Types of Liquid Chromatography

I. Ion Exchange Chromatography

A. Factors influencing retention
B. Suppressed ion exchange
II. Partitioning Chromatography
A. Normal phase/ reverse phase
III. Size Exclusion Chromatography
IV. Supercritical Fluid Chromatography/ SFE
V. Capillary Electrophoresis

Factors Influencing retention in Ion

Exchange
Ionic strength: not real
important in selectivity
pH: anion exchange pH
retention cation
exchange pH
retention
Temperature: T
efficiency
Flow rate: Slightly
slower than other
HPLC methods to
maximize resolution &
improve mass transfer
kinetics

Buffer salt: Influences pH

& selectivity
Organic Modifier: Solvent
strength increases with
increases in modifier

Suppressed Ion Chromatography

Partitioning Chromatography
Analyte interacts with mobile and
stationary phase, differential interaction
leads to selectivity
Interactions that are important
Proton accepting ability * most important
Dipole interaction
Proton Donor * most important
e- pair donating ability
Van der Waals dispersion forces

Types of Partitioning
Chromatography
Normal Phase
Stationary phase: Polar
with short carbon
chains
Mobile phase: Non-polar
such as hexane
Polar things are retained
on column
Applications: oil soluble
vitamins, nitrophenols

Example
Stationary
Phase

Types of Partitioning
Chromatography
REVERSE PHASE
More common

Stationary Phase: Hydrophobic C18 or C8

Mobile Phase:
Polar usually aqueous
Polar substance elute first

Solvophobic Theory
Water has a lot of intermolecular interactions in the liquid
phase
Solute dissolved in water disrupts those intermolecular
interactions
Solute is forced out of aqueous phase not because of
favorable interactions between analyte and stationary
phase but because of unfavorable interactions between
solute and water when solute is dissolved in aqueous
phase hence: SOLVOPHOBIC THEORY
Polar functional groups such as OH would increase the
favorability of interaction and thus decrease retention (in
mobile phase longer)
Polar things elute
non-polar things elute

Sample

Column Mobile
Packing phase
C-18
C-8
C-2

Size Exclusion Chromatography

Molecules partition into bead
Large molecules cant get in and
are unretained, small molecules
get in and never get out, medium
size will differentiate
Need at least 10% difference in
MW to differentiate
GPC organics
Gel filtration chromatography
aqueous

Size Exclusion Chromatography

Advantages
1) Short, well defined retention times
2) Narrow bands high sensitivity
3) No sample loss b/c no interaction with stat. phase
4) No column destruction b/c no interaction with stat.
phase

Size Exclusion Chromatography

Disadvantages
1) Only limited # of
peaks can be
separated b/c time
scale of separation is
short
2) Not good for
separating
compounds of similar
size

Summary
Phase/ Mode
Reverse phase
Normal phase
Ion Exchange
Size Exclusion
Chiral
Hydrophobic

% Use
50.6
24.1
14.1
6.6
3.5
1.1

Supercritical Fluids

Supercritical Fluid Chromatography

Instrumentation

Properties of Mobile Phases Used

in Chromatography
Mobile Density (g/mL)
Phase

Viscosity Diffusion
(poise
coefficient
10-4)
(cm2/sec)

Gas

0.5 3.5 0.01 1.0

0.6 2.0 x 10-3

Super- 0.2 0.9

critical
fluid

2.0 9.9 0.5 3.3 x 10-4

liquid

30 -240

0.8 1.0

0.5 2.0 x 10-5

Fluid

Dipole
mome
nt

Tc(oC)

Pc(atm) Density Densit

c(g/mL) y400(g/
mL)

CO2

31.3

72.9

0.47

0.96

N2O

0.51

36.5

72.5

0.45

0.94

NH3

1.65

132.5

112.5

0.24

0.40

N-C5

196.6

33.3

0.23

0.51

N-C4

152.0

37.5

0.23

0.50

SF6

45.5

37.1

0.74

1.61

Xe
0
CCl2F2 0.17

16.6
111.8

58.4
40.7

1.10
0.56

2.30
1.12

CHF3

25.9

46.9

0.52

1.47

Supercritical fluid extraction (SFE)

Used instead of soxhlet
extraction
Advantages
1. Fast: rate of diffusion
between sample matrix
& extraction fluid 10-60
min vs. days
2. Solvent strength can
be varied by changes
in P & T

3. Less Harmful solvent

4. Many SCF are gases at RT,
recovery of analytes is easy
5. Many SCF are cheap, inert,
and non-toxic
6. On-line extraction

Supercritical fluid extraction

Disadvantages
1. Method development
is more complex
2. Limited # of mobile
phases
3. Capital equipment &
CO2 expensive
4. Requires more
operator time to do 1
at time

 Insert Hawthorne paper