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Chromatography

It is the colour distinguishing technology

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4 views

Chromatography

It is the colour distinguishing technology

Uploaded by

faizanrana80200
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Chromatography and its applications

Reference

Introduction to chromatography theory and practice

Instrumental methods of chemical analysis by H.Kaur


INTRODUCTION

There are many methods which have been used to obtain and identify
the substances in the high state of purity
Those methods worked quite satisfactorily in many cases but great
amount of difficulty was realised with the compounds which had the
composition of the individual components of very similar physical and
chemical properties. Chromatography plays a very important and
significant role in solving all such problems. The complicated separations
are achieved very rapidly and efficiently by chromatography. This method
is fruitful and common by this time chromatography has been used in
almost every type of compounds and fields. Medicine, biology, art and
painting and even intelligence department have used this method to
their greatest advantage.
History
 Chromatography (Greek : Khromatos – Colour and graphos wtiting) a relatively new
separation technique , was discovered by Dr.Michael Tswett (1906) in Warsaw for the
separation of complex mixtures by the process of adsorption .The earliest work in the
field of chromatography dates back to 1855 when Friedrich Runges obtained a coloured
chromatogram by impregnating a filter paper with ferric sulphate, drying and adding a
drop of potassium ferrocyanide solution
CLASSIFICATION OF CHROMATOGRAPHIC METHOD

Paper chromatography: substances are applied as a small


spot on filter paper dipped in an organic solvent. The
mixtures are partitioned between paper(stationary phase)
organic solvent (mobile phase).

Partition column chromatography: The column is packed


with a porous solid of high surface area The components
of a mixture are separated by passing an organic
solvent(mobile phase)through the column.
Ion exchange chromatography: Ionised compounds are
separated in aqueous solutions differences in affinity for
ionised compounds

Thin layer chromatography: The adsorbent (stationary


phase) is spread over a glass plate in a thin film of even
thickness. The solvent(mobile phase) moves up the plate
by capillary action and thus affects separation .
HIGH PERFORMANCE LIQUIQ CHROMATOGRAPHY

ALL forms of liquid chromatography (LC) are differential


migration.LC covers a variety of separation techniques,
such as liquid-solid, liquid-liquid, ion-exchange and
exclusion chromatography. The technique of high
pressure liquid chromatography or high speed liquid
chromatography, later termed high performance
chromatography (HPLC) attained greater significance in
separation science. The technique of HPLC was developed
by Csaba Horvath(1964) Kirkland and Huber in 1969.
The first mixture to be separated Horvath group were
nucleic acid components associated with thyroid
function.
CHARACTERISTIC FEATURES OF HPLC

 High resolving power and speedy separation.


 Accurate quantitative measurement
Repetitive and reproducible analysis using the same
column
 HPLC is able to separate macromolecules and ionic
species
 It can determine multiple components in a single analysis
INSTRUMENTATION FOR HPLC

Solvent Resorvoir : HPLC is usually a mixture of polar and


non-polar liquid components whose respective
concentrations are varied depending on the composition
of the sample.
Pump : A pump aspirates the solvent resorvoir and forces
it through the syste ’s colu a d detecter.
a) Gas Displacement pump These are pumps offer non
pulsating flow but have limited solvent capacity
b) Pneumatic pump which is contained in a collapsible
container and placed in a vessel.These pumps are
inexpensive and pulse free but depend on solvent
viscosity and column back pressure
C) Syringe pump work on the principle of solvent
displacement by a piston mechanically driven at
constant rate. These pumps generate pluse less flow
with high pressure

D) Reciprocating pump provide accurately controlled flow


rates of 1-15 cm3 min -1 against a column back
pressure
Columns : Columns are usually made of polished stainless
steel, are between 50 and 300 mm long and have an
internal diameter of between 2 and 5 mm. They are
commonly filled with a stationary phase with a particle
size of 3–10 µm. Columns with internal diameters of less
than 2 mm are often referred to as microbore columns.

Detector : The HPLC detector, located at the end of the


column detect the analytes as they elute from the
chromatographic column. Commonly used detectors are
UV-spectroscopy, fluorescence, mass-spectrometric and
electrochemical detectors.
 Data Collection Devices : Signals from the detector may be collected on chart recorders
to process, store and reprocess chromatographic data. The computer integrates the
response of the detector to each component and places it into a chromatograph that is
easy to read and interpret.
 Recorder
A recorder, now more precisely termed a potentiometric recorder, is a device that draws
the chromatogram that results from a chromatographic process onto chart paper and
provides a visual representation of the separation that has been achieved. Most modern
chromatographs no longer employ recorders to present the chromatogram and the
results are automatically processed by a computer and presented on the computer
monitor or printed out as required.
Effect of the temperature

There are two other significant effects of separation under


the elevated temperature.

Stabilization of the column and absence of local


temperature fluctuations due to the solvent friction lead
to the more uniform adsorption-desorption process.

Another effect is the increase of the column efficiency. At


the elevated temperature viscosity of liquids decrease
and the diffusion coefficient increaseoptimum efficiency.
Applications of hplc

In inorganic chemistry:


(i)The chromatographic separation of anions can be
effectively carried out by using ion pair chromatography
(ii)For the separation of cations ,sulphonated inert
polymer resins have been used.

 In organic chemistry:
(i) Separationof lipids:Lipids range from hydrocarbons and
wax esters to highly polar sugar of phosphoric acid
conataining glycol and phospholipids.The polar head
group interact with a polar stationary phase
Pharmaceutical Applications
1. To control drug stability.
2. Tablet dissolution study of pharmaceutical dosages
form.
3. Pharmaceutical quality control.
Environmental Applications
1. Detection of phenolic compounds in drinking water.
2. Bio-monitoring of pollutants.
Applications in Forensics
1. Quantification of drugs in biological samples.
2. Identification of steroids in blood, urine etc.
3. Forensic analysis of textile dyes.
4. Determination of cocaine and other drugs of abuse in
blood, urine etc.
Food and Flavour
1. Measurement of Quality of soft drinks and water.
2. Sugar analysis in fruit juices.
3. Analysis of polycyclic compounds in vegetables.
4. Preservative analysis.

Applications in Clinical Tests


1. Urine analysis, antibiotics analysis in blood.
2. Analysis of bilirubin, biliverdin in hepatic disorders.
3. Detection of endogenous Neuropeptides in
extracellular fluid of brain etc.

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