1 / 23

Introduction HPLC Process

Introduction HPLC Process. Lecture 1. Yuri Kazakevich Seton Hall University. HPLC History. Chromatography was discovered by M.S.Tswett in 1903. Chromatographic Column. HPLC Retention. Major parameters ,

agrata
Download Presentation

Introduction HPLC Process

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. Introduction HPLC Process Lecture 1 Yuri Kazakevich Seton Hall University

  2. HPLC History Chromatography was discovered by M.S.Tswett in 1903.

  3. Chromatographic Column

  4. HPLC Retention Major parameters, • VRis retention volume, depends on the column type, size, and the instrument parameters • Vois dead volume, volume of the liquid phase inside the column • k’ is retention factor (capacity factor), independent of the column size and instrument setup

  5. Retention Characteristics • Retention factor, k’ • Selectivity, a • Efficiency, N • General recommendations: • Optimize retention factor between 1 – 10 • HPLC selectivity should be > 1.2 • Maximize efficiency

  6. e Dead Volume Vo, Dead volume is the volume of the liquid phase in the column Simple rule: Column dead volume = 65% of the volume of empty column

  7. Retention Parameters

  8. HPLC Selectivity

  9. Efficiency Parameters

  10. Efficiency Reduced HETP is a measure of how well the column is packed.

  11. Column Efficiency • Column length is a compromise between the efficiency and backpressure • Column efficiency is proportional to the column length • Specific efficiency (# of particles per one plate) decreases with an increase of column length

  12. Efficiency

  13. Resolution

  14. Factors Influencing HPLC Separation • Parameters affecting efficiency: • Flow rate • Column length • Particle diameter • Particle size distribution • Parameters affecting retention factor: • Eluent type • Eluent composition • Stationary phase type • Analyte nature • Parameters affecting selectivity: • Stationary phase type • Analyte nature • Eluent additives • Temperature • Eluent composition (ionizable analytes)

  15. Reversed Phase Separation Principle • Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4) • Difference in analyte sorption affinities results in their separation • More polar analytes retained less • Analytes with larger hydrophobic part are retained longer • Almost no separation of structural isomers

  16. Retention Process(Surface Equilibria)

  17. Retention Factor, k’ Retention factor is a measure of the analyte competitive interactions with the stationary phase %MeCN 70% 80% 90% 100%

  18. Reversed-Phase HPLC Retention: Neutral Analytes Dependencies of retention of alkylbenzenes, alkylphenones, and alkylparabenes plotted against the number of carbon atoms in alkyl chain. ln(k’)= m (#carbon atoms in alkyl chain) + b

  19. Selectivity • Eluent composition • Ideally does not have any effect on the selectivityfor neutral compounds

  20. 90% MeCN 80% MeCN 70% MeCN 60% MeCN Eluent Composition Effect on Selectivity

  21. 1 2 3 4 5 Eluent Composition Effect on Selectivity

  22. Eluent Composition Effect MeOH/Water Alkylpyridines vs. eluent composition on Luna-C18

  23. Eluent Composition Effect MeCN/Water

More Related