Introduction To Chromatography
Introduction To Chromatography
Introduction To Chromatography
Definition
Chromatography is a separation technique based on the different interactions of
compounds with two phases, a mobile phase and a stationary phase, as the compounds
travel through a supporting medium.
Components:
mobile phase: a solvent that flows through the supporting medium
stationary phase: a layer or coating on the supporting medium that interacts with the
analytes
supporting medium: a solid surface on which the stationary phase is bound or coated
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
Types of Chromatography
Liquid Chromatography
Name of LC Method
Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography
Affinity chromatography
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
Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):
chromatogram - concentration versus elution time
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
A similar plot can be made in terms of elution volume instead of elution time. If volumes
are used, the volume of the mobile phase that it takes to elute a peak off of the column is
referred to as the retention volume (VR) and the amount of mobile phase that it takes to
elute a non-retained component is referred to as the void volume (VM).
Capacity factor (k): more universal measure of retention, determined from tR or VR.
k = (tR tM)/tM
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.
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.
A (mobile phase)
KD
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
k = KD
Volumestationary phase
Volumemobile phase
G = -RT ln KD
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 () of peaks
Wb = 4
Wh = 2.354
Dependent on the amount of time that a solute spends in the column (k or tR)
Number of theoretical plates (N): compare efficiencies of a system for solutes that have
different retention times
N = (tR/)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
a.) Eddy diffusion a process that leads to peak (band) broadening due to the presence
of multiple flow paths through a packed column.
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.
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).
d.) Stationary phase mass transfer band-broadening due to the movement of solute
between the stagnant phase and the stationary phase.
e.) Longitudinal diffusion band-broadening due to the diffusion of the solute along the
length of the column in the flowing mobile phase.
H = A + B/ + C
where:
phase
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
optimum
Optimum linear velocity (opt) - where H has a minimum value and the point of maximum
column efficiency:
opt = B/C
opt is easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
k = (tR tM)/tM
where:
k1 = the capacity factor of the first solute
k2 = the capacity factor of the second solute,
with k2 k1
A value of 1.1 is usually indicative of a good separation
Does not consider the effect of column efficiency or peak widths, only retention.
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