Reversed phase HPLC added salts

The Step 1 product (0.25 mmol) was added to the well of a reaction block containing 5 ml THF and then A-benzyl-A-ethylamine (0.5 mmol) was added neat. The reaction block was shaken 24 hours then filtered, concentrated, and purified using reverse-phase HPLC with water/acetonitrile (0.1% TFA) and the product isolated as a trifluoroacetate salt. 

To overcome the problem of tailing in reversed-phase HPLC, salts can also be added to the mobile phase - so-called ion-suppression. Ammonium carbonate, sodium acetate and sodium phosphate have been used for this purpose in the analysis of alkaloids. Also, ion-pairing has proved to be successful in alkaloid analysis. It allows analysis of alkaloids under acidic conditions, thus avoiding the problem of chemisorption of the basic compounds on the acidic silanol groups. 

To a stirred solution of 23.3 mg tosyl oxime (0.0236 mmol) in 3.5 mL EtOH at 0" C was added 450 /xL 50% aqueous KOH dropwise over 1 min. The reaction mixture was stirred at 0°C for 3 h then 5 mL 6 N aqueous HCl was added. The reaction mixture was heated to 60°C for 10 h, cooled to 23°C, and purified by loading to a reversed-phase Sep-Pak column with water containing 0.1% TEA (w/v) and by washing with 15% acetonitrile in water containing 0.1% TEA (w/v) to remove salts and then in 70% acetonitrile in water containing 0.1% TEA (w/v) to collect the crude product. The solvents were removed under reduced pressure to afford the hemiaminal intermediate, which was immediately mixed with 60 mg K2CO3 (0.434 mmol) in 2 mL THE 200 /xL H2O was added at 23°C. The reaction mixture was stirred for 10 min and purified by a similar procedure for the hemiaminal intermediate to afford the crude product. After the solvents were removed under reduced pressure, the crude material was further purified by reversed-phased HPLC. Concentration under reduced pressure provided 15.0 mg amino ketone as an orange/red oil, in a yield of 96%. 

Since it is necessary to remove solvent from the product, the mobile phase buffer must be considered. Some popular reverse-phase HPLC buffers, such as phosphates or zwitterion organic buffers, are nonvolatile. They must be replaced by a volatile buffer such as formic acid or ammonium acetate. Otherwise, a desalting step must be added. Trifuouroacetic add is another common buffer. Although it is fairly volatile, it forms a salt with the basic product and therefore cannot be completely removed from the final product. 

A solution of potassium nitrosodisulfonate (Fremy s salt) (0.56 mmol) dissolved in 5 ml water was added to a solution of the product from Step 7 (0.18 mmol) dissolved in 5 ml dioxane, the mixture stirred at ambient temperature 2 hours, and concentrated. The material was purified by reverse phase chromatography and the product isolated in 54% yield having a HPLC retention time of 1.75 minutes. 

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