Stationary phase and column technology

The column is often called the heart of the HPLC separation process, and the availability of stable, high performance stationary phases and columns is critical to the development of mgged, reproducible and robust methods. Modem commercial columns can differ widely among suppliers and these differences can sometimes affect the development process of the desired HPLC method. Specifically, different columns can vary in terms of plate numbers (Af), retention characteristics (X) and resolution (i s). For these reasons, column and stationary phase manufactures have developed technologies to help ensure that these separation materials are produced in a more consistent and reproducible maimer. An excellent reference by Snyder etal. [1] provides a comprehensive overview of modem stationary phases and column technology. 

Most HPLC column packing materials use silica particles as the basis of the support. Silica is known for its reliable strength and rigidity, relative inermess and ability to be modified chemically. Significant scientific effort has been devoted to developing and improving the morphological and physico-chemical characteristics of these silica materials (Fig. 2.4). 

One unfavourable characteristic of silica is its solubility at extreme pH values and at elevated temperatures, thus leading to reduced lifetime and deterioration of peak shape and resolution. With the advent of particles formed by the aggregation of silica sols, so-called sol-gel silicas, this property of silica has been significantly reduced. The recent development of hybrid silica particles also address this problem [59]. 

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The fast HPLC development in our work has closely followed the availability and improvements in chromatographic stationary phase and column technology. The initial methodology, based on Luna material, was centered on a 5- m particle size. These analyses with columns of 75 mm length and conventional 4.6 mm internal diameter gave analysis times of the order of 3.5 min (Fig. 3) with an overall cycle time, injection to injection, of almost 6 min. For such a cycle time the analysis of a 96-well microtiter plate (MTP) would be completed in 8 h, permitting the analysis of three MTPs per day. 

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