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While reversed phase is the most widely used mode of retention in HPLC, very polar compounds are often not sufficiently retained in low percent organic, or even in 100% aqueous mobile phase.
By using a strongly polar bonded phase such as TSKgel Amide-80, polar compounds can be retained by a normal phase or hydrophilic interaction chromatography (HILIC; see references) retention mechanism using an mobile phase mixture of acetonitrile and ammonium acetate buffer. In contrast to the retention behavior in reversed phase, in HILIC solutes are retained longer by increasing the percent acetonitrile. |
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Features |
Benefits |
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Stable bonded phase |
- Amide-80 bonded phase does not react with reducing sugars (unlike amino columns).
- Can be used with evaporative light scattering (ELS) and mass spec (MS) detectors.
- Max. operating temperature: 80°C.
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TSKgel Amide-80 |
Unique
polar phase |
- Retain polar drug candidates and drug metabolites
- Handles a wide spectrum of sample polarities.
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Stable in 100% organic eluents
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Separates many different types of polar molecules | |
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The 'normal' or traditional way of doing liquid chromatography, normal phase liquid chromatography (NPC) uses a polar stationary phase and a non-polar mobile phase. The polar surface can either be obtained by derivatizing the support with a ligand containing a polar functional group, or it can be the original (polar) support, as in the case of silica gel.
The popularity of bare silica gel declined after the introduction of bonded phases, since bonded phases feature shorter equilibration times and allow the use of gradient elution. Cyano, amino, and diol bonded phases are the most common normal phase column types.
The order of elution in normal phase is opposite that found in RPC for the same mixture of compounds. Although non-polar organic mobile phases and a silica stationary phase were used traditionally in normal phase LC, today most normal phase separations are performed with aqueous-organic mobile phases and a more polar-bonded stationary phase. This mode of HPLC is now commonly referred to as HILIC, hydrophilic interaction chromatography.
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