SOP FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1.   Objective:  To lay down the procedure for the operation of HPLC. 

2. Responsibility: It is the responsibility of the Technical Assistant, Technical
Manager, Quality Manager, and all those individuals who are working on the
instrument.
3.   Instrument Details:
     Make- .................., 
Model- .................................... 

4.   Procedure: Enter in the log book.

4.1.  Purpose: The following document describes the procedure for the Operation of
the HPLC.
4.2.  Scope: This instrument is used to separate and quantify organic compounds in
pharmaceutical formulations and biological fluids.
5.   Precautions:
· Always use a guard column to avoid clogging of HPLC analytical column.
· Filter the mobile phase with 0.45µm filter paper. 
6.   Procedure: 
6.1.  Degassing Solvents 
·     Ensure there are enough degassed solvents before running a sample.
·     Get a 500 mL or 1000 mL Erlenmeyer filter flask.
·     Sonicate the solvent/Mobile phase for 5-10 mins
·     DO NOT HEAT.
·     The solvent is degassed when bubbles no longer appear
6.2. Starting the Instrument 
·     Turning on the Instrument
·     Turn on the Column oven
·     Turn on the Liquid Chromatographs
·     Turn on the UV Detector
·     Starting the Computer
 ·     If the computer is off, log in to the system
·     Open the program by Clicking on the HPLC Icon.
·     Click OK (No password) 
·     Click Empower LC Icon
6.3.Creating a Method
·     Click the Method drop-down menu
·     Click LC Set-Up
·     Set up the instrument mode
·     Set the run Time of your method
·     Define the flow rate
·     Percent Concentration Adjusts the graph limits.
·     Click parameters, Adjust flow rate, and Adjust the pressure.
·     Click OK.
·     Click the File drop-down menu Click Exit
6.4.Saving the Method
·     Click the file drop-down menu
·     Click save as
·     Enter desired name of the method
·     Click Save
6.5.Aquisition Set-Up
·     Click the set-up icon
·     Select Sample Frequency
·     Ensure your frequency is 1 unit greater than your sample requires
·     Enter Run Time
·     Default is 5 minutes
·     Save the method again
6.6.Running the sample
·     Select the run single icon (Which has a blue arrow pointing to the right)
·     Enter sample identification name
·     Select the desired method (Select from browse button)
·     Select the file destination (Select from browse button)
·     Click start
6.7.Injecting the sample
 ·     A blank should be run first
·     Make sure the knob is turned to load
·     Insert sample using a blunt tip syringe
·     Quickly turn the knob up to inject the sample
7.   Select analyze icon
·     This will display retention times and peak areas
8. Shutting down the instrument
·     Wash the column with 50% water and 50% methanol or acetonitrile for 10
min.
·     To Stop the pump change the flow rate to 0 ml/min.
·     Click on the pump icon.
·     Turn off the Column oven & Turn off the Liquid Chromatography
·     Turn off the UV Detector
·     Exit from the HPLC software.
·     Shut down the computer.
END OF THE DOCUMENT