This document discusses various types of liquid chromatography. It describes ion exchange chromatography and factors that influence retention such as ionic strength, pH, temperature, and buffer salt. It also discusses suppressed ion exchange. It explains partitioning chromatography and normal versus reverse phase, as well as factors that influence analyte interaction. Size exclusion chromatography is summarized, noting its advantages and limitations. Supercritical fluid chromatography is also covered, along with its instrumentation and properties of mobile phases used. Supercritical fluid extraction is defined as an alternative to soxhlet extraction.

1. Types of Liquid ChromatographyTypes 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

2. Factors Influencing retention in IonFactors Influencing retention in Ion
ExchangeExchange
• 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

3. Suppressed Ion ChromatographySuppressed Ion Chromatography

4. Partitioning ChromatographyPartitioning 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

5. Types of PartitioningTypes of Partitioning
ChromatographyChromatography
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

6. Types of PartitioningTypes of Partitioning
ChromatographyChromatography
REVERSE PHASE
More common
Stationary Phase: Hydrophobic C18 or C8
Mobile Phase:
Polar usually aqueous
Polar substance elute first

7. Solvophobic TheorySolvophobic 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

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

9. Size Exclusion ChromatographySize Exclusion Chromatography
• Molecules partition into bead
• Large molecules can’t 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

10. Size Exclusion ChromatographySize 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

11. Size Exclusion ChromatographySize 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

12. SummarySummary
Phase/ Mode % Use
Reverse phase 50.6
Normal phase 24.1
Ion Exchange 14.1
Size Exclusion 6.6
Chiral 3.5
Hydrophobic 1.1

13. Supercritical FluidsSupercritical Fluids

15. Supercritical Fluid ChromatographySupercritical Fluid Chromatography
InstrumentationInstrumentation

16. Properties of Mobile Phases UsedProperties of Mobile Phases Used
in Chromatographyin Chromatography
Mobile
Phase
Density (g/mL) Viscosity
(poise
10-4
)
Diffusion
coefficient
(cm2
/sec)
Gas 0.6 – 2.0 x 10-3
0.5 – 3.5 0.01 – 1.0
Super-
critical
fluid
0.2 – 0.9 2.0 – 9.9 0.5 – 3.3 x 10-4
liquid 0.8 – 1.0 30 -240 0.5 – 2.0 x 10-5

17. Fluid Dipole
mome
nt
Tc(o
C) Pc(atm) Density
c(g/mL)
Densit
y400(g/
mL)
CO2 0 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 0 196.6 33.3 0.23 0.51
N-C4 0 152.0 37.5 0.23 0.50
SF6 0 45.5 37.1 0.74 1.61
Xe 0 16.6 58.4 1.10 2.30
CCl2F2 0.17 111.8 40.7 0.56 1.12
CHF3 1.47 25.9 46.9 0.52

18. Supercritical fluid extraction (SFE)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

19. Supercritical fluid extractionSupercritical 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

20. • Insert Hawthorne paper