Chromatography Basics

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Chromatography Basics
Last lecture covered Membrane separations
Concentration profiles in membranes Membrane
separation factors
This lecture will cover Chromatography
Sorption Basic configurations Basic
mechanisms
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Chromatography
A gas or liquid mixture carrying several
components is passed as a mobile Phase through a
stationary phase configured as a packed bed, a
plate, etc.
Species which have higher binding to the mobile
phase have their average Velocities reduced. The
affinity is based on interactions such as
Chemi or physi sorption to surfaces  Partitionin
g into a immobile liquid.  Ionic interactions.
Chromatography is often run as a batch process.
Continuous operation Is sometime performed for
frontal analysis.
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Sorption
Adsorption, ion exchange and chromatography are
all sorption processes Solutes are
selectively transferred to insoluable, immobile
particles. Sorbed species are called
sorbates. Sorbing agent is called sorbent. In
adsorption, solute ions or molecules diffuse to
the surface of a solid where they are weakly
bound (adsorbed). Highly porous solids are often
used to increase the specific surface area of
the sorbent. In ion-exchange, ions in a liquid
displace dissimilar ions with the same charge
froman ion exchange solid (resin). The resin
also contains permanently bound co-ions (to
create the affinity for the exchange ions). In
chromatography, the sorbent may be a solid, or an
insoluble, nonvolatile liquid contained in the
pores of the solid. The solutes move through the
separator withan inert eluting fluid.
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Sorption
In sorption separation is achieved based on the
residence time of a component in separator. The
separation consists of a mobile phase of the
mixture of solutes to be separated which moves
past a stationary phase. The solutes bind to
the stationary phase with different affinities.
This causes a difference in the lifetimes of
the bound states and consequently a difference in
average velocities and thus residence times for
the different solutes in the mixture.
Mobile Phase (mixture)
Stationary Phase
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Sorbents

• Sorbents should
• Be highly selective
• Have high capacity
• Have fast kinetics
• Be chemically and thermally stable
• Be hard and mechanically strong
• Be free flowing
• Resist fouling
• Have no tendency to promote unwanted reactions
• Be easy to regenerate
• Be relatively low cost

Adsorption maybe be either physisorption or
chemisorption. Physical adsorption is generally
due to van der Waals interactions while
chemical adsorption is due to the formation of
chemical bonds.
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Chromatography Affinities
The larger the difference in affinities
(attraction of solutes to the stationary phase)
the greater the degree of separation. The more
tightly bound a solute binds to a substrate, the
longer it stays bound and the less time it spends
flowing. This reduces its mean velocity and
increases its residence time.
Mobile Phase (mixture)
R
Stationary Phase
Strongly bound
Weakly bound
E
E
R
R
Eb
The ratio ofbound speciesto unbound speciesat
temperature T.
Eb
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Frontal Analysis
Frontal analysis involves continual addition of
the mixture to the start of thechromatography
column. Of course, faster moving species will
catch up to slower moving species introduced
earlier so this is an analysis method, not a
separations method.
Response
Time
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Equilibrium Analysis
Although the use of a K-value is not strictly
valid, it works in a semi-quantitative sense
even for solid supports. It can be extremely
useful for the initial selection of stationary
and mobile phases. Ideal choice of phases one
that maximizes separation factor
If K-value ratio is large, in other words the
solutes are very dissimilar, then it is easy to
get a separation. The problem is that making the
K-value ratio large implies that the K-value for
one species becomeslarge and binds so tightly
that it has a very long residence time. This
causes a long elution time andlow productivity.
c
Typical concentration versus time or position for
chromatography
x
T2
T1
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Types of Chromatography
There are several types of chromatography which
depend on the chemistry of the separation.
However, the physics and designs are relatively
similar. The stationary phase is often a
packed bed of solid particles. These may be a
solid support with anactive material on the
particles. Glass plates, paper, plastic and metal
sheets are also used. The mobile phase is
either a liquid or a vapor. It is very thin to
ensure short equilibration times betweensolutes
and the mobile and stationary phases. One can
often make the assumption of instantaneous
localequilibrium. Chromatography is most well
suited for batch processes.
Packed Column
Packed Column
Packed Column
Liquid in
Liquid in
Vapor out
Pump
Vapor in
Liquid out
Liquid out
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Gas-Liquid Chromatography
In this case, a liquid film is the stationary
phase and a gas is the mobile phase.
Equilibrium is given by Henrys Law
Henrys Constant
Mole fractionin liquid
Partial pressureof gas
Packed Column
Advantages Gives rise to exquisite
separations Disadvantages 1) Requires high
pressures 2) Small scale primarily analytical
Vapor out
Liquid filmon solid support
Vapor in
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Adsorption Chromatography
In this case, a solid is the stationary phase
and a gas or liquid is the mobile phase.
Affinity is due to weak binding forces, typically
H-bonding or vdW forces. Equilibrium given by
the Langmuir equation
C for max.coverage
Equilibrium Constant
Packed Column (Gas-solid case)
Advantages 1) Supports are relatively
inexpensive 2) Large scales possible Disadvantages
1) Relatively poor selectivity
Vapor out
Adsorbedspecies on solid particle
Vapor in
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Partition Chromatography
In this case, a liquid is the stationary phase
and a different liquid is the mobile phase.
Based on equilibrium partitioning of species
between liquid phases (similar to liquid-liquid
extraction) Equilibrium given by Liquid-Liquid
equilibrium relationship Examples of stationary
liquid phases Diatomaceous earth impregnated
with ethylene glycol (old method, poor
selectivity) Covalently linked monolayers on
solid supports octyl silica, octadecyl silica,
phenyl silica, cyano silica, perfluorenated
silica.
Packed Column (Gas-solid case)
Liquid in
Linked species with absorbed sorbate
Advantages Good selectivity with moderately
more expensive supports than silica.
Liquid out
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Normal Phase and Reverse Phase Chromatography
In the case of Partition and Adsorption
Chromatography two different terms are used
Normal phase chromatography The stationary phase
is more polar (larger dipole moment) than the
mobile phase. ethyl acetate / silica
hexane / cyano silica Reverse phase
chromatography Where the stationary phase is
less polar than the mobile phase. water /C18
silica water / cyano silica
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Ion-Exchange
In ion exchange, which is the technique most
often usedfor water softenerse, ions are removed
from the mobilephase by replacing less strongly
bound ionic species attachedto the stationary
phase.
Packed Column
Liquid in (includes Ca2)
Liquid out (includes Na)
Separation works by displacement.
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2-D Methods
Species have differingaffinities for bindingto
the plate (stationaryphase).
Coated plate or paper
Driving force is the relative competition between
solvation and adsorption or absorption.
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Capillary Chromatography
Relative to Packed Bed Chromatography Capillary
Chromatography Columns are much shorter Have
much longer effective distances Internal
diameters are are much smaller Head pressures
are similar Have smaller capacity  The result
is a capillary peak which is much higher
andnarrower which increases sensitivity.
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Example Capillary Chromatography Spectrum
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Multidimensional GC
Disadvantages More equipment Requires more
control. Takes longer than single
column Advantages Can separate tight peaks
Takes less time than two separate column runs.
One GC setup can do the entire assay.
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Multistage or Multidimensional GC
GC is well optimized to its separation limits.
However, this may still not be good enoughfor
exquisite separations or for separations
involving many species. Using multiple stages
can improve the separation.
Mass spectroscopy can help identify the
components in a peak. However, it cannot tell
the difference between different stereo isomers
or cis-trans conformers, etc.
Column 1
Column 2
Flow switch
Detector
Detector
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Multidimensional GC
Case 1 Single column GC separates
effectively Case 2 Multiple column are used or
multiple assays with different conditions Case 3
Multidimensional GC is used.
Column 1
Column 1
Column 2
Column 2
Flow switch
Flow switch
Detector
Detector
Detector
Detector
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Summary
This lecture covered Chromatography
Sorption Basic configurations Basic
mechanisms.
Next lecture will cover Additional details of
chromatography.