1. INTRODUCTION
Although the benefit of using small particle sizes in combination with higher operating pressures in high-performance liquid chromatography (HPLC) was already predicted in the 1960s byGiddings (1964, 1965a)andKnox and Saleem (1969), the standard format for HPLC columns from the early 1980s were 4.6 mm250 mm columns packed with spherical particles of 5e10mm diameter. These columns were operated with HPLC instruments with an upper pressure limit of 400 bar and produced plate numbers of c. 25000 with a column dead time of 2e5 min.
In the late 1990s, studies were reported from the groups of Jorgenson (MacNair et al., 1997;
MacNair et al., 1999) and Lee (Wu et al., 2001) that made use of ultrasmall particles and high operating pressures. The first sub-2-micron particles were introduced by Agilent (Barber and Joseph, 2004) and Waters in2004; at this time also the first commercial instrument with an operating pressure up to 1000 bar was introduced by Waters (Swartz and Murphy, 2005). Since then, a wide range of ultra-high performance LC (UHPLC) instruments have become commercially available from almost every major instrument vendor, and a new termdUHPLCdwas coined for HPLC instruments and columns capable of operation above 400 bar.
In parallel to the progress in instrument development, new stationary phase morphologies were developed, with the promise of producing higher efficiencies with similar particle sizes compared to conventional particles. In particular, coreeshell particles have become very successful and are now available in various particle sizes (between 1.3 and 5mm) and chemistries (Gonza´lez-ruiz et al., 2015;
Guiochon and Gritti, 2011; Hayes et al., 2014; Tanaka and McCalley, 2016). Certain coreeshell materials are also available with larger pore sizes for the separation of biomolecules (Chen et al., 2015;
Fekete et al., 2012a, 2013; Wagner et al., 2012).
Simultaneously, the development of stationary phase chemistries has made huge progress. More and more phases are being developed for specific separation tasks: hydrophilic interaction liquid chromatography (HILIC) phases for the separation of polar compounds, pH stable and temperature stable phases, low bleed phases for use specifically with mass spectrometry (MS), and phases suitable for supercritical fluid chromatography (SFC).
Although many columns are offered in different length and diameters, HPLC users are faced with a choice of hundreds of columns to select from for a specific separation problem.
The requirements for separation speed and resolution range from ultrafast separations of only a few compounds, as used in the second dimension of two-dimensional LC, to ultrahigh resolution of complex mixtures, such as in proteomics or metabolomics. As the use of MS coupled to LC for complex separation problems has dramatically increased over the past decade, the properties of stationary phase materials with respect to compatibility with MS detection have also become an important point of interest.
In this chapter we will discuss the latest developments in stationary phase technology with respect to particle morphology, as well as particle and phase chemistries.
The first part of this chapter will focus on mass transfer properties of coreeshell particles compared to fully porous particles, covering the fundamental aspects of column performance as a function of morphology and particle size. The second part of the chapter will discuss new developments in stationary phase chemistry that are particularly important with respect to LC/MS analysis.
2. CORE e SHELL PARTICLES
One of the most important trends over the past 10 years was the development of high efficiency coreeshell or superficially porous particles. This type of particle holds the promise to deliver higher efficiency compared to a fully porous particle of the same size. The concept of using a solid core covered by a thin porous layer to enhance mass transfer was initially introduced by Horvath and Lipsky in the late 60s (Horvath and Lipsky, 1969a,b; Horvath, 1967) followed by various approaches in the 1970s to create pellicular particles such as Zipax, Corasil, or Pellicosil (Done and Knox, 1972;
Kennedy and Knox, 1972; Kirkland, 1972). The porous layer was impregnated with a liquid serving as the stationary phase, and the porous shell comprised only 5%e10% of the particle volume. Because of the limited particle porosity, the loading capacity of these particles was very low. The large size of these particles (c. 50mm) and the insufficient stability of liquid stationary phase limited the use of this type of particles.
A second generation of coreeshell particles was introduced in1992by Jack Kirkland and later commercialized by Agilent Technologies. These particles had an average size of 5mm, with a shell thickness of 0.25mm, and an average pore size of 300 A˚ . The general approach to produce these particles involved co-spray-drying an aqueous silica sol mixture and dense silica beads, so that a uniform porous shell formed around the solid core. The particle with the porous shell was sintered to give it strength and then rehydroxylated for subsequent surface chemical modification. These particles were mainly suited for the separation of large molecules such as proteins and peptides (Kirkland et al., 2000; Wang et al., 2006a,b).
The real breakthrough of coreeshell particles for high efficiency separations of small molecules started in 2007 with the introduction of the HALO particles (2.7mm with a shell thickness of 0.5mm) by Advanced Materials Technology (Destefano et al., 2008; Kirkland et al., 2007). To date, many column vendors have introduced coreeshell particles in sizes ranging from 1.3 to 5mm.
2.1 PRODUCTION OF CORE e SHELL PARTICLES
The production of modern coreeshell particles usually starts with producing a nonporous core using the Sto¨ber process (Sto¨ber et al., 1968). This process yields nonporous silica particles with a tightly controllable size distribution. Subsequently, a porous shell is added by either a layer-by-layer approach (Kirkland and Langlois, 2007) or by a one-step coacervation process (Chen et al., 2015; Chen and Wei, 2010).
2.1.1 Layer-By-Layer Process
In this method (Kirkland, 1970; Kirkland et al., 2000), large sol particles inw50e100 nm size range are used for coating. Only one layer of sol particles is coated at a time. To get a 0.25mm thick shell, five to six coatings must be applied. Between each coating, the excess polymer and sol particles must be washed out by filtration or centrifugation. This method is good for coating the large size sol and thin shell particles used for large molecule separations. Using this process to coat small sol particles of 10e16 nm would take 40e50 coats to get a 0.5mm thick shell. This would not be efficient or practical. To overcome this issue, a multi-multilayer method was developed (Kirkland and Langlois, 2007). One layer of polymer, usually poly (diallyldimethylammonium chloride), is applied on the cores. This polymer, depending on molecular weight, can absorb several layers of sol particles, and
the porous shell grows 5e10 layers at a time, and so the shell grows much faster. It takes fewer coating steps but still needs more than 10 coating steps to get a 0.5mm thick shell. It is still a labor intensive and time-consuming approach because of the numerous centrifugation steps that are needed to remove the extra material and loosely bound species in each coating cycle. Details of this process are shown inFig. 6.1.
2.1.2 Coacervation Process
In this method, the surface-modified solid silica cores are suspended in coacervation reaction mixtures of urea, formaldehyde, and colloidal silica sol under acidic conditions. A coacervate of ureae formaldehyde polymer and the ultrapure silica sol particles is formed and is then coated on the solid cores. The ureaeformaldehyde polymer is removed by burning in an oven, and the particles are then strengthened by sintering at higher temperatures. This process is shown inFig. 6.2and is described as follows:
1. Modifying the solid core surface with a proper functionality, such as ureaeformaldehyde polymer.
2. Coating the dense silica cores with a ureaeformaldehyde/silica sol coacervate film.
3. Eliminating the ureaeformaldehyde polymer from the outer coating by heating.
4. Sintering to increase particle strength and eliminating undesirable micropores.
5. Rehydroxylating the surface of the coreeshell particles.
6. Size classification to remove fines and aggregates.
FIGURE 6.1
Schematic representation of layer-by-layer process for synthesis of coreeshell particles.
Reprinted with permission from Chen, W., Jiang, K., Mack, A., Sachok, B., Zhu, X., Barber, W.E., Wang, X., 2015. Synthesis and optimization of wide pore superficially porous particles by a one-step coating process for separation of proteins and monoclonal antibodies. J. Chromatogr. A 1414, 147e157.