Experiment 63 HPLC Separation of Nucleotides

Prepare 500 mL of a mobile phase that is 25 mM KC1, 5 mM MgCl2, and 50 mM tris-(hydroxym-ethyljamino methane (TRIS or THAM). Adjust the pH to 7.2 using concentrated HC1. Filter and degas this mobile phase using a vacuum filtration apparatus equipped with 0.45-/LL filters. 

The first part of this experiment uses an HPLC instrument with an anion exchange column installed. Flush the flow path with the mobile phase at a flow rate of 1.25 mL/min for 10 min. 

Prepare separate solutions of four nucleotides (concentration not important). Either DNA or RNA nucleotides maybe used. Filter approximately 2-mL quantities of each nucleotide solution into small vials. Also prepare and filter a mixture of all four by combining small quantities of each in one vial. 

Inject 20 pL of each sample and observe the retention times. Then inject 20 pL of the mixture. Record all experiment parameters, e.g., flow rate, attenuation, pressure (if pressure gauge is installed), etc. Be sure to print hard copies of all chromatograms. When finished, flush the flow path for 10 min with filtered and degassed methanol. 

Install a reverse phase column and repeat. Do not change the parameters recorded in step 4. When finished, again flush the flow path with filtered and degassed methanol for 10 min. 

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