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Chromatography is a physical method of separation in which

the components to be separated are distributed between two
phases, one of which is stationary while the other moves
(mobile phase or eluant) in a definite direction.

Chromatography may be preparative or analytical.

Preparative chromatography seeks to separate the
components of a mixture for further use (and is thus a form
of purification). Analytical chromatography normally

operates with smaller amounts of material and seeks to measure the relative proportions of
analytes in a mixture. There are two basic types of chromatography : planar &
column chromatography.
Basic diagram showing chromatography
HIGH PERFORMANCE LIUID CHROMATOGRAPHY
y
THIN-LAYER CHROMATOGRAPHY
y
Chromatographic techniques
y
GAS CHROMATOGRAPHY
y
HYPHENATED CHROMATOGRAPHY (GC-MS)
y
THINLAY ER CHROMATOGRAPHY
(TLC)
Thin layer chromatography(TLC) is a

chromatography technique used to separate mixtures.

Thin layer chromatography is performed on a sheet of
glass, plastic, or aluminum foil, which is coated with
the a thin layer of adsorbent material, usually silica gel,
aluminium oxide, or cellulose. This layer of adsorbent
is known as the stationary phase.
detection of pesticides or insecticides in food
y
examining the radiochemical purity of
radiopharmaceuticals
y
determination of the pigments a plant contains
y
APPLICATIONS OFTLC
Thin layer chromatography finds many applications,
including:
y
analyzing the dye composition of fibers in forensics, or
y
identifying compounds present in a given substance
y
monitoring organic reactions.
TLCPICTURES
The R
f
)
y
The retention factor, or R
f
, is defined as the distance traveled by
the compound divided by the distance traveled by the solvent.
y
For example, if a compound travels 2.1 cm and the solvent front
travels 2.8 cm, the R
f
is 0.75:

CALCULATIONSOF TLC
TLC - Retention Factor (R
f
for a compound is a constant from one experiment to the
next only if the chromatography conditions below are also
constant:
o
solvent system
o
adsorbent
o
thickness of the adsorbent
o
amount of material spotted
o
temperature
=D
y
The retention factor, is defined as
R
f
= distance the solute (D
1
) moves divided by the
distance traveled by the solvent front (D
2
)
y
R
f
CALCULATIONSOF TLC
(CON¶T)
1
/D
2
W
here
y
D
1
= distance that color traveled, measured from center
of the band of color to the point where the food color
was applied
y
D
2
= total distance that solvent traveled
Diagrams showing Retention factor
HIGHPERFOMANCE LIQUID
CHROMATOGRAPHY (HPLC)
High-performance liquid
chromatography (HPLC) is a form of
column chromatography used

frequently in biochemistry and

analytical chemistry. It is also
sometimes referred to as high-pressure
liquid chromatography.
Methodology &Components of
HPLC
ION-EXCHANGE CHROMATOGRAPHY
y
PARTITION CHROMATOGRAPHY
y
ADSORPTION CHROMATOGRAPHY
y
TYPESOF HPLC
y
SIZE-EXCLUSION CHROMATOGRAPHY
y
AFFINITY CHROMATOGRAPHY
y
CHIRAL CHROMATOGRAPHY
GAS CHROMATGRAPHY
(GC)

This is an instrumental method for the

separation and identification of chemical
compounds. In gas chromatography the mobile
phase is an inert carrier gas and the stationary
phase is a solid or a liquid coated on a solid
contained in a coiled column. A sample is
introduced into a heated injector, carried through
a separating column by an inert gas, and detected
as a series of peaks on a recorder when
components leave column.
METHODOLOGY &
COMPONENTS
OF GC
y
Mass spectrometry

This is an analytical technique that identifies the

chemical composition of a compound or sample based
on the mass-to-charge ratio of charged particles. A
sample undergoes chemical fragmentation forming
charged particles (ions). The ratio of charge to mass of
the particles is calculated by passing them through
electric and magnetic fields in a mass spectrometer.
Mass spectrometers can be divided into three
fundamental parts, namely the ionization source,
theanalyzer, and thed e te cto r.
y

The sample has to be introduced into the ionization

source of the instrument. Once inside the ionization
source, the sample molecules are ionized, because ions
are easier to manipulate than neutral molecules. These
ions are extracted into the analyzer region of the mass
spectrometer where they are separated according to
their mass (m) -to-charge (z) ratios (m/z). The
separated ions are detected and this signal sent to a
data system where the m/z ratios are stored together
with their relative abundance for presentation in the
format of a m/z spectrum.
m/z spectrum
Basic arrangement of MS
apparatus
Hyphenated chromatographic
techniques
The most common of these type is the
GC-MS. Gas chromatography-mass
spectrometry (GC-MS) is a method
that combines the features of gas-
liquid chromatography and mass
spectrometry to identify different
substances within a test sample.
y
y
drug detection,
y
fire investigation,
Applications of ms
Applications of GC-MS include
environmental analysis,
y
explosives investigation
y
identification of unknown samples.
y
The GC-MS is composed of two major

building blocks: the gas-chromatograph and

the mass spectrometer. The gas
chromatograph utilizes a capillary column
which depends on the column's dimensions
(length, diameter, film thickness) as well as
the phase properties. The difference in the
chemical properties between different
molecules in a mixture will separate the
molecules as the sample travels the length of
the column.
y

The molecules take different amounts of

time (called the retention time) to come out
of (elute from) the gas chromatograph, and
this allows the mass spectrometer
downstream to capture, ionize, accelerate,
deflect, and detect the ionized molecules
separately. The mass spectrometer does this
by breaking each molecule into ionized
fragments and detecting these fragments
using their mass to charge ratio.
y
y

State: Organic compounds must be in solution for

injection into the gas chromatograph. The solvent
must be volatile and organic (for example, hexane or
dichloromethane).
Samples
Amount: Depending on the ionization method,
analytical sensitivities of 1 to 100 pg per component are
routine.
y
Preparation: Sample preparation can range from
simply dissolving some of the sample in a suitable
solvent to extensive cleanup procedures using various
forms of liquid chromatography.

Mobile phase  this moves over or through the stationary

phase, carrying with it the analyte mixture. The mobile
phase may be a gas, a liquid or a supercritical f luid.
y
Stationary phase - this is fixed in place either in a column
or on a planar surface.
y
IMPORTANT TERMS TO
REMEMBER
y
Absorbent  chromatographic materials which cause
separation through physical and chemical interactions with
the sample components.
y
Bonded  term which implies that the stationary phase is
chemically bonded to the surface of the supporting
material.
y
y
Super-critical chromatography
OtherChromatographic
techniques
Electro-chromatography (planar)
y
Paper chromatography (planar)
Sorbent  the porous, chemically modified silica used for
extraction of chemical species from liquids.

Elution  removal of a chemical species from a sorbent by

changing the solvent or matrix chemistry to disrupt the
isolate/sorbent interaction. Strength of an elution solution refers
to the effectiveness of the solvent for eluting an isolate or
interference from a particular sorbent. For example, non-polar
solvents are strong eluters for non-polar sorbents, whereas polar
solvents are weak elution solvents for non-polar sorbents.
y
y
y
Isolate  the compound(s) of interest to be isolated from the
sample matrix.
y

Interaction  attraction or repulsion between two chemical

species in a specific chemical environment. In sorbent
extraction, the three principal interactions are isolate/sorbent,
matrix/sorbent, and isolate/matrix. Specific possible
interactions include non-polar, polar, ion-exchange, covalent
and a variety of others.
y
Retention time  the time it takes from the moment of injection
until a compound elutes and is detected at the peak maximum.
y

Retention  the attraction of a chemical species for a sorbent

such that the species is immobilized on the sorbent. The degree
of this attraction is referred to as the strength of the retention.
y

Selectivity  the degree to which discrimination of one chemical

species relative to others. Selectivity of a sorbent for a species is
the ability of that sorbent to retain only that species. Selectivity
of an extraction is the degree of isolate purity achieved as a result
of the extraction. Selectivity of a counter-ion is the degree to
which anionic sorbent prefers that counter-ion relative to other
counter-ions. Selectivity is important in sorbent extraction since
the more selective each step in the extraction is, the fewer steps
are required. Also, the more selective each step, the smaller the
mass of sorbent required.
yW
y
Different types of ion exchange
Questions
hat is void volume?
yW
hat is partition co-efficient
Chromatography
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