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HPLC Columns and Stationary Phases

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  1. HPLC Columns andStationary Phases Lecture 2 Yuri Kazakevich Seton Hall University

  2. Outline • Packing material • Particle type • Particle geometry • Surface chemistry • Bonded Layer • Chemistry • Conformational freedom • Interaction with solvent

  3. Silica from : Journal of Chromatography A, 1006 (2003) 207–228 • rigid porous (or nonporous) particles • wide variety of particle and pore sizes • soluble in water at pH > 8

  4. HPLC Adsorbent Particles • Average particle diameter is 5 m • Average pore diameter is 100 Å • Average surface area is 300 m2/g Most of the adsorbents have cylindrical pore shape. What is the ratio of particle diameter to the pore diameter? What is the total length of all pores in 1 g of adsorbent?

  5. Packing MaterialPore size, pore volume, surface area • Assuming cylindrical pore model one can get: • The larger the pore diameter, the smaller the surface area. • The larger the surface area the greater the retention. • The smaller the pore diameter the greater the steric hindrance effect. Analyte retention in HPLC is proportional to the surface area of packing material

  6. Bonded Phase • Bonded phase shields polar silica surface, making it inaccessible for analyte molecules. • Suppressing strong polar interactions with silica surface and substituting them with weak dispersive forces is a key factor of reversed-phase HPLC. • BP types - C18, C8, C5, C1, Phenyl, CN, NH2, etc.

  7. Selected Types of Bonded Ligands C1 C8 Oxy-Phenyl C-18 PFP

  8. C18 Ligands C18 chains have ~21 Å length in all-trans conformation Their molecular volume is ~700 Å3 Maximum bonding density is 2.5 chains/nm2 or 4.1 mmole/m2 on flat surface

  9. Methylene selectivity vs. eluent composition 0.6 0.5 Zorbax-C8 0.4 Allure-C18 0.3 Slope ln (k') Allure-PFP 0.2 0.1 0 50 60 70 80 90 100 MeCN/Water %v/v Methylene Selectivity of Different Bonded Phases

  10. Monomeric and Polymeric Bonding

  11. Endcapping Secondary bonding with trimethylchlorosilane

  12. Bonding Density Propyl-Phenyl ligands at 2.7 group/nm2 (left) and 1.9 group/nm2 (right)

  13. Bonding Density • The only measurable parameter related to the quality of bonded phase is Carbon Loading (%w/w of carbon atoms bonded on the silica surface). • Bonding density is the number of bonded ligands per unit of silica surface.

  14. Arrangement of the Bonded Phase Chains on Silica Surface

  15. Bonded Layer Thickness For concave surface For flat surface A B

  16. Column Pore Volume

  17. Volume and Thickness of Adsorbed Layer on All Studied Adsorbents for Three Adsorbates

  18. Eluent Type Effect MeCN [v/v%] MeOH/Water MeCN/Water

  19. Retention Model HPLC analyte injected in the column equilibrated with binary eluent Assumption: Small amount of analyte does not significantly disturb eluent equilibrium in the column Overall retention is a composition of two concurrent processes

  20. Column PerformancepH stability • The main parameter affecting pH stability of packing material is Bonding Density • Low pH (<2.5) causes hydrolysis of the siloxane bonds destroying bonded layer • The higher the bonding density the lower hydrolysis effect. • High pH (>8.5) causes silica dissolution • High bonding density shield silica surface which makes it stable up to pH 13.

  21. Column Testing • Good reversed-phase column should • exclude acidic components (benzoic acid should come out before Vo), • show low retention and tailing for basic components (pyridine) • show complete separation and very symmetrical peaks for naphthalene and ethylbenzene. • Testing conditions: Acetonitrile/water 70/30, 1 ml/min.

  22. Column Testing Benzoic acid Pyridine Benzoic acid Pyridine SupelcoSil-LC18 Prodigy-ODS

  23. Column Cleaning Solvent front disturbs phase equilibrium Release of trapped impurities

  24. Summary • Surface area • Analyte retention is proportional to the adsorbent surface area • Pore size • Effects the conformational freedom of bonded ligands • Restricts the pore volume accessibility for large molecules • Minor effect on the amount of accessible residual silanoles • Type of bonded ligands • Determines the adsorbent retentive power and selectivity • Bonding density • Determines the accessibility of residual silanoles • Minor effect on the selectivity