2122, 10:08 AM HPLC Calibration Procedure : Pharmaceutical Guidelines HPLC Calibration Procedure Kw the procedure ocarale the High Perfomance Laid Cromaography {HPC incting leakage lst, Bow ate, reproducibly and ineary amp eneray and ur posture dp in Pharmaceutesl Quty Conte Check HPLC chromatography (Pump) forthe following |. Checkpoint: Leakage test (By Pressure Drop) 1 Flow rate calibration (CHECKPOINT: LEAKAGE TEST (BY PRESSURE DROP) 1. Ensure thal, the instruments ready for calbration and StarLup procedure i flowed. 2 Place inlet tubing of the Pump into the Water HPLC grade through a suction Alter. 3. low the mobile phase to low for about 5 rin 4. Block Pump outlat with the black serow. 5. Tho pressure rises and on crossing the 300 bar, "ERROR P-MAX" appoars on the display window. Note the imo, Press “CE” key and observe the pressure drop for 5 min 6. After 5 min, record the pressure inthe Calibration Log 7. Make erty ofthe HOLC colursn usage in the Colum Usage Log Register. 8, Make anty ofthe user into the Instrument Usage Lag Resist 8, Compare the result for is compliance with the limit given in the Calibration Log and put the remark regarding the HPLC chromatography Caloration Status 10. n case of non-compliance, folw the Maintenance Program, Related: Principle and Working of HPLC Chromatograghy FLOW RATE CALIBRATION 1. Ensuro that, the instruments ready for calibration and StarLup procedure i flowed. 2. Ensure that, the Pump is passing th "Leakage Test (By Pressure Drop 3. Keep the Drain tube in such a way thatthe mobile phase (Water) drops falls nto 10 ml clean, dry volumetric ask without touching the walls ofthe flask and start immediatly the stopwatch when the fst drop falls int the flask 4, Wait til the cote fed mobile phase ches 10 ml mark of the valumatic flask and Stop the stopwat, 5, Record the tie required to colect the 10 ml mabile phase inthe calbraion log, 6, Repeat the procedure for 1.0m 1.5 mil and 2.0 ml min. ow rates. 7. Repeat stop 3 to 6 but using methanol HPLC grade as mobile phase instead of water £8, Compare the resus forits complies with iits given inthe Calibration Log and pu the remark regarding the Calibration Status. 9. Make erty ofthe usage of he instrument and HPLC column in the Instrument Usat Log Register and Column Usage Log Register 10. Prepare Calibration Status Label and display on the instrument atthe designated place. 11 In ease of non-compliance, flow the Maintenance Program. REPRODUCIBILITY AND LINEARITY OF INJECTION VOLUME Solution Mixer Preparation 1. Take clean ane dry 50 ml volumetric ask. 2 Pipette out 1.0 mi of Benzene and Toluene int the clean ans dry 50 ml volumetric flask 3. Make up the volume to 50 mlwith Methanol and mix wel ‘Chromatographie Condition HPLC Column: ODS C18, (25 em x 4.6 mm 1D, 5 um) Mobile Phase: Mothanot: Water (70 30) Flow Rate: 1.0 milmin, Wavelength: 254 nm Injection Volume: 20 ui Calibration +. Ensure that, the instruments ready for calloration and Startup procedure i followed. 2. Ensure that, the instrument is set according to the Chromatographic conditions 3. Follow the Instument Operating procedure Inject 10 pin trpiate and record the chromatograms. 4. Repeat the injection ofthe above solution by injecting 15,20, 25, 30 lin tripeale, 5. Take the printout ofthe chromatograms and attach tothe Calibration Log, 6, Record the Area and Retention times ofthe of the Benzene and Toluene peaks in the Callration Log. 7. Make erty ofthe usage ofthe instrument and HPLC column i he Instrument Usage Log Register and Column Usage Log Regster respectively hitpsswww-pharmaguidline.com/2011/01/mple-caibraton html 42122, 10:08 AM HPLC Calibration Procedure : Pharmaceutical Guidelines 8. Plot the curve for the area corresponding to Benzene to Toluene peaks vis inlecton volume, Find out the RSD (reproducillly) and record inthe Calaration Log, 8. Find out he Conelation coefficient “2” foreach peak al five levels ana 10. Compare the result for is compliance with the lt given inthe Calibration Log and put the remark regarding the Callraion Status, 11, Prepare Calibration Status Label and display onthe instrument atthe designated place. cord inthe Calibration Log 12. Incase of non-compliance, folow the Maintenance Program, (CHECKPOINT: 02 LAMP ENERGY CHECK (DETECTOR) 4. Ensure that, the instruments ready for calloration and Startup procedure i flowed. 2. On the Detector’ display window, some vahies at the funetions "inmy’,"abs(AUY’, “range(AUFS)” and “lamp” appears. 3. On the cisplay, previously set value blinks at“inm)" function, enter the wavelength to 254 nm by pressing numeric keys 4. Press "Func Back” key and select lamp” functions, enter 1 to select D2 lamp. 5. Further pross “Func Back” key tll"REF EN appears. 6, Record the Reference Energy af the D2 Lamp at 254 nm Inthe Callbration Log 7 Compare the result for ts compliance with the limit gven in the Callration Log and put the remark regarding the Calbration Status, 8. In case of non-compliance, follow the Maintenance Program. LINEARITY OF DETECTOR RESPONSE Solution Mixer Preparation: 4. Take three clean and dry 50 ml volumetric asks. 2 Pipette out ml of Benzene and Toluene as per the following table ino the clean, dry 50 ml volumetric flask separately. 3. Make up the volume to 50 mith Methanol and mix wel ‘otaton oven Tn of Benzene ob taken ‘Wot Thane tobe taken Tobe Died wih Mathanolto Chromatographic Condition 4. Set each of he equipment othe following parameters as per the Equipment’ Parameter Setup Procedure, HPLC Column: ODS C18, (25 em x 4.6 mm ID, 5m) Mobile Phase: Methancl : Water (70 : 20) Flow Rate: 1.0 mimin, \Wavelengt: 254 nm Injection Volume: 20 pi Callbration 1. Ensure that, the instruments ready for cllbration and Startup procedure is flowed. Ensure that, the instrument is set according tothe Chromatographic condtions. Follow the Instrument Operating procedure, Inject each of the Solution in wipleate and record the chromatograms. ‘Take the print out ofthe chromatograms and attach tothe Calibration Log Make entry of the usage ofthe instrument and HPLC column in the Instrument Usage Log Register and Column Usage Log Ragsler respectively. CCaleulate the Avea Ratio of Benzene to Toluene, Find out the Mean value ofthe rato ang recordin the Calibration Log, 2 3 4 5, Record the Area and Retention times of he of the Benzene and Toluene peaks in the Caliration Log. 6 r. 8, Find out the Correlation coefficient “2” from the Mean area rato values ofthe three levelsand record inthe Calbation Log 8 Plot the curve for area ratio coresponding to Benzene to Toluene peaks vis concentration, Find out the RSD (reproducibly) and recordin the Calioration Log 8, Compare the result forts compliance agenst iit given in the Calibration Lag and put the remark regarding the Calioraton Status 10. Prepare Galtration Status Label and display on te instrument atthe designated place. 11 In caso of non-compliance, flow the Maintonance Program. ‘CALIBRATION OF HPLC ( Liquid Chromatography ) PUMP. [A Chachpont: Laskage Check Test (Preasure Crop) tps: pharmaguidline.com/2011/01/mple-caibration html 28,2122, 10:08 AM HPLC Calibration Procedure : Pharmaceutical Guidelines (nie) aca "a wih ‘awh Wana (see) ‘CALIBRATION OF MUECTOR Moc! Make: Intrument Cade No. ral (1.0 ml) of the (Benzene) and (Toluene) in to a_i (50 mi clean, dry volumetric Nask, make up the volume with methanol, xed well Tes Pass athe Wate (7-30) TELE Cabin (ODS C18, (25 emx 4.8 mm ID, Sum) tare in 208) CCoeficient of corlations :r2 (Limit: NLT 0,999) (CALIBRATION OF oeTECTOR itpsswww-pharmaguidline.com/2011/01/mple-caibraton html2122, 10:08 AM HPLC Calibration Procedure : Pharmaceutical Guidelines ivr HPLC ge), mised we, alles 1 ozo room temperate Fi re yeu) CASO 2 mane Set, deesse orn by voewun snc. Ms Pass Wath Waar (720) (@orzeraToene) Linearity of Rato : Correlation Co-ofcent r2 Linearity of Rata : Correlation Co-afcint F2 : Not ess than 0.988 Casati Status = Satisfactory / Not Satisfactory Next Catbration Due Calrated By Checked By Date Date Related: SOP for Galation of HPLC Ankur Chowdnay is forthe roe gma update for your di hitps Aww: pharmaguidline.com/20"1/01/yple-caibraton Atm harmacoutial bog 45,2122, 10:08 AM HPLC Calloraton Procedure : Pharmaceutical Guidlines hitps uw pharmaguidline.com/20"1/01/yple-caibraton Atm 95