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

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

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

4. 4 HPLC Adsorbent Particles Average particle diameter is 5  Average pore diameter is 100 Å Average surface area is 300 m 2 /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. 5 Packing Material Pore 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. 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, NH 2, etc.

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

8. 8 C18 Ligands C18 chains have ~21 Å length in all-trans conformation Their molecular volume is ~700 Å 3 Maximum bonding density is 2.5 chains/nm 2 or 4.1  mole/m 2 on flat surface

9. 9 Methylene selectivity vs. eluent composition 0 0.1 0.2 0.3 0.4 0.5 0.6 5060708090100 MeCN/Water %v/v Slope ln (k') Zorbax-C8 Allure-C18 Allure-PFP Methylene Selectivity of Different Bonded Phases

10. 10 Monomeric and Polymeric Bonding

11. 11 Endcapping Secondary bonding with trimethylchlorosilane

12. 12 Propyl-Phenyl ligands at 2.7 group/nm 2 (left) and 1.9 group/nm 2 (right) Bonding Density

13. 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. 14 Arrangement of the Bonded Phase Chains on Silica Surface

15. 15 For flat surface For concave surface AB Bonded Layer Thickness

16. 16 Column Pore Volume

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

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

19. 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 Performance pH 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 Prodigy-ODS SupelcoSil-LC18 Benzoic acid Pyridine

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

24. 24 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 Summary