Isocratic Elution of Analytes Differing Significantly in Polarity on Discovery® HS F5

By: Dave Bell, Reporter EU Volume 11

Dave Bell

Role of Stationary Phase Chemistry Toward Retention

Retention and selectivity observed in chromatographic processes are the manifestation of the multiple types of interactions between the analytes and the stationary phase. Traditional alkyl phases such as C18 are designed to operate primarily by hydrophobic interactions and exhibit minimal polar interactions. These properties often provide for predictable method development and smooth operation, however, in many cases analytes that vary significantly in polarity require gradients to perform the analysis efficiently. Although gradient elution is acceptable practice for some applications, isocratic solutions are still favoured.

Polar reversed phase columns, by design, impart polar interactions of retention as well as the hydrophobic interactions available on traditional C18 phases. The additional polar interactions can be utilised to alter the selectivity for a given set of analytes. In this report, such manipulation of the polar and hydrophobic interactions provided by a pentafluorophenylpropyl stationary phase (Discovery HS F5) is demonstrated using the antiparkinsonian drug, selegiline, and two of its polar metabolites.

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Retention and Separation on Discovery HS F5

Selegiline and two of its metabolites (amphetamine and methamphetamine) vary significantly in polarity (Figure A). Using alkyl stationary phase chemistries, low percentages of organic solvents are required to retain the polar amphetamines. Under the same conditions the relatively non polar selegiline solute is highly retained resulting in an overall run time of about 30 minutes.

Due to the polar interactions available on the Discovery HS F5 phase, the amphetamines can be retained using greater percentages of organic modifier. Under these conditions, selegiline and the amphetamine analytes elute in less than 5 minutes as shown in Figure A.

Figure A Retention of Selegiline and Polar Metabolites on Discovery HS F5 (567513-U)

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The availability of polar interactions provided by modern stationary phases such as the Discovery HS F5 provides new opportunities for optimal HPLC method development. In those cases where C18 phases provide too much resolution or inadequate retention of polar analytes, polar reversed-phases are likely to provide superior performance.

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