High performance liquid chromatography (HPLC) is a very
efficient separation technique, that is, it yields excellent separation in a
very little period of time.
The
inventors of modern chromatography, Martin and Synge (at 1941), in theory, the
stationary phase requires very small particles and hence a high pressure
is essential for forcing the mobile phase through the column. As end result,
HPLC is also sometimes referred to as high-pressure liquid chromatography.
High
performance liquid chromatography (HPLC) is a type of column Chromatography and
it is used commonly in biochemistry and analytical chemistry’ to
separate components of a mixture by using a variety of chemical interactions
between the substance being analyzed (sample) and the constituent elements of
column of HPLC.
Fig: High performance liquid chromatography (HPLC)
Principle of HPLC
The principle of HPLC is to force the sample through the
column of the stationary phase by pumping the mobile phase at high pressure.
The sample to be analyzed is introduced in small volume to the stream of mobile
phase and the sample molecules are retained by specific chemical and physical
interactions with the materials of the stationary phase as it travels the
length of the column. The amount of retention depends on- (1) the nature of the
sample, (2) the nature of the composition of the stationary phase and mobile
phase. The time taken by the sample to travel from the point of injection to
the end of the column is called the retention time and is considered an unique
identifying characteristic of a given sample because no two compounds will
have the same retention time (like no two persons can sit in a single seat / No
single person can have two heads). The use of pressure increases the linear
speed giving the components less time to diffuse within the column, leading to
improved resolution in the resulting chromatogram. Common solvents used include
any miscible combinations of water and various organic liquids (the most common
are methanol and acetonitrile).
In HPLC separation, the compound that tend to reside in the
mobile phase, move more quickly than those that prefer to reside in stationary
phase. Phase preference can be expressed by distribution co-efficient, K:
Cstat
Kx=-------------
Cmob
Where,
Cstat= Concentration of compound X in the
stationary phase
Cmob= Concentration of X in mobile phase
K= Partition coefficient
Band
Broadening
First Cause: Eddy Diffusion- The column is packed with small
stationary phase particles. The mobile phase passes through and transports the
sample molecules with it . Some molecules are fortunate and leave the
column before most others by traveling an almost directly line path. Other
sample molecules undergo several diversions along the way.
Second Cause: Flow distribution-The
mobile phase passes in a laminar flow between the stationary phase particles
(Fig 6). The flow is faster in the channel center than it is near the particle.
Eddy diffusion and floe distribution may be reduced by packing the column with
evenly sized particles (First remedy).
Third Cause: Sample molecule
diffusion- in the mobile phase- It is also called longitudinal diffusion effect.
Sample molecules spread out in the solvent without any external influence (Just
like salt or sugar dissolves slowly in water). The longitudinal diffusion has a
harmful effect if
(I) Small stationary phase particles (<10
micron)
(ii) Low mobile phase velocity
and
(iii) Large sample diffusion
co-efficient etc coincide in the HPLC
system. This can be overcome by following Second remedy.
Fourth cause: Mass transfer
between mobile, stagnant mobile and stationary phase- Pore structure of a
stationary phase particle explain us that the channels are both narrow and
wide, some channels pass through the whole of the particle while others are stopped off (half way open
and then closed). Half way open pores are filled with mobile phase and are stagnated.
The recovery process may occur in two ways-(a) the molecules may diffuse back
to the mobile flux phase .It depends on the size of pores and viscosity of the
solvents used. (b) The molecules may interact with stationary phase and is
adsorbed and then after sometime desorbed. Hence mass transfer takes place. In
both cases band broadening decrease with increaseing mobile phase flow
velocity. These effects can be overcome by Third and Fourth principles.
Remedy:
First remedy: The packing should be composed of particles
with as narrow a size distribution as possible (5:7.5=1:1.5)
Second remedy: The mobile phase flow velocity should be
selected so that longitudinal diffusion has no adverse effect
Third remedy: Small particles or those with a thin, porous
surface layer should be used as the stationary phase
Fourth remedy: Low viscosity solvents should be used
Mathematical expression of principle of HPLC
Mathematically the principle of HPLC is based on van Deemter
theory (The rate theory) expressed as van Deemter equation. This theory employs
a kinetic approach and explains band broadening in terms of a number of rate
factors.
The van Deemter equation is:
2γDG 8 k’ df2ū
HEPT= 2λdp + ------------ + ---
---------- -------
ū π2 (1+k’)2 DL
Eddy Molecular Non-equilibrium effect
diffusion diffusion
Axial
diffusion
Where,
HEPT=Height Equivalent to Theoretical Plates
λ = a
measure of the packing irregularities
dp= Particle diameter, γ=Tortuosity (Full of twists and turns)
factor
DG=Coefficient of gaseous diffusion
DL=Co-efficient of liquid diffusion
ū=Average linear velocity (Flow rate)
K’=ratio of the capacity of the liquid phase to that of the
gas phase. For capillaries, k’=2kdf/r
df-=Average film thickness, r=radius of the
capillary
A = Eddy-diffusion, B =Longitudinal diffusion, C =
Resistance to mass transfer
Fig: High performance liquid chromatography (HPLC)
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