CHEM 240: Introduction to
Bioanalytical Chemistry

 J. D. Cronk
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24. header

Lecture 24. Principles and applications of chromatography

Monday 26 March 2007

"Classical" liquid chromatography. Resolution and spreading of bands. The van Deemter equation. High-performance liquid chromatography (HPLC). Normal phase vs. reversed phase; columns, solvents, detectors. LCMS. Sample preparation.

Reading: Harris, Ch.22 - p.479-489.

  
24. Summary

Lecture 24 summary

The chromatogram, "peaks" or "bands" and their idealized Gaussian shapes; theoretical plates, and resolution.

"Classical" liquid chromatography. Flow rate u. Longitudinal and volume flow rate.

Three factors account for the spreading of bands in chromatography, longitudinal diffusion, equilibration time, and multiple flow paths. Broadening by longitudinal diffusion is minimized by decreasing flow rate u. Broadening by finite rate of mass transfer (equilibration time) is exacerbated by decreasing flow rate u. Broadening by multiple flow paths is independent of flow rate u. The van Deemter equation incorporates all three factors in a mathematical expression

Rules for scaling chromatographic protocols. (i) Cross-sectional area of column should be proportional to the mass of analyte. (ii) keep column length constant. (iii) The volume flow rate proportional to the cross-sectional area of column. (iv) sample volume proportional to the mass of analyte.

 

 
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