1. Analytical Separations Chromatographic Principle

2. Chromatographic Separations • Column Chromatography －the stationary phase is held in a narrow tube. －the mobile phase is forced through the tube under pressure or by gravity. • Planar Chromatography － the stationary phase is supported on a flat plate or in the pores of a paper － the mobile phase moves through the stationary phase by capillary action or by gravity.

3. Types of Chromatography • Adsorption Chromatography • Partition Chromatography • Ion Exchange Chromatography • Molecular Exclusion Chromatography • Affinity Chromatography

4. One of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibrium between the mobile and stationary phase accounts for the separation of different solutes. Adsorption Chromatography

5. Based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid. Partition Chromatography

6. The use of a resin (the stationary phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Ion Exchange Chromatography

7. Molecular Exclusion Chromatography • Lacks an attractive interaction between the stationary phase and solute. • The mobile phase passes through a porous gel which separates the molecules according to its size. • The pores are normally small. • The larger molecules pass through the column at a faster rate than the smaller ones.

8. Separating Ions by Ion Exchange • Ion exchange resin －high molecular weight polymers －large numbers of an ionic functional group －cation exchange resin －anion exchange resin

9. Ion Exchange Equilibria K↑strong tendency for binding Ca+2

10. Elution in Column Chromatography

11. Distribution of solutes between phases • Kc: distribution constant (partition coefficient ) • Define as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase.

12. Retention Time(滯留時間) • The time taken for the mobile phase to pass through the column is called tM (dead time) • The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR ).

13. Relating Migration Rates to Distribution Constants

14. Relating Migration Rates to Distribution Constants

15. Retention Factor • An important factor is widely used to compare the migration rates of solutes on columns. kA=(tR-tM)/tM • kA<1 → elution is so fast that accurate determination of the retention time is very difficult. • High retention factors, kA>20 → elution takes a very long time. • Ideally, 1<kA<5

16. Tailing and Fronting Peaks • Tailing is caused by sites on the packing that have a stronger-than normal retention for the solute. • Fronting is related to the shape of the sorption isotherm or that sample introduced onto a column is too large.

17. Column Efficiency • Two related terms, (1) plate height H and (2) number of theoretical plates N, are widely used to measure the column efficiency. • N=L/H • H=σ2/L

18. Theoretical Plate Model of Chromatography • Suppose that the chromatographic column contains a large number of separate layers, called theoretical plates. • It is important to remember that the plates do not really exist. • Measure column efficiency

19. W: width of the peak at its base W1/2: peak width at half-height Calculate Theoretical Plate Numbers

20. Height Equivalent to Theoretical Plate (HETP) • Van Deemter equation for plate height HETP = A + B / u + C u • u:average velocity of the mobile phase • A, B, and C are factors which contribute to band broadening.

21. Molecules move through different paths Larger difference in pathlengths for larger particles This will cause broadening of the solute band, because different paths are of different lengths. A - Eddy diffusion

22. B - Longitudinal diffusion • The concentration of analyte is less at the edges of the band than at the center. • Analyte diffuses out from the center to the edges. • Inversely proportional to flow rate - high flow, less time for diffusion • High velocitydecreases the effects of longitudinal diffusion.

23. C - Resistance to mass transfer • The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. • The high velocity of the mobile phase lead the analyte to have a strong affinity for the stationary phase. • The analyte in the mobile phase will move ahead of the analyte in the stationary phase. • The higher the velocity of mobile phase, the worse the broadening becomes.

24. Van Deemter Plots

25. Column Resolution • Resolution: ability to separate two analytes High resolution: △ Z↑&W↓

26. Optimization of Column Efficiency Resolution

27. Optimization of Column Efficiency Resolution

28. Resolution and Zone Broadening Factors • u－low flow favors increased resolution • H (plate height) －use smaller particles, lengthen column • a - vary temperature, composition of column/mobile phase • kA' (capacity factor) - vary temperature, composition of column/mobile phase