Chromatography

The ideal HPLC hardware is one that measures both the UV absorbance of the eluate and the radioactivity with an on-line isotope detector. The two sets of data are then superimposed by the computer to allow for the identification of individual peaks (Fig. 2). Chemicals should be HPLC grade and buffers degassed before use.

1. Use a 5-|im encapped C18 column (Lichrospher, Merck, Darmstadt, Germany) with an equivalent precolumn. The precolumn prevents any particulate matter, such as phospholipids, from entering the column and is changed at regular intervals.

2. Inject the sample onto the column.

3. Elute at 1 mL/min using the following mobile phases. Solvent A, 1% acetic acid in MilliQ water. Solvent B, acetonitrile/methanol 3:1. The initial conditions are 60% solvent B rising linearly to 100% solvent B over 25 min.

4. Monitor the eluent at 351 nm with a UV detector and the radioactivity with an isotope detector.

Fig. 2. (continued) (peak 3) and tRA (peak 4) are clearly identifiable. Peak 2 coelutes with authentic [3H] tRA, suggesting that the mouse embryo contains significant quantities of tRA and a good deal of retinol. The peak on the extreme right of the chromatogram is an unknown, and the peak to the left of arrow 5 is butylated hydroxy-toluene, the antioxidant added to the extraction solvent.

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