1. Introduction to Chromatographic Separations Due to lack of analytical specificity, separations are often necessary Chromatography is about separations –need minimum of two phases –stationary phase –mobile phase –analyte and matrix must have differing affinities for one or more phases

2. Example of Chromatographic Experiment In this example, compound B is more attracted to the stationary phase than is compound A The compounds A and B which are attracted to the stationary phase form bands, or zones, along the length of the stationary phase

3. Intro to Band Broadening, I As analytes migrate farther through the stationary phase, band broadening occurs

4. Intro to Band Broadening, II Band broadening decreases chromatographic resolution  we need to optimize the chromatographic method

5. Chromatographic 'Figures of Merit' Table 26-5 in text, 'Calculation of Derived Quantities' –a series of formulas that describe, in various ways, aspects of chromatographic performance –we will not examine the derivation of these here, but you may find it helpful to follow the derivation in the text as an aid to understanding Table 26-5 –many of the derived quantities in Table 26-5 are themselves functions of derived quantities –the question becomes, what can we measure? (and how does that relate to the derived quantities)

6. What we can measure Experimental Quantities (see Table 26-4) migration time of unretained species –(dead time) t M retention time (species A and B) (t R ) A, (t R ) B adjusted retention time (species A) (t R ) A - t M peak width (species A) W A length of column packing L flow rate of mobile phase F volume of stationary phase V S concentration of analyte in mobile and stationary phases c M, c S –actually, c M, c S aren't practical to measure Chromatographic figures of merit resolution capacity factor selectivity

7. Resolution From experimental data As a derived quantity (rearranged from last equation in Table 26-5) –u is the linear velocity of the mobile phase, also related to the Height Equivalent of a Theoretical Plate (HETP) or H –  is the selectivity factor –k' is the capacity or retention factor The Capacity or Retention Factor Experimentally gives relative value for attraction of analyte to the stationary phase Experimentally Derived Info on the preferential attraction of A for the stationary phase relative to B (1     ) The Selectivity Factor

8. Plates, u and the van Deempter Equation plate – conceptually separations unit the more plates, N, the better the separation the shorter the plate, the more that can be stuffed onto fixed length, L, column –experimentally –empirically (the van Deempter equation)

9. van Deempter Plots Gas Chromatography Liquid Chromatography

10. Laundry List of Experimental Parameters of Concern number of plates, HETP  L stationary phase  column type, head pressure mobile phase  F, head pressure, mixture, gradient temperature (programmable)  T quality of column packing  column type

11. The 'General Elution Problem'