Chromatography

Separates components in mixture: Based on - polarity - boiling point - ionic strength - size

Chromatography
Mobile phase: phase which sample is dissolved in may be gas, liquid, or supercritical fluid Stationary phase: phase which mobile phase is forced through Mobile and stationary phases are chosen so the analyte will distribute itself between the two phases

Partition Chromatography
Movie Used in GC & LC Molecules will partition into the stationary phase based upon affinity for stationary phase & eventually partition into mobile phase again Thin layer is coated onto inside of GC column or on small particles on LC column

Adsorption Chromatography
Very similar to partition chromatography Adsorption just on surface, partition into thin layer Not used as widely as partition used mainly in TLC & very small particles in LC Movie

Ion Exchange Chromatography

Movie Separation of either cations or anions Separtion based on relative strength of ionic bond Anion exchange has cations on surface Used in LC exclusively

Molecular Exclusion Chromatography

Separation based on size Small molecules get trapped in pores & take longer to get out Movie

Gel Electrophoresis
Separation based on size and charge Smaller molecules will migrate further, less tangled Movie

Affinity Chromatography
Very selective Specific binding site is used to concentrate analyte on column Used a lot in biological applications Movie

Typical Gas Chromatogram

Typical Liquid Chromatogram

Introduction to Chromatography - Theory

General Relationships 1. Distribution constant a. Craig counter current experiment 2. Retention time 3. Relationship between distribution constant and retention time 4. Capacity factor k 5. Selectivity factor a

Introduction to Chromatography - Theory

Peak Broadening 1. Shapes 2. Column efficiency a. plate height b. number of plates 3. Kinetic factors Van Deemter equation

Craig counter current

movie

2. Retention time tr
Time it takes for analyte to reach detector after sample injection Tm = retention time for material to come through column which is not retained also called dead time or void volume

tm rate of migration is the same as the average rate of motion of the mobile phase molecules u = L/tm

3. Distribution constant & retention time

v = u x moles of analyte in mobile phase

total moles of analyte

v=ux v=ux

CmVm CmVm + CsVs 1 1 + KVs/Vm

1 1 + CsVs/CmVm

4. Capacity factor k
Describes migration rates of analytes in column

For a species A
k = KAVs

v = u x 1/(1 + k)

kA = (tr- tm)/tm For separations involving few components ideal capacity factors are between 1 - 5
What is k for this peak?

5. Selectivity factor a
Ability to distinguish between 2 species, A & B

Purpose of Chromatography
Achieve separation

Elution movie

Peak Broadening

Peak Broadening

 Is peak broadening a good or bad thing?

BAD Why?

Column Efficiency
Plate height (H) # theoretical plates (N) N = L/H Efficiency of a column goes up as N increases and H decreases Typical 250 10,000 plates

Plate Height

3. Kinetic Factors: The Van Deemter Equation

Reality: column efficiency is affected by kinetic factors

What variable do you think are important in determining the efficiency of a separation?

In your notebook predict what the effect of increasing linear velocity (flow rate) will have on column efficiency (H)

Van Deemter Equation

Van Deemter Equation

H = A + B/u + Cu
A = Eddy diffusion: Due to different paths molecules can take as they go through particles B/u = longitudnal diffusion Band diffuses in and against direction of mobile phase movement Cu often broken into 2 terms Csu + Cmu Mass transfer coefficient: Time it takes for analyte to diffuse into stationary phase

How can band broadening be reduced? (and thus column efficiency be enhanced)
1. Decrease particle diameter 2. Decrease column width 3. Lowering temperature in GC (reduces diffusion coefficient) 4. Minimize thickness of liquid stationary phase

Resolution
This is called General Elution Problem

Rs = 2((tr)B (tr)A) wA + wB