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SOP for Calibration of HPLC (Low Pressure Quaternary Gradient Liquid Chromatograph)

Standard operating procedure of calibration of HPLC and its components like PUMP, Detector, Column Oven and injector.


       To lay down the procedure and frequency for calibration of HPLC.

2.0  SCOPE

       This procedure is applicable to check the calibration of HPLC Quaternary Gradient system
(Model : LC 2010 C, LC 2010 C HT Make : Shimadzu) installed in Quality Control laboratory.


3.1  Doing       : Technical Assistant / Executive
3.2  Checking  : Executive / Manager   


       Head of the department



6.1  Operate the instrument as per respective standard operating procedure.

6.2  For PUMP calibration

6.2.1  Set the flow rate to 1.0 ml/min.
6.2.2  Connect Resisto tubing 2 meter x 0.1 mm ID. in place of column.
6.2.3  Start the pump and wait till equipment stabilised or a baseline is achieved.
6.2.4  Weigh the dry empty A grade measuring cylinder. Weigh & record the same.
6.2.5  Collect the water from outlet of Resisto tubing for 5 minutes and weigh the measuring cylinder. Record the value.
6.2.6  Repeat the step 6.2.5 and continue the process for four repetitions.
6.2.7  Calculate the flow rate accuracy.

6.3  Wavelength accuracy of detector

6.3.1  Switch on the LC 2010 C, LC 2010 C HT by pressing function key.
6.3.2  Press VP key.
6.3.3  Press Func Key and then Press F5.
6.3.4  Press for detector.
6.3.5  Press Right Arrow Key, to go to next screen (2/5).
6.3.6  Press Down Arrow Key seven times (make sure the cursor is blinking in ‘z wave’ parameter) press 1 enter.
6.3.7  Press Func and Press F5 for exit.
6.3.8  Press Met Key.
6.3.9  Flush the flow cell with Methanol.
6.3.10  Open the port of Detector and pass the methanol with using the syringe for cleaning.
Press Zero Key and ensure that the absorbance is zero.
6.3.12  Inject 5 ml of 2 mg/100 ml solution of caffeine in methanol and changing the wavelength from 266 nm to 277 nm with the step of 1nm and record the respective absorbance.
6.3.13  Record the wavelength that produces the maximum absorbance.
6.3.14  Record the results.

6.4  Reproducibility of Results

6.4.1  Prepare the mobile phase consisting of 70 % Methanol: 30 % Water.
6.4.2  Prepare a mixture of 0.1 % v/v  Benzene and 0.1 % v/v Toluene in Methanol.
6.4.3  Set the flow rate of 1 ml/min.
6.4.4  Allow the system to be saturated with the mobile phase for at least 15 minutes before injecting the sample preparation.
6.4.5  Record the area and Relative Retention time of both benzene and toluene.
6.4.6  Calculate the RSD for both area and Relative Retention time respectively.
6.4.7  RSD of Area should not be more than 2.0 %. & RRT of  Benzene & Toluene should not be more than 2.0 %. 
6.4.8  Calculating the Tailing Factor ( Asymmetry), Resolution and Theoretical Plate and record.

6.5  Program files Check

6.5.1  Click start menu in the computer.
6.5.2  Go to Programs and select Chromatography.
6.5.3  Click Program Alteration Test.
6.5.4  Program files check window will open and Click Execute.
6.5.5  Software Checks all the files and gives the result.
6.5.6  If all the files are found good, Check is finish <<Integrity Check Passed>> will display.
6.5.7  Click notepad to see the e status of files.
6.5.8  Click files in notepad window and select print for print out.
6.5.9  If some files are found not good, Check is finish <<Integrity Check Failed>> will display.
6.5.10  Click notepad to see the status of files.
6.5.11  In such case call service engineer.
6.5.12  Record the status of program file.

6.6  Linearity test :

6.6.1  Prepare the mobile phase consisting of 70% Methanol: 30 % water.    
6.6.2  Stock solution preparation: Prepare a mixture of 0.2 % Benzene and 0.2 % Toluene in methanol.
Make dilution in following manner.
6.6.3  Take 5 ml, 10 ml, 15 ml, 20 ml of stock solution respectively in 25 ml of volumetric solution and make up with methanol.  
6.6.4  Set flow rate at 1.0 ml/min.
6.6.5  Allow the system to be saturated with mobile phase  for at least 15 minutes before injecting the different solutions.
6.6.6  Record the area of both benzene and toluene.
6.6.7  Calculate the Linearity for both Benzene and Toluene respectively.

6.7  Carry Over test :

6.7.1  After completion of the Linearity test, inject 20┬Ál of methanol.
6.7.2  Calculate the Carry Over test.

6.8  Quaternary gradient accuracy test :

6.8.1  Connect Resisto tubing ( 2 meter x 0.1 mm I.D.) to the instrument.
6.8.2  Prepare 0.3% Acetone in Purified Water.
6.8.3  Put mobile phase as follow in to system.
         A : Purified Water
         B, C, D : 0.3 % Acetone in Purified Water.
6.8.4  Set flow rate to 1.0 ml / min.
6.8.5  Set wave length to 254 nm.
6.8.6  Set time programme.
6.8.7  Adjust the base line level to fit the full scale of the integrator.
6.8.8  Start the time programme for gradient performance.
6.8.9  Determine the signal level of 0% (B conc 0), 10 % (B conc 10), 50 % (B conc 50), 90% ( B conc 90), and 100 % (B conc 100).
6.8.10  Determine the signal level of 0% (C conc 0), 10 % (C conc 10), 50 % (C conc 50), 90% ( C conc 90), and 100 % (C conc 100).
6.8.11  Determine the signal level of 0% (D conc 0), 10 % (D conc 10), 50 % (D conc 50), 90% ( D conc 90), and 100 % (D conc 100)
6.8.12  Calculate concentration of B10, B50, B90, C10, C50, C90 and D10, D50, D90.
6.8.13  The performance of the instrument is satisfactory if the obtained RSD is within the given limit, otherwise follow the SOP.

6.9  Temperature Accuracy Test For Column Oven & Sample Cooler Tray.

6.9.1  Use a calibrated thermometers to measure the actual temperature in the oven & of sample cooler tray and compare it to set value
6.9.2  Procedure for Temperature Accuracy test for Column Oven.  Set the temperature accuracy is measured by using “ Performance Check ”  Press [VP]  [F3 (VALID)]   [2]   [Enter] key, and select “ 5 oven Temperature” from the “ Performance Check ” menu.  Select the 40°C and 50°C temperature to be measured as instructed on the screen.  Use the clip to set a calibrated temperature sensor at the center of the lower step of heat block inside the device .  Press the [OVEN] key to start temperature adjustment. After the temperature has stabilized the temperature fluctuation amplitude will be automatically measured for 5 minutes.  Record the Actual value against the calibrated thermometer value for different time intervals of 30, 45 and 60 minutes.
6.9.3  Procedure for Temperature Accuracy test for sample cooler tray  Press the [Seq], [F1] key, and enter “4” to “cooler temp” ( auto temperature setting parameter).  Enter 1 to “ SMPL.C” in other parameter section of Method screen.  Set the cooling rack ( R ) in the main unit & put the probe of calibrated thermometer inside the sample cooler tray.
Wait to stable at the set temperature since it normally takes around 90 min.
Press [VP]   F3 (VALID)]   [2]   [Enter] key, and select “ 11. Cooler  Temperature” from the “ Performance Check”.  Record the  Actaul value against the calibrated thermometer value for different time intervals of 30, 45 and 60 minutes.


RRT  :  Relative Retention Time
%      :  Percentage.
ml     :  millilitre
RSD  :  Relative Standard Deviation.
RT    :  Retention Time
v/v    :  Volume / Volume

Also see: Relative Response Factor (RRF) Calculation in HPLC

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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1 Comment so far : Add yours...

Suresh Pottella said...

In detector calibration only checking the wavelength accuracy by using caffeine(266nm-277nm),what about remaining nm,becasue my product having 385 nm wavelength shall i use the instrument by this calibration procedure

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