CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient (equilibrium
distribution) of sample components between 2
different phases.
One of these phases is a mobile phase and
the other is a stationary phase.
Distribution Coefficient (Equilibrium Distribution )
Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to stationary
phase causes the separation.
Retention Time
• The retention time of a solute is taken as the elapsed time
between the time of injection of a solute and the time of
elution of the peak maximum of that solute.
• The corrected retention time of a solute is the retention
time minus the retention time of a completely unretained
solute.
• By multiplying the corrected retention time of a solute by
the exit flow rate then the corrected retention volume can
be obtained.
• If the mobile phase is compressible (i.e. the mobile phase is
a gas) a pressure correction must be applied which is a
function of the column inlet-outlet pressure ratio.
Chromatogram – Basic Parameter
tR = retention time
tm = dead time
1/2H
unretained
W1/2
H
Peak Shape
• The peak shape in chromatography is the name given to the type of
curve resulting from plotting the concentration of solute in the
mobile phase (or the mass of solute per unit time) eluted from a
chromatographic column, against time.
• Its shape can be described mathematically or empirically.
• If the curve can be described by the error function then it is often
described as a Gaussian or Error Function curve.
• If the adsorption isotherm is not linear the peak will be distorted. A
non linear isotherm can cause different types of peak distortion: if
the non-linearity results from column overload, then the peak will
have a sloping front and a sharp tail. For obvious reasons this is
called an overload peak.
• If the support in a packed column has adsorptive properties, the
peak will exhibit a long tail and, again for obvious reasons, is
called a tailing peak.
Peak Shape
• As the speed of migration of the peak is inversely proportional
to the distribution coefficient, the front to the peak will migrate
a little faster through the column than the rear of the peak and,
thus, the front of the peak will be compressed and the rear of
the peak extended, producing a slightly asymmetrical peak.
• Most chromatographic peaks will exhibit slight asymmetry
resulting from thermal effects. In the front of the peak there is
a net amount of the solute dissolving into the stationary phase
which results in the heat of solution being continuously
released.
• Breadth of a band increases as it moves down the column
because more time is allowed for spreading to occur. Zone
breadth is directly related to residence time in column and
inversely related to velocity at which mobile phase flows.
Column efficiency
• The efficiency of a chromatographic column is a measure of
the capacity of the column to restrain peak dispersion and thus,
provide high resolution.
• The higher the efficiency, the more the peak dispersion is
restrained, and the better the column.
• The column efficiency will vary with the retention of the peak
• The expression for calculating the column efficiency can be
derived from the plate theory.
• The Height Equivalent to the Theoretical Plate (HETP) or the
Variance per unit Length of a Column is calculated as the ratio
of the column length to the column efficiency.
Variables that affect column efficiency
•Linear velocity of mobile phase
•Diffusion coefficient in mobile phase
•Diffusion coefficient in stationary phase
•Capacity factor
•Diameter of packing material
•Thickness of liquid coating on stationary phase
Theories of Chromotography
• Basic theories applicable to chromatography,
1. Plate Theory
2. Rate Theory
• The Plate theory describes,
Mechanism of retention
Calculation of the retention volume of a solute and the
column efficiency.
• The Rate theory describes,
Process of peak dispersion (band spreading)
Calculation of the variance per unit length of a column
(the height of the theoretical plate, HETP)
Mobile phase velocity
Other physical chemical properties of the solute
Distribution system.
Plate Theory
• A number of different peak dispersion processes are proposed
• Describes the contribution of each process to the total variance
of the eluted peak.
• The final equation gives an expression for the variance per unit
length of the column.
•
The processes proposed are
– Eddy diffusion,
– Longitudinal diffusion,
– Resistance to mass transfer in the mobile phase and
– Resistance to mass transfer in the stationary phase.
Rate theory
• The rate theory has been developed differently by a number of
well established scientists in the field.
• This has resulted in a number of different equations; viz.
Van Deemter Equation,
Giddings Equation,
Huber Equation,
Horvath Equation and
Knox Equation.
• All the equations give a type of hyperbolic function
– Predicts a minimum plate height ,
– Optimum velocity,
– Maximum efficiency.
• At normal operating velocities it has been demonstrated that
the Van Deemter equation gives the best fit to experimental
data.
Rate Theory
• Based on a random walk mechanism for the
migration of molecules through a column
• takes into account:
– band broadening
– effect of rate of elution on band shape
– availability of different paths for different
solute molecules to follow
– diffusion of solute along length
Plate height (cm)
Chromatography – van Deemter Plot
H
Cu
Mass transfer
A
Multipath effect
B/u Diffusion (Longitudinal)
Mobile phase velocity
Van Deemter factors:
Conclusions:
 Minimum value for H is achieved
when:
– stationery phase thickness is minimal
– column packed with the smallest particles
– capillary columns have the smallest
internal diameter
– mobile and stationary phases have low
viscosity and high diffusion coefficient
Chromatogram – Basic Parameter
tR = retention time
tm = dead time
1/2H
unretained
W1/2
H
Chromatography - Resolution
Column Resolution
The resolution Rs of a column provides a quantitative measure of its
ability to separate two analytes. A chromatogram for species A and B
on three columns having different resolutions. Column resolution
can be defined as
Rs = (tR)B - (tR)A/ WA/2 + WB/2
= 2[(tR)B – (tR)A]/WA + WB
Effect of capacity and selectivity factor on resolution:
Rs = N½/4 (α-1/α)(K'B / 1 + K'B )
N= 16 R2s (α-1/α)2(1 + K'B / K'B )