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What is Chromatography
1) Solvent Extraction :
transfer of a solute from phase 1 phase 2
S (in phase1) S (in phase 2)
Partition coefficient
K
s 2
s 1
What is Chromatography
The more it interacts with the stationary phase, the slower it is moved along a
column.
Xm Xs
Xm
Ks =
Xs
Solutes with a large Ks value will be retained more strongly by the stationary
phase.
Interaction Between Solutes, Stationary
Phase, and Mobile Phase
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary
Mobile phase
phase
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The analytes interacting most
strongly with the stationary
phase will take longer to pass
through the system than those
with weaker interactions.
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How do we describe a chromatogram
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Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):
Wh
Wb
Inject
Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units 9
Wh = half-height width of the peak in time units
Note: The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i.e., a difference in their time
or volume of elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for
the separation system)
Peak width & peak position
determine separation of peaks
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2.) Solute Retention:
or
k = (VR VM)/VM
capacity factor is useful for comparing results obtained on different
systems since it is independent on column length and flow-rate. 11
The value of the capacity factor is useful in understanding the retention mechanisms for a
solute, since the fundamental definition of k is:
moles Astationary phase
k =
moles Amobile phase
k is directly related to the strength of the interaction between a solute with the stationary
and mobile phases.
Moles Astationary phase and moles Amobile phase represents the amount of solute present in each
phase at equilibrium.
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A simple example relating k to the interactions of a solute in a column is illustrated for
partition chromatography:
KD
A (mobile phase)
A (stationary phase)
Volumestationary phase
k = KD
Volumemobile phase
As KD increases, interaction of the solute with the stationary phase becomes more
favorable and the solutes retention (k) increases 13
Volumestationary phase
k = KD
Volumemobile phase
since DG = -RT ln KD
peak separation also represents different changes in free energy
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How do we describe a chromatogram
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How do we describe a chromatogram
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3.) Efficiency:
Wb = 4s
Wh = 2.354s
Dependent on the amount of time that a solute spends in the column (k or tR)
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Number of theoretical plates (N): compare efficiencies of a system for solutes that have
different retention times
N = (tR/s)2
N = 16 (tR/Wb)2
N = 5.54 (tR/Wh)2
The larger the value of N is for a column, the better the column will be able to separate
two compounds.
- the better the ability to resolve solutes that have small differences in retention
- N is independent of solute retention
- N is dependent on the length of the column
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Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of
columns with different lengths:
H = L/N
Note: H simply gives the length of the column that corresponds to one theoretical plate
H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,
etc.) to the kinetic processes that give rise to peak broadening:
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a.) Eddy diffusion a process that leads to peak (band) broadening due to the presence
of multiple flow paths through a packed column.
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b.) Mobile phase mass transfer a process of peak broadening caused by the
presence of different flow profile within channels or
between particles of the support in the column.
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c.) Stagnant mobile phase mass transfer band-broadening due to differences in the
rate of diffusion of the solute molecules between the
mobile phase outside the pores of the support
(flowing mobile phase) to the mobile phase within
the pores of the support (stagnant mobile phase).
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Van Deemter equation: relates flow-rate or linear velocity to H:
H = A + B/m + Cm
where:
One use of plate height (H) is to relate these kinetic process to band broadening to a
parameter of the chromatographic system (e.g., flow-rate).
This relationship is used to predict what the resulting effect would be of varying this
parameter on the overall efficiency of the chromatographic system.
H = L/N 25
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of
the mobile phase
m optimum
Optimum linear velocity (mopt) - where H has a minimum value and the point of maximum
column efficiency:
mopt = rB/C
mopt is easy to achieve for gas chromatography, but is usually too small for liquid
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chromatography requiring flow-rates higher than optimal to separate compounds
4.) Measures of Solute Separation:
separation factor (a) parameter used to describe how well two solutes are separated by
a chromatographic system:
Does not consider the effect of column efficiency or peak widths, only retention. 27
resolution (RS) resolution between two peaks is a second measure of how well two
peaks are separated:
tr2 tr1
RS =
(Wb2 + Wb1)/2
where:
tr1, Wb1 = retention time and baseline width for the
first eluting peak
tr2, Wb2 = retention time and baseline width for the
second eluting peak
2.) Further divisions can be made based on the type of stationary phase used in the system:
Gas Chromatography
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support
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Types of Chromatography
Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized support
Partition chromatography liquid-coated or derivatized support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support
Affinity chromatography support with immobilized ligand
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3.) Chromatographic techniques may also be classified based on the type of support material
used in the system:
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Mobile Phase: Liquid
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Column Chromatography and Planar
Chromatography
Separation column
Paper or a
substrate coated
with particles
Packing material
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PARTITION CHROMATOGRAPHY
Early partition chromatography was the liquid-liquid type;
now the bonded-phase method has become predominate
because of certain disadvantages of liquid-liquid systems.
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Columns for Bonded-Phase Chromatography
The supports for the majority of bonded-phase
packings for partition chromatography are
prepared from rigid silica, or silica-based,
compositions.
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The most useful bonded-phase coatings are
siloxanes formed by reaction of the hydrolyzed
surface with an organochlorosilane.
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Reversed-Phase and Normal-Phase Packings
Two types of partition chromatography are
distinguishable based upon the relative polarities of
the mobile and stationary phases.
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Ion Exclusion Chromatography
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Factors Affecting Retention in IC
Degree of ionization of the solute
- solute pKa
- eluent pH
- organic modifier content
hydrophobic interactions between solute
and sp
molecular size of the solute
degree of X-linkage of the sp
system temperature 44
Affinity Chromatography
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Chiral Chromatography
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Gas Chromatography (GC)
GC is currently one of the most popular methods for separating and analyzing
compounds.
This is due to its high resolution, low limits of detection, speed, accuracy and
reproducibility.
GC can be applied to the separation of any compound that is either naturally volatile (i.e.,
readily goes into the gas phase) or can be converted to a volatile derivative.
This makes GC useful in the separation of a number of small organic and inorganic
compounds (They can be big compounds if you can make them small before separation!)
Carrier gas main purpose of the gas in GC is to move the solutes along the column, mobile
phase is often referred to as carrier gas (MUST BE INERT!).
Carrier Gas or Mobile phase does not affect solute retention, but does affect:
Solid adsorbents
Liquids coated on solid supports
Bonded-phase supports
- same material is used as both the stationary phase and support material
- common adsorbents include:
alumina
molecular sieves (crystalline aluminosilicates [zeolites] and clay)
silica
active carbon
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Magnified Pores in activated carbon
Gas-Solid Chromatography
Advantages:
- ability to retain and separate some compounds not easily resolved by other
GC methods
geometrical isomers
permanent gases
Disadvantages:
Based on polarity, of the 400 phases available only 6-12 are needed for most separations.
The routinely recommended phases are listed below:
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