1 / 29

Chromatography General

Chromatography General. Chromatographic Process. Chromatographic Systems. Chromatographic Techniques. TLC/PC. PC-Paper Chrom. HPLC. GC/SFC. Chromatography – Separation Mechanism. Adsorption Partition Ion - Exchange & Ion - Interaction Size Exclusion

spencer
Télécharger la présentation

Chromatography General

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

  11. Chromatography – Peak Broadening

  12. 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

  13. Chromatography – Packing Effect on Broadening

  14. Chromatography - Equilibrium Amobile Astationary

  15. 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

  16. 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)

  17. 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

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

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

  20. 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

  21. Chromatography - Resolution

  22. 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

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

  24. 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

  25. END

  26. Chromatography - Methodology

  27. Peaks Broadening

More Related