High Performance Liquid Chromatography

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Purpose

This protocol is used to purify various biomacromolecules. This particular method is for purifying a protein that has antibiotic activity from Dr. James Smith's lab.

Materials Needed

-HPLC Machine

-10 mL syringe with needle

-10 mL syringe without needle

-waste containers

-99.9% distilled water/ 0.1% Trifluoroacetic acid (Buffer B)

-99.9% acetonitrile/ 0.1% Trifluoroacetic acid (Buffer A)

-graduated cylinder

-stopwatch

-collection tube for desired sample

Protocol

-Insure that there are no solid particles in the sample to be used by centrifuging the sample and collecting the supernatant. Solid particles can clog the injector and column, causing blocks and leaks

-Insert the appropriate tubes for buffers A and B into their respective containers

-Use a syringe without a needle attached to take out some of each buffer from the tubes to get rid of any air bubbles that may have been formed while transferring the tubes to the buffer containers, this is done at the base of the machine

-In the Biologic Duoflow software, set the machine to purge and hit start, this will flush out the tubes and column with buffers A and B to insure there is no extra material still in the system, stop it after about 30 seconds of purging

-Set the machine back to load, the flow rate to 3mL (when using a 3 mL column) and the percentages of buffers A and B to 50%

-Start the machine and use a graduated cylinder and stopwatch to make sure the flow is not blocked and 6 mL of fluid has been collected after 2 minutes

-If the flow rate is not correct, then set the machine to 100% A and 0% B and check again, if its still not right then A is clogged, if it flowing at the correct rate now, then so 100% B and 0% A to see if B is clogged

-When the flow rate is correct, stop the machine and select the protocol you wish to run

-Click "New Run", title the run desired and click "Ok"

-Collect 5 ml of the sample in a syringe and inject it into the injector port

-Click "Start" and let the machine run

-Collect the liquid when the peak that represents the material desired appears on the screen

-When finished, always wash the tubes and column with methanol using the same method used at the beginning to test the flow rate, but with both buffer tubes in the methanol, let it run for a few minutes

Notes

A basic understanding of the HPLC machine is needed to follow this protocol. Refer to the owner's manual of your particular machine for more information on how your machine works

References

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