User:Jordan L. Metsky/Notebook/Phosphorylation/2012/01/27

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Objective

To isolate t-butyl ammonium ATP.

Description

1. 1 L of a 50 mmol solution of tert-butylammonium chloride was made.* 2. A column was elluted with 100 mL of the previously prepared 50x10^-3 solution of tert-butylammonium chloride. Beads swelled for 20 minutes. 3. After 20 minutes, the column was elluted with 400 mL of the tert-butylammonium chloride solution. Fractions were poured off. 4. 0.5 g of ATP was massed, and dissolved in about 100 mL of tert-butylammonium chloride solution. The mixture was added to the column using a Pasteur pipette. 5. The column was elluted with an additional 300 mL of tert-butylammonium chloride solution. 2 fractions were collected as follows: Fraction A was 150 mL-hypothetically containing the ATP solution and saved; Fraction B was 200 mL and poured off. 6. Fraction A was filtered using filter paper to remove solid matter (the mesh media from the column that had escaped into the solution), and transferred to a 500 mL flask. 7. The solution was distilled using a rotovap with H2O. 8. The filtrate obtained was a beige, opaque, oily residue.

  • Steps 1-5 are based off of the procedure of Hoard and Ott's "Conversion of Mono- and Oligodeoxyribonucleotides to 5' Triphosphates" from the Journal of the American Chemical Society, 1964.

Data

1 L of 50 mmolar soln of tertbutylammonium chloride (C16H36ClN): (277.95 g/1 mol C16H36ClN) x (x g/50 x 10^/3 mmol) =13.896 g in 1 L H2O

Notes

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