Friday, December 10, 2010

Operation And Calibration Of HPLC SYSTEM (SIMADZU )

1.0.            OBJECTIVE:

The objective of this SOP is:
1.1               To ensure that the instrument performs satisfactorily and gives accurate and reproducible data. 
2.0.            RESPONSIBILITY:

2.1               The Chemist/ Executive - Quality Control shall be:
2.1.1.        Responsible for operating the HPLC as per the SOP.
2.1.2.        Responsible for timely calibration of HPLC System as per the SOP.
2.1.3     Responsible for ensuring the adherence of SOP.

3.0.            ACCOUNTABILITY:
          
Head - Quality Control

4.0.            PROCEDURE:

4.1              PROCEDURE FOR GENERAL CLEANING:

Ø      Ensure that the power supply to the instrument is switched off and main cord is removed from supply.
Ø      Clean the instrument with a clean dry cloth everyday. A wet cloth dipped in dilute soap solution may be used occasionally.
Ø      Precaution must be taken to clean the instrument immediately with dry cloth.
                       
4.2              OPERATING INSTRUCTIONS:

Ø      Ensure that the instrument is properly connected to the power supply.
Ø      Fix the column and prepare the mobile phase. 


4.3              PREPARATION OF MOBILE PHASE

Ø      Use HPLC grade solvent / water & AR/Excel/ HPLC grade reagent only.
Ø      Filter the mobile phase through 0.45 membrane filter after mixing it in required proportion and de-gas on ultrasonic bath for about 5 minutes.
4.4              PROCEDURE TO GET STARTED

Ø      Switch on pump, auto injector, detector and then system controller.
Ø      Turn the drain valve knob to 180 in anti- clockwise to open the drain valve to run the purge system.
Ø      After the purge is over close the drain valve.
Ø      Set desired flow rate by pressing function key.
Ø      Press pump key. The pump will run and indicator will glow.
Ø      Purge the auto injector by pressing purge key on auto sampler display.
Ø      Enter the desired wavelength on detector using function key

4.5              PROCEDURE TO OPERATE CLOSE UP

Ø      Switch on the computer monitor and printer.
Ø      Double click on the main Menu of SHIMADZU Icon.
Ø      It will show Confrigration,Real Time ,Sample Shedule & Post Run Analysis.
Ø      Open Real Time.
Ø      From the “file menu” create a new method or load the existing method.
Ø      Create a new sample shedule and feel sample name ,batch number,,sample volume , method name and file name ,after then save the shedule .
Ø      Run Sample Shedule .
Ø      For system suitability test, inject six continuous injections of same standard and RSD should not be more than 2.0 %, otherwise it is specified in standard test procedure.
Ø      After every ten injection of sample (5 samples in duplicate), the standard solution shall be injected 3 times and the RSD shall be calculated and ensure that it is within the limit. 

4.6              SHUT DOWN PROCEDURE


Ø      Close Sample Shedule ,Real Time and then CLASS LC 10 to Software.
Ø      From Start button select “ Shut down” and click yes.
Ø      Switch-off the computer.
Ø      Switch-off CBM .
Ø      Switch-off Detector then, auto sampler.
Ø      Stop pump by pressing, “Pump” key and switch off the pump module.

4.7              CALIBRATION PROCEDURE.

4.7.1          FOR PUMP:

Ø      Disconnect the column and connect the inlet and outlet tubing’s with a union.
Ø      Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles.
Ø      Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry preweighed  beaker and collect the mobile phase for 10 min. Weigh the beaker to get the weight of mobile phase.
Ø      Calculate the flow rate by dividing the weight obtained with weight per ml and 10 (run time).
Ø       Calculate the corresponding flow rate. Carry out the experiment in duplicate.
Ø      Repeat the same procedure for Pump- B
Ø      Record the observation in Annexure -1
Ø      Acceptance criteria : Flow rate should be in between 0.99 to 1.01 ml / min..


4.7.2          FOR GRADIENT VALVE:

Ø      Install union in place of column & flush solvent lines (A&B) at flow rate of 2ml/min with water.
Ø      Prepare the mobile phase.
Ø      Prepare 0.3% acetone with HPLC grade water.
Ø      Fill reservoir A with 100% HPLC grade water & reservoir B with 0.3% acetone in HPLC grade water as mobile phase.

4.7.3          INSTRUMENT SET UP:

Ø            Enter the following time program:
 TIME
FUNCTION
VALUE
0.01
B CONC
10
10.00
B CONC
10
10.01
B CONC
50
20.00
B CONC
50
20.01
B CONC
90
30.00
B CONC
90
30.01
B CONC
100
40.00
B CONC
100
40.01
B CONC
0
50.00
B CONC
0

Ø            Use detector at wavelength of 254 nm.
Ø            Record the printout of gradient valve test as per Annexure - 2.
Ø            The gradient valve test shall be accepted if actual concentration with ±1% of set concentration.


4.7.4          CALIBRATION OF INJECTOR:
4.7.4.1    CHECK FOR PRECISION:
Ø      Purge the injector system with 100% water to ensure the complete washing of injector.

4.7.5          STANDARD PREPARATION:
Ø      Transfer about 50 mg of Uracil to a 250ml volumetric flask. Add 100ml of Methanol, sonicate to dissolve and make up the volume with Methanol to obtain a solution containing about 0.02 mg/ml of Uracil.
Ø      Filter the solution through 0.45m membrance filter. 
Ø      Use HPLC grade Methanol as the mobile phase.
Ø      Instrumental Set Up
                        Column                         :  Hypersil ODS or equivalent 150 x 4.6 mm, 5.0m
Flow rate                      :  1.0 ml/min
Injector Volume            :  20m litre
Detector                       :  254 nm

Ø      Inject the standard preparation six times in the system. The peak areas observed shall be consistent.
Ø      The relative standard deviation for area counts calculated shall not be more than 1.0%.
Ø      Record the observation in the format as mentioned in Annexure – 3.

4.7.6          CHECK FOR LINEARITY:

Ø      Inject 10, 20, 30, 40 & 50m litre of standard preparation in duplicate. Calculate the average area counts corresponding to each set of injection.
Ø      Tabulate the average area against each injection. Plot a graph for area counts vs mlitre the resulting graph shall be linear.
Ø      The correlation co- efficient calculated shall be not less than 0.99.  
Ø      Record the observation as per the Annexure - 3.


4.7.7          CALIBRATION OF DETECTOR:
Ø      Standard preparation. Mobile phase, Instrument set- up same as mentioned in calibration of Injector.
Ø      Run the chromatograph at different wavelength (252 nm – 262 nm with 2 nm increment)
Ø      The largest peak response shall be at 258 nm ± 2nm.
Ø      Record the results in the detection calibration record as per the Annexure - 4.
Ø      Affix a calibration status label on the instrument containing “Calibrated On”, “Due On” and “Signature”.
Report to Head – QC, if any discrepancy observed during calibration or operating the instrument and affix ‘Under Maintenance’ label on the instrument



-----Pharmaceuticals Limited
UNDER MAINTENANCE
EQUIPMENT             :

SINCE             :

SIGNATURE:



    FREQUENCY OF CALIBRATION:

Ø      Detector :      Once in three months and after each maintenance job.
Ø      Pump     :       Once in three months and after each maintenance job.
Ø      Injector  :       Once in six months and after each maintenance job.
Ø      Gradient :       Once in six months and after each maintenance job in the pump.

         


1 comment:

  1. Great thoughts you got there, believe I may possibly try just some of it throughout my daily life...


    calibration company

    ReplyDelete

Related Posts Plugin for WordPress, Blogger...
Subscribe to Quality Assurance and GMP by Email