One would think that the mass of an extremely pure nucleotide could be reliably determined on a laboratory balance. Not so, because during the manufacturing process, nucleotide prepara- tions typically accumulate molecules of water(via hydration) and counter-ions (lithium or sodium, depending on the manufacturer) which signficantly contribute to the total molecular weight of the nucleotide preparation. Unless you consider the salt form and the presence of hydrates, you're adding less nucleotide to the solution than you think. The presence of salts and water also contribute o the molecular weights of oligo-and polynucleotides, which are also most reliably quantitated by spectroscopy H Adjustment The pH of a solution prepared by dissolving a nucleotide in water will vary, depending on the ph at which the nucleotide triphosphate was dried. An aqueous solution of nucleotide triphosphate prepared at Amersham Pharmacia Biotech will have a pH of approximately pH 4.5. The ph may be raised by addition of Naoh (0. 1n NaOH for small volumes, up to 5n NaOH for larger volumes). Approximately 0.002 mmol NaoH per mg nucleotide triphosphate is required to raise the pH from 4.5 to neutral pH. If the pH needs to be lowered, addition of a H* cation exchanger to the nucleotide solution will lower the pH without adding a counter-ion. The amount of cation-exchanger resin per volume of 100 mM nucleotide solution varies greatly depending on the starting and ending pH For very small volumes(<5 ml) of icleotide solutions, a 50% slurry of SP Sephadex can be added dropwise. For larger volumes(>5ml), solid cation exchanger can be added directly in approximately 0.2cm'increments The cation exchanger can be removed by filtration when the desired ph is obtained The triphosphate group gives the solution considerable buffer ng capacity. If an additional buffer is added, the ph should be checked to ensure that the buffer is adequate. The ph should be adjusted when the solution is at or near the final concentration. A significant change in the concentration will change the pH.An increase in concentration will lower the pH, and dilution will raise he ph, if no other buffer is present Similar results will be obtained for all of the nucleotide triphos- phates. Monitor the pH of the solutions as a precaution; purines are particularly unstable under pH 4.5, and all will degrade at acid ph 274 GersteinWeighing One would think that the mass of an extremely pure nucleotide could be reliably determined on a laboratory balance. Not so, because during the manufacturing process, nucleotide prepara￾tions typically accumulate molecules of water (via hydration) and counter-ions (lithium or sodium, depending on the manufacturer), which signficantly contribute to the total molecular weight of the nucleotide preparation. Unless you consider the salt form and the presence of hydrates, you’re adding less nucleotide to the solution than you think. The presence of salts and water also contribute to the molecular weights of oligo- and polynucleotides, which are also most reliably quantitated by spectroscopy. pH Adjustment The pH of a solution prepared by dissolving a nucleotide in water will vary, depending on the pH at which the nucleotide triphosphate was dried. An aqueous solution of nucleotide triphosphate prepared at Amersham Pharmacia Biotech will have a pH of approximately pH 4.5. The pH may be raised by addition of NaOH (0.1 N NaOH for small volumes, up to 5 N NaOH for larger volumes). Approximately 0.002mmol NaOH per mg nucleotide triphosphate is required to raise the pH from 4.5 to neutral pH. If the pH needs to be lowered, addition of a H+ cation exchanger to the nucleotide solution will lower the pH without adding a counter-ion. The amount of cation-exchanger resin per volume of 100 mM nucleotide solution varies greatly depending on the starting and ending pH. For very small volumes (<5 ml) of nucleotide solutions, a 50% slurry of SP Sephadex can be added dropwise. For larger volumes (>5ml), solid cation exchanger can be added directly in approximately 0.2 cm3 increments. The cation exchanger can be removed by filtration when the desired pH is obtained. The triphosphate group gives the solution considerable buffer￾ing capacity. If an additional buffer is added, the pH should be checked to ensure that the buffer is adequate. The pH should be adjusted when the solution is at or near the final concentration. A significant change in the concentration will change the pH. An increase in concentration will lower the pH, and dilution will raise the pH, if no other buffer is present. Similar results will be obtained for all of the nucleotide triphos￾phates. Monitor the pH of the solutions as a precaution; purines are particularly unstable under pH 4.5, and all will degrade at acid pH. 274 Gerstein