A Practical Handbook of Preparative HPLC by Donald A Wellings

By Donald A Wellings

A distillation of over 20 years of the author's useful adventure of this technique, this publication intentionally steers away from complicated theoretical features and concentrates extra at the 'black paintings' components of the concepts concerned. accordingly is a publication for common use within the laboratory, specially of curiosity to these within the pharmaceutical, agrochemical and biotechnology industries and to somebody excited about the research and purification of substances, small molecules, proteins and DNA.

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Analysis and subsequent purification of peptides at high pH can be a useful tool for the chromatographer. It is not uncommon for impurities co-eluting at low pH to be completely resolved at high pH and the ability to operate at high pH is particularly advantageous for the separation of peptides rich in aspartic and glutamic acid. Probably one of the major advantages of polymerics lies in the extreme base stability, which allows cleaning in place (CIP) using concentrated sodium hydroxide solutions.

However, as the diameter gets much larger the effective depth of the cooling gets smaller in proportion to the diameter, so the effect on band broadening is lessened. 4 28 Fluid Dynamics, Mass Transport and Friction The effect can be counteracted by heating the column walls and nowadays it is commonplace to use jacketed columns. If a jacketed column is not available, the effect can be minimized by either lagging the column or by winding a tube around the column and circulating a heating fluid in the direction of the outlet to the inlet of the column.

The stationary phases prepared in the early stages of development of the technique suffered due to incomplete reaction of the silica surface. 1) are both hydrophilic and mildly acidic, resulting in a mixed interaction with the analyte and consequently leading to band broadening. This was particularly prominent for basic compounds due to cation exchange with the free silanol groups. Understandably, the first major development in this mode of separation was to block the unreacted groups with smaller chloroalkylsilanes such as chlorodimethylethylsilane in a process commonly termed ‘end-capping’.

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