CHROMATOGRAPHY

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CHROMATOGRAPHY
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CONTENTS

1. Introduction
2. Classification of chromatographic methods
3. Principle of chromatography
4. High performance liquid chromatography (HPLC)
5. Gas chromatography (GC)
6. Thin layer chromatography (TLC)

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Introduction
What is chromatography ?
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)
• Liq/Sol (Adsorption)
• Gas/Liq (Partition)
• Gas/Sol (Adsorption)

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Classification of chromatographic methods
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III. PRINCIPLE OF CHROMATOGRAPHY
A. Concept of Chromatography
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 time
  versus detector response)
• Partition coefficient K
• k Cs/CM

A
B
Response
time
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Thin-Layer Chromatography
TLC
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Principles of (TLC)
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Basic Steps of TLC Technique
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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
?
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Locating of the Spots
For Colored Compounds
Rf b/a
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For Colorless Compounds
Rf b/a
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Applications of TLC Technique
Identification of Unknown Compounds
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Analysis of Reaction Mixture
?
?
?
Product
Start. mat.
Rxn. mixt.
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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 moving
  solvent and the stationary phase.

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Determination of the Purity of a Product Compound
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Quantitative Determination of an Unknown
Concentration
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Instrumentation of HPLC
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HPLC Detector
Characteristics of Typical HPLC Detectors
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HPLC Recorder
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What is the Applications of HPLC ?
Peaks correspond to individual components
Qualitative Analysis
Separation of Mixture Components
Quantitative Analysis
Purification of Compounds
Identification of Compounds
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Quantitative Analysis
External Standard Method
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GC
Gas Chromatography
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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 mm 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)

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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
Identification of Compounds
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 results from its thermal
dissociation and fragmentation.
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Quantitative Analysis
External Standard Method
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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- et
hane (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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Good Luck
mahmoud sheha