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

2. 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

3. 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

4. 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

5. 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

6. Molecular Exclusion Chromatography
Separation based on size
Small molecules get trapped in pores & take longer to get out
Movie

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

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

10. Typical Gas Chromatogram

11. Typical Liquid Chromatogram

12. 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

13. Introduction to Chromatography - Theory
Peak Broadening
1. Shapes
2. Column efficiency
a. plate height
b. number of plates
3. Kinetic factors – Van Deemter equation

14. Craig counter current
movie

17. 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

18. 3. Distribution constant & retention time
v = u x moles of analyte in mobile phase
total moles of analyte
v = u x CmVm =
CmVm + CsVs 1 + CsVs/CmVm
v = u x
1 + KVs/Vm

19. 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?

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

21. Purpose of Chromatography
Achieve separation
Elution movie
When analytes migrate down a column there will be differential separation
Different molecules have different affinities for the stationary phase so will stay with stationary phase for different lengths of time but some analytes even though they are the same will travel at different rates within the column, show movie elution This leads to band broadening

22. Peak Broadening
Draw Gaussian curve of his location if he was staggering for 5 minutes, 10 minutes, 30 minutes

23. Peak Broadening
Wider peaks at end of run

24. Is peak broadening a good or bad thing?
BAD Why?

25. 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

26. Plate Height
Do notes from notebook on white board

27. 3. Kinetic Factors: The Van Deemter Equation
Reality: column efficiency is affected by kinetic factors

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

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

30. Van Deemter Equation

31. 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
Which type of chromatography would you expect longitudnal diffusion to be more pronounced in? Why? GC because gases have a higher diffusion coefficient.

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

33. This is called General Elution Problem
Resolution
This is called General Elution Problem
Rs = 2((tr)B – (tr)A) wA + wB