HPLC Columns andStationary Phases Lecture 2 Yuri Kazakevich Seton Hall University
Outline • Packing material • Particle type • Particle geometry • Surface chemistry • Bonded Layer • Chemistry • Conformational freedom • Interaction with solvent
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
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?
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
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.
Selected Types of Bonded Ligands C1 C8 Oxy-Phenyl C-18 PFP
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
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
Endcapping Secondary bonding with trimethylchlorosilane
Bonding Density Propyl-Phenyl ligands at 2.7 group/nm2 (left) and 1.9 group/nm2 (right)
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.
Bonded Layer Thickness For concave surface For flat surface A B
Volume and Thickness of Adsorbed Layer on All Studied Adsorbents for Three Adsorbates
Eluent Type Effect MeCN [v/v%] MeOH/Water MeCN/Water
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
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.
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.
Column Testing Benzoic acid Pyridine Benzoic acid Pyridine SupelcoSil-LC18 Prodigy-ODS
Column Cleaning Solvent front disturbs phase equilibrium Release of trapped impurities
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