By Donald A Wellings
A distillation of over 20 years of the author's sensible adventure of this approach, this ebook intentionally steers away from complicated theoretical features and concentrates extra at the 'black paintings' parts of the suggestions concerned. consequently is a booklet for common use within the laboratory, in particular of curiosity to these within the pharmaceutical, agrochemical and biotechnology industries and to someone all for the research and purification of substances, small molecules, proteins and DNA.
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Extra resources for A Practical Handbook of Preparative HPLC
However, stationary phases from different manufacturers will behave differently, so it is important Modes of Chromatographic Separation 35 to reiterate the need to screen seemingly identical materials to get the optimum separation required. 1 Normal Phase Chromatography The IUPAC Compendium of Chemical Technology defines ‘Normal Phase’ as ‘an elution procedure in which the stationary phase is more polar than the mobile phase’. In practice, the most widely used stationary phases for preparative HPLC are based on silica and the polarity of the underlying silyl ether and silanol provides the required hydrophilic surface.
The control of pore size and structure during preparation is a complex process that has been mastered by few manufacturers, but it is true to say that pore size distribution and uniformity is more readily achieved in silica particles than it is in polymerics. This is not to say that polymerics are inferior, because in contrast these stationary phases have a more uniform particle size. Remember the forest? The branches of an Oak are more diverse than those of a Spruce! In practice, the less efficient mass transfer within polymeric stationary phases results in a lower optimum flow rate being obtained following a van Deemter plot.
There is also a range of alkyl chain lengths available but the most popular remain to be C8 and C18. 1 so long-term operation at high pH can be limited. The introduction of macroporous polymeric stationary phases has broadened the pH range for reversed phase HPLC since these media are stabile from pH 1 to pH 14. The most commonly used polymeric reversed stationary phases are based on copolymers of styrene and divinyl benzene. The hydrophobicity of these media is derived from the polymer backbone and pendant benzene rings, so the elution profiles are often expected to be different Modes of Chromatographic Separation 41 from a typical C18 coated silica for instance.
A Practical Handbook of Preparative HPLC by Donald A Wellings