Shimadzu Prominence HPLC Quaternary System(PDA-Detector) Operation and Calibration

1.0 OBJECTIVE 

To lay down a procedure for operation and Calibration of Shimadzu Prominence HPLC Quaternary system with PDA-detector. 

2.0 SCOPE

This SOP is applicable to Operate and Calibrate of Shimadzu Prominence HPLC Quaternary system with PDA-detector in Company Name

3.0 BACKGROUND 

NIL

4.0 RESPONSIBILITY

4.1 All Analytical research personnel whoever is operating the Equipment.

4.2 Group Leader Analytical research to ensure proper and safe operation of the Equipment.

4.3 Head - Analytical research or his designee to ensure overall compliance.

5.0 PROCEDURE

5.1 Safety Precautions:

5.1.1 Don’t switch on the equipment with wet hands.

5.1.2 Use goggle while using the Equipment.

5.1.3 Do not use highly acidic or basic solvents (seals, Plungers etc can be damaged by extreme PH Condition).

5.1.4 Don’t open the vial tray while instrument is in operation.

5.2 Operating Procedure:

5.2.1 Switch on the Power of LC-2030C unit.

5.2.2 Switch on the Power of Computer, Monitor and printer.

5.2.3 Double click on Lab Solution icon, a Login screen appear. Enter user ID and Password

5.2.4 Then, Double Click on desired instrument to open and connect with the hardware.

5.2.5 For New Method creation go to the file and click on new method file.   

5.2.6 Then in advance mode go to Data Acquisition and enter the run time of your method.

5.2.7 Then Click on Pump, select low pressure gradient (or) isocratic based on your method. Then input maximum pressure range of system. In low pressure gradient give concentration of solutions individually.

5.2.8 Then click on LC time Prog and enter the time and select pump module and solvent concentrations in Command and enter the % concentration value in value.

5.2.9 Then click on detector and enter the wavelength.

5.2.10 Then in the column oven, enter column temperature.

5.2.11 Then click on the controller, in controller tick mark the degasser (for reverse phase analysis). Where as in normal phase analysis do not tick (do not switch on) degasser.

5.2.12 Then click on Auto sampler and click on detect rack and enter the coller temperature if required.

5.2.13 Then click on Auto purge, give purging if your method requires purging after every analysis. Otherwise leave it. Then, go to file click on save method file as

5.2.14 Then enter the file name where to save it in

5.2.15 Click on download and so that instrument will get the method.

5.2.16 Then go to Real-time batch and enter vial number, tray number and sample name, sample ID, method file, Date file, injection volume.

5.2.17 Right click on batch table and click on Add row and then input number of rows.

5.2.18 Fill the information and click on start Real-time batch on left side assistant bar.

5.2.19 Open the Existing Method:

5.2.19.1 Click file, select open method file, then click the method file you want to select and click open.

5.2.20  Post Run Analysis

5.2.20.1 Go to the main window of Lab solution and then double click on  icon Postrun.

5.2.20.2 select the folder where you have saved the data and double click on folder to open the data. 

5.2.20.3 Then double click on data file in data explorer window to open the data file.

5.2.20.4 Then click on wizard for auto-integration and input slope height, area drift value for Integration.

5.2.20.5 Then click on program to integrate using integration bar. Finally click on simulate and then ok. 

5.2.20.6 Click on next and select the peaks (tick mark peaks), click on ‘next’, again click ‘next’ and again click ‘next’ , type the name of the compound and click ‘finish’

5.2.20.7 Click ‘Apply to method’ on the Assistant Bar, save a new method in the respective folder, click ‘ok’ to save the processing method.

5.2.21 Reporting

5.2.21.1 Click Data report in assistance bar.

5.2.21.2 Default report template will open.

5.2.21.3 To open existing report template Click file, then select open report format file.

5.2.21.4 Click and drag the data to report format file from data explorer.

5.2.22 Enter the details in usage log book in (Annexure-1)

5.3  Calibration:

5.3.1 Flow rate accuracy test:

5.3.1.1 Remove the column and connect a resistance tube 0.065MMID. In parameter setting of the pump “ON” the degassing unit. If degassing unit is OFF, turn “ON”

5.3.1.2 Select the pump in the performance check screen and press flow rate accuracy, click next. Select mobile phase port used for the test, flow rate 0.5ml/min and press next.

5.3.1.3 Purge the channels thoroughly. Stabilize for about 10 minutes with flow rate of 0.5ml/min.

5.3.1.4 Take a dried and calibrated 10ml volumetric flask and insert at the end of outlet tube and immediately start calibrated stopwatch.

5.3.1.5 Stop the stopwatch when the bottom of the meniscus reaches the 10ml mark on the volumetric flask.

5.3.1.6 Record the elapsed time in seconds.

5.3.1.7 Calculate the flow rate using the following equation

Flow rate accuracy(%) =Actual flow rate/set flow rate – 1 x 100

5.3.1.8 Similarly Test the flow rate accuracy with flow rate 1.0ml/min and 2.0ml/min.

5.3.1.9 Record the details in “Calibration record of flow accuracy” (Annexure- 2)

5.3.1.10 Acceptance Criteria: ±2%

5.3.1.11 Calibration Schedule:  once in six months and after any major maintenance job.

5.3.2 Column oven Set Temperature: 

5.3.2.1 Select oven temperature accuracy in performance check screen.

5.3.2.2 Place calibrated temperature sensor on to the center of the column holder in the column oven using aluminum tape.

5.3.2.3 Set the oven temperature to 40oc.

5.3.2.4 Wait approximately 20 minutes after the temperature stabilized at the set temperature. After measurement has finished press “next”.

5.3.2.5  Based on the result of the temperature measurement error described in the test report for the temperature sensor, correct the temperature and enter the measured temperature. Press “next”.

5.3.2.6 When the measurement results of the temperature accuracy and the range of temperature are displayed, press ”logged” to record the results.

5.3.2.7 Set the oven temperature to 50oc and repeat the procedure from point 5.3.2.4 to 5.3.2.6.

5.3.2.8 Record the details in “calibration record of Temperature accuracy”(Annexure-3)

5.3.2.9 Acceptance Criteria: ±2.0 oc.

5.3.2.10 Calibration schedule:  Once in a 6months and after any major maintenance job.

5.3.3 Repeatability test:

5.3.3.1 Perform the Repeatability teat by using Standard Caffeine solution.

5.3.3.2 Wash the flow channels with suitable solvent. Use 1.6 C coupling with no column connected. Pump the mobile phase and then replace the coupling with column.     

5.3.3.3 Instrumental parameters as follows:

Mobile Phase : 80:20 (Water : Methanol)

Column       : shim-pack XR-ODS (3.0mm i.d  X 50mm)

Sample       : Caffeine solution (20mg/L)

B.con        : 20%

P.max        : 40 Mpa

Oven temperature: 40 0C

Flow         : 1.000 mL / Min

Run time     : 5.0 minutes

Sample aspiration rate:5µl (for 50µl sample loop)15µl (for other sample loop)

PDA- Detector:

Sampling rate: 60ms

Time constant: 0.32s

Lamp:D2

Start wavelength:190nm

End wavelength:800nm

Slit: 8nm

Multi-chromatogram:Ch1: 272nm, band width: ±4nm, Ref. correction: OFF

5.3.3.4 Use the default values for other parameters.

5.3.3.5 Start the LC system. 

5.3.3.6 Wait until the baseline stabilizes. Inject 5µl mobile phase and check that no peak is detected on the chromatogram.

5.3.3.7 Inject 5µl test solution. Repeat the analysis 6 more times. Record the results.

5.3.3.8 Calculate the retention time and the coefficient of variation (CV%) of the retention time and the peak area based on six analyses.

5.3.3.9 Acceptance criteria: RSD should be NMT 1.0% for peak area.

5.3.3.10 Record the Corresponding observed values in “Calibration Record for Repeatability” ( Annexure-4).

5.3.3.11 Calibration Schedule: once in 6 months and after any major maintenance job.

5.3.4 Injected volume accuracy test: 

5.3.4.1 Set the measurement conditions as:

Mobile phase and auto sampler rinse solution:  Water 

Sample : Water

Resistor tube PEEK(0.065 mm ID):Standard accessory S228-38994 

Sampler cooler: OFF

Injection volume: 5µl, 10µl, 50µl

Analysis time: 0.1 min

No. of injections: 5µl: 20 times

10µl: 10 times

50µl: 10 times

5.3.4.2 Thoroughly flush the mobile phase port to be used and rinse the solution with water.

5.3.4.3 On Performance check screen. Press the auto sampler tab and then press injection volume accuracy.

5.3.4.4 Put 1 ml of water into the 1.5ml sample vial, and measure and record the mass (W1) using a calibrated balance(enter the measured mass).

5.3.4.5 Set the weighed sample vial containing water on sample rack (position 1 of plate 1 of left side), and then press “Next”. 

5.3.4.6 Select the mobile phase port to be used and press “Next”. 

5.3.4.7 Repeated injections of either 5, 10, 50µl is automatically started.                

5.3.4.8 When injection is completed, remove the 1.5ml sample vial and measure the mass (W2) using calibrated balance.

5.3.4.9 Press “Next” automatically calculates the injected volume accuracy and displays results. Press”logged” to record the results.

5.3.4.10 Injected volume accuracy can be obtained using the equation

Injected volume accuracy(%) = (W1-W2)/ (set injection volume x no. of injections) x 100

5.3.4.11 Acceptance criteria:  ±2.00%.

5.3.4.12 Record the details in “Calibration Record for Injector Volume Accuracy” - (Annexure-5)

5.3.4.13 Calibration Schedule: Once in 6 months and after any major maintenance job.

5.3.5 CARRY OVER TEST:

5.3.5.1Set Instrument Parameters as 5.3.3.3 except 

Sample: 250mg/L caffeine

Injection volume:    5µl

5.3.5.2 Inject 5µl of 250mg/L caffeine solution sample.

5.3.5.3 Then inject 5 µl of mobile phase as a blank sample. 

5.3.5.4  Calculate the Carry over % using the following formula:

     % Carry over=(Area observed in blank / Average area of Caffeine peak) X 100

5.3.5.5 Record the details in “Calibration record for Injector Carryover” -(Annexure-6)

5.3.5.6 Acceptance criteria: NMT 0.010% 

5.3.5.7 Calibration Schedule: Once in 6 months and after any major maintenance job.

5.3.6 ABSORBANCE LINEARITY:

5.3.6.1 Prepare 5 Caffeine standard solutions (20.0 mg/L, 30.0 mg/L, 40.0 mg/L, 50.0 mg/L,60.0 mg/L).

5.3.6.2 Record the details in work sheet in “WORK SHEET FOR  PREPARATION OF STANDARD CAFFEINE SOLULTIONS” (Annexure-7).

5.3.6.3 Install the resistor tube and set water of HPLC grade.

5.3.6.4 Instrument Parameters as follows:

Pump flow rate:0.3mL/min

Auto sampler sampling speed :15µl/s

Column oven temperature:40 0c.

PDA Wavelength:272nm

Bandwidth : ±4nm

Reference correction: OFF

Lamp:D2

Slit width:8nm or 1.2nm

Sampling:160ms

Time constant:320ms

5.3.6.5 Analyze the 5th solvent to determine the injection volume. Inject 35µl. if the height of the detection peak does not fall between 1.4 AU and 1.7 AU, adjust the injection volume.

NOTE: changing the injection volume by 10% cause the peak height to vary by about 10%.

5.3.6.6 inject  1st solvent to 5th solvent under the condition determined in procedure 5.3.6.3 and read the peak height.

5.3.6.7 Calculate the absorbance deviation using the following procedure

Relative sensitivity = (Absorbance value)/(sample concentration)

Standard relative sensitivity = { (relative sensitivity of solution 1) +(relative 

sensitivity of solution 2) + (relative sensitivity of solution 3 )}/3

Deviation (%) = (Relative sensitivity-standard relative sensitivity)                                      

5.3.6.8 Report the results in “Calibration  Record  for Absorbance Linearity”(Annexure-8)

5.3.6.9 Acceptance criteria: Error is within ± 5.0% for each concentration.

5.3.6.10 Calibration Schedule: Once in 6 months and after any major maintenance job.

5.3.7 GRADIENT CONCENTRATION ACCURCY:

5.3.7.1 This test is executed using Auto validation function. This test is not executed if the target system is in isocratic mode.

5.3.7.2 Perform low pressure gradient (A/B, A/C AND A/D) using 10 mg/l caffeine solution.

5.3.7.3 Measure absorbance at 0%, 10%, 50%, 90%, 100% .

5.3.7.4 Report the results in “Calibration record of Gradient Concentration Accuracy” (Annexure-9).

5.3.7.5 Acceptance criteria: A/B, A/C, A/D accuracies at concentration of 10%, 50%, 90% are within ± 2.00%.

5.3.7.6 Calibration Frequency: Once in 6 months and after any major maintenance job.  

5.3.8 Test for Noise and Drift:

5.3.8.1 Ensure the instrumental parameters as follows,

Column: shim-pack XR-ODS (3.0mm i.d  X 50mm)   

Flow rate:0.000 ml/min

Injection volume:0mL

Run Time:15 minutes

Wave length:250 nm  

5.3.8.2 Test is executed using auto validation function.

5.3.8.3 Acceptance criteria: Drift ≤2.00 Mau/h, Noise≤ 0.0500mAU

5.3.8.4 Report the results in “Calibration Record of Noise and Drift” (Annexure-10)

5.3.8.5 Calibration Frequency: Once in 6 Months and after any Major Maintenance job.

5.3.9 WAVELENGTH ACCURACY

5.3.9.1 This test is executed using auto validation function:

5.3.9.2 Using emission line of D2 lamp and absorption spectral line of internal Ho filter, wavelength accuracy shall be checked.

5.3.9.3 Record the Corresponding observed values in “Calibration Record for Wavelength Accuarcy ( Annexure-11).

5.3.9.4 Acceptance criteria :Ho filter ± 1.00nm, D2 (656.1nm) ± 1.00nm

5.3.9.5 Calibration Schedule: once in 6 months and after any major maintenance job.

NOTE: All calibration parameters shall be performed within + 7 days of due date.

5.4  Maintenance:

5.4.1 Maintenance shall be done by in-service engineer.

5.4.2 Remove the suction/inlet filters and sonicate in 100% methanol followed by water, if physically damaged needs to replace with new one.

5.4.3 Remove the pump check valves, line filters and cartridges and sonicate in 50:50 (methanol: water),if physically damaged needs to replace with new one.

5.4.4 Remove the autosampler port valves sonicate the stator and rotor seal with 50:50 (methanol: water) if physically damaged needs to replace with new one.

5.4.5  check needle and needle seal at regular intervals, if physically damaged needs to replace with new one.

6.0 ANNEXURES