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Hybrid particle technologyand its use in HPLC

1st April 2013


High performance liquid chromatography (HPLC) has been a mainstay of the laboratory scientist for many years. However,there is a new chromatographic base material which is leadingto dramatic performance improvements, including an extended useful pH range, all without sacrificing the benefits of silica-based chromatographic supports.

The majority of high performance liquid chromatography (HPLC) separations today are performed using silica-based reversed-phase columns. Such columns have been favoured for more than 30 years because they deliver the best combination of desired chromatographic properties ­ high efficiency, high bed stability and ruggedness.

In addition, they are available in a wide range of chemistries, particle sizes and pore diameters. However, silica-based reversed-phase columns suffer from two major shortcomings. Firstly, they give broad, tailing peaks for many basic compounds. In addition, they have short lifetimes when the mobile phase pH is outside the 2­8 range. Below pH2 the bonded phase may be stripped off, while above pH8 the underlying silica support begins to dissolve.

Over the past 20 years considerable research has gone into developing chromatographic media that have improved performance for basic compounds and that can operate over a wider pH range. However, only moderate improvements in performance for basic compounds and pH stability have been realised through modifications of the silica bonding processes.

For the past two years, research efforts at Waters have focused on the development of a new chromatographic base material that offers dramatically improved performance for basic compounds and an extended useful pH range, without sacrificing the benefits of silica-based chromatographic supports. The result is new hybrid particle technology.

The goal with hybrid technology was to take a quantum leap to a higher level of performance by radically-changing the composition of the underlying chromatographic particle itself.

Traditional silica-based reversed-phase packings are manufactured by derivatising the surface of silica particles using silane reagents that react with silanol (Si-OH) groups. Because the silanes are considerably larger than silanols, not all of the silanols are derivitised. In addition, the small size of the pores in many silica particles limits the accessibility of the entire surface to bulky reagents such as octadecylsilanes. As a consequence, surface derivatisation is always incomplete and may leave large patches of unbonded surface. The residual silanols are acidic and cause peak tailing for basic compounds due to ion-exchange interaction.

The patent-pending hybrid particle is a member of a class of materials known as organic/inorganic hybrids. These materials contain both inorganic (such as silica) and organic (such as organosiloxane) elements and thus share the advantages of both. One route (Fig. 1) to creating hybrid particles is to use a mixture of two high-purity monomers: one that forms SiO2 units during the particle formation process and another that forms RSiO1.51.5 (organosiloxane) units. The resulting particles contain organosiloxane groups incorporated throughout their internal and surface structure.

Optimum performance is obtained using a methyl R-group with a 2:1 mole ratio of SiO2 to CH3SiO1.5 units. These particles are then additionally surface-bonded to attach a variety of different reversed-phase groups (such as C8 and C18). As described below, bonded phases based on hybrid particles deliver the sharpest, most symmetrical peaks for all basic compounds, low-pH stability and dramatically improved stability in high-pH mobile phases.

Basic compound performance

The peak shapes obtained for strongly-basic compounds in reversed-phase HPLC are dependent upon both the concentration and acidity of the residual silanol groups that are present after bonding. As already mentioned, steric hindrance limits the extent of derivatisation for traditional surface-bonded packings. However, because the new hybrid particles contain methylsiloxane groups in place of a third of the SiO2 units, they yield bonded phases with reduced concentrations of residual silanols. As a consequence, bonded phases based on hybrid particles deliver exceptional peak shape for basic compounds.

The most sensitive methods for measurement of residual silanol activity are chromatographic tests using highly basic probes at a mobile phase pH of 7. At this pH, many of the residual silanols are in the ionised form (SiO-) and the basic probes are completely protonated (BH+). The protonated bases will then interact with the ionised silanols by an ion-exchange mechanism, with the degree of tailing a direct measure of silanol activity. To determine the silanol activity of a number of C18 columns, a series of six strongly-basic probes were used together with an acetonitrile/water mobile phase buffered at pH 7 with K2HPO4. A C18 bonded hybrid particle column was benchmarked against the best brands on the market. The results demonstrate excellent peak symmetry for all probe compounds on the C18-bonded hybrid particle columns. In contrast, the benchmark silica-based columns show severe tailing of the compounds.

The low-pH stability of silica-based reversed-phase columns is determined by two factors: the rate of cleavage of the siloxane (Si-O-Si) bonds that attach the bonded phase to the particle, and the rate of removal of the products of this cleavage by the mobile phase.

For a surface-bonded hybrid particle, both the stability of the bonded phase and the stability of the integral methylsiloxane groups in the hybrid particles need to be considered. The methylsiloxane groups are found to have exceptional low-pH stability. Even after exposure to 1M HCl at for 16 hours, no loss of methyl groups was detected using carbon analysis. This stability is believed to be due to the fact that most of the methylsiloxane units are attached by three siloxane bonds, as determined using 29Si cross-polarisation/magic angle spinning nuclear magnetic resonance spectroscopy.

Because of the exceptional stability of the hybrid particles, low-pH stability is determined by the stability of the surface-bonded groups. Bonded phases prepared using tri-functional silanes are known to give much better low-pH stability than those based on mono-functional silanes. To compare the stability of C18 bonded hybrid particle columns to a series of benchmark columns, an accelerated stability test was used. In this test, columns are stored at 50oC in one per cent trifluoroacetic acid (TFA). The columns are periodically flushed with water and methanol and tested to determine the retention factor for acenaphthene. Retention decreases with increasing time exposed to one per cent TFA, due to loss of bonded phase (Fig. 2). One of the benchmark columns (which is mono-functionally bonded) showed large retention losses after several days of exposure. In contrast, the tri-functionally bonded C18 hybrid particle column shows a much smaller retention loss in this accelerated test.

Improved high-pH stability

The high-pH stability of silica-based reversed columns is determined by the rate of dissolution of the underlying silica particle. After dissolution has proceeded to a critical point, the packed bed abruptly collapses, causing voids which result in catastrophic loss of efficiency.

Because dissolution requires access of hydroxyl ions to the silica surface, the rate of dissolution depends on the amount of underivitased silica surface. Bonded phases based on hybrid particles have an extremely low area of underivitised silica surface because of the methylsiloxane units incorporated throughout their structure.

Accordingly, columns containing these particles show exceptional lifetimes in high pH mobile phases.

For more information about hybrid particle technology contact Waters Corporation, 34 Maple Street, MA 01757, USA, tel 1 508 478 2000, www.waters.com





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