High Performance Liquid Chromatography (HPLC) Protocol

First-time users, please contact Mr Pengjun Xia (pjxia@udel.edu ) in the Parashar laboratory or Aakash Koppula (aakash@udel.edu) from Batish laboratory. They will demo the operation of HPLC using Chromeleon software, including connecting, purging, cleaning, designing protocols, running, and shutting down.

Materials

• Buffers

Protocol

1. Prior to your experiment, OPEN the column module and make sure you are using the correct column. If you need to switch the column, install it in the correct Direction.

2. Operate HPCL using Chromeleon software.

3. Connect all modules, turn on the UV and Vis, and turn the “acquisition” on.

4. Switch the tubing to your buffers, A to Buffer A (aqueous) and B to Buffer B (organic) and set the flow rate.

5. Unscrew the valve in Pump module, the pressure will become 0 bar, and start purging.

6. Purge will run for 5min. Screw the valve once the purge completes.

7. Washing the column with 90% Buffer B for 2-5 min.

8. Run the “cleaning” protocol by injecting Buffer A as your sample.

9. Now you can start loading and running your samples with

10. When you finish, run the “cleaning” protocol again.

11. For the storage of column, switch tube A back to H2O, purge, and run 30% Buffer B for at least 5min. If you will use the same column the next day, it can be stored in your buffers.

Tips:

1. Always filter your buffers (buffer A).

2. Deproteinization: make sure no proteins (or other big molecules) are loaded to the columns.

3. Components in your buffer may form insoluble chemicals

4. Don’t leave both ends of your column open.

5. Cleaning protocols should be your first and last run, loading and injecting buffer A as your samples. Cleaning is strongly suggested in between of two runs.

6. Don’t forget but the “inject” back to “load” when you finish

7. Storage of column: in water + Buffer B (methanol or acetonitrile)

8. Record the pressure of your column when you’re running the last cleaning protocol.

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