A New Method for Measuring Charged Analytes in Complex Mixtures

Measuring by HPLC a particular component of complex samples such as natural products, body fluids, environmental probes is complicated due to interference from other components. Different sample matrices require different sample cleaning procedures prior to separation and as a result, one procedure cannot be used across a variety of products. The column’s operating life is usually short, even with sample cleaning, due to irreversibly retained contaminants. Thus, long cleaning procedures, guard columns, and expensive and complex compound specific detection techniques such as MS are often required. An example of such problems appear in the measuring of nitrates. All of the existing methods are complicated and take significant time to execute. Usually, the methods are specific only for the particular sample type.

FlipLC™ is an alternative method to avoid the interference of most of the contaminants by the use of an isolation column and a high pressure switching valve before the separation column. This method allows sample cleaning and analyte separation in one automated process.The isolation column and the separation column should have orthogonal retention characteristics to operate efficiently in this setup. Mixed-mode columns with reverse phase and ion-exchange characteristics were used in this analysis. The setting below employs a high pressure 6-port, 2-position switching valve which is placed between an autosampler and analytical column and actuated by the chromatography system by a defined time after the injection. 

An additional isolation column is connected to the valve’s ports and allows the switch of the position of this column in the chromatography flow path. In this setup, a sample is introduced to the isolation column first. The isolation column passes analytes through and retains the late eluted components of the sample. When the valve is actuated at a defined time after the injection (this time depends on the nature of the analyte) the whole plumbing system of the setting changed without flow interruption. After the valve switching (takes fraction of a second) the isolation column flip in reverse relative to the mobile phase flow and become connected to the detector outlet.  The late eluted components, while still in the isolation column, are back-flushed to waste by the flow coming from the detector outlet. The original plumbing arrangement restored prior to next injection.

FlipLC  has been used for measuring different charged compounds in complex mixtures.

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