Types of Liquid ChromatographyTypes of Liquid Chromatography

I. Ion Exchange ChromatographyA. Factors influencing retentionB. Suppressed ion exchange

II. Partitioning ChromatographyA. Normal phase/ reverse phase

III. Size Exclusion ChromatographyIV. Supercritical Fluid Chromatography/ SFEV. Capillary Electrophoresis

Factors Influencing retention in Ion Factors 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

Suppressed Ion ChromatographySuppressed Ion Chromatography

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

Types of Partitioning Types of Partitioning ChromatographyChromatography

Normal PhaseStationary 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 Types of Partitioning ChromatographyChromatography

REVERSE PHASEMore common

Stationary Phase: Hydrophobic C18 or C8

Mobile Phase:

Polar usually aqueous

Polar substance elute first

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

Sample Column Packing

Mobile phase

C-18

C-8

C-2

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

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

Size Exclusion ChromatographySize Exclusion Chromatography

Disadvantages1) Only limited # of

peaks can be separated b/c time scale of separation is short

2) Not good for separating compounds of similar size

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

Supercritical FluidsSupercritical Fluids

Supercritical Fluid Chromatography Supercritical Fluid Chromatography InstrumentationInstrumentation

Properties of Mobile Phases Used Properties 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

Fluid Dipole moment

Tc(oC) Pc(atm) Densityc(g/mL)

Density400(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

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

Supercritical fluid extractionSupercritical fluid extraction

• Disadvantages1. Method development

is more complex2. Limited # of mobile

phases3. Capital equipment &

CO2 expensive4. Requires more

operator time to do 1 at time

• Insert Hawthorne paper