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.
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:
stationary phase - stationary
mobile phase - moves in a definite direction.
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 quantitatively 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
Chromatograhic processes/ techniques
Adsorption:
stationary phase is solid on
which sample components is
adsorbed. components
distribute between two phases
through a combination of
sorption and desorption
process
http://www.rpi.edu/dept/chem-eng/BiotechEnviron/CHROMO/be_types.htm
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
Chromatograhic processes/ techniques
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)

http://www.rpi.edu/dept/chem-eng/BiotechEnviron/CHROMO/be_types.htm
Elution chromatography on columns
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 columns
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
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
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, N2).
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
HPLC
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.
HPLC
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.
HPLC - Normal-Phase Chromatography
Highly polar stationary phases such as triethylene
glycol or water
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
HPLC - 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