operating procedure for HPLC

Operation And Calibration Of HPLC SYSTEM

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               Officer or above of Quality control  - 

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/ 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 software which is you use for your hplc system .

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

    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.