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High performance liquid chromatography hplc (shimadzu)

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  • 1. High Performance Liquid Chromatography-HPLC (Shimadzu)<br />1.0   Purpose     :  The purpose of this SOP is to lay down the operating for HPLC.2.0   Objective    :  To  provide a guideline how the HPLC used  in QC department.3.0   Scope       :  This SOP shall be applicable for calibration of HPLC and train and qualify the person in HPLC.4.0     Responsibility:·        Primary       :   QC Officer·        Secondary   :   Overall: QC Manager5.0     Procedure:Instrument details:               Name             : HPLC            Make              : ShimadzuModel No.      : LC-2010 CHTI. D. No.          :5.2       Operation:5.2.1    Before starting analysis, ensure that the instrument is clean.5.2.2    Switch ‘ON’ power supply of PUMP, DETECTOR & COLUMN OVEN.5.2.3       Initialization will occur for 2 minutes, Self check up of the instrument will occur.5.2.4       Fix the Column, required for the analysis, to the instrument. Initially, wash the column by   flushing with water: acetonitrile (90:10) or appropriate solvent if specified in the specification which is filtered through 0.45 micron filter for at least 30 minutes.  5.2.5       Prepare the mobile phase as per the Standard Analytical Procedure5.2.6       After initial check up is over and mobile phase is connected with system, open the drain valve and press PURGE KEY.5.2.7       Do drying for 2 minutes, the flour line is rinsed, air bubbles come out, again press purge, to stop purging close drain valve.5.2.8       Set the flow rate, Pmax, Pmin in case of Pump, required for that particular analysis.5.2.9       Set the Wavelength, Range, in case of detector, required for that particular analysis.5.2.10  Switch ‘ON’ the pump by pressing the PUMP KEY.5.2.11  Back pressure initial increases first, and get stabilized, after some times.5.2.12  After the mobile phase is allowed through the column, depend on the column used and also on the mobile phase used, is taken some times for column stabilization.5.2.13  If column gets stabilized, the abs display of detector shows you zero.5.2.14  Press zero key of the detector to make the absorbance of mobile phase zero.5.2.15  Before inject sample give the sample name & rum time, select method file name.5.2.16  Set required temperature in column oven.5.2.17  As soon as baseline gets stabilized the system is ready for inject.5.2.18  Inject the System suitability solution and if all system suitability parameter are passed, then only carry out further analysis.5.3             System Suitability: The following requirements should be fulfilled for system suitability unless otherwise stated in the individual specification.5.3.1       Symmetry factor for principle peak should be in between 0.8 and 2.0 unless otherwise specified in the individual specification.5.3.2       The Relative Standard Deviation of five replicate injections of standard solution or prescribed reference solution should not be more than 2%.5.3.3       If prescribed in the specification, inject Resolution solution in to chromatograph. The resolution between two specified peaks should not be less 1.0 or as specified in specification.5.4    For assay, allow the chromatogram to run for at least 1.5 times and for related substances or chromatographic purity, allow the chromatogram to run for at least 3 times the retention time of principle peak or as specified in individual specification.5.5       Take the Mean of area of these Standard solutions for calculation.5.6      After every 8 injections of the sample or 2 hours from the standard injections or change in RT by more the ±1 minute whichever is earlier, once again inject standard solution for system suitability. The Relative Standard Deviation of first five injection of standard solution and last injection of standard solution should not be more than 2%..5.7     If it fails in system suitability, repeat the injection of standard solution once again and analyze previously analyzed sample to confirm the validity of results.5.8   While doing analysis for impurity or in the analysis of related substances or chromatographic purity, inject the mobile phase or solvent used for preparation of sample intermediately to avoid carry over contamination in to the next sample injection.5.9   After completion of analysis, flush the column with water: acetonitrile (90:10) or appropriate solvent for sufficient time so as to completely remove the mobile phase used for analysis.5.10     After flushing, store the column in appropriate solvent.5.11   Take electronic backup of all chromatograms and methods on compact disk.  Keep all CD’s with proper labeling of date in CD cabinet under lock and key.5.12          Destroy all CDs after 5 years.5.13          Make the entry of relevant analysis in respective log book. Make also entry in HPLC  column log book for respective column use.6.0              Calibration:6.1             Calibrate the instrument monthly and record the results of calibration in form  F-1 7.0    Copies to Distribution Sites:§          QA   = Quality Assurance·         QC   = Quality Control8.0    Abbreviations & Important definition   :§         QA  = Quality Assurance·        QC = Quality Control·        SOP = Standard Operating Procedure9.0  Enclosure:                8.1  Calibration report of HPLC:  F-1                8.2  Log Sheet of HPLC : F-2CALIBRATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPH (LC 2010)F-1Instrument : High Performance Liquid ChromatographCALIBRATION DONE ON:CALIBRATION DUE ON:Manufacturer      : SHIMADZUModel                 : LC 2010A HT  &   RID  DetectorSoftware            :  CLASS- VPChromatographic conditionsMobile phase - A filtered and degassed mixture of Methanol and Distilled water  99:1Column        : 250 X 4.6 mm C18.Flow rate    : 1 ml/min.Detector wavelength: 190 nm. To 600 nm.Injection volume    : 1 µl to 50 µl1.      Precision of Injection:AIM : To check the precision of response produced by the 5 PPM solution  of Anthracene  in methanol.Stock solution of anthracene (100 ppm): Accurately weigh and transfer about 10 mg of Anthracene to a 100 ml volumetric flask. Dissolve and make up the volume with Methanol and mix well.Anthracene solution (5 ppm):  Dilute 5 ml of this stock solution to 100 ml with methanol and mix well to get 5 ppm solution of anthracene.Procedure: Make 5 consecutive injections of the above solution (5ppm) of Anthracene and record the chromatograms.Calculate the Relative Standard Deviation of area and retention time of anthracene.Injection No.Area of AnthraceneRetention Time of Anthracene1.2.3.4.5.RSD=RSD=Conclusion : The RSD of the area of 5 injections of Anthracene 5 ppm solution is less than 1 % / more than 1% and the RSD of the retention time of 5 injections of anthracene 5 ppm solution is less than 0.5% / more than 0.5% indicates  / does not indicates consistency of the  performance of the  pump and whole HPLC system.2    Wavelength Calibration Of Detector:Inject 20 µl of 5 ppm Anthracene solution 5 times, each time setting the detector at following 5 different wavelengths - 240nm, 245nm, 250nm, 255nm, 260nm.Record the Areas of the responses due to Anthracene.Wavelength  (in nm)Response (Area under Anthracene peak)240245250255260Conclusion  : The detector stands calibrated / does not stands calibrated with respect to wavelength  as it shows / does not shows maximum response at 250 nm  which  is  the wavelength of  maximum absorption for  Anthracene.3.      Linearity of response:AIM: To evaluate the performance of the detector.Dilute 10 ml of stock solution of anthracene to 100 ml with methanol and mix  well to get10 ppm solution of anthracene.Prepare a series of solutions having following concentrations by diluting the 10 ppm anthracene solution as follows.Solution          Concentration                                    DilutionSolution 1       2 ppm             2 ml of 10 ppm solution-->10 ml with MethanolSolution 2       3 ppm             3 ml of 10 ppm solution-->10 ml with MethanolSolution 3       4 ppm             4 ml of 10 ppm solution-->10 ml with MethanolSolution 4       5 ppm             5 ml of 10 ppm solution-->10 ml with MethanolSolution 5       6 ppm             6 ml of 10 ppm solution-->10 ml with MethanolSolution 6       7 ppm             7 ml of 10 ppm solution-->10 ml with MethanolProcedure: Inject above 6 solutions separately twice to obtain chromatograms and record the areas of Anthracene responses.Solution No.           Concentration (in ppm )Response( Area under Anthracene peak )Injection - 1Injection - 2Mean area1.              22.               33.               44.               55.              66.              7Plot a graph of Area responses against concentration.Conclusion : The area responses produced by the Anthracene  solutions are found to beLinear / not found to be linear with correlation coefficient, indicates / does not indicates the satisfactory performance of the HPLC system.Aim: To check the performance of the pump.Procedure: Set the flow rate to 0.5 ml, 1.0 ml, 1.5 ml and 2.0 ml respectively and record the volumes of solvent   collected in 10 minutes.Observation: The actual volumes of the solvent collected should be within the tolerance limits specified.Flow rate set atVolume collected (per 10 min.)Tolerance0.5 ml/min4.8 ml-5.2  ml1.0 ml/min9.8 ml-10.2 ml1.5 ml/min14.8 ml-15.2 ml2.0 ml/min19.8 ml-20.2 mlConclusion: Pump is working / not working satisfactory.Remark: Hence the instrument stands / does not stands calibrated w.r.t.1.      Precision of injection.2.      Detector performance.3.      Linearity of responses.4.      Pump performance.Calibrated By:                           Checked By:                           Manager Q.A :           Date:                                         Date:                                       Date:<br />You might also like:<br />OPERATION AND CALIBRATION OF HPLC SYSTEM (SIMADZU)<br />OPERATION, CLEANING, STORAGE, CALIBRATION AND REGENERATION OF HPLC COLUMN<br />Cleaning of HPLC columns<br />Calibration for Gas Chromatograph with Head Space-Shimadzu<br />LinkWithin<br />Posted by Quality Assurance and GMP and ICH at 3:31 PM <br />Email ThisBlogThis!Share to TwitterShare to FacebookShare to Google Buzz<br />Reactions: <br />0 comments:<br />Post a Comment<br />