1. 1 Introduction HPLC Process Lecture 1 Yuri Kazakevich Seton Hall University

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

3. 3 Chromatographic Column

4. 4 HPLC Retention Major parameters, V R is retention volume, depends on the column type, size, and the instrument parameters V o is 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. 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. 6 Dead Volume V o, Dead volume is the volume of the liquid phase in the column Simple rule: Column dead volume = 65% of the volume of empty column e

7. 7 Retention Parameters

8. 8 HPLC Selectivity

9. 9 Efficiency Parameters

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

11. 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. 12 Efficiency

13. 13 Resolution

14. 14 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) Factors Influencing HPLC Separation

15. 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. 16 Retention Process (Surface Equilibria)

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

18. 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. 19 Selectivity –Eluent composition Ideally does not have any effect on the selectivity for neutral compounds

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

21. 21 Eluent Composition Effect on Selectivity c%  Peak numbers 2,13,24,3 701.751.61.45 801.721.581.4 901.71.651.43 1001.711.621.42 1234512345

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

23. 23 Eluent Composition Effect MeCN/Water