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 C 18 or C 8 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

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

Size 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 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% Use Reverse phase50.6 Normal phase24.1 Ion Exchange14.1 Size Exclusion6.6 Chiral3.5 Hydrophobic1.1

Supercritical Fluids

Supercritical Fluid Chromatography Instrumentation

Properties of Mobile Phases Used in Chromatography Mobile Phase Density (g/mL)Viscosity (poise ) Diffusion coefficient (cm 2 /sec) Gas0.6 – 2.0 x – – 1.0 Super- critical fluid 0.2 – – – 3.3 x liquid0.8 – – 2.0 x 10 -5

FluidDipole mome nt T c ( o C)P c (atm ) Densit y c (g/m L) Densit y 400 (g/ mL) CO N2ON2O NH N-C N-C SF Xe CCl 2 F CHF

Supercritical fluid extraction (SFE) Used instead of soxhlet extraction Advantages 1.Fast: rate of diffusion between sample matrix & extraction fluid 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