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CHAPTER6:CHROMATOGHRAPH

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General Principle
Chromatography encompasses a diverse and important
group of methods that allow the separation, identification
and determination of closely related components of
complex mixtures.
commonly used in the pharmaceutical and biotech
industries, R&D, manufacturing, and quality control.
Also applications in food, water, and environmental
monitoring. For instance, tragedy that occurred in China
in 2008 in which baby formula was found to be
contaminated with melamine
A physical method of separation in which the
components to be separated are distributed between
two phases, one of which is stationary (stationary
phase) while the other moves in a definite direction
(mobile phase).
In all chromatographic separations, the sample is
dissolved in a mobile phase which may be a gas, a
liquid or a supercritical fluid (a substance is heated
above its critical temp which a distinct phase cannot
exist, regardless of pressure.

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Two phases are chosen so that the
components of the sample distribute
themselves between the mobile and
stationary phase to varying degrees. (Based
on establishment of an equilibrium between a
stationary phase and a mobile phase.)
Those components that are strongly retained
by the stationary phase move slowly with the
flow of mobile phase.
In contrast, components that are weakly held
by the stationary phase travel rapidly.
As a consequence of these differences in
mobility, sample components separate into
discrete bands/zones that can be analyzed
qualitatively and/or qualitatively.

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.
Chromatography can be characterized by:
Physical means by which stationary phase and
mobile phase are brought into contact
column chromatography: stationary phase in narrow
tube through which mobile phase is forced under
pressure
planar chromatography: stationary phase is
supported on flat plate. Mobile phase moves through
stationary phase by capillary action or under influence
of gravity
Types of mobile and stationary phases and the
kinds of equilibria involved in the transfer of
solutes between phases.
Liquid chromatography
gas chromatography
supercritical-fluid chromatography

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Chromatograhic processes
Adsorption: stationary phase is solid on which
sample components is adsorbed. components
distribute between two phases through a
combination of sorption and desorption process
Partition: stationary phase is liquid supported
on inert solid. Polar stationary phase and non
polar mobile phase retention of polar
compounds and elution of non polar compounds
Ion exchange: stationary phase is an ion
exchange resin. Separation mechanism based
on ion exchange equilibria .
Size exclusion: molecules separated according
to size by ability to penetrate a seivelike
structure stationary phase. (small molecules
longer elution time)


Elution chromatography on columns.

Elution chromatography on
columnsCont
Elution involves washing a species through a
column by continuous addition of fresh solvent.
A small volume of sample is placed at the top of the
column which is filled with the chromatographic
particles (stationary phase) and solvent.
The sample components will then distribute
themselves between the two phases (The individual
components interact with the stationary phase to
different degree)
Introduction of additional mobile-phase forces the
solvent containing a part of the sample down the
column where further partition between the mobile
phase and fresh portions of the stationary phase
occurs.
Simultaneously, partitioning between fresh solvent
and the stationary phase takes place at the original
sample.
Continued addition of solvent carry solute molecules
down the column in a continuous series of transfer
between the mobile and stationary phases.

Elution chromatography on
columnsCont
Solute movement can only occur in the mobile phase, at which
a solute zone migrates down the column depends upon the
fraction of time it spends in that phase.
This fraction is small for solutes that are strongly retained by
the stationary phase
It is large where retention in the mobile phase is more likely.
The resulting difference in rates causes the components in a
mixture to separate into bands/zones located along the
column.
Isolation of the separated species is then accomplished by
passing a sufficient quantity of mobile phase through the
column to cause the individual zones to pass out to the end
where they can be detected or collected.
Detector that responds to solute concentration is placed at the
end of column (signal is plotted against time) - a series of
peaks obtained called chromatogram.
Retention time of sample is obtained. Must be compared with
standard.
Chromatogram useful for qualitative and quantitative analysis


Thin Layer Chromatography

Thin Layer Chromatography-
Cont..
Planar chromatography
simple, rapid versatile sensitive, inexpensive
analytical technique for the separation of substance.
The mobile phase is a liquid
The stationary phase consists of a thin layer of
absorbent material, which is usually a silica gel,
aluminium oxide or cellulose immobilized onto a flat
inert carrier sheet.
The developing solvent or the mobile phase is the
transport medium for the solutes to be separated as it
is migrated through the stationary phase by capillary
forces
The movement of substances during TLC is the result
of two opposing forces, the driving force of the mobile
phase and the resistive or retarding action of the
sorbent stationary phase
The driving force tends to move the substances from
the origin in the direction of the mobile phase flow

Thin Layer Chromatography-Cont..
The resistive force impedes the movement
of the substances by dragging them out of
the flowing phase back onto the sorbent.
Each molecule alternates between a
sorbed and unsorbed condition, following a
stop- and-go path through the sorbent
At the end of development, each substance
has migrated a certain mean distance
Substance that move slower are more
attracted to the absorbent material,
whereas those that move quickly spend a
smaller fraction of their time in the layer
because of less affinity to it.


GAS
CHROMATOGRAPHY

Separates gaseous substances based on adsorption on
or partitioning in a stationary phase from a gas phase
Mobile phase is a gas (Ar, He, N
2
).
Sample is converted to the vapor state by injection into a
heated port and the eluent is a gas.
Stationary phase is a nonvolatile liquid supported on a
capillary wall or inert solid particles.


LIQUID CHROMATOGRAPHY(HPLC)

HPLC-Schematic
Diagram
HPLCCont
The system is made up of a high pressure solvent
pump, an injector, a column, a detector and a data
recorder
elevated pressure is applied to force a liquid or a
liquid mixture through a packed bed of the stationary
phase, which is the column, to separate the sample
mixture
The pressurized mobile phase passes through the
injector and into the column where it equilibrates with
the stationary phase
Under the optimal conditions the components to be
separated passes through the stationary phase at
different velocities and leave the column at different
times.
The components are then registered by a detector.
This information is passed on to the data evaluation
unit, recorder, and the output is a chromatogram.

HPLCCon
t
The number of peaks is equal to the number of
separated components in the sample and the
area is proportional to the amount of component

The key to changing the separation is to change
the difference in polarity between the column
packing and the mobile phase.

Increasing the difference in polarities between
column and mobile phase makes compounds
stick tighter and come off later. The longer a
mixture stays on the column, the better the
chance for a separation to occur.


HPLCCont
Normal-Phase Chromatography:
Highly polar stationary phases such as
triethylene glycol or water,a relatively
non-polar solvent such as hexane or i-
propyl ether served as the mobile
phase.
The least polar component is eluted
first; increasing the polarity of the
mobile phase then decrease the
elution time

HPLCCon
t
Reverse-Phase Chromatography:
the stationary phase is non-polar such
as hydrocarbons and the mobile
phase is a relatively polar solvent such
as water, methanol, acetonitrile or
tetrahydrofuran
The most polar component elutes first,
and increasing the mobile-phase
polarity, increases the elution time
(refer fig 28-14, pg 829)

HPLCCont
Case Study HPLC- Determination Arsenic in Urine
Sample
Case Study HPLC- Determination Arsenic in Urine
Sample
While arsenic is often considered to be the synonym for toxin,
arsenic toxicity depends strongly on the species being present.
Humans are exposed primarily through ingestion via diet (drinking
water, seafood) or inhalation and workplace exposure. Arsenic is
used in the manufacture of glass, pigments, medicinals,
pesticides, wood preservation and semiconductor products. Once
ingested, arsenic gets metabolised to a certain degree depending
on speciation end level of exposure and is predominantly excreted
in the urine
In order to buffer the changing matrix between different urine
samples but also avoid excessive total salt concentrations that
would compromise long term stability, a mixture of sodium acetate
and sodium nitrate was used as the mobile phase. For enhancing
the sensitivity, 1% of ethanol was added as a modifier.
All five main arsenic species present in human urine (As(III),
As(V), AB, MMNA, DMA) were resolved from each other and from
chloride within a separation time of 12 min. Detection limits were
excellent and ranged from 0.05 to 0.1 g/l depending on species.
Reproducibility of peak area was better than 3% at 10 g/L and
reproducibility of the retention time was better than 0.6%. The
arsenic species determined in the CRM NIES No. 18 agreeed well
with the certified values vor DMA and AB.
Sample: Analysis
Condition