CALIBRATION OF ALLIANCE HPLC WITH EMPOWER SOFTWARE

• PROCEDURE:
• Alliance HPLC with EMPOWER software
• FLOW ACCURACY TEST:
• Fix the Union/Restriction Capillary in the column compartment.
• Keep degassed water (Milli Q) in channel ‘A’ and prime the channel thoroughly.
• Keep the degasser mode on and stabilize for about 1 hour with a flow rate of 1 ml /min.
• Take a 10 ml calibrated volumetric flask and dry it.
• When the flow and pressure are stable, insert the outlet tubing into the volumetric flask and immediately start a calibrated stopwatch.
• Stop the stopwatch when the bottom of the meniscus reaches the 10 ml mark on the flask.
• Record the elapsed time in seconds.
• Calculate the flow rate using the following equation:
• Calculated flow rate = (10.00 ml/ measured time in seconds) x 60
• Similarly test the flow rate accuracy with flow rates 0.5 mL/min and 2.0 mL/min and record the calculated flow rate.
• Acceptance criteria:
• ± 0.02 mL/min of set flow rate.Record the results in calibration record as per Format – I.
• Calibration schedule:
• Once in 6 months and after any major maintenance job.
• TEMPERATURE ACCURACY:
• Set column oven temperature to 30ºc and stabilize for about half an hour.Keep the temperature probe of Digital thermometer inside the oven compartment and allow to stabilize the reading.
• After stabilization of the temperature, note down the reading as per Format-II.
• Repeat the steps above 3 steps for the temperatures 40°C and 50 °C respectively.
• Acceptance criteria: ± 2ºC of set temperature.Record the results in calibration record as per Format-II.
• Calibration schedule: Once in 6 months and after any major maintenance job.
• DETECTOR CALIBRATION:
• Wavelength Accuracy:
• Operate the instrument as per SOP
• Weigh accurately 50 mg of caffeine working standard into a 50ml volumetric flask, dissolve and dilute to 50 ml with water. Dilute 2 ml of the above solution to 100 ml with water (0.02 mg/ml).
• Prepare filtered and degassed mobile phase mixture of Water and methanol in the ratio of 70: 30.

• Fix the Column (Symmetry C18, 4.6mm x 75mm 3.5µm) in the column compartment.
• Place the mobile phase and stabilize for 30 minutes at a flow rate of 1.0 ml/min.
• Set the wave length as 201 nm and Column temperature as 35°C.
• Inject the 0.02mg/ml solution each time with the following parameters by varying the wavelength from 201 nm to 209 nm, 241 nm to 249 nm and 269nm to 277 nm with 1 nm frequency. Run time 5 min and injection volume 10µl, Perform duplicate injections at all wavelengths.
• Inject 0.02 mg/ml of caffeine in duplicate and after completion of the run, extract the spectrum from 201 nm to 209 nm, 241 nm to 249 nm and 269nm to 277 nm.

Acceptance Criteria: The maximum peak area response should be at 205 ± 2 nm. The minimum peak area response should be at 245 ± 2 nm. The maximum peak area response should be at 273 ± 2 nm.

• Enter the results in calibration record as per Format-III.
• Calibration schedule: Once in 6 months and after any major maintenance job.

Note: If the calibration is performed continuously, no further stabilization is required for injector linearity, detector response linearity and wavelength accuracy.

• DETECTOR LINEARITY:
• Fix the Column (Symmetry C18, 4.6mm x 75mm 3.5µm) in the column compartment.
• Prepare the degassed mobile phase mixture of Water and methanol in the ratio of 70: 30, Prepare caffeine working standard solution of 25ppm, 50ppm, 75ppm, 100ppm, and 125ppm concentration from stock solution using mobile phase as diluents.

• Preparation of Caffeine Standard Stock solution (1000ppm): Weigh accurately about 100 mg of caffeine working standard and transfer in to 100 ml volumetric flask dissolve and dilute to volume with methanol.
• Operate the instrument with the following chromatographic conditions:
• Wavelength: 274 nm.
• Flow rate: 1.0 ml / min.
• Injection volume: 10ml.
• Wavelength: 274 nm.
• Flow rate: 1.0 ml/min
• Run time: 4mins.
• Temperature: 25°C.
• Preparation of Caffeine Standard solutions:
• Dilute2.5 ml of stock solution to 100 ml with mobile phase (25ppm).
• Dilute 5.0 ml of stock solution to 100 ml with mobile phase (75ppm)
• Dilute 10.0 ml of stock solution to 100 ml with mobile phase (100ppm).
• Dilute 12.5 ml of stock solution to 100 ml with mobile phase (125ppm)
• Separately inject the above solutions in duplicate and calculate the average peak area response.
• Plot the graph of detector response VS concentration and calculate the detector linearity in terms of correlation coefficient.
• Acceptance criteria:
• The correlation coefficient should be not less than 0.99.Enter the results in calibration record as per Format-IV.
• Calibration schedule: Once in 6 months and after any major maintenance job.

• INJECTOR PRECISION AND LINARITY TEST:
• PRECISION
• Keep mobile phase (Water and methanol in 70:30 ratio) in channel A stabilize for 10 minutes at a flow rate of 1.0 ml/min.
• Fix the column (symmetry C18, 4.6 mm x 75 mm, 3.5µm) in the column compartment.
• Place the 10ppm caffeine solution (Dilute 10 ml of 100ppm caffeine standard solution to 100 ml with mobile phase) sample in carousel.
• Follow the chromatographic conditions for precession.
• Wavelength: 274 nm.
• Flow rate: 1ml/min.
• Injection volume :10µl (For 100 and 200µl loop) 20µl (For 500µl loop)
• Run time: 4 minutes.
• Temperature: 25°C.
• Inject standard solution five times in to the chromatogram and measure the peak area response.
• Acceptance criteria:
• The %RSD area of replicate injections is not more than 2.0 % The %RSD RT of replicate injections is not more than 1.0 %.
• LINEARITY
• Wavelength: 274 nm
• Flow rate: 1 ml /min
• Injection volume:
• For 100µl loop: 2µl, 5µl, 10µl, 20µl, 50µl and 100µl.
• For 200µl loop: 5µl, 10µl, 20µl, 50µl, 100µl, 150µl, 200µl.
• For 500µl loop: 20µl, 50µl, 100µl, 200µl, 300µl, 400µl & 500µl.

• Run time: 4 minutes.
• Temperature: 25°C
• Separately Inject standard solution with above mentioned injection volume in duplicate and measure the average peak area response.
• Plot the graph of detector response VS injection volume and calculate the injector linearity in terms of correlation coefficient.
• Acceptance criteria:
• The correlation coefficient should be not less than 0.99.Enter the results in calibration record as per Format-V.
• Calibration schedule:
• Once in 6 months and after any major maintenance job.
• GRADIENT PROPORTIONING VALVE TEST:
• Preparation For the GPV Test:
• Fill the reservoir for A and B lines with methanol.
• Fill the reservoir for C and D Lines with 500 ml of methanol containing 5.6 mg/L propylparaben / or 1 ampoule of GPV test solution (ref: waters part no WAT042876, per 500 ml solution).

Note: Preparation of 5.6 mg /L propylparaben solution: Take 5.6 mg of propylparaben in1000 ml dry volumetric flask, dissolve and dilute to volume with methanol. Sonicate the solution for 20 minutes.

• Prime the pump.
• Run the inline degasser for 10 minutes.
• Create an instrument method with following parameters.
• Wavelength – 254nm.
• Flow – Gradient (program give below)

• Accept default settings for other parameters.

Time	Flow	%A	%B	%C	%D	Curve
Initial	2.00	50	50	0.0	0.0
2.00	2.00	0.0	0.0	50.0	50.0	11
6.00	2.00	50	50	0.0	0.0	11
10.00	2.00	45	45	10.0	0.0	11
12.00	2.00	50	50	0.0	0.0	11
14.00	2.00	45	45	0.0	10.0	11
16.00	2.00	50	50	0.0	0.0	11
18.00	0.00	50	50	0.0	0.0	11

• RUN THE GPV TEST:
• Run the sample in single injection mode.
• Run the single injection with 18 minutes run time using the above created method set and prepare and injection icons.
• Select Run only in the Run sample window.
• Acceptance criteria: 10 ± 0.5 %.
• Record all the results in the calibration record as per Format-VI.

Note: Peak height is based on the average height along the flat area at the top of the peak. If not adjust as necessary.

• TEST FOR NOISE AND DRIFT:
• Stop flow from the separations module. Purge the detector flow cell with nitrogen and allow staying for 1 hour.
• Create new instrument method, click on 2690/5 module and select flow as 0.00 ml and accept default settings for all other parameters.
• Click on W2489 detector icon and select wavelength as254 nm.

• Come to single inject, select injection volume 0 µL, vial no.=1 and run time = 60 minutes.
• After the instrument has stabilized, click on prepare for injection icon and after 2 minutes click on inject icon.
• Acceptance criteria: The noise should not be more than 0.125 Au and drift should not be more than 5.000 Au.Enter the results in calibration record as per Format-VII.
• Calibration schedule: Once in 6 months and after any major maintenance job.

• ANNEXURES: 

ANNEXURE NO.	TITLE OF ANNEXURE
Annexure-I	Wavelength accuracy calibration record
Annexure-II	Detector Linearity calibration record
Annexure-III	Flow Accuracy Calibration record
Annexure-IV	Temperature accuracy calibration record
Annexure-V	Linearity and precision calibration record
Annexure-VI	Gradient proportioning valve calibration record
Annexure-VII	Drift and noise calibration record

Annexure-I
WAVELENGTH ACCURACY CALIBRATION RECORD

Annexure-II
DETECTOR LINEARITY CALIBRATION RECORD

Annexure-III
FLOW ACCURACY CALIBRATION RECORD

Annexure-IV
TEMPERATURE ACCURACY CALIBRATION RECORD

Annexure-V
LINEARITY AND PRECISION CALIBRATION RECORD

Annexure-VI
GRADIENT PROPORTIONING VALVE CALIBRATION RECORD

Annexure-VII
DRIFT AND NOISE CALIBRATION RECORD