SOP for HPLC Analysis & Documentation

To lay down a procedure for analysis to be carried out by HPLC and its documentation. 

This procedure is applicable for analysis to be carried out by High Performance Liquid Chromatography in Quality Control Laboratory.

Execution : Chemist – QC department. 
Checking : Chemist and above – QC department 

  • Collect the sample along with the protocol/datasheet for analysis from section in charge 
  • Read the STP and protocol/datasheet carefully for tests to be performed by HPLC, reagents used, HPLC parameters, System suitability parameters, standard preparation and test preparation etc. 
  • Select the HPLC system and column (as specified in STP/protocol/datasheet) for analysis. 
  • Use only HPLC grade solvents in the preparations. 

Preparation of Mobile Phase: 
  • Use appropriate grade of reagents as stated in the STP. 
  • Weigh the reagents accurately, dissolve and dilute with solvent as per the individual STP.  
  • Adjust the pH with appropriate reagent as per the STP. 
  • Mix thoroughly the buffer and solvents with the ratio mentioned in the individual STP and adjust the pH if it is mentioned in the STP. 
  • Follow the same procedure for gradient buffer also and do the preparation separately. 
  • Prepare the quantity of buffer according to the sample load. 
  • Filter the mobile phase with appropriate membrane filter paper. 
  • Do the filtration without delay. 
  • Degas the mobile phase for about 5 minutes by using sonicator. 
  • Use the mobile phase within 5 days from the date of preparation. 
  • Label the mobile phase with following information, product name, Test prepared on and use before as per the annexture-QCD30/A01-00. 
  • If any visual impurities are found within 5 days discard the mobile phase and prepare freshly.  

Sequence of Injections: 
  1. System suitability (if applicable) 
  2. Standard Solution (In replicates, if mentioned in STP) 
  3. Sample Solution (In replicates, if mentioned in STP) 
  • Before starting the analysis trial run to be carried out is required to check the Baseline noise, RT, system suitability parameters etc 
  • On completion, flush the system with lab water for 15minutes followed by organic solvent used in mobile phase: water (50: 50) 
  • After complete analysis of one sample, make the entries in Column usage log book and in instrument log book.

SOP - Standard Operating Procedure 
STP - Standard test procedure 
QC - Quality Control 
HPLC - High Performance Liquid Chromatography 



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