GC)

VI.
VI. Thin layer chromatography (TLC)
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 Definition:
Chromatography is defined as a procedure
by which solutes are separated by
dynamic differential migration process in a
system consisting of two or more phases,
one of which moves continuously in a given
direction and in which the individual
substances exhibit different mobilities by
reason of differences in adsorption,
partition, solubility, vapor pressure,
molecular size, or ionic charge density.
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Mobile Phase:
The Phase that travels through the
column (gas or liquid) – transport
sample through the column.

Stationary Phase:
Immiscible solid or liquid phase that
fixed in place in the column or on a
solid support – retain analytes within
the column.

Band or Zone:
-Area across which analyte is
distributed on column
-Zones of different analytes gradually
separate as bands progress down
column

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Column Chromatography Thin Layer
chromatography
Sample

Mobile phase (eluant)

Stationary Phase
Detection method
Chromatogram

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 Method to separate components in a mixture based on
different Distribution coefficients between the two phases.
 Chromatography categorized on the basis of interaction
between solute and stationary phase
 Mobile phase either gas or liquid
 Stationary phase either liquid or solid
– Liq/Liq (Partition) Liquid
– Liq/Sol (Adsorption) Chromatography
– Gas/Liq (Partition) Gas
– Gas/Sol (Adsorption) Chromatography

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 According to methodology

Planer Column
chromatography chromatography

Thin Layer Paper

HPLC GC
TLC PC

Electrophoresis

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 Sample
Mobile
Figure:
Schematic diagram showing the separation of
compounds A and B. and the output of the detector
response at various stages of elution

The process of:

Addition of sample
Mobile elution process
Separation mechanism
Retention time ?
Detection by, UV lamp, UV detector,
other detectors.
Eluted bands / collection
 Chromatogram? (function of retention A
time versus detector response) B
Response
 Partition coefficient K’
 k’ = Cs/CM
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time
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 Principles of (TLC) TLC
Chromatography carried out on
active particulate material (silica
gel or alumina) dispersed on an
Inert support (flat glass plates)

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 Basic Steps of TLC Technique

Preparation of the Plate

Sample Application

Chromatogram Development

Locating of the Spots

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 Preparation of the Plate

Slurry of the active material is uniformly

spread over the plate by means of a
commercially available spreader.

Air-drying overnight, or oven-drying at

80-90 C for about 30 minutes.

Ready to use thin layers (pre-coated plates)

are commercially available.

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 Sample Application

1-2 cm 1-2 cm
 Base line
2-2.5 cm

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 Locating of the Spots

For Colored Compounds:

Solvent front

Rf = b/a a

b
Base line 

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 For Colorless Compounds:
Where is the spots ??
We do not know.

Solvent front

a Rf = b/a

 Base line

•Iodine or sulphuric acid is used for most organic mixtures.

•Ninhydrin is used for amino acids.
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•2,4-Dinitrophenylhydrazine is used for aldehydes and ketones
 Applications of TLC Technique

Identification of Unknown Compounds

     

Co-spot
Co-spot
Unknown

Unknown
Authentic

Authentic
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 Analysis of Reaction Mixture

  
Start. mat.

Rxn. mixt.
Product

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 Chromatogram Development

Avoid direct contact between the sample and the solvent system.
The tank or chamber is preferably lined with filter paper.
As the developing solvent travels up the plate, it dissolves the
sample and carries it up; the sample distributing itself between the
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moving solvent and the stationary phase.
 Determination of the Purity of a Product Compound

Product compound

Impurities


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 Quantitative Determination of
an Unknown Concentration

Standard Unknown
conc.

Signal
      

Concentration

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Calibration curve 20
 Instrumentation of HPLC

Sample
injection
Solvent valve
mixing Pump
valve n
m
lu
Co

HPLC
Chart
Detector Recorder

Mobile phase
reservoir Waste

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 HPLC Detector

Characteristics of Typical HPLC Detectors :

Type Response Sensitivity

(ng/mL)
Refractive index Universal 1000

Conductimetric Selective 100

UV/visible absorption Selective 10

Mass-spectrometry Selective 0.1

Fluorescence Selective 0.001

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 injection

HPLC Recorder Solvent valve

mixing
valve Pump

n
m
u
ol
C Chart
Detector Recorder

Mobile phase
reservoir Waste

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 What is the Applications of HPLC ? Peaks correspond to
individual components

Qualitative Analysis
Separation of Mixture Components

Quantitative Analysis Compound

Impurity

Purification of Compounds

Authentic

Unknown

Identification of Compounds
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 Quantitative Analysis Calibration curve

External Standard Method

Peak hight
Concentration

0 10 0 10 0 10 0 10 0 10 0 10 0 10
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100 g/mL 75 g/mL 50 g/mL 25 g/mL 10 g/mL 5 g/mL Unknown
 GG
CC

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 Instrumentation of GC

Flow meter
Injector Vent
Detector GC
Septum Chart
Pressure Flow
Recorder
regulator controller


Oven

Gas
supply Column

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 GC Column

 Packed column
* ~ 3-6mm inner diameter tubing, 1-5
m long
* used for preparative separations or to
separate gases that are poorly
retained
* lower resolution
* small, uniform particle size decreases
Eddy diffusion (requiring higher
pressures)
 open tubular (more common):
* 0.1-0.5 mm inner dia., 10-100 m long
* 0.1-5 m thick sp coated on inner walls
* higher resolution, shorter analysis times,
greater sensitivity compared to packed columns

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 Detectors
 Flame Ionization Detector (FID):

cathode (collects
CHO+ ions)
anode

air
H2

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 Detectors
 Flame Ionization Detector (FID):
– organic solutes are burned in flame producing CH
radicals and eventually CHO+
– CH . + O .  CHO+ + e-
– CHO+ ions are collected by cathode, produces
current as the response

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 Applications of GC ? Peaks correspond to
individual components

Qualitative Analysis
Separation of Mixture Components:
Quantitative Analysis
Authentic

Identification of Compounds: Unknown

Retention time comparsion

Pyrolysis gas chromatography

It is used for the identification of non-volatile materials (plastics,

natural and synthetic polymers, and some microbiological materials.

It is based on the fingerprint chromatogram for the sample, which

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results from its thermal dissociation and fragmentation.

 Quantitative Analysis Calibration curve

External Standard Method

Peak hight
Concentration

0 10 0 10 0 10 0 10 0 10 0 10 0 10
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100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown
 Aspects of GC Applications:

Food Analysis
Analysis of foods is concerned with confirm the presence
and determination the quantities of the analytes (lipids,
proteins, carbohydrates, preservatives, flavours, colorants,
and also vitamins, steroids, and pesticide residues).

Drug Analysis
GC is widely applied to identification of the active
components, possible impurities as well as the metabolites.

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Environmental Analysis
The environmental contaminants; e.g. dichlorodiphenyltrichloro-
ethane (DDT) and the polychlorinated biphenyls (PCBs) are present
in the environment at very low concentrations and are found among
many of other compounds.

GC, with its high sensitivity and high separating power, is mostly
used in the analysis of environmental samples.

Forensic Analysis
In forensic cases, very little sample is available, and the
concentration of the sample components may be very low.

GC is a useful due to its high sensitivity and separation efficiency.

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