Chromatography Theory

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CHROMATOGRAPHY

Chromatography

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

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Partition Chromatography 

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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

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

Adsorption Chromatography Chr omatography 

 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

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Ion Exchange Chromatography 

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 Separation

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

Gel Electrophoresis   Separation

based on size and charge  Smaller molecules will migrate further, less tangled  Movie

Molecular Exclusion Chromatography 

 Separation

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

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Affinity Chromatography 

 Very selective

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 Specific

binding site is used to concentrate analyte on column  Used a lot in biological applications

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Typical Gas Chromatogram

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Typical Liquid Chromatogram 

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Introduction to Chromatography Theory

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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

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Introduction to Chromatography Theory

Craig counter current

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

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2. Retention time t

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 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

4. Capacity factor k’

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k’ = KAVs

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kA’ = (tr- tm)/tm For separations involving few components ideal capacity factors are between 1 - 5

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v  =

u x moles of analyte in mobile phase total moles of analyte

v  =

ux

CmVm

=

1

CmVm + CsVs ux

1 + CsVs/CmVm

1 1 + KVs/Vm

Describes migration rates of analytes in column For a species A v 

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v  =

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

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3. Distribution constant & retention time

= u x 1/(1 + k’)

5. Selectivity factor a   Ability to

distinguish between 2 species, A & B

What is k’

for this peak?

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Purpose of Chromatography

Peak Broadening

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 Achieve separation

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Peak Broadening 

  Is peak

broadening a good or bad thing?

BAD Why?

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Column Efficiency •

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Plate Height 

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

3. Kinetic Factors: The Van Deemter Equation 

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

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Reality: column efficiency is affected by kinetic factors

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

Van Deemter Equation

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Van Deemter Equation

How can band broadening be reduced? (and thus column efficiency be enhanced)

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 C su + Cmu Mass transfer coefficient: Time it takes for analyte to diffuse into stationary phase

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1. 2. 3. 4.

Decrease particle diameter Decrease column width Lowering temperature in GC (reduces diffusion coefficient) Minimize thickness of liquid stationary phase

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Resolution is called General ThisElution Problem

Rs = 2((tr)B – (tr)A) wA + wB

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