30 May / June 2016
Recent Developments in Support Materials for use in High Performance Liquid Chromatography
by David McCalley, Centre for Research in Biosciences, University of the West of England, Bristol BS16 1 QY U.K.
A number of advances have taken place in the last decade in the area of support materials for high performance liquid chromatography (HPLC). These developments have included the use of very small porous particles (< 2.0 µm diameter), new types of superficially porous or ‘shell’ particles and the development of a second generation of silica monolithic columns with increased efficiency compared with earlier versions. This article will consider these developments mostly with regard to their use in the analysis of small molecules.
Very Small Particle/ Shell Particle Columns
The potential of very small particles for use in HPLC has been recognised for a long time. However, only in the last 10 years or so, since the introduction of commercial instruments capable of high pressure operation together with low extra-column band spreading properties, has it become possible to exploit the advantages of these particles. Small particles give rise to higher effi ciency for the same column length, but their benefi t is principally in generating similar effi ciencies to larger particle columns in a shorter analysis time. A 25 cm column of 5.0 µm totally porous particles may generate 25,000 theoretical plates, which should also be readily achieved on an 8.5 cm column of similar 1.7 µm particles, assuming a reduced plate height h=2 in both cases. As the 1.7 µm column is only about one third of the length, it should produce results 3x faster than the large particle column. The optimum fl ow rates of small particle columns are also higher, so considerably greater increases in speed are possible. Shorter retention means less solvent consumption. As high pressure instruments are designed with low extra-column bandspreading, narrower columns can also be used e.g. a 2.1 mm i.d. column operated at the same fl ow velocity uses approximately one fi fth the solvent of a 4.6 mm column. Thus further reductions in solvent consumption result. The smallest commercial particles have a particle diameter of 1.3 µm but optimum fl ow may not be reached due to pressure limitations even if relatively short (e.g. 5 cm) columns are used. The advantages of small particle columns can be further enhanced by the use of small shell particles instead of totally
Figure 1. (Upper) Layer by layer process and (lower) coacervation process for production of shell particles.
porous materials, which have a coating of porous material surrounding a solid non- porous core [1].
The use of very small particle columns is, however, not without practical diffi culties. The most obvious consequence is the higher back pressure generated. As pressure is inversely proportional to the particle diameter squared, a 1.7 µm column requires ~ 9 times the pressure compared with a 5.0 µm particle column operated at the same fl ow velocity, necessitating pumps of improved capability. Other diffi culties,
such as frictional heating and selectivity differences may also result
Core shell particles. Preparation. These particles are now available from many HPLC column manufacturers. Although methods of preparation differ in their details, two common approaches are outlined in Figure 1 [2]. The layer by layer process consists of adding a cationic polymer to solid silica cores (made by the Stöber process) held at high pH such that the silanols are negatively charged. After elimination of excess material, a suspension
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