A New Generation of Silica-Based Monoliths HPLC Columns with Improved Performance

Apr 01, 2012
Volume 30, Issue 4, pg 30–35

With the advent of the second generation of commercial silica monolith columns, liquid chromatographers now have an alternative to the sub-2-µm and core–shell packings for high-throughput, high-efficiency separations. In this paper, the preparation of the new-generation silica-based monolithic high performance liquid chromatography columns is described in detail and the first- and second-generation columns are compared based on physical characteristics, with application examples given. The future prospects for silica-based monoliths also are explored.

Monolithic high performance liquid chromatography (HPLC) columns are made of one single piece of either porous silica or organic polymer. These columns have a unique chromatographic feature: high column efficiency and high column permeability at the same time. Therefore, there is an increased interest in these materials, driven by the never-ending demand for faster and more efficient HPLC methods. Both types of monolithic columns are now commercially available and under intensive investigations. Their chromatographic properties and applications have been described in several recent reviews (1–11). Two features primarily differentiate the silica-based monoliths from the organic polymeric-based monoliths: pH stability and separation efficiency (plate count per meter = N/m). While pH stability is definitely better for organic polymeric monoliths (pH 1–12), separation efficiency is undoubtedly better for silica monoliths (N/m up to 200,000). This article will focus on the latest advancements in the development of silica-based monoliths while organic polymeric monoliths are reviewed by others (7–12).

The first silica-based monolithic HPLC column (Chromolith, Merck Millipore) became commercially available in 2000 and attracted a lot of attention because of its novelty. This type of HPLC column consists of a porous silica rod that is encapsulated in a mechanically strong and solvent-resistant PEEK polymer and equipped with low-volume endfittings. The high porosity of silica-based monoliths (total porosity > 80%) is caused by macro- or through-pores that offer significant advantages compared to classical particle-packed columns — for example, low column back pressures, operation with higher flow rates, faster analysis, and direct applications of "dirty" samples without prior sample preparation (2). However, in the last 5 years, particle-packed columns filled with either sub-2-µm silica particles or with superficially porous particles (that is, core–shell particles) have been developed in parallel with the monolithic columns (13,14). As chromatographic theory has predicted, separation efficiency is inversely proportional to the particle diameter. This is why columns filled with smaller porous particles show very high separation efficiencies. Since the beginning of HPLC in the 1970s, the predominant particle size has constantly decreased from 10 µm to 5 µm and later to 3.5 and 3 µm. Recently, manufacturers have been able to produce fully porous sub-2-µm silica particles or core–shell particles and columns packed with these materials achieved impressive separation efficiencies above 200,000 N/m (15–18).

However, HPLC columns packed with sub-2-µm particles show some disadvantages. The resulting column back pressure of these columns is above 400 bar (~6000 psi). Therefore, the effective use of such columns, especially with lengths in excess of 100-mm, may require dedicated ultrahigh-pressure chromatography (UHPLC) instrumentation, which permits operation at pressures over 1000 bar. In addition, the frictional heat generated during the operation of these columns at very high pressures might lead to problems in overall chromatographic performance. Furthermore, blockage of the column bed is often observed in columns packed with sub-2-µm particles because of small porosity frits (to contain these particles), as well as their smaller interstitial volumes. Unless column protection devices (for example, guard columns, in-line filters, or sample membrane filtration) are employed, column lifetime may be jeopardized.

The more recent success of core–shell materials with particle sizes of 2.5–3 µm is associated with the fact that they offer high separation efficiencies (as high as sub-2-µm particles) with a much more favorable column permeability and corresponding lower column back pressures (15,16). The improvement of separation efficiency is because of an improvement in all terms of the van Deemter equation:

where H represents the theoretical plate height, A the eddy diffusion (great improvement here because of their narrow particle size distribution resulting in a more homogeneous packed bed), B the axial diffusion (which is inversely proportional to the linear velocity u), and C is the mass transfer depending on u.

The more favorable correlation of column back pressure and separation efficiency shown by core–shell particles as compared to fully porous particles also can be achieved with a new generation of silica-based monolithic HPLC columns (Chromolith HighResolution [HR]). Compared to the first generation of Chromolith columns, these new columns possess a more homogeneous porous silica network based on a well-designed silica skeleton in combination with a tailor-made bimodal pore structure (that is, macro- and mesopores).The chromatographic performance of this new generation of monolithic columns demonstrates improved separation efficiency and peak symmetry, especially for basic compounds, (for example, amitryptiline). In the following sections, the preparation and applications of these new columns is described.

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