Chromatography General

1. ChromatographyGeneral

2. Chromatographic Process

3. Chromatographic Systems

4. Chromatographic Techniques TLC/PC PC-Paper Chrom HPLC GC/SFC

5. Chromatography – Separation Mechanism • Adsorption • Partition • Ion - Exchange & Ion - Interaction • Size Exclusion • Affinity (antibody-antigen interactions; chemical interaction; attraction) • Complexation - Chelation • Ion – exclusion (Separation of weak acids)

6. Sorption problems ADsorption ABsorption Different sorptions explained

7. Chromatograhy – Mechanism of Separation Partition Ion exchange Adsorption

8. Chromatography – Mechanism of Separation Size Exclusion Affinity

9. Chromatogram – Basic Parameter tR = retention time tm = dead time H W1/2 1/2H unretained

10. Chromatographic Theories • Adjusted retention time: tR’ = tR – tM • Plate theory – distillation – plate number N = 5.54[(tR – tM)/w1/2]2 • Plate height H = L/N • This theory did not include interaction of analytes with stationery phase

12. Chromatography – Peak Broadening

14. Chromatographic Theories • Rate Theory – kinetic factors – van Deemter H = B/u + Cu (+ A) Where: u – velocity of mobile phase B – effect of molecular diffusion C – Resistance to mass transfer A – Spreading related to different distance traveled by molecules in packed columns

15. Chromatography – Packing Effect on Broadening

16. Chromatography - Equilibrium Amobile Astationary

17. Van Deemter factors: Molecular diffusion (B)– in mobile phase • proportional to time analyte spends in a column • affected by diffusion coefficient of analyte in mobile phase • affected by temperature and pressure • not important in LC – low diffusion coefficient • inversely affected by mobile phase velocity

18. Van Deemter factors: Resistance to mass transfer (C): • Mass transfer in mobile and stationary phase • Lack of equilibrium – moving phase • Affected by thickness of liquid phase • Affected inversely by the diameter of particles or inner diameter of capillary column • Lower at higher temperatures (viscosity)

19. Van Deemter factors: Conclusions: • Minimum value for H is achieved when: • stationery phase thickness is minimal • column packed with the smallest particles • capillary columns have the smallest internal diameter • mobile and stationary phases have low viscosity and high diffusion coefficient

20. Chromatography – van Deemter Plot H Plate height (cm) Cu Mass transfer Multipath effect A Diffusion (Longitudinal) B/u Mobile phase velocity

21. Chromatography - Resolution DtR tR1 tR2 R = 2(tR1 – tR2)/Wb1 – Wb2 Response 100% Baseline resolution for Gaussian shape peaks = 1.5 Wb1 Wb2

22. Chromatography - Resolution • Resolution equation where separation parameters are included: Rs = ½ x (a-1/a+1) x k’2/1+k’2x (L/h)1/2 Where: a – selectivity factor (separation) a = tR1/tR1 k’ – migration term, capacity factor; k’ = ms/mm L – column length h – plate height

23. Chromatography - Resolution

24. Chromatography • Qualitative Analysis • Retention data – RT; Rf; RRT; Kovacs Index • Quantitative Analysis • Peak area and height usually proportional to the amount of component • Calibration • Internal Standard method • External Standard method • Area Normalization method

25. Chromatogram – Basic Parameter tR = retention time tm = dead time H W1/2 1/2H unretained

26. 4 RRT1 = RT1/RTIS RRT2 = RT2/RTIS RRT3 = RT3/RTIS 1 3 Cholesterol Accurate to e few digits (2) at fourth Decimal Point IS α - Cholestane 2 5

27. END

28. Chromatography - Methodology

29. Peaks Broadening