Biomolecule Detection Using Fluorous Separations

The Yamaguchi group at Fukuoka University has published a new review of their work in biomolecule detection in J. Chromatography B.  They have been focused on the use of fluorescence derivatization and/or fluorous enrichment for the detection of molecules of interest by LC.  Within the review they divide their approaches into what they call detection-selective derivatization or separation-selective derivatization.  The former are derivatizations which increase the fluorescence signal of the analytes of interest.  The latter are derivatizations which enrich a sample in the analytes of interest.  Both methods result in greater sensitivity by essentially increasing signal-to-noise, or perhaps more precisely, signals of interest compared to signals of non-interest.

From a fluorous viewpoint of course, we’re interested in the separation-selective derivatization and the authors supply three examples from their lab using fluorous methods.  Each of these was previously published and we have covered them in F-Blog.  Despite that it is nice to see all three briefly described in one paper making it easier to compare and contrast.  The three approaches were each a little different.

Approach 1) Fluorous Derivatization: Pretty simple and straightforward.  In this case the researchers used a fluorous amine to derivatize carboxylic acids.  The sample is then enriched for the fluorous derivatized acids by fluorous HPLC.  This was best done on native fluorescent compounds so that any non-fluorescent acids are not detected.  The authors have demonstrated this approach by measuring NSAID levels from human plasma.

Approach 2) Fluorous Trap Fluorescence Derivatization:  This strategy was designed to fluorescence label analytes of interest, but provide a method by which excess labeling reagent can be easily removed so as to not intefere with detection.  The authors achieved this by using a fluorous Marshall resin acylating agent.  Upon reaction with an amino group containing compound, the amine gets fluorescence labeled and is non-fluorous.  Any excess labeling agent can then be easily removed by FSPE prior to LC analysis.

Approach 3) Fluorous Scavenging Derivatization:  Similar to approach 2 but in this case the excess derivatizing agent is reacted with a fluorous scavenger.  Carboxylic acids were derivitized by a fluorescent amine and excess amine was scavenged by a fluorous isocyanate.  The fluorous scavenger and fluorous-tagged excess derivatizing agent are then removed by FSPE.  For this example, the authors used fluorous silica gel in a micro-spin column format to conduct the FSPE.  Once again the removal of the excess reagents led to easier analysis by LC.

These three approaches are basically three of the major approaches used in fluorous based syntheses also; fluorous tagging, fluorous reagents and fluorous scavengers.  They’re just being applied to analytical applications instead.

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