Calibration of HPLC, Purpose:
Calibration of HPLC, The purpose of this SOP is to describe the operation, calibration and cleaning of High Performance Liquid Chromatography system with DAD & RID (Brand: Agilent; Model: 1260 Infinity) used in the quality control laboratory at XX Pharmaceuticals Limited.
Calibration of HPLC, Scope:
This procedure is applicable for Agilent PC based Quaternary Gradient Automated High Performance Liquid Chromatography System, installed in the quality control laboratory of XX Pharmaceuticals Limited.
Definitions / Abbreviation:
[][]SOP : Standard Operating Procedure.
[][]QC : Quality Control.
[][]HPLC : High performance liquid chromatography
[][]DAD : Diode Array Detector
[][]RID : Refractive Index Detector
Responsibilities:
The roles and responsibility is as follows:
Sr. Executive/Executive, QC
[][]To ensure that this procedure is followed.
[][]To maintain the records properly as per SOP.
Manager, Quality Control
[][]To ensure that this procedure is kept up to date.
[][]To confirm that the SOP is technically sound and reflects the required working practices.
[][]To arrange training on the SOP to all concerned personnel and to ensure implementation of the SOP after training.
[][]Schedule calibration of the instrument at the defined intervals.
Procedure:
General Precaution(s):
[][]Prior to use, user must ensure that instrument is calibrated.
[][]Be sure that the drain valve is open before purging and tightly closed after purging.
[][]Before shutting down the system ensure that column is thoroughly washed and kept in appropriate solvent.
[][]Mobile phase, Diluting solvents and other solutions, which are used in HPLC, should be freshly prepared.
[][]Degas the mobile phase before using.
[][]Rinse the flow lines after completion of analysis as per solvent used in the system.
[][]Make sure that the solvent filter/diffuser is completely immersed in solvent.
[][]Update the solvent bottle before run.
[][]During set up the flow rate at priming, do not press Run/Stop to start the pump.
[][]Purge the pump with fresh diluent (filtered and degassed).
[][]Clean the solvent diffuser/filter once in a week.
[][]Fill up & attach Labels of the solvent reservoirs properly.
Operation of DAD:
[][]Switch ‘ON’ the power of Quaternary Pump, Autosampler, Thermostatic Column Compartment, Detector, CPU, Monitor if already not switched on.
[][]Wait until all the modules are initialized and are ready for next operation.
[][]Set specified column in the column compartment.
[][]Computer and Software Log in
[][]Double click on the icon of HPLC_DAD (online) from Desktop.
[][]Click Download to instrument/Upload from instrument/New method from instrument.
Purging Lines
[][]Insert the solvent tubing into the appropriate reservoirs.
[][]Gently shake the filters in the reservoirs to remove any bubbles that may be trapped.
[][]Open the purge valve of pump by half (1/2) turning anticlockwise.
[][]Go to the Agilent modular controller screen.
[][]Click Switch On to switch on all the modules.
[][]Right Click on the mouse over Quat. Pump module.
[][]Click Method.
[][]Enter flow rate 5.00 ml/min.
[][]Enter the % of composition of the desired purging line.
[][]Press OK/Apply.
Update the Solvent Bottle
[][]Right Click on the mouse over Quat. Pump module.
[][]Click Bottle Fillings.
[][]Enter the Solvent Volume of respective Bottles and enter the Total volume of the Bottle.
[][]Click OK.
[][]Create a new Method
[][]Click Method from menu.
[][]Click New Method.
[][]Click Method from menu.
[][]Click Entire Method…
[][]Click OK > OK >OK.
[][]Select Quart. Pump.
[][]Enter Flow rate, % of solvent ratio, stop time and maximum pressure Limit (Max. 400).
[][]For Gradient flow enter the time program.
[][]Select Sampler.
[][]Click OK.
[][]Click desire method.
[][]Click Add Method.
[][]Click OK>OK> OK>OK> OK.
[][]Click Method from menu.
[][]Click Save Method As…
[][]Select location and enter Method Name.
[][]Click OK.
Create a new Sequence
[][]Click Sequence from menu.
[][]Click New Sequence Template.
[][]Click Sequence from menu.
[][]Click Sequence Table…
[][]Enter Sample Location, Sample Name, Method Name and Data File.
[][]Click OK.
[][]Click Sequence from menu.
[][]Click Sequence Parameters…
[][]Locate the Data File Path.
[][]Click Post-Sequence Command/macro.
[][]Select STANDBY from dropdown list.
[][]Click OK.
[][]Click Sequence from menu.
[][]Click Save Sequence Template As…
[][]Select location and enter Sequence Name.
[][]Click OK
Run Samples
[][]Select the created Method.
[][]Select the created Sequence.
[][]Insert the solvent tubing into the appropriate reservoirs.
[][]Click Switch On to switch on all the modules.
[][]Wait for Base line stable.
[][]After getting the Baseline stability click RunControl from menu.
[][]Click Run Sequence.
[][]Instrument will be run as per selected Method and Sequence; and data will save as per selected location.
Data Processing
[][]Double click on the icon of HPLC DAD (offline) from Desktop.
[][]Select desire Sequence file from Data Analysis window.
[][]Double click a file of Navigation Table.
[][]Click Graphics form Menu.
[][]Click Signal Options.
[][]Click Compound Names.
[][]Click OK.
[][]Click Edit or Set Integration Events Table.
[][]Remove the unexpected peak either using Area or Height rejection or using time integration event program.
[][]Click OK.
[][]Click Calibration from Menu.
[][]Click Calibration Settings
[][]Enter Amount Units.
[][]Click OK.
[][]Click Calibration from Menu.
[][]Click New Calibration Table…
[][]Enter Default Amount (Standard wt. in mg).
[][]Click OK.
[][]Enter Compound Name in Calibration Table.
[][]Click OK.
[][]Click Method from Menu.
[][]Click Save Method As Save As New Master Method.
[][]Select location and enter Method Name.
[][]Click OK.
[][]Click Sequence from menu.
[][]Click Sequence Table
[][]Select the desire processing Method and Fill down.
[][]Select Sample Type as Calibration using drop down list for standard injections.
[][]For first Standard select Replace both for Update RF & Update RT using drop down list.
[][]Enter Multiplier.
[][]Click Reprocess.
[][]Click OK.
[][]Sequence will be processed as per selected processing method.
Report Print
[][]Select Data/Set of Data.
[][]Select Review.
[][]Click File from Menu.
[][]Clock Load Template.
[][]Select Template.
[][]Click Open.
[][]Click Print Report from Menu.
Operation of RID:
[][]Switch ‘ON’ the power of Quaternary Pump, Autosampler, Thermostatic Column Compartment, Detectors, CPU, Monitor if already not switched on.
[][]Wait until all the modules are initialized and are ready for next operation.
[][]Set specified column in the column compartment.
[][]Computer and Software Log in
[][]Double click on the icon of HPLC_RID (online) from Desktop.
[][]Click Download to instrument/Upload from instrument/New method from instrument.
Purging Lines
[][]Flow the operation steps
[][]Update the Solvent Bottle
[][]Flow the operation steps
Stabilization of RID detector
[][]Insert the solvent tubing into the appropriate reservoirs. Be sure that the detector waste lines and the sample loop waste line drain into the appropriate container.
[][]Right Click on RID window.
[][]Click Open Purge Valve.
[][]Right Click on RID window.
[][]Click Switch Recycling On.
[][]Wait for baseline stability.
[][]After baseline stability right Click on RID window.
[][]Click Close Purge Valve.
[][]Right Click on RID window.
[][]Click Switch Recycling Off.
Create a new Method
[][]Flow the operation steps
[][]Select DAD.
[][]Deselect Signals and UV Lamp if they are not previously deselected.
[][]Click OK.
[][]Click RID.
[][]Enter the Optical Input Temperature.
[][]Click OK.
[][]Select RID1 A, Refractive Index Signal.
[][]Click Add Method.
[][]Flow the operation steps
Create a new Sequence
[][]Flow the operation steps
[][]Run Samples
[][]Flow the operation steps
[][]Data Processing
[][]Double click on the icon of HPLC_RID (offline) from Desktop.
[][]Flow the operation steps
[][]Click File from menu.
[][]Click Load Signals…
[][]Select a standard file.
[][]Select RID1 A, Refractive Index Signal.
[][]Click OK.
[][]Flow the operation steps
Report Print
Flow the operation steps
Shut Down
[][]Click “Off” icon to stop Instrument.
[][]Carryout system suitability test every day before analysis.
[][]Fill up the column information logbook after analysis.
[][]Wash the column following as per HPLC Column Washing Procedure
[][]Exit from the “OpenLab Chemstation’’ software.
[][]Put off the power of equipment and computer.
[][]Record in the Logbook for operation of HPLC (as per Annexure-IV).
Operation of Queue Planner:
[][]When two or more sequence to be run with the same column and same mobile phase/mobile phase composition; Queue Planner is required.
[][]Save individual method and sequence for individual operation.
[][]Click RunControl from menu.
[][]Click Queue Planner.
[][]Insert/Append sequence when required. Use Up/Down button to rearrange the sequence.
[][]Click Save As.
[][]Select location and enter file name.
[][]Click Add to back of queue.
[][]During using of Queue Planner keep wash method and STANDBY at the last sequence only.
Calibration of HPLC:
Calibrate the HPLC at every 6 month’s frequency either by following procedure or as per supplier’s protocol:
[][]Quaternary Pump : 1260 Qart Pump VL
[][]Check the LED is on.
[][]Turn on the power.
[][]Set Flow Rate at 0.500 ml/min and 5.000 ml/min.
[][]Check flow rate, average value, STD, accuracy and precision.
[][]Record the value in Calibration Information Sheet for Pump Flow and Accuracy of Quaternary Pump VL (Annexure-I).
Set the following parameters:
Injection Volume : 0.0 µl
Flow Rate : 2.0 ml/min
Wavelength : 265 nm
Run Time : 26.0 min
Column Temperature : 40.0 °C
Evaluated Compound : Acetone
[][]Set the following Gradient Program and record the data:
Gradient Pump A/ Pump B
Initial 00% /100%
Step 1 20% /80%
Step 2 40% /60%
Step 3 60% /40%
Step 4 80%/ 20%
[][]Check the height, noise, drift and composition for various compositions.
[][]Record the Height, Noise, Drift and Composition in Calibration Information Sheet for Gradient Composition (Annexure-I).
[][]Thermostatic Column Compartment: 1260 TCC
[][]Check the LED is on.
[][]Turn on the power.
[][]Set Flow Rate 1.0 ml/min.
[][]Set column temperatures at 80.0°C and 40.0°C.
[][]Check the Column Temperature Accuracy and Column Temperature Stability from Column Compartment Display in 4 munities interval.
[][]Record the value in Calibration Information Sheet for Thermostatic Column Compartment (Annexure-II).
[][]Variable Wavelength Detector: 1260 DAD VL
[][]Check the LED is on.
[][]Turn on the power.
Set the following parameters:
Pump Flow Rate : 1.0 ml/min
Run Time : 2.0 min
Injection volume : 15 µl
Column Temperature : 40.0°C
Sample : Caffeine Std
Evaluated Compound : Caffeine
[][]Evaluating Standard Concentration : 25.0 µl/ml (form Certificate of Analysis)
[][]Set wavelengths at 205 nm, 245 nm and 273 nm.
[][]Check the area of the chromatograms for respective wavelengths.
[][]Record the value in Calibration Information Sheet for Variable Wavelength Detector (Annexure-III).
[][]Set the following parameters:
Flow Rate : 1.0 ml/min
Run Time : 24.0 min
Noise Evaluation Start Time : 3.0 min
Noise Evaluation Duration : 20.0 min
Injection Volume : 0.0 µl
Column Temperature : 40.0°C
[][]Check the Noise and Drift.
[][]Record the Noise and Drift in Calibration Information Sheet for Noise and Drift of Variable Wavelength Detector (Annexure-III).
Set the following parameters:
Flow Rate : 1.0 ml/min
Column Temperature : 40.0°C
Run Time : 7.0 min
Injection Volume : 10.0 µl
Noise Evaluation Start Time/Duration : 3.00 min/3.00 min
Evaluation Standard Concentration : 25 µg/ml (Certificate of Analysis)
Sample : Caffeine Std
Evaluation Compound : Caffeine
Detector Path Length : 10 mm
Wavelength : 273 nm
[][]Check the signal to noise ratio.
[][]Record the Signal to Noise in Calibration Information Sheet for Signal to Noise of Variable Wavelength Detector (Annexure-III).
Set the following parameters:
Flow Rate : 1.0 ml/min
Column Temperature : 40.0°C
Run Time : 2.0 min
Injection Volume : 20.0 µl
Sample : Caffeine Std
Wavelength : 273 nm
Evaluation Compound : Caffeine
[][]Check the area and height and find the R/F, R/F average, R/F STD and Coefficient of Determination.
[][]Record the linearity in Calibration Information Sheet for Response Linearity of Variable Wavelength Detector (Annexure-III).
[][]Refractive Index Detector: 1260 RID
[][]Check the LED is on.
[][]Turn on the power.
Set the following parameters:
Flow Rate : 1.0 ml/min
Run Time : 24.0 min
Noise Evaluation Start Time : 3.0 min
Noise Evaluation Duration : 20.0 min
Injection Volume : 0.0 µl
Column Temperature : 40.0°C
[][]Check the Noise and Drift.
[][]Record the Noise and Drift in Calibration Information Sheet for Noise and Drift of Refractive Index Detector (Annexure-IV).
Set the following parameters:
Flow Rate : 1.0 ml/min
Column Temperature : 40.0°C
Run Time : 7.0 min
Injection Volume : 10.0 µl
Noise Evaluation Start Time/Duration : 3.00 min/3.00 min
Evaluation Standard Concentration : 25 µg/ml (Certificate of Analysis)
Sample : RID Std kit
Evaluation Compound : Glycerin
Detector Path Length : 10 mm
[][]Check the signal to noise ratio.
[][]Record the Signal to Noise in Calibration Information Sheet for Signal to Noise of Refractive Index Detector (Annexure-IV).
Set the following parameters:
Flow Rate : 1.0 ml/min
Column Temperature : 40.0°C
Run Time : 2.0 min
Injection Volume : 20.0 µl
Sample : RID Std kit
Evaluation Compound : Glycerin
[][]Check the area and height and find the R/F, R/F average, R/F STD and Coefficient of Determination.
[][]Record the linearity in Calibration Information Sheet for Response Linearity of Variable Wavelength Detector (Annexure-IV).
[][]High Performance Autosampler: 1260 ALS
[][]Check the LED is on.
[][]Turn on the power.
Set the following parameters:
Injection Volume : 20 µl
Run Time : 2.0 min
Flow Rate : 1.0 ml/min
Column Temperature : 40.0°C
Wavelength : 273 nm
Sample : Caffeine Std
Evaluated Compound : Caffeine
Standard Concentration : 25 µg/ml (from Certificate of Analysis)
[][]Check the Area and Height of six chromatograms and calculate average value, standard deviation and %RSD.
[][]Record the value in Calibration Information Sheet for High Performance Autosampler (Annexure-V).
Set the following parameter:
Injection Volume : 20 µl
Run Time : 2.0 min
Flow Rate : 1.0 ml/min
Column Temperature : 40.0 °C
Wavelength : 273 nm
Sample : Caffeine Std
Evaluated Compound : Caffeine
Standard Concentration : 25 µg/ml (from Certificate of Analysis)
[][]Check the Carry Over of Injection.
[][]Record the Carry Over in Calibration Information Sheet for Injection Carry Over of High Performance Autosampler (Annexure-V).
[][]Cleaning procedure
[][]Clean the instrument daily as follows
[][]Switch ‘OFF’ the instrument and switch ‘OFF’ the mains.
[][]Clean the outer surface of the instrument with dry clean cloth.
[][]Clean the surrounding areas of instrument with a cloth dampen in water then wipe with a dry cotton cloth.
[][]Remove any dirt or spot with isopropyl alcohol and then dry with tissue paper.
[][]After completion of each analysis, clean the HPLC as follows
[][]Transfer the used glassware for washing.
[][]Clean the plunger seal by injecting distilled water three to four times with syringe.
[][]Remove the column and replace with joints for the cleaning of tubings & flush with 6N nitric acid, distilled water and then with methanol.
[][]Remove the air in suction pipe by purging the line and flushing with distilled water for some time.
[][]Clean the suction filter by sonicating it into nitric acid for 10 to 15 minutes and then wash it with water, when required.
[][]Replace the joints with column and wash the column first with distilled water and then with methanol.
[][]Switch “OFF” the pump, detector(s), injector and column compartment.
[][]Finally enter fill-up the Logbook for Operation of HPLC (Annexure-VI).
Annexure: Calibration of HPLC
Annexure-I: Calibration Information Sheet for Quaternary Pump VL
Annexure-II: Calibration Information Sheet for Thermostatic Column Compartment
Annexure-III: Calibration Information Sheet for Variable Wavelength Detector
Annexure-IV: Calibration Information Sheet for Refractive Index Detector
Annexure-V: Calibration Information Sheet for High Performance Autosampler
Annexure-VI: Logbook for Operation of HPLC