STANDARD OPERATING PROCEDURE Title: HPLC (SYKAM)

1. Purpose
a) To define the activities which need to be followed for working on the HPLC system in the Quality Control department.
2. Scope
a) It is applicable for chemical testing.
3. Responsibilities
a) QC Manager
b) Asst. QC Manager
c) Senior Analyst
4. Procedure
Operation of HPLC is based on its four parts.
1. Solvent Delivery System(PUMP)
2. Detector
3. Rheodyne Injector
4. Computing System( Signal Channel & computer)

1. Solvent Delivery System:
1.1 Insure that the instrument is clean and free from dust.
1.2 Put the power plug in the socket and switch it ON.
1.3 Switch on the pump by pressing “On/Of” button, located on back side of the instrument.
1.4 Press program button to set parameter e.g. flow rate, minimum pressure, maximum pressure, constant and analysis time.
1.5 Using up and down button adjust the required parameters and press stop button to exit the program.
1.6 Dip the inlet filter into the solvent (Pre-filtered and degassed HPLC grade water or Mobile phase or washing solvent etc)
1.7 Connect the syringe to the adapter in the waste port; twist the syringe slightly to make a leak free connection. Open the bypass valve by turning it anticlockwise.
1.8 Slowly pull the syringe plunger back, solvent steadily appears in the syringe collect two or three syringe volumes and ensures that no air bubbles are present in the solvent line, close the bypass valve.
2. Detector:
2.1 Switch on the detector by pressing “On/Of” button, located on back side of the instrument, after completion of self test the main window will display.
2.2 Press menu button twice to set wavelength, using “up () & down ()” button set your required wavelength and press “Start Status” button once to start the detector.
2.3 Press “Auto zero” button before going to run a sample.
3. Column & Injection System:
3.1 Join a required column (ensure the direction of flow of column) to the outgoing line of solvent delivery system to the upper part of the column and down ward of it is join with the line going to the detector.
3.2 Injection system is used to inject the sample into system.
3.3 Turn the injector (anti-clock wise) to load position, enter the needle to injector & inject the sample & rotate the valve (Clock wise) to inject position.
Note: Rotation of the injector valve & pressing of space bar of the computer key board will be done at the same time.
4. Computing System:
4.1 Switch on computer system.
4.2 Double click “Peak Simple” icon from the desktop of the computer, after completion of self test the main window will display.
4.3 Open file in which we want to work.
4.4 Run sample by pressing “Space Bar” of keyboard or by clicking Run from acquisition menu at top of the page.
4.5 Stop sample by pressing the end key of the key board or by clicking Stop from acquisition menu at top of the page.
4.6 Print Out result from file option.
Flow Diagram:

Switch off the whole system at the end of the working.
5. Precautions:
5.1 Clean the system before and after use.
5.2 Handle with care.
5.3 Give washing to the system with water first for at least 30 minutes when mobile phase containing buffer is used & then with methanol: water(70:30) for another 30 minutes , at the end give washing to the system with pure methanol for 20-30 minutes.
5.4 If mobile phase is free from water & buffer then give washing directly with methanol for 40-45 minutes.