Chromatograph yfinal

CHROMATOGRAPHY

 A laboratory technique that separates
components within a mixture by using the
differential affinities of the components
for a mobile medium and for a stationary
adsorbing medium through which they
pass.

CHROMATOGRAPHY

 Introduced first by the Russian botanist
Mikhail Semenovich Tswett.
 Mixtures of solutes dissolved in a common
solvent are separated from one another by
a differential distribution of the solutes
between two phases.

CHROMATOGRAPHY

Two phases in chromatography are:
 mobile phase is part of the chromatographic
system which carries the solutes through the
stationary phase.
 stationary phase is the part which the mobile
phase flows where the distribution of the
solutes between the phases occurs.

PRINCIPLE

 Fractionalism of mixtures of
substances
 In the operation of the
chromatogram, a mobile gaseous or
liquid phase is use to wash the
substances to be separated through a
column of a porous material.

PRINCIPLE

 Capillary Action – the movement of
liquid within the spaces of a material
due to the forces of adhesion,
cohesion, and surface tension.

PRINCIPLE

 The rate of migration of the solute
depends upon the rate of interaction
of the solute with the two phases, one
being the mobile phases and the
other stationary phase as the
compounds travel through the
supporting medium.

MECHANISMS OF SEPARATION
IN CHROMATOGRAPHY
• Based on the interactions of solutes with
mobile and stationary phases.

ADSORPTION (LIQUID-SOLID)
CHROMATOGRAPHY
 Based on the competition
between the sample and the
mobile phase for binding
sites of the solid (stationary)
phase. Molecules that are
soluble in the mobile phase
move fastest.

CHROMATOGRAPHY
Advantages
 An extensive separation
literature is available on thin
layer chromatography
methods that are readily
transferable to adsorption
 The flexibility, speed, and low
cost of TLC allow its use in
experimental development.

CHROMATOGRAPHY
Advantages
 TLC has great value for use in
the preliminary investigation
of samples of unknown
constituents
 Adsorption chromatography,
particularly with silica gel, has
been widely used for the
separation of drugs in both the
HPLC and TLC modes.

PARTITION (LIQUID-LIQUID)
CHROMATOGRAPHY
 separates molecules on
the basis of sample
volatility.
 Depends on the solubility
of the solute in nonpolar
(organic) or polar
(aqueous) solvents

CHROMATOGRAPHY
 Advantage: the
stationary phase does not
leave the solid support
and bleed into the
detector, and a uniform
monomolecular layer of
the stationary phase is
obtained through the
bonding procedure.

CHROMATOGRAPHY
 Since the chemical
influence of the solid
support may be largely
ignored, the adhering
film behaves essentially
like a liquid stationary
phase.

ION-EXCHANGE CHROMATOGRAPHY
∞ Based on the net charge
of molecules
∞ It is one of the common
types of separation
mechanism which
depends on the nature of
the stationary phase.
∞ It has 2 prinicipal types
of ion- exchanger is
cationic and anionic.

∞ Ion exchange matrices
can be further
categorized as either
strong or weak.
∞ It separates amino acid
by electric charges based
on their respective
changes

∞ Usually performed in
columns
∞ Uses a charged
stationary phase to
separate charged
compounds including
anions, cations, amino
acids, peptides and
proteins.

∞ PRINCIPLE:
∞ Relies on charge
to charge
interactions
between proteins
in the sample and
the charges
immobilized on
the resin.

∞ PRINCIPLE:
∞ Once the solutes are bound, the
column is washed to equilibrate it in
the starting buffer,which should be of
low ionic strength
∞ Then the bound molecules are eluted
off using a gradient be of low ionic
strength

∞ TWO PRINCIPAL
TYPES:
∞ Anion exchange
∞ Cation exchange

Factors to be considered in Ion Exchange
Chromatography:

⓭ Buffers – use anionic buffers for cation exchange
and cationic buffers for anion exchange to avoid
difficulty.
⓭ pH- influence the charge on the macromoloecules
in solution.

Factors to be considered in Ion Exchange
Chromatography:
⓭ Salts to use for elution
Ions of the eluting salt must displace other
molecules from the charged groups on the stationary
phase with either a gradient or step in the 0 to 1.0
range.

Most molecules have a net
charge within a pH range of 2 to
10. When the pH is altered, the
net charge on molecules can
change drastically.

In this experiment, a mixture of two
chemicals is absorbed onto a solid
support ion-exchange column and
separated during elution under
conditions that influence their net
charge.

 Instruments
 Eluent Generator
 Water Separation
Column
 Sample Injector
 Electrolytic Eluent
Suppressor
 Detector
 Computer

APPLICATIONS:
 SOFTENING OF WATER
 DEMINERALIZATION OF WATER
 PURIFICATION OF SOLUTIONS FREE FROM
IONIC IMPURITIES
 SEPARATION OF INORGANIC IONS
 SEPARATION OF SUGARS,AMINO ACIDS


ADVANTAGES: DISADVANTAGES:
 Long Life of Resins  Nature and properties of
 Cheap maintenance ion exchange resins
 Environmental friendly  Nature of exchanging ions
because it deals only with  There are substances (such
substances occurring in as organic matter or Fe3+
water. occurring in some water
which can foul the resin.


FACTORS AFFECTING THE
INSTRUMENTALIZATION
1. Column Packing
2. Detectors
3. Flow Rate
4. Sample Size
5. Solvent and Temperature

Applications:

 Serves as liquid chromatography detectors
and as quality control monitors in drug
manufactures
 Also occurs in air and water quality, medical
and clinical laboratories and industrial
laboratories

TYPES OF CHROMATOGRAPHY

A.By Chromatographic bed shape

B. By Physical State of Mobile Phase

By chromatographic bed shape
A. Column Chromatography

• A separation technique in which the
stationary bed is within a tube
• It works on a much larger scale by
packing the same materials into a
vertical glass column.

A. Column Chromatography

B. Plane Chromatography

• A separation that takes place on a flat surface or
a plane
Example:
• Paper Chromatography
• Thin Layer Chromatography

Paper chromatography
 Based on nature of
solvent, solubility of
solute and rate of
diffusion.
 Uses paper as the
stationary phase and a
solvent as the mobile
phase.

 Solvent moves through
the paper by a capillary
action
 Separation depends on
the solubility of solute
and solvents, the polarity
of solvent, and polarity of
solutes in the sample.

 Visualization of the
separated sample
occurs by chemical
reaction, which
produces a color
change.

 Considered to be the simplest
and the most widely used of
the chromatographic
techniques because its
APPLICABILITY TO THE
FOLLOWING:
 ISOLATION
 IDENTIFICATION
 AND QUANTITATIVE
DETERMINATION OF
ORGANIC AND INORGANIC
COMPOUNDS

Instrumentation
of Paper chromatography

1) Lid
2) Paper
3) Solvent
Front
4) Solvent

THIN-LAYER CHROMATOGRAPHY
 Used as a semi-quantitative screening test
screening test
 Uses as thin layer of silica gel, alumina gel,
polyacrylamide gel, or starch gel attached
to glass plate as stationary phase and the
mobile phase is liquid solvent.

 Fractions in the sample are generally quite
soluble in the solvent and move with it up the
stationary phase by capillary action.
 Separated fractions are also developed in TLC by
applying a chemical reaction with the separated
fractions to produce color changes


ADVANTAGE: DISADVANTAGE:
 Simple and economical  Spots are often faint
 Easy to perform since it  TLC is difficult to
only involves spotting
reproduce
the stationary phase
with the sample &  Not typically
placing one edge of the automated
stationary phase plate in
the mobile phase
reservoir.

By Physical State of Mobile Phase

Gas Chromatography
• It can separate nanograms or pictograms of
volatile substances.
• It is principally a method for the separation
and quantitative determination of gases and
volatile liquids and substances.

Gas Chromatography

 Volatile compounds can be separated in a
gas chromatograph, in which the mobile
phase is usually a relatively unreactive
carrier gas such as helium, nitrogen or
hydrogen.
 Separations can be carried out in the vapor
phase, most parts of a gas chromatograph
are temperature controlled; selection of
temperature is based on the composition of
the sample.

Gas Chromatography

 It uses a special detector according to
the different kinds of compound and
the most widely used are:
A. Mass spectrophotometer
B. Thermal Conductivity
C. Flame Ionization Detector
D. Electron Capture Detector

GAS CHROMATOGRAPHY

Applications:
 Most effectively used for analyses of organic
compounds, space related, complex mixtures
of volatile substances at column temperature
of less than -40 °C to greater than 550° C.
 Geochemical research projects such as
determination of various environmental
pollutants at extremely low concentrations.

GAS CHROMATOGRAPHY

 Ability to provide  LIMITED to volatile
qualitative information and samples
quantitative information  Not suitable for
thermally labile samples
 FAST ANALYSIS
 Fairly difficult for large
 Efficient, providing high preparative samples
resolution  Requires spectroscopy
 Sensitive usually mass
 Nondestructive spectroscopy for
confirmation of peak
 Requires small samples identity
 Inexpensive

COMPONENTS
 Autosampler- provides the means to introduce a
sample automatically into the inlets. Automatic
insertion provides better reproducibility and time-
optimization.
 Column inlet (or injector)- provides the means to
introduce a sample into a continuous flow of
carrier gas. The inlet is a piece of hardware
attached to the column head.

COMPONENTS

 Carrier Gas (mobile phase) - must be
chemically inert, include helium, hydrogen
and nitrogen. It should be of high purity, and
the flow must be tightly controlled to ensure
optimum column efficiency and
reproducibility of test results.
 Detector

GAS CHROMATOGRAPHY
GAS-LIQUID GAS-SOLID
CHROMATOGRAPHY CHROMATOGRAPHY
 Separates molecules on  Uses a solid material as an
the basis of sample absorbent
volatility  Based upon a solid
 Mobile phase is a gas such
stationary phase on which
retention of analytes is the
as helium and the consequence of physical
stationary phase is a high adsorption
boiling point liquid
 Relies upon a large
absorbed onto a solid. granular surface to aid in
the separation of the
substances.

GAS CHROMATOGRAPHY

Interferences
 Volatility of compound
 Polarity of compounds
 Column temperature
 Column packing polarity
 Flow rate of the gas
 Length of the column

Application

 Use in biomedical research, routine clinical
determination and drug researching
programs

LIQUID CHROMATOGRAPHY

 The mobile phase is percolated through the
column by means of either gravity , under
pressure generated by a suitable pump or
centrifugal force.

SIZE EXCLUSION CHROMATOGRAPHY

 Particles of different size
will elute (filter) through
a stationary phase at
different rates. This
results in the separation
of a solution of particles
based on size. Provided,
that all the particles are
loaded simultaneously or
near – simultaneously of
the same size should
elute together.


 The support material has certain range of
pore sizes. As solutes travel through, the
small molecules can enter the pores, whereas
the larger ones cannot and will elute first
from column.
 The determination of molecular weight, e.g. ,
of enzymes, and estimation of equilibrium
constants can be achieved with relative ease


ADVANTAGES DISADVANTAGES
 RAPID ROUTINE ANALYSIS  FILTRATIONS MUST BE
PERFORMED BEFORE USING
 IDENTIFYING HIGH MASS THE INSTRUMENT BAD
COMPONENTS EVEN IN RESPONSE FOR VERY
LOW CONCENTRATION SMALL MOLECULAR
WEIGHTS
 CAN ANALYZE  STANDARDS ARE NEEDED
POLYDISPERSED
 SENSITIVE FOR FLOW RATE
SAMPLES,BRANCHING VARIATION. INTERNAL
STUDIES CAN BE DONE, STANDARD SHOULD BE
ABSOLUTE MOLECULAR USED WHENEVER POSSIBLE
WEIGHTS CAN BE  HIGH INVESTMENT COST
OBTAINED.


 Instruments
 Eluent
 Degasser
 Pump
 Auto Injector
 Size Exclusion Column
 Computer


High-performance liquid
chromatography (HPLC)
 Uses a pressure for the pumping of aqueous
or organic solution through a column.
 The mobile phase is forced under pressure
through a long, narrow column, yielding an
excellent separation in a relatively short time.
 Highly sensitive and specific.


High-performance liquid
chromatography (HPLC)
 Become the primary means of monitoring the
use of drugs and of detecting drug abuse.
 Also used to separate the compounds
contributing to the fragrance of the flowers.

High-Performance Liquid Chromatography
An automated process that Difficult to detect coelution,
takes only a few minutes to which may lead to inacurrate
produce results. compound categorization.
Uses gravity instead of high High cost for equipment
speed pump to force
compounds through the needed to conduct HPLC.
densely packed tubing. Operation is complex, requiring
Results are of high resolution a trained technician to operate.
and are easy to read. Equipment has low sensitivity
Can be reproduce easily via to some compounds because
automated process. of the speed of the process.

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 Instruments
 Fraction Collector
 Auto Sampler
 Pumping systems
 Columns & Packing
 Detectors
 Control Data & Processing

Application

 Use in monitoring the use of therapeutic
drugs and detecting drug abuse.
 also use to separate compounds contributing
to the fragrance of flowers

Chromatograph yfinal

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