R&D is creative work undertaken on a systematic basis in order to increase the stock of knowledge, including knowledge of man, culture and society thus in R&D investigative activities that a business chooses to conduct with the intention of making a discovery that can either lead to the development of new products or procedures, or to improvement of existing products or procedures.
ØFilter the mobile phase through 0.45 membrane filter after mixing it in required proportion and de-gas on ultrasonic bath for about 5 minutes.
4.4PROCEDURE TO GET STARTED
ØSwitch on pump, auto injector, detector and then system controller.
ØTurn the drain valve knob to 180 in anti- clockwise to open the drain valve to run the purge system.
ØAfter the purge is over close the drain valve.
ØSet desired flow rate by pressing function key.
ØPress pump key. The pump will run and indicator will glow.
ØPurge the auto injector by pressing purge key on auto sampler display.
ØEnter the desired wavelength on detector using function key
4.5PROCEDURE TO OPERATE CLOSE UP
ØSwitch on the computer monitor and printer.
ØDouble click on the main Menu ofSHIMADZUIcon.
ØIt will show Confrigration,Real Time ,Sample Shedule & Post Run Analysis.
ØOpen Real Time.
ØFrom the “file menu” create a new method or load the existing method.
ØCreate a new sample shedule and feel sample name ,batch number,,sample volume , method name and file name ,after then save the shedule .
ØRun Sample Shedule .
ØFor 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.
ØAfter 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.
4.6SHUT DOWN PROCEDURE
ØClose Sample Shedule ,Real Time and then CLASS LC 10 to Software.
ØFrom Start button select “ Shut down” and click yes.
ØSwitch-off the computer.
ØSwitch-off CBM .
ØSwitch-off Detector then, auto sampler.
ØStop pump by pressing, “Pump” key and switch off the pump module.
ØDisconnect the column and connect the inlet and outlet tubing’s with a union.
ØPrime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles.
ØSet the flow rate at 1ml / min and collect the mobile phase (water) in a dry preweighedbeaker and collect the mobile phase for 10 min. Weigh the beaker to get the weight of mobile phase.
ØCalculate the flow rate by dividing the weight obtained with weight per ml and 10 (run time).
ØCalculate the corresponding flow rate. Carry out the experiment in duplicate.
ØRepeat the same procedure for Pump- B
ØRecord the observation in Annexure -1
ØAcceptance criteria :Flow rate should be in between 0.99 to 1.01 ml / min..
4.7.2FOR GRADIENT VALVE:
ØInstall union in place of column & flush solvent lines (A&B) at flow rate of 2ml/min with water.
ØPrepare the mobile phase.
ØPrepare 0.3% acetone with HPLC grade water.
ØFill reservoir A with 100% HPLC grade water & reservoir B with 0.3% acetone in HPLC grade water as mobile phase.
4.7.3INSTRUMENT SET UP:
ØEnter the following time program:
ØUse detector at wavelength of 254 nm.
ØRecord the printout of gradient valve test as per Annexure - 2.
ØThe gradient valve test shall be accepted if actual concentration with ±1% of set concentration.
4.7.4CALIBRATION OF INJECTOR:
22.214.171.124CHECK FOR PRECISION:
ØPurge the injector system with 100% water to ensure the complete washing of injector.
ØTransfer 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.
ØFilter the solution through 0.45mmembrance filter.
ØUse HPLC grade Methanol as the mobile phase.
ØInstrumental Set Up
Column:Hypersil ODS or equivalent 150 x 4.6 mm, 5.0m
Flow rate:1.0 ml/min
ØInject the standard preparation six times in the system. The peak areas observed shall be consistent.
ØThe relative standard deviation for area counts calculated shall not be more than 1.0%.
ØRecord the observation in the format as mentioned in Annexure – 3.
4.7.6CHECK FOR LINEARITY:
ØInject 10, 20, 30, 40 & 50mlitre of standard preparation in duplicate. Calculate the average area counts corresponding to each set of injection.
ØTabulate the average area against each injection. Plot a graph for area counts vsmlitre the resulting graph shall be linear.
ØThe correlation co- efficient calculated shall be not less than 0.99.
ØRecord the observation as per the Annexure - 3.
4.7.7CALIBRATION OF DETECTOR:
ØStandard preparation. Mobile phase, Instrument set- up same as mentioned in calibration of Injector.
ØRun the chromatograph at different wavelength (252 nm – 262 nm with 2 nm increment)
ØThe largest peak response shall be at 258 nm ± 2nm.
ØRecord the results in the detection calibration record as per the Annexure - 4.
ØAffix 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:
ØDetector :Once in three months and after each maintenance job.
ØPump:Once in three months and after each maintenance job.
ØInjector:Once in six months and after each maintenance job.
ØGradient :Once in six months and after each maintenance job in the pump.