To use column as stationary phase .
Based on nature of stationary phase it classified into two
•Column adsorption chromatography(solid)
•Column partition chromatography(Liquid)
Adsorption is a principle (if using solid stationary phase)
The liquid mobile phase move from top of the column
•The lesser affinity compounds more faster.
•More affinity compounds more slow.
The rate of movement of component is calculated by
RF= Distance travelled by solute
Distance travelled by solvent
•Preparation of column
•Introduction of sample
•Detection of compounds
•Recovery of components
STATIONARY PHASE: Adsorbent used as a stationary phase.
CHARACTERISTIC OF ADSORBENT:
The particle should have uniform size. The range 60-200 u.
•It should have high mechanical stability.
•Not react with solute
•Insoluble in solvents or mobile phase
•It should be colorless.
•It allow free flow of mobile phase.
•It useful for wide variety of compounds.
TYPES OF ADSORBENT:
It classified according to activity.
•Weak – sucrose, starch ,inulin, talc
•Medium –caco3, ca3(po4)2,mgco3,mgo.
•Strong –Activated mg silicate, Activated magnesia.
SELECTION OF STATIONARY PHASE:
To select the stationary phase according to following characteristic.
•Removal of impurities:
Small quantity of impurity-use weak adsorbent.
•Number of components:
- To separate few no/of components-weak
-To separate large no/of components-strong
•Affinity difference between components:
-Affinity difference is more –use weak
-Affinity difference is less –use strong.
•Length of the column:
Short – strong adsorbent.
Long – weak adsorbent.
PREPARATION OF THE COLUMN:
•The bottom portion of the column is packed with cotton wool or
•Then keep aspestas pad or sheet.
•Then keep whatmann filter paper.
•To pack the column with adsorbent.
• Top of adsorbent layer keep the paper disc.
•Introduce the sample.
•Dry packing technique.
•Wet packing technique.
•DRY PACKING TECHNIQUE:
To pack the adsorbent in dry form.
Then allow to pass mobile phase up to equilibrium.
•Entrapment of air bubbles between particles.
•Column is not packed uniformly.
•Cracks may be present.
•Clear bands not obtained.
WET PACKING TECHNIQUE:
•It is ideal technique.
•First adsorbent mixed with mobile phase then packed in to column.
•No entrapment of air between particles.
INTRODUCTION OF SAMPLE:
The mixture of components mixed with mobile phase then
introduce in to of the column.
To use two types.
•Isocratic elution technique
•Gradient elution technique
To use same solvents and same ratio –for whole process
To change the mobile phase solvent or ratio
Eg : First to use benzene then cHcl3,meoH
DETECTION; For color compounds- separated as different band and
For colorless compounds to use any of the following
•Absorption of light(uv/visible)
•Fluorescence or light emission characteristic
•Flame ionization detector
•Refractive index detector
•Evaporate the solvent and collect residue.
RECOVERY OF COMPONENTS:
To collect the component by cut the column according to zone then dissolve in mobile phase.
Solvent – Eluent, substance – Eluate
FACTORS AFFECTING COLUMN EFFICIENCY:
The following factors are affected the column efficiency.
DIMENSIONS OF THE COLUMN:
• The length of the column- better separation
•Ideal -20:1 or 30:1
PARTICLE SIZE OF THE ADSORBENT:
Adsorbent activity depend upon the surface area decrease the particle size
- the adsorbent activity.
NATURE OF SOLVENT:
Less viscous solvent –Better separation.
TEMPERATURE OF THE COLUMN:
For particular Temperature limit it is directly proportional, later it will be
decrease separation rate.so to maintain temperature.
PRESSURE: Top of the column to maintain high pressure
Bottom of the column to maintain low pressure
Separation of mixture of compounds.
•Removal of impurities
•Isolation of active constituent
•Isolation of metabolite from biological fluids
•Estimation of drugs in formulation
Eg : 1.@2.glycosides.
Phytomenodione in injection or tablets.