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HPLC Protocol

March 2004

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Prepare your buffers/solvents. Filter before using. Turn on instruments in this order: computer, pump, degasser, UV detector. Login to Millenium software: system->manager CHECK YOUR SOLVENT LEVELS! For NTP purification (Vydac Ion Exchange semipreparative column): Install column. Right click Run, select akUVpump->NTPseparation50min Put solvent tubing in ddH2O bottle Select Method Set NTPseparation50min Select Instrument Method ntpseparation50min Make sure injection volume is 200uL, run time is 50min. Injection loop should be 200uL. Click SetUp below Instrument Method, watch window for messages at bottom of screen. When says Idle, click Monitor to observe pressure and UV. 13. CHECK YOUR SOLVENT LEVELS! 14. Click faucet on icon (bottom left of screen), edit to run 50% A, 50% B at 6mL/min. Click OK. 15. Flush lines with 50% Buffer A, 50% Buffer B for 15min. Column is stored in 50% methanol. This step flushes both lines of residual solvent and flushes methanol out of column. Collect MeOH waste in hazardous waste storage. 16. After 15 min click faucet on icon, edit to run 100% A at 6mL/min. Click OK. 17. Flush lines with 100% A for 2 min. 18. Stop flow by clicking faucet off icon. 19. Now put solvent tube A in Buffer A (12.5mM Na2HPO4/12.5mM NaH2PO4 pH 2.8) and tube B in Buffer B (62.5 mM Na2HPO4/62.5 mM NaH2PO4 pH 2.9) 20. Start flow by clicking faucet on icon. Edit to run 50% A and 50% B at 6mL/min. Click OK. Let run 15 min. 21. After 15 min, click faucet on icon, edit to run 100% A at 6mL/min. Click OK. Let run 10 min. 22. Prepare for injection: Click Red Abort icon at top left of screen. Wait until message at bottom of screen says User aborted. (Pump will continue to run, but screen no longer monitors pressure and UV absorbance in real time.) 23. Click the Prepare Injection icon. Draw sample into flat-bottomed Hamilton syringe. Make sure Rheodyne manual injector is in Load position. Gently inject sample. Leave syringe in place. When screen gives message Single injection waiting turn Rheodyne manual injector to Inject position, wait for beep and keep in Inject for 15 sec or as long as it takes given your flow rate to ensure sample gets loaded onto column, and return to Load position. Remove syringe. Screen message should say Single injection running and pressure and UV monitoring should start automatically. 24. Experiment runs for 50min. After exp, begin next injection (repeat steps 21-22). Check your solvent levels at start of every run. 25. At end of all runs, flush lines with ddH2O. (Click faucet off icon. Put both solvent tubes into ddH2O. Click faucet on icon. Edit to run 50%A and 50% B. Click OK. Let flush 20 min. 26. Click faucet on, edit to run 100%A. Click OK. Let run 15 min.) 27. Click faucet off. Put both solvent tubes into 50% MeOH. Click faucet on and edit to run 50% A. Click OK. Let run 20 min. 28. Click faucet off and put both tubes back into ddH2O. Now flush lines with ddH2O to remove MeOH. Open reference valve, disconnect line to column and seal up column. Click faucet on, edit to run 100% A, 5mL/min for 10 min. 29. In this order, turn off UV detector, pump, and degasser. Log out of software.

General use: 30. Flush lines with water. Open reference valve and disconnect column. Flush lines at rate 5-10mL/min for 5 min. Measure flow rate out of pumps. Prime pumps. If output less than the rate you specified, check for consistency and note the actual flow rate. 31. Stop pump, close reference valve, reconnect column. Flush lines at lower rate 1-3.5mL/min for 10 min.