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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 ,

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Introduction HPLC Process

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  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

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