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CHAPTER6:CHROMATOGHRAPHY

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.

HPLCCont
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

HPLCCont
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