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- 1. There are two theories to explain chromatography Plate theory - older; developed by Martin & Synge in 1941 Rate theory - currently in use Proposed by van Deemter in 1956 Accounts for the dynamics of the separation
- 2. View column as divided into a number (N)of adjacent imaginary segments calledtheoretical plates Within each theoretical plate analyte(s) completely equilibrate between stationary phase and mobile phase Column Theoretical plate
- 3. Chromatographic principle Mobile phaseSamplemixture Equilibrium establishes at each point (ideally) Stationary phase The molecules of the Retardation of ratemixture interact with the of movement ofmolecules of the Mobile molecules and Stationary Phase Each molecule Different distribution interacts differently coefficients and different with MP and SP net rates of migration
- 4. Greater separation occurs with: –greater number of theoretical plates (N) –as plate height (H or HETP) becomes smaller L = N H or H = L / N where L is length of column, N is number of plates, and H is height of plates or height equivalent to theoretical plate (HETP)
- 5. The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution by various methods like- half-height method- USP method
- 6. N is a ratio of tR and σ of Wb which is 4σ•N = 5.55 tR2/ w1/22 = 16 tR2/ w2where: tR is retention time w1/2 is width at h0.5 w is width measured at baseline
- 7. Where:N = Number of theoretical platesVe = elution volume or retention time (mL, sec,or cm)h = peak heightw1/2 = width of the peak at half peak height(mL, sec, or cm)
- 8. • Nmax = 0.4 * L/dp where: Nmax - maximum column efficiency L - column length dp - particle size• So, the smaller the particle size the higher theefficiency!
- 9. Band spreading - the width of bands increases as their retention time (tR) or retention volume (VR) increases
- 10. A band exhibiting a width of 4 mL and aretention volume of 49 mL, is eluted from acolumn. What width is expected for a bandwith a retention volume of 127 mL elutingfrom the same analyte mixture on the samecolumn?ANS: 10.4 mL
- 11. The smaller HETP, the narrower theeluted peak
- 12. • It is not unusual for a chromatography columnto have millions of theoretical plates• Columns often behave as if they havedifferent numbers of plates for differentsolutes present in same mixture

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