Friday, 7 September 2018

Introduction to chromatography: Notes for Pharmacy

Chromatography is defined as a separation technique of a mixture, which is based on the interactions of the components with two phases i.e. mobile and stationary phase, as the mixture is allowed to run through a supporting medium.

  • Mobile phase: Mobile phase is the solvent which is made to run through the supporting medium.
  • Stationary Phase: This is a coating or layer on the supporting medium which remains stationary and allowed to interact with the components of the mixture.
  • Supporting medium: It is the solid surface on which stationary phase is bonded.

Classification of Chromatography
Most common basis of the classification of the chromatography is based on the type of mobile phase.

Type of chromatography
Type of mobile phase
Gas Chromatography (GC)
Liquid Chromatography (LC)

Further these chromatography techniques can be classified on the basis of the stationary phases used in these techniques:

Gas Chromatography technique
Type of Stationary Phase
Gas Solid Chromatography
Gas Liquid Chromatography
Bonded Phase Chromatography
Chemically derivatized solid support

Liquid Chromatography technique
Type of Stationary Phase
Adsorption Chromatography
Solid underivatized support
Ion Exchange Chromatography
Fixed charges on a solid support
Partition Chromatography
Liquid coated support
Size Exclusion Chromatography
Porous support
Affinity Chromatography
Immobilized ligands with support

The overall classification of the chromatography techniques can be summed up as follows:
Primary Classification
Secondary Classification
Stationary Phase
Types of Equilibrium
Liquid Chromatography
Liquid supported on solid
Liquid-bonded phase
Organic groups bonded to solid surface
Partition between liquid & Bonded phase
Resins with charged species
Ion Exchange
Size Exclusion
Polymers having liquids in the voids
Gas Chromatography
Liquid on solid
Gas-Bonded Phase
Organic species bonded on solid surface
Partition between liquid & Bonded phase
Superficial Fluid Chromatography

Organic species bonded on solid surface
Partition between Super-critical fluid & Bonded phase


The theory of the chromatography can be best understood with the following points:

1     1. Chromatogram: Chromatogram is the graph between the volume of the analyte eluted versus the time taken for the elution.
Basic Chromatogram
             2. Separation of the solute: Separation of the solute in the chromatographic system depends on the two factors:

a.       Solute retention: Solute retention or retention time/retention volume best describes the interaction of the solute with the stationary and the mobile phase.
·         In a particular system, the retention of solute depends upon the length of the column & the flow rate of the mobile phase.

Average migration rate, µ = L (Column Length)/ tR

·         Capacity factor (k’): Capacity factor is more acceptable variable for studying the solute retention as it is independent of the column length & flow rate.

                        K’ = (tR – tM)/tM or (VR-VM)/VM
·        In the simplest definition, K’ can be defined as:

K’: Moles of A in stationary phase/ Moles of A in Mobile phase

K’ ≤ 1, separation is poor
K’ > 30, separation is poor
K’ = 2-10, separation is optimum

b.  Efficiency of the chromatography technique: Efficiency is the term used to define how efficiently an chromatography technique can separate the mixture of compounds.

·        It is dependent on the width of the peak.
·        Narrow the peak more efficient will be the technique.
·        Mathematically, efficiency is measured in the terms of the Number of theoretically plates (N),

N = 16(tR/Wb)2  or 5.54(tr/Wh)2  or L/H (H= height equivalent of theoretically plate)

3.       Broadening of the peaks: Broadening of the peak in a chromatogram is attributed to the following factors:

a.      Eddy’s diffusion: This occurs due to the different path traveled by the solute molecules column.

b.      Mobile phase mass transfer: Mobile phase transfer causes for the broadening of peaks due to the different travel path of the mobile phase when travels through the support material of the column.

The degree of broadening due to the eddy’s diffusion & Mobile phase transfer depends upon:
·        Size of the particles of material.
·        Diffusion rate of the solute.

c.      Stagnant mobile phase mass transfer: Sometimes a mobile phase get stagnated in the support column resulting in the difference in the retention time of the solute and ultimately the broadening of the peaks.

d.      Stationary phase mass transfer: This is attributed due to the different time spend by the solute molecule is stationary phase and the stagnant mobile phase resulting in the broadening of the peak.

e.      Longitudinal diffusion: Broadening of the peak is also attributed due to the diffusion of the solute molecules while travelling through the column.

Van-Deemter equation
It relates the linear velocity/flow rate to the Height equivalent of the theoretical plates (H).
H = A + B/µ +Cµ

Where, A = Constant for eddy’s diffusion & mobile phase transfer
             B = Constant for longitudinal diffusion
             C = Constant for stagnant mobile phase & stationary phase mass transfer

4.      Solute Separation: This Phenomenon is studied with the help of following two factors:
a.      Separation factor (α) : It represents that how well two solutes are separated by any chromatographic technique.

α = k’2/k’1

where, k’1  = Capacity factor of the first solute
            k’2 = Capacity factor of the first solute with k’2 >  k’1
For a good separation, α should be greater than 1.

b.      Resolution (RS): Resolution is given by the following equation:

RS = 2(tr2 –tr1)/(Wb2 + Wb1)

Where, RS ≥ 1.5, ideal separation
             RS ≥ 1, adequate separation

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