Do's and Don'ts in HPLC Analysis

 

Introduction:

                    HPLC (High Performance Liquid Chromatography) is an advance chromatographic technic used in almost all the chemical and Pharmaceutical industry. Handling of HPLC and UPLC are very sensitive. More care must be taken by the analyst who handling these sensitive instruments. Proper training must be given to the analyst/scientists before starts the analysis in HPLC, which is required to reduce the analytical error and breakdown of the instrument.

                             1. What we should do in HPLC?

                             2. What we should not do in HPLC?

            So there are some important key points need to know before start handling of HPLC.               

 Do's: 

              1. Grade of Chemicals and Water: Always use HPLC grade chemicals and Water need to be used for mobile phase and sample preparation. Low grade chemicals and water leads to problem later. Use clear mobile phase bottles for preparation.

            2. Mobile phase Filtration:  Filter your mobile phase through 0.45µm (use 0.22µm if UPLC) membrane filter using filtration assembly.             

            3. Column & Instrument Washing: Wash the column for every end of analysis by suitable solvent for at least 30 column volumes. Instrument also need to be washed before and after analysis to eliminate buffer deposition in the lines.

           4. Column Storage: Column must be stored in the suitable solvent after washing. Columns should be properly stored with end cap. 

                  

              Proper Storage with End-capped

           5. Suction Filter: Use Suction filter and make sure that suction filters are always completely dipped in mobile phase. Remove and clean the suction filters weekly once to avoid microbial contamination.

     Suction Filter

            6. Sample Filtration & Vials: Sample solution must be filtered through 0.45µm (use 0.22µm if UPLC) syringe filter before injecting into the column. Always use new vials and caps with septa.

 Syringe Filter                                                   Vials with Cap and Septa

           7. Purging of System: Purge the system to remove the air bubble in tubing before starting the analysis.

           8. Degassing the Mobile phase: Remove dissolved gasses in the mobile phase by online degasser or sonicate the mobile phase to remove.

Don'ts: 

          1. Don't run the empty Pump. 

Dried pump          

           2. Don't use dirty mobile phase bottle.

Dirty/contaminated mobile phase bottle

          3. Don't use pH of the column out side the range of column that being used for the analysis.

          4. Don't use high concentration buffer and ion pair reagents in the mobile phase.

          5. Don't store the column and instrument in water for long time, will leads to microbial growth. 

          6. Don't leave the column without end capped.

Improper Column storage

           7. Don't leave the buffer in the column or instrument for long time.

  

           8. Don't use low grade chemicals or water for mobile phase preparation

        

           9. Don't use mobile phase without filtration and degassing.

        

         10. Don't use used vials and caps without septa.

         11. Don't inject the sample without filtration.