STANDARD OPERATING PROCEDURE CALIBRATION OF SHIMADZU HPLC (LC-2010CHT)

STANDARD OPERATING PROCEDURE

SUBJECT:                      CALIBRATION OF SHIMADZU HPLC (LC-2010CHT)

DEPARTMENT           : QA SOP NO.                      : Page No                       : Effective Date             : Next Review                : SOP VERSION            :

1.0       OBJECTIVE:

             To provide guidelines for Calibration of Shimadzu HPLC.

2.0       SCOPE:  

             To establish a procedure for calibration of Shimadzu HPLC.

3.0       PROCEDURE:

3.1       Switch on the instrument and connect to LC solution software.

3.2       Press the Purge button, select the required port and purge by using display panel present on instrument. Purging can also be performed by gradually increasing the flow rate of the pump by using software.

 3.3      Solvents used are Methanol, IPA, and Water.

3.4.      Calibration of the pump

3.4.1    Check the flow rate of the filtered and degassed Purified water as follows.      

3.4.2    Disconnect any column if connected to the system and connect flow restrictor. Set the flow of the

             Channel “A” at 0.500 ml per minute. Keep the system at this flow rate for 10 minutes to equilibrate the

             system.

3.4.3    Set the flow to 1.000 ml per minute and 2.000ml per minute respectively and repeat the above experiment, allowing the system to equilibrate at least for 10 minutes at each flow set. 

3.4.4    Repeat the steps for setting the flow using channel “B, C and D”                             

                                   

 3.4.5   Acceptance criteria:

            Flow accuracy:  Between 95% and 105% of the set flow rate.

           

5          Wavelength accuracy (Using Caffeine in purified water)

           

3.5.1    Caffeine Solution:

            Set the following chromatographic conditions.

Column	: C18, 250 mm x 4.6 mm, 5 microns
Mobile phase	: Methanol: Purified water (40: 60 v/v). Filter and degas
Flow rate	: 1.0 ml / min.
Wavelength	: 272 nm
Injection volume	: 5, 10, 15, 20 µl
Column oven temp	: 30°C

3.5.2    Preparation of standard solution: Weigh accurately 100mg of caffeine, transfer into a 100 ml volumetric flask, dissolve and dilute to volume with purified water. Further dilute 2 ml of this solution to 100 ml with purified water. (Concentration 20ppm Caffeine)

3.5.3    Install the flow restrictor in the column thermostat, equilibrate and keep sample solution in auto sampler.

3.5.4    Switch ‘ON’ the detector UV lamp for at least 30 minutes.

3.5.5    Inject the Caffeine solution & record the results.                                                        

           

3.5.6    Acceptance Criteria:

               Acceptable range: 270-276 mm.

3.6       Accuracy of auto injector and linearity of UV- Detector response.

           Chromatographic conditions same as above:

3.6.1    Preparation of standard solution: Weigh accurately 100mg of caffeine, transfer into a 100 ml volumetric flask, dissolve and dilute to volume with purified water. Further dilute 2 ml of this solution to 100 ml with purified water. (Concentration 20ppm Caffeine)

3.6.2    Theoretical plates of caffeine peak should not be less than 3000 and tailing factor should not be more than 2.0.

3.6.3    Calculate the % RSD of the peak areas & retention time of caffeine from the 6 replicate injections at each level. Calculate the Mean peak area of caffeine from the 6 replicate injections at each level.

3.6.4    Plot a linearity graph between injections volumes on the X-axis V/S mean area of caffeine at each level on the Y-axis (instrument linearity graph can be considered for the same).

3.6.5        Acceptance Criteria:

1)      The % RSD of retention time of caffeine from the six replicate injections at each level should be less than 2% and % RSD of Area of caffeine from the six replicate injections at each level should be less than 2%

2)    The graph of caffeine for different levels should be Linear.

                       

3.7       Carry Over Test:

 3.7.1   Chromatographic conditions same as above:

3.7.2    Preparation of standard solution: Weigh accurately 100mg of caffeine, transfer into a 100 ml volumetric flask, dissolve and dilute to volume with purified water. Further dilute 2 ml of this solution to 100 ml with purified water. (Concentration 20ppm Caffeine)

3.7.3    Inject blank followed by standard in triplicate and again blank form fresh vial. 

Calculate any carryover of caffeine in the blank.

3.7.4    Calculate % carryover of Caffeine peak, in the blank chromatogram after 20 µl of standard solutions,    

             by using formula:

                            Area of Caffeine in blank  

                 = ------------------------------------------------ x 100

                          Mean area of Caffeine standard

3.7.5    Acceptance Criteria: Carry over should not be more than 0.1%.

3.8       Gradient composition:

3.8.1    Set the following chromatographic conditions.

Column                                           : C18, 250 mm x 4.6 mm, 5 microns

Solvent for channel A                 : Filtered and degassed purified water.

Solvent for channel B / C / D       : 0.1 % Acetone in purified water

Flow rate                                      : 2.0 ml/min

Wavelength                                  : 265 nm

Gradient programming:

Step	Time in minute	% Solvent – A	% Solvent – B / C / D
1	0.0	100.0	0
2	1.0	100.0	0.0
3	4.0	99.0	1.0
4	8.0	50.0	50.0
5	12.0	1.0	99.0
6	15.0	0.0	100.0
7	24.0	100.0	0.0

3.8.2    Procedure: Set the flow rate of at 2.0 ml per minute. Keep the system in this flow rate for some time to equilibrate. Start the gradient programming.

3.8.3    Acceptance Criteria:

            As per the fed gradient program the system functioned.

Result: The accuracy of Gradient composition passes as per the desired program.

3.8.4    The instrument is covered under annual service contract and it is serviced as and when there is a breakdown or requirement.

4.0       FREQUENCY:  Internal Calibration:

Once in three months for flow rate and Gradient composition. Once in six months for Wavelength accuracy, Accuracy of auto injector, linearity & Carry Over test.

External Calibration:

Once a year for wave length accuracy check flow rate Accuracy, Injection volume accuracy Oven Temperature accuracy Repeatability and check of program files by SPINCO LABORTORY PVT LTD Service Engineer.

5.0       RESPONSIBILITY: G.M (Q.A)/ Executive Q.C

                                                                                                Version No   : 13

6.0       REVIEW GUIDE: Calibration record.

7.0       ANNEXURE: --------

8.0       SOPs DISTRIBUTION:

            This SOP shall be distributed to following Departments:

Name of the Department	Master Copy	Circulatory Copy
QA/QC	√	√
Production	x	x
Warehouse	x	x
General	x	x
Maintenance	x	x