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
You may like to read these links:
1. List of All SOPs and Documents for Laboratory Instruments and Equipment
2. List of All SOPs and Documents for Chemistry Laboratory
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