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SEPARATION TECHNIQUE
Why this name ?
First used to separate plant pigments
Such as Chlorophyll and Xanthophylls
By passing solutions of their compounds through glass column packed with
CaCO3
Separated species appeared as colored bands in the column
CHROMA Color
GRAPHEIN Writing
GENERAL DESCRIPTION
Chromatography is
A group of different methods which allows
CHROMATOGRPAHIC SEPARATION
INVOLVES
The sample dissolved in a mobile phase
-
Gas
Liquid
Supercritical fluid
Mobile Phase
Gases
(only column)
Liquid Chromatography(LC)
Liquids
Supercritical Fluids
In MODERN CHROMATOGRAPHY
Continuous addition of mobile phase achieved by
using pump in LC and SFC
application of pressure in GC
With introduction of fresh mobile phase ELUENT
Sample moves down the column and gets partitioned between the mobile
and stationary phase
Rate at which a solute zone migrates down the column depends on the
fraction of time it spends in mobile phase (t1)
For solutes Strongly retained by stationary phase Fraction is small
For solutes Not strongly retained by stationary phase Fraction is large
Separation into Bands
Resulting difference in rates cause components in a mixture to separate into
bands/ zones
Chromatogram
A detector that responds to solute concentration placed at the end of the
column whose signal is plotted as a function of time
-
Small peak for species not retained by the column (often mobile phase
contains one or added)
tm time for un retained species to reach detector --------- Dead / Void time
All components spend tm in mobile phase
Large peak corresponds to Analyte time required for this zone to reach
Detector after injection Retention time tR
tR = tS + tM
Average linear rate of solute migration = L / tR
Average linear velocity u of mobile phase, u = L / tM
Retention factor
Important experimental quantity used to compare migration rates of solutes
in column
k independent of column geometry / flow rate
ka =
tRtM
tM
( t R ) Bt M
( t R ) A t M
H=
2
L
R=
PARTITION CHROMATOGRAPHY
Stationary phase is a second liquid
Immiscible with the liquid mobile phase
i.
ii.