HPLC/UHPLC Technical Tip
Level: Basic
Basic analytes and the benefits of modified silica supports in reversed-phase liquid chromatography
Silica-based stationary phases are currently the most frequently used phases in liquid chromatography. Traditional C18 columns often suffered from poor peak shape and tailing of basic analytes at low pH, and limited stability due to gradual silane hydrolysis at high pH. Through cross-linking and end-capping technologies, silica‘s undesired secondary interactions and limited stability have been significantly improved.
The most recent development introduced for analysis of basic analytes at low pH levels is the production of C18 columns with a residual positive charge on the silica support. This combination allows for 100% aqueous conditions to be used, enhancing retention of basic compounds. It also minimizes (ionic) secondary interactions between positively charged bases and silanol groups due to charge repulsion at the silica surface. The result is that basic compounds can be retained and eluted with good peak shape under acidic conditions, which are generally favored. Figure 1 shows an example of enhanced peak shape of chlorpheniramine, a basic antihistamine, using a Kinetex™ PS C18 column with this technology.
Figure 1. Improved reversed-phase retention and peak shape of chlorpheniramine with the Kinetex PS C18.
However, when high pHs are needed, new hybrid materials have been produced that greatly extend the high pH stability of HPLC sorbents. This new development allows users to use high pH to deprotonate basic compounds, increasing their hydrophobicity (and hence retention) whilst also removing the possibility of ion exchange. This approach is particularly useful when considering polar basic compounds as the combination of good peak shape with enhanced retention is useful, especially in LC-MS/MS where elution under higher organic mobile phase conditions has been shown to be beneficial for peak response. Columns such as Gemini™-NX and Kinetex EVO can be used in the pH range 1-12. Figure 2 shows the successful analysis of basic compounds at pH 8.2 using a Kinetex EVO C18 column with this technology.
Basic Compounds by LC/MS using a High pH Mobile Phase
We investigate the effectiveness of using high pH mobile phases for the quantitation of basic compounds in ESI+ LC/MS/MS by comparing limits of detection (LOD), limits of quantitation (LOQ), linearity ranges, precision, and accuracy observed in high and low pH mobile phases.
Using pH-LC to Control Selectivity of Acidic and Basic Compounds by HPLC
We investigate the effectiveness of using pH-LC techniques in the separation of a mixture of acidic (naproxen), basic (amitriptyline), and neutral (toluene) compounds using a Gemini 5 um NX-C18 column.
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