Introduction to Chromatographic Separations

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

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 measureExperimental Quantities (see Table 26-4) • migration time of unretained species • (dead time) tM • retention time (species A and B) (tR)A, (tR)B • adjusted retention time (species A) (tR)A-tM • peak width (species A) WA • length of column packing L • flow rate of mobile phase F • volume of stationary phase VS • concentration of analyte in mobile and stationary phases cM, cS • actually, cM, cS 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 The Selectivity Factor • Experimentally • Derived • Info on the preferential attraction of A for the stationary phase relative to B (1   )

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 Liquid Chromatography Gas 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'