Difference between revisions of "HPLC - Plumbing Setup"

Revision as of 16:29, 14 May 2010

For Use in Non-Radioactive Applications

• Check that the Tubing from the Union Goes to the Column and not the radioactive injector. If not, disconnect the tubing at the union and connect the column tubing. Do not disconnect at the injector.
• Check the sample loop. You will need a sample loop at least 2X the volume of your sample. If your sample is > 5mL load using one of the pumps.
• If using a sample loop, inject at least 10X the volume of the loop through the loop twice using equillibration buffer. Make sure that you do not inject air into the sample loop.

Column Connections

• If the correct nonradioactive column is not connected, and you need to remove the current column do the following steps (this assumes the column is already in an appropriate storage buffer, ie 20% ethanol. If it is not then put into ethanol first. You do not need to do anything with the radioactive (metal) column):
  • Turn on the pump at 0.1 mL per minute and 100% equillibration buffer to get some liquid flowing through the system.
  • Gently disconnect the tubing from the top of the column and put the adaptor into a beaker to collect the buffer.
  • Put a stopper onto the top of the column (this is a finger tight fitting with no hole through it).
  • Disconnect the tubing from the bottom of the column and put a stopper on there as well.
• To connect a new column do the following:
  • With the pump running at 0.1 mL per minute, disconnect the stopper from the top and botom of the column and place the tubing (with fluid flowing through it) onto the column top.
  • Let the top of the column fitting fill with fluid (to avoid air bubble) and then tighten the fitting.
  • Wipe the top of the column with a kimwipe and check for leaks. If fluid is leaking out, either tighten the fitting or ensure the tubing is pushed as far down as possible.
  • Once the top is connected with no leaks, connect the bottom of the column to the appropriate tubing. For nonradioactive columns, this should be the piece of tubing which goes to the UV monitor. Tighten and check for leaks.

For Use in Radioactive Applications

• Check that the Tubing from the Union Goes to the radioactive injector and not a column. If not, disconnect the tubing at the union and connect the column tubing. Do not disconnect at the injector.
• Check the sample loop. You will need a sample loop at least 2X the volume of your sample.
• Inject at least 10X the volume of the loop through the loop twice using equillibration buffer. Make sure that you do not inject air into the sample loop.

Column Connections

• If the correct radioactive column is not connected (it should always be connected), and you need to remove the current column do the following steps (this assumes the column is already in an appropriate storage buffer, ie 20% ethanol. If it is not then put into ethanol first. You do not need to do anything with the non-radioactive (glass) column):
  • Turn on the pump at 0.1 mL per minute and 100% equillibration buffer to get some liquid flowing through the system.
  • Gently disconnect the tubing from the top of the column and put the adaptor into a beaker to collect the buffer.
  • Put a stopper onto the top of the column (this is a finger tight fitting with no hole through it).
  • Disconnect the tubing from the bottom of the column and put a stopper on there as well.
• To connect a new column do the following:
  • With the pump running at 0.1 mL per minute, disconnect the stopper from the top and botom of the column and place the tubing (with fluid flowing through it) onto the column top.
  • Let the top of the column fitting fill with fluid (to avoid air bubble) and then tighten the fitting.
  • Wipe the top of the column with a kimwipe and check for leaks. If fluid is leaking out, either tighten the fitting or ensure the tubing is pushed as far down as possible.
  • Once the top is connected with no leaks, connect the bottom of the column to the appropriate tubing. For radioactive columns, this should be the piece of tubing which goes to the radioactive monitor. Tighten and check for leaks.