This document discusses separation by chromatography. It describes: 1) Chromatography separates components of a mixture based on differences in how they interact with a stationary and mobile phase. 2) There are different types of chromatography including column chromatography which uses a stationary phase in a tube, and planar chromatography which uses a stationary phase on a flat surface. 3) Chromatography can also be categorized based on the mobile phase, including gas chromatography using a gas, liquid chromatography using a liquid, and supercritical chromatography using supercritical fluids.

1. Analytical Chemistry-II
BS-V, CH-311
Department of Chemistry, SNS, NUST
By
Dr. Musammir Khan
Separation by Chromatography

2. Separation by chromatography
o In which the components of a mixture are separated based on
differences in the rates (polarity) through the stationary phase
by a gaseous or liquid mobile phase.
o Stationary phase: Phase that is fixed in place either in a
column or on a planar surface.
o Mobile phase: Phase that moves over or through the stationary
phase carrying with it the analyte mixture.
o Classification:
o Column chromatography: Here the stationary phase is held in
a narrow tube, and the mobile phase is forced through the tube
under pressure or by gravity.
o Planar chromatography: Where stationary phase is supported
on a flat plate or in the pores of a paper and mobile phase
moves by capillary action or under the influence of gravity.

3. 3 Categories: Based on mobile phase
I. Gas chromatography (G-C): In which liquid is
bonded/adsorbed to solid stationary surface, while gas eluent
sample partition between gas and this liquid surface.
II. Liquid chromatography (L-C): Several types: a) Liquid-
liquid: In which sample partition between two immiscible
liquids, b) Liquid-solid: Adsorption occur on solid stationary
phase, c) ion exchange: In which ion-exchange occur ionic
resin, d) size exclusion: In which partition or sieving occur
between interstices of polymer solid/matrix, e) Affinity: In
which functional group specific liquid in solid surface cause
partition between two liquids.
III. Supercritical chromatography (SC):
In which supercritical liquid/fluid (at above Tc/Pc: CO2) is used
as mobile phase and organic species bonded to a solid stationary
surface causes the separation of sample between them.

4. Elution in chromatography
• “Elution”: The process in which the solute molecules are
washed/carried by mobile phase through the stationary phase.
• Consider the elution of two components in a mixture: A+ B, by
introduction of mobile phase is eluted gradually through the
column by gravity or pressure successively: to, t1, t2, t3…t4

6. Chromatogram
• A plot of some function i.e. concentration (signal) vs elution
time, useful for qualitative and quantitative analysis of
analytes.
• Signal is plotted vs elution time.
• Factors effecting separation: Several physical + chemical
• Using long column (“L”)
• Selective solvent/mixture
• Clean separation require less band broadening and good band
separation.

7. Migration rates of solute_ Derivation of equation
• The effective chromatographic separation of two solutes A & B
(mixture) depends on the relative rates at which the two species are
eluted.
• Distribution constant (Kc): The ratio of the molar concentration of
solute in the stationary phase to mobile phase i.e.
• A (mobile phase_A (stationary)
• Kc = (aA)S/(aA)M or CS/CM = Ideally constant over a wide range C
• Retention time (tR): The time required for the solute molecules to
reach the detector (R = retained by st. phase).
• Dead/void time tM: The time taken by an un-retained solute specie
to pass through. It provide an average time taken by the mobile
phase.
• This can be achieved by adding an un-retained specie into the
sample if not already present e.g. LHS peak Figure---next page

8. • LHS peak represent un-retained specie or average time taken by
mobile phase, while peak at RHS represent time taken by sample
through the stationary phase:
tR = tM + tS
• Average linear velocity of solute migration: (L =length of column)
ν = L/tR
• Average linear velocity of mobile phase =
µ = L/tM

9. Volumetric flow (F=cm3/min)
• Volumetric flow rate is related with the linear velocity
µo at the outlet of mobile phase by:
F = µo × A
--- ( A= πr2 = cross-sectional area of column)
F = µo × πr2
F = ε µo × πr2
--- (ε =fraction of area (pores/available to liquid)
• Migration rate of solute/Linear velocity
ν = µ ×fraction of time solute molecules spend in mobile
phase
Mole fraction = nM/nT ----- (M = mobile phase, T =total)

10. Retention factor kA = Kc VS/ VM
ν = µ × number of moles of solute in mobile
phase/total number of moles of solute
Putting ν = L/tR
& µ = L/tM
L/tR = L/tM × CM × VM/CS × VS +CM × VM
After re-arranging and dividing by CM × VM
tM/tR = 1/CS × VS/CM × VM + 1 ------{Kc = CS/CM }
tR = tM × Kc VS/ VM + tM
tR – tM/ tM= kA
tS/ tM= kA -----------1-5 usually

11. Retention factor