HPLC - Running a Sample or Series
From Bridges Lab Protocols
Column and Plumbing Setup
- To connect or disconnect a column or sample loop see HPLC - Plumbing Setup
- Prepare and filter buffers. There is no need to degas the buffers (the system has inline degassing). If you need your sample to be run cold then pre-chill buffers and fill the buffer container with ice to keep the buffers cold (this will also keep the column cold).
- Prepare sample (either filter if > 2 mL or centrifuge at 14 000 RPM for 15 min if < 2 mL). If your sample is being injected via a pump, make sure your sample is in one of the buffer tanks.
- If using the fraction collector, load the appropriate rack with tubes and set the fraction collector parameters.
- Rack should be 0 for the eppendorf block, 1 for the 13 mm tube rack and 15 for the 96 well plate rack.
- Set the time of collection
- If there is a TEV1 remote start signal then you dont need to do anything else. Otherwise you will have to manually press start.
Initial Software Setup
- Turn on TotalChrom Navigator and login.
- Click Hands On
- Set the pump to 0.1 mL/min and 100% equillibration buffer
- Click 'Start Pump
- Under Detector Click Turn Lamp On if it is not on already. There is no need to set the wavelength (this will be defined in the method).
Method Setup
- Click on Setup and select either method or sequence
- Pick your Method (see HPLC - Designing a Method or Sequence )
- Select a filename for saving the raw results file and then click OK
Injection - Only if using sample loop
- If sample is loaded via pump, skip this section.
- With valve set to load (turn all the way counter-clockwise), rinse sample loop twice with 10X volumes of equillibration buffer. If using the Hamilton syringe remove the luer lock syringe fitting first. Make sure not to inject air into the sample loop.
- Suck up your sample and inject into sample loop. This volume should be less than half of the loop volume.
Run Start
- On TotalChrom Navigator, wait until the status is colored green and says ready (not NREADY). After you load your method, you need to wait for the column to equillibrate (step 0 in your method).
- To start run, turn the valve counter clockwise until it stops. The valve must remain in this position for at least as long as the injection time (ie for a 1 mL injection at 1 mL/min you need to keep it on inject for at least one minute). There is no problem with leaving it on inject, but you need to put it back into the load position to reload or run another sample, so turn it back at some point during the run.
- Data will be collected, saved and processed automatically.
