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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
s 2
K
s 1
What is Chromatography
A solute equilibrates between a mobile and a stationary phase.
The more it interacts with the stationary phase, the slower it is moved along a
column.
Xm Xs
Ks =
Xm
Xs
Solutes with a large Ks value will be retained more strongly by the stationary
phase.
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary
phase
Mobile phase
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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
Wh = half-height width of the peak in time units
The value of the capacity factor is useful in understanding the retention mechanisms for a
solute, since the fundamental definition of k is:
k =
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.
Equilibrium is achieved or approached at the center of a chromatographic peak.
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KD
A (mobile phase)
A (stationary phase)
k =
k = KD
Volumestationary phase
Volumemobile phase
As KD increases, interaction of the solute with the stationary phase becomes more
favorable and the solutes retention (k) increases
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k = KD
Volumestationary phase
Volumemobile phase
DG = -RT ln KD
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3.) Efficiency:
Efficiency is related experimentally to a solutes peak width.
- an efficient system will produce narrow peaks
- narrow peaks smaller difference in interactions in order to
separate two solutes
Efficiency is related theoretically to the various kinetic processes that
are involved in solute retention and transport in the column
- determine the width or standard deviation (s) of peaks
Wh
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
or for a Gaussian shaped peak
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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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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d.) Stationary phase mass transfer band-broadening due to the movement of solute
between the stagnant phase and the stationary phase.
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e.) Longitudinal diffusion band-broadening due to the diffusion of the solute along the
length of the column in the flowing mobile phase.
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H = A + B/m + Cm
where:
m = linear velocity (flow-rate x Vm/L)
H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer
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.
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
chromatography requiring flow-rates higher than optimal to separate compounds
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k = (tR tM)/tM
where:
Does not consider the effect of column efficiency or peak widths, only retention.
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resolution (RS) resolution between two peaks is a second measure of how well two
peaks are separated:
RS =
tr2 tr1
(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
Types of Chromatography
1.) The primary division of chromatographic techniques is based on the type of mobile phase
used in the system:
Type of Chromatography
Gas chromatography (GC)
Liquid chromatograph (LC)
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
Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography
Affinity chromatography
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3.) Chromatographic techniques may also be classified based on the type of support material
used in the system:
Packed bed (column) chromatography
Open tubular (capillary) chromatography
Open bed (planar) chromatography
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Stationary Phase
- Solid
- Liquid Layer
- Ion exchange resin
- Microporous beads
- Chemically modified resin
Separation Mechanism
Adsorption
Partition
Ion exchange
Size Exclusion
Affinity
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Paper or a
substrate coated
with particles
Packing material
Column Chromatography
Paper Chromatography
Thin Layer Chromatography (TLC)33
PARTITION CHROMATOGRAPHY
Partition chromatography can be subdivided into
(i) liquid-liquid chromatography and
(ii) bonded-phase chromatography.
With liquid-liquid, a liquid stationary phase is retained on
the surface of the packing by physical adsorption.
With bonded-phase, the stationary phase is bonded
chemically to the support surfaces.
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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.
One of these disadvantages is the loss of stationary
phase by dissolution in the mobile phase, which requires
periodic recoating of the support particles.
Furthermore, stationary-phase solubility problems prohibit
the use of liquid-phase packings for gradient elution.
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Reversed-phase chromatography
The stationary phase is nonpolar, often a
hydrocarbon, and the mobile phase is relatively
polar (such as water, methanol, or acetonitrile).
In normal-phase chromatography, the least polar
component is eluted first because it is the most
soluble in the mobile phase; increasing the polarity
of the mobile phase has the effect of decreasing
the elution time.
In contrast, in the reversed-phase method, the most
polar component appears first, and increasing the
mobile phase polarity increases the elution time. 40
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Ion Chromatography
Separation of inorganic cations and anions
and low molecular weight water-soluble
organic acids and bases
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Affinity Chromatography
Separation of proteins and biomolecules
through the use of a biologically specific
immobilized ligand (sp).
Complexation Chromatography
Separation based on the rapid
reversible formation of a complex
between metal ions and ligands
Suitable ligands are immobilized on sp
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Chiral Chromatography
Separation based on the use of chiral
stationary phases which interact to
varing degrees with optical isomers of
the solute
Systems differ little from conventional
HPLC systems
Limited applications for a given sp
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GAS CHROMATOGRAPHY
Mobile phase is a gas!
Stationary phase could be anything
but a gas
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Carrier gas:
Column:
Oven:
Detectors:
He (common), N2, H2
Pinlet 10-50 psig
Flow = 25-150 mL/min packed column
Flow = 1-25 mL/min open tubular column
2-100 m coiled stainless steel/glass/Teflon/fused silica, packed vs. unpacked
0-400 C ~ average boiling point of sample
Accurate to <1 C
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FID, TCD, ECD, NPD, FPD, AED, PID, MSD. (SINGLE OR TANDEM)
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Mobile Phase
GC separates solutes based on their different interactions between mobile and stationary
phases.
solutes retention is determined mostly by its vapor pressure and volatility
solutes retention is controlled by its interaction with the stationary phase
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!).
Common carrier gas: include He, Ar, H2, N2
Carrier Gas or Mobile phase does not affect solute retention, but does affect:
1.) Desired efficiency for the GC System (Van Deemter!)
- low molecular weight gases (He, H2) larger diffusion coefficients
- low molecular weight gases faster, more efficient separations
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Mobile Phases
Stationary phase in GC is the main factor determining the selectivity and retention
of solutes.
There are three types of stationary phases used in GC:
Solid adsorbents
Liquids coated on solid supports
Bonded-phase supports
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Magnified Pores in activated carbon
Gas-Solid Chromatography
Advantages:
- long column lifetimes
- ability to retain and separate some compounds not easily resolved by other
GC methods
geometrical isomers
permanent gases
Disadvantages:
- very strong retention of low volatility or polar solutes
- catalytic changes that can occur on GSC supports
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Gas-Liquid Chromatography
2.) Gas-liquid chromatography (GLC) LIKE DISSOLVES LIKE!
- stationary phase is a liquid coated on a solid support
- over 400 liquid stationary phases available for GLC
- material range from polymers (polysiloxanes, polyesters, polyethylene glycols) to
fluorocarbons, and liquid crystals etc.
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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