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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
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
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
v=ux v=ux
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
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
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)
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