Operational SOPs for HPLC (High Performance Liquid Chromatography)

In this post, we will study Operational SOPs for HPLC (High Performance Liquid Chromatography).  All the related outline, scope, responsiblity and procedure you will study here.

1.      PURPOSE

To outline work instruction for “HPLC” in Quality Control Department.

2.      SCOPE

To separation of different material in solution in Quality Control Department.


All analysts are responsible to follow the instruction.   


4.1   Instrument Information

  1. Name  HPLC
  2. Identification No.
  3. Model (Pump)                   L-2100/2130
  4. Model (UV-VIS Detector)   L-2420
  5. Range wave length           190-900 nm
  6. Operating Temp. Range     15 ºC to 25 ºC          

4.2 Method of Operation

  • Connect the instrument to power supply.
  • Switch on the instrument by pressing “on/off” button.
  • Do purge for 5min maximum with solution of methanol: water with ratio of 20:80. Now keep running pump for washing for at least 20-30minutes.
  • The detector will go through the process of wave length and lamp initialization by itself and main menu will appear with six functions i.e. (a) time (b) wave length (c) Abs. (d) Prog. (e) Lamp (f) [ I ]
  • Press ‘wl’ detector, now select your desired wave length and put value in it and press ‘ENT’ button.
  • Pump menu will appear with three functions i.e. (a) time (b) flow (c) pressure.
  • Press ‘Manual’ button, will be selected with two functions (a) Flow = 1, (b) Press = 2.
  • Again Press ‘1’ Flow = 1 will be selected; now press ‘ENT’ button and select your desired rate of flow and put value in it. Also desired max and min pressure will be selected and put value in it.
  • Similarly select other functions and feed their values.
  • 4.3   Software:
  • First ON HPLC instrument
  • Then ON software of HPLC in PC.
  • Enter in “injection” press “add” then “new injection” and then set changes.
  • Press “New” and enter “sample name” and “stop time”.
  • Now set path and select desired location where chromatogram should save
  • After pressing “Add In” it will visible in software.
  • Press “Method” and select desired method.
  • To start analysis press “Analysis” and “start analysis”
  • for print: enter in Injection History.
  • Print preview then press print.


  • Rinse suction filter with new mobile phase.
  • Put suction filter in new mobile phase.
  • Open Drain Valve of pump (Anticlockwise)
  • Set flow rate of pump 2~3 ml / min and start the flow.
  • After 5 minutes stop the flow.
  • Select the required flow rate and press Enter
  • Close drain Valve (Clockwise) and wait for stable base line.


Use of buffer solution as the mobile phase will produce crystalline substances dehydration or evaporation of the solution, which damage the plunger and plunger seal and shorten their service life. When using a buffer solution, accordingly, it is recommended to wash the flow line frequently, after completing the analysis.

  • Suck washing solution with the disposable syringe and insert the needle end into the rinsing pipe.
  • Feed the washing solution to the washing flow line slowly.
  • Remember it is possible to conduct the washing irrespective to whether the pump is in or out of operation.
  • If Mobile phase contains the buffer solution, minimum 100ml of D. I. water is used as washing solution or in other case solvent of the mobile phase can be used instead of water.

4.6 Precaution

  • Place the instrument in a smooth, vibration less, dustless smooth place.
  • Always operate the instrument in a area with humidity range 45 to 85%.
  • Always operate the instrument in a area with temperature range 5 to 350C, but in order to perform measurement under stable condition, it is recommended that instrument should be used in air conditioned room with temperature range 20 to 25 0C.
  • 500 voltage is hazardous for instrument, similarly 100 voltage is also hazardous.
  • Instrument atmosphere should be free of acidic or alkaline gas or other gas that can corrode metal.
  • Instrument atmosphere should be free of vapor from organic solvents like benzene or thinner that may dissolve paint.
  • Avoid exposing the instrument to direct sunlight.
  • A strong vibration that is felt by human body is transmitted into this instrument, so avoid from such incident.
  • Avoid vicinity of a heat generating apparatus such as gas burner, electric heater, or oven.
  • Avoid vicinity of instruments that generate strong electric field.

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