Page 292 - Tandem Techniques

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level in each case is almost impossible to discern the actual difference in sensitivity can not be
determined.

Yamaguchi et al. [1] used optimized fluorescence in a similar way to measure fatty acid binding
proteins (FABP) in rat liver. Fatty acid binding proteins are thought to facilitate the transport of long
chain fatty acids in cells and may protect specific enzymes against inhibition by acyl-CoA esters. The
authors derivatized the column eluent with dansylundecanoic acid that binds to the FABP to give it
fluorescent properties. The reaction was carried out post-column, using the apparatus shown in Figure
7.16

Figure 7.16
Post Column Reactor for the Detection of
FABP with Dansylundecanoic Acid (ref.1)

The rat liver cytosolic fraction was injected directly into a Tosoh TSK gel G2000SW XL column (30
cm long, 7.8 mm I.D.) and eluted with 0.1 M potassium phosphate buffer (pH 7.2) at a flow rate of 0.5
ml/min. Subsequent to the column, the eluent was mixed with the dansylundecanoic acid reagent in a
low volume mixing T and then passed through a coil for the derivatizing reaction to complete. The
derivatized eluent was monitored with an Hitachi F1000 fluorescence spectrometer fitted with a 12 µl
flow cell. This cell volume might appear rather large, but it should be recalled that the column was
nearly 8 mm I.D and thus the peak volume was also fairly big. Consequently, an oversized cell volume
could be tolerated without adversely affecting the separation. The excitation wavelength was