Chemistry
by Richard A. Henry, Paul Ross, William R. Betz, Gaurang Parmar and Wayne K. Way
The Importance of Monodisperse Silica in the Evolution of UHPLC and HPLC Column Performance
T
here is growing interest in the use of monodisperse particle-size distribution or PSD (here defined as having 10% RSD or less) to prepare modern HPLC columns because good correlation has been observed between narrow PSD and higher efficiency, longer life and lower pressure drop. HPLC evolution has included synthesis of spherical silica particles with uniform diameters. Monodisperse, non- porous silica (NPS) has been commercially available for 15 years or more; however, columns with NPS particles have not found acceptance in HPLC methods because surface area is too low to retain and resolve most samples. Fused-Core spherical silica with monodisperse PSD was introduced by Advanced Materials Technology (Wilmington, Del.) in 2006. Featuring a uniform, impervious core surrounded by multiple layers of colloidal-size microspheres that provide increased sample ca- pacity, Fused-Core silica has become widely adopted in HPLC methods to improve separation speed.
Another recent example of this trend toward narrower PSD was the intro- duction of Titan monodisperse, totally porous, spherical silica in 2012 by the Supelco Division of Sigma-Aldrich (St. Louis, Mo.). This article describes the performance of monodisperse silica particles for HPLC and UHPLC and offers thoughts about future direction in HPLC technology.
Introduction to modern HPLC particle design Use of smaller particles to increase HPLC column efficiency and achieve
faster separation was predicted in 1969 by Knox.1 to develop2
However, it was slow because small particle columns and instruments designed
to use them were not widely available. Modern era small-particle HPLC, or UHPLC, with its emphasis on faster separation at high-flow velocity and pressure, began with the successful introduction of 1.8-µm Zorbax Rx particle columns from Agilent Technologies (Santa Clara, Calif.) in the early 2000s and continued in 2005 when Schwartz3
porous silica particles with classic stationary phases are being rapidly incorporated into new HPLC and UHPLC methods that provide much higher sample throughput.6
Figures from Kirkland’s landmark paper4 emphasized two performance
advantages for modern core-type particles, including higher efficiency at any velocity and the ability to better maintain efficiency as velocity in- creases. Figure 1 compares van Deemter plots (see Appendix) for core-type and traditional porous silica of the same particle size for a medium-sized, polar drug, while Figure 2 shows Fused-Core particles breaking the reduced plate height barrier of 2 for the first time (hmin
= 1.5) with small molecules.
The practical significance of a lower reduced plate height is that higher efficiency can be achieved with larger particle columns that operate at lower pressure. Note in Figure 2 that naphthalene and lorazepam, with different physical and chemical properties, achieve maximum efficiency at different flow velocities and therefore could not be optimized together in the same mixture. During method development, all critical pairs should be tested for efficiency because some pairs may lose resolution faster than others and limit separation speed. In addition to H or N, plotting selectivity (α) and resolution (Rs) versus flow velocity can be a valuable tool for identifying solutes that limit separation speed. Operation at ex- treme flows and pressures can damage column performance; however, if columns are not rugged at the desired flow velocity, they should not
described the com-
mercial, turnkey Acquity system (Waters Corp., Milford, Mass.); it included a low-dispersion, higher-pressure instrument (ca. 1000 bar) designed to use small i.d. columns filled with 1.7-µm, totally porous silica particles.
An alternate approach to higher HPLC column efficiency and separation speed was described in 2007 by Kirkland,4
who introduced a 2.7-µm
superficially porous silica particle under the trade name Fused-Core silica. The larger particles, with an optimized porous layer and extremely nar- row PSD, generated efficiency comparable to sub-2-µm porous particles at lower operating pressure. Fused-Core columns with larger i.d.s could transform traditional instruments having 400–600 bar pressure limits into much higher-performance systems. Only minor modification to sample inlet and outlet tubing of traditional instruments was required to reduce extra-column bandspreading. An excellent paper by Chester5 lished on this topic in 2009. Modern columns using new solid-core and
was pub-
Figure 1 – Plots (h-u) demonstrating a performance advantage for the monodisperse 2.7-µm Fused-Core C8 silica column over a broader PSD 2.5-µm porous column. Sample: cephalosporin antibiotic, Vantin (MW 557.6); columns: 50 × 4.6 mm; mobile phase: 35% acetonitrile/65% 20 mM sodium phosphate, pH 3.5; temperature: 24 °C. Reproduced with permission from Ref. 4.
AMERICAN LABORATORY • 8 • AUGUST 2015
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