6. Select an appropriate
chromatographic technique
Gas-Liquid Gas-Solid
(GLC) (GSC)
Gas Chromatogr. (GC) Liquid Chromatogr. (LC)
Reversed Phase
(RPC)
Normal Phase
(NPC)
Ion Exchange
(IEC)
Affinity
(AC)
Size Exclusion
(SEC)
…..
7. How an analyte interact with the phases?
(types of interaction force)
8. Interactions in chromatography
♦ Dispersion force (non-polar compounds, e.g.
hydrocarbons)
♦ Polar force (dipole-dipole/dipole-induced dipole)
♦ Ionic force (ion-ion)
Electrostatic nature!
9. ♦ Dispersion force
2 molecules interacting
and held together
by dispersion force
Interacting plane
Charge fluctuation
Hydrocarbons
aliphatic, aromatic
10. ♦ Polar forces Two molecules interacting and held together
by dispersive forces and polar forces from
permanent / induced dipoles
Permanent dipole e.g.
alcohols, esters, amines, nitrile
Hydrogen bonding
Induced dipole e.g. benzene
+
+
11. ♦ Ionic force
Two molecules interacting
and held together by dispersive
forces and ionic forces between
net ionic charges
+
Sodium dodecyl sulfonate
Cetyl trimethyl ammonium bromide
17. The time an analyte take to pass
a chromatographic column (retention time)
is a function of…
18. Factors governing the retention
Partition Coefficient, K
The difference in partitioning of an analyte between the
mobile and stationary phases is governed by the partition
coefficient K
CS: concentration in stationary phase
CM: concentration in mobile phase
K = CS / CM
20. Plate theory
A chromatographic column
- is envisioned as repetitive liquid-liquid extraction process
or a distillation column
- composed of a series of discrete, contiguous horizontal layers
Question: draw the concentration profiles of A and B in the
mobile phase after they pass through 5 theoretical plates?
A → KA = 1
B → KB = 2
21. Concentration profiles of A, B, C (KA = 1/9, KB = 1, KC = 2)
after passing a) 10 and b) 20 theoretical plates
Which is A, B or C?
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0 2 4 6 8 10
-0.2
-0.1
0
0.1
0.2
0.3
0 5 10 15 20
a) b)
22. The column efficiency increases with
the number of successive equilibrations,
and the number of theoretical plates,
N, has become a way of determining the
column separation efficiency.
23. Solute transferring
from the MP to the SP
at the front of the peak
profile
Solute transferring from
the SP to the MP at the
back of the peak profile
How does a solute migrate along a column?
25. Is K (partition coefficient) always constant
regardless the concentration of analytes?
26. Linear and non-linear chromatography
Isotherm effect on peak shape
(1)
(2)
(3)
Cs
CM
(1): linear Gaussian peak
(2): concave fronting (due to e.g. solute-solute interaction in the case of overloading)
(3): convex tailing (heterogeneous surface of stationary phase with strong active sites)
29. n A (S) , n A (M) : number of moles of A in the SP and MP, respectively
VS , VM : volume of the SP and MP, respectively
β : phase ratio
k’ = nA(S) / nA(M) = K (VS/VM) = K / β
k’ = t’R / t’0
Factors governing the retention
Capacity/retention factor, k’ (k)
The role of phase ratio !
31. van Deemter equation
CCSS, C, CMM: mass transfer coefficients related to the: mass transfer coefficients related to the
properties of the phases and the soluteproperties of the phases and the solute
• A term: eddy diffusion
• B term: longitudinal diffusion
• C term: mass transfer resistance
in stationary and mobile phases
H: plate height
u: linear velocity (cm/s)H = A + B/u + Cu
C = CS + CM
33. Factors contributing to band broadening
• Eddy Diffusion (A)
Band broadening arises in part from a multitude of pathways that
a solute molecule can find through a packed column.
Rate theory
Well-packing particles with narrow size distribution is preferred !
dp: particle diameter
λ: packing properties
narrower size distribution of particles → smaller λ
(0.5 – 1.5)
He = 2λdp
He = 0 in open tubular column (GC)
34.
35. • Longitudinal Diffusion (B/u)
Results from the tendency of molecules to diffuse from regions of
high concentration to regions of low concentration
Rate theory
Longitudinal diffusion occurs both in the mobile and the stationary phase,
but it is significant only in the mobile phase, and only when this is a gas.
t1 < t2 < t3
Ψ: obstruction factor
~ 0.6 for a packed bed and 1 for an open tube
DM: diffusion coefficient of solute in the mobile phase
B /u = 2ΨDM / u
37. • Mass transfer TO and FROM stationary phase (CSu)
Influenced by the rate at which analyte molecules can be transferred
to and from the stationary phase.
Rate theory
THIN stationary liquid films in open tubular column are advantageous !
df : stationary film thickness
q : shape factor, ~2/3 for uniform film
DS: diffusion coefficient of solute in stationary phase
for GC!!!
CSu = u [qk’df
2] / [(1 + k’)2.DS]
38. “CSu” term in LC is more complex, dependent on the surface (pore) morphology
and stationary phase film thickness.
Remember that the surfaces are usually porous!
Solutes get into stagnant mobile phase “pool” to interact
with functional groups by diffusion
Stagnant
mobile phase
Stationary phase
film
• Mass transfer To and From Stationary Phase (CSu)
Thick film of stationary phase, too small, deep and
tortuous pores on the surface increase CS
39. Rate theory
The contribution of CM u to plate height is not linear,
but bears a complex dependency on mobile phase velocity.
Mass transfer in mobile phase
CM u = u × f (dp
2, u) / DM
40. Comparison of different efficiencies of carrier gases in OT column GC
Which carrier gas do you prefer regarding efficiency and analysis time?
41. Band broadening in open tube and
porous media
Mobile phase flow profile for an open tube and a packed column
with pressure-driven and electroosmotic flow
Flow profile in
inter-particle space
particle particle
42. Number of theoretical plates –
an indicator of column quality
• HETP (Height Equivalent to a Theoretical Plate), H
The column length corresponding to one theoretical plate
• Number of theoretical plates, N
N = (tR / σ)2
N = 5.54 (tR / W1/2)2
N = 16 (tR / Wb)2
• Assuming a Gaussian peak shape
1.000
0.882
0.607
0.500
0.324
0.134
0.044
σ
2σ
W1/2=2.354 σ
W1/2
3σ
4σ
5σ
Wb=4σ
Gaussian peak
L: column length
44. Classical chromatographic theory considers that a separation process take place
by a succession of equilibrium steps, the more equilibrium steps in the column
the greater column efficiency with less band broadening (σ), therefore
In practice the proportionality constant is 1, therefore
Where , the standard deviation of the Gaussian peak, describes the spread of
the molecules in the band. Band broadening is also a function of time, the longer
the band takes to elute the more time the molecules have to spread out, therefore
45. Selectivity factor, α
α = K2 / K1
= k’2 / k’1
= t’2 / t’1
Shows how much difference in (relative) interaction strength of
two solute 1 and 2 with the stationary phase
48. Resolution -
Relationship to column properties
Purnell’s equation for resolution factor of two closely spaced peaks
How to vary α , k’, N (in practice) to improve resolution?
RS = [N1/2 /4] [(α -1) / α] [k’2 / (1+k’2)]
How to optimize a separation regarding resolution and analysis time?
k’2
k’
2/(1+k’
2)
α
49. Effect of k’, α, N
on resolution
k’ = 3.0; α = 1.10; N = 3500; Rs = 1.00
k’ = 3.0; α = 1.20; N = 3500; Rs = 1.83
k’ = 3.0; α = 1.10; N = 7000; Rs = 1.42
k’ = 6.0; α = 1.10; N = 3500; Rs = 1.16
(B)
(D)
(C)
(A)
51. to
tR, VR
t’R, V’R
Retention time/volume
Vo
to: dead time (time an un-retained solute spends in the column)
tR: retention time (total time a retained solute spends in the column)
t’R: corrected retention time (time a solute spends in the stationary phase)
F: flow rate (mL/min)
t: time (min)V = F.t
