Calibration of HPLC

TITLE: CALIBRATION of HPLC

Calibration of HPLC
SOP NO.:	QC00X-01	EffectiveDate:	DD/MM/YYYY	Company Logo
Supersedes :	NA	Page No.:	01 of end
Department:	Quality Control	Review Date:	DD/MM/YYYY

I           OBJECTIVE 

• To provide a procedure for calibration of the HPLC (High-Performance Liquid Chromatography).          

II         SCOPE

•  This procedure is applicable for the HPLC series, used in the Quality Control department of          ABC Ltd, Location. 

III        RESPONSIBILITY 

•  It is the responsibility of all the QC personnel involved in the analysis using the system.    

IV        DOCUMENT REFERENCE(s)

            SOPs               :  Policy on calibration (Current version of QA 0XX)

            Forms              :

S.No	Details	Format No. (Current version)
1	Calibration of the HPLC system	QC0XX/F0X-XX

 

 

  

V         SCHEDULE:

•   Calibration shall be carried out once every 6 months.

VI        PROCEDURE  

1.0       Calibration Procedure

1.1       To Perform the Calibration of HPLC as follows.

1.2       Calibration shall be carried out with the below parameters.

1.2.1    Pump flow calibration

1.2.2    Wavelength accuracy

1.2.3    System precision

1.2.4    Carryover

1.2.5    Detector linearity

1.2.6    Injector linearity

1.2.7    Temperature verification for oven and sample cooler.

1.2.8    Gradient Performance Check

1.2.9        Signal Noise and Drift

1.3             Enter the calibration details in the tag and appear the same to the respective instrument

1.4              Calibration procedure shall be carried out as follows

            Conditions:

   Column  : Zorbax SB-C8 150mm x 4.6mm, 3.5u or equivalent

            Wavelength                 : 273 nm

            Flow Rate                   : 1.0ml/minutes

            Injection Volume        : 20 μl

            Runtime                      : 10 min

            Temperature                : Ambient temperature

1.5       Mobile Phase Preparation

A filtered and degassed mixture of Methanol and Water in the ratio of 60:40V/V.

1.6       Preparation of Caffeine stock solutions

Weigh accurately 100 mg of Caffeine into 100 volumetric flasks and make up to the mark with the mobile phase.

1.7       Sample Solutions Preparation:

1.7.1   Sample solution-1 (0.02mg/ml concentration) preparation: Take 2.0 ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.

1.7.2   Sample solution-2 (0.04mg/ml concentration) preparation: Take 4.0 ml of Caffeine stock solution in 100ml volumetric flask and make it to the mark with the mobile phase

1.7.3   Sample solution-3 (0.06mg/ml concentration) preparation: Take 6.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.

1.7.4     Sample solution-4 (0.08mg/ml concentration) preparation: Take 8.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it up to the mark with the mobile phase.

1.7.5     Sample solution-5 (0.1mg/ml concentration) preparation: Take 10.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.

1.8       Pump flow rate calibration

1.8.1    Measure the flow rate ua sing 10 and ml, 25 ml volumetric flask, and calibrate the ted stop clock. Measure the flow rates of 1.0ml/min, 1.5 ml/min, 2.0 ml/min, and 2.5 m/min.               

                                           Volume in ml  X 60

         Flow Rate     =  ___________________________________                                                   

                                 Actual time taken to collect the volume in seconds

1.8.2    Acceptance Criteria    :  ± 2.0%.

1.9       Wavelength Accuracy

1.9.1    Chromatographic conditions  

            Column                   :    Zorbax SB-C8 150mm x 4.6mm, 3.5 micron or equivalent

            Flow rate                 :   1.0 ml/min.

            Injection volume     :   20 ml

            Run time                 :   10 min

1.9.2  Preparation of Mobile phase: Prepare a filtered and degassed mixture of methanol and water in the ratio of 60:40 v/v.

1.9.3 Prepare 0.1 mg/ml caffeine solution in the Mobile phase. Inject caffeine solution at each length rise from 200nm – 210nm, from 239nm – 249nm, and from 268nm-278nm (increment of   1nm), record the details in HPLC calibration format record as per format No: QC0XX/F0X-0X

1.9.4   Acceptance Criteria

•    Maximum peak response must be at wavelength: 205 ± 2 nm for200 nm-210 nm 
•   Maximum peak response must be at wavelength: 273 ± 2   nm for 268 nm-278 nm
•   Minimum peak response must be at wavelength: 245 ± 2 nm for 239nm-249nm               

1.10   Detector Linearity

1.10.1  Inject Sample solution-1 to Sample solution-5 into the chromatographic system.

1.10.2  Record the area of Caffeine in each sample solution and calculate the Correlation coefficient. Draw the linearity graph with area vs. concentration.

1.10.3    Acceptance Criteria: The Correlation coefficient should be not less than 0.99.

1.11     System Precision:

1.11.1  Inject 5 times sample solution-5 into the Chromatographic system and calculate the           %RSD for Retention time and %RSD for Areas of the sample solution in 5 injections.

1.11.2  Acceptance Criteria: %RSD for Area and Retentions times should be less than 2.0.

1.12     Carryover:

1.12.1  Applies only if carry-over is run immediately after precision.

1.12.2  Inject diluent as a blank. And record the chromatogram.

1.12.3  Calculate the carryover percent using the fifth injection area of precision.

                                                                Blank area/height

                            Carryover (%) =      ------------------------    x 100

                                                               Sample area/height

1.12.4   Acceptance Criteria: NMT 0.2 % for the area and NMT 0.4 % for height. 

1.13     Temperature verification for Oven and Sample cooler:

1.13.1  Check the oven temperature at different set temperatures at 30°C, 40°C, 50°C, 60°C using calibrated Digital thermometer.

1.13.2 Check the Sample cooler temperature at 5°C and 10°C

1.13.3 Acceptance Criteria: ±1°C

1.14     Injector Linearity:    

1.14.1  Inject Sample solution-5. 10µl, 20µl, 30µl, 40µl, 50µl into    Chromatographic system.

1.14.2  Record areas of Caffeine in each injection and draw the linearity graph with area vs injection volume.

1.14.3  Calculate correlation coefficient.

1.14.4  Acceptance Criteria: The correlation Coefficient should not be less than 0.99. 

1.15    Gradient performance check:( GPV Test)

1.15.1 Preparing the Instrument:

1.15.2 Connect with union 

1.15.3 Filter and degas methanol, 0.5% acetone in methanol

1.15.4 Method Parameters:

1.15.5 Detector wavelength of 254 nm

1.15.6 Binary gradient with a total flow of 2.0 ml/min

1.15.7 Gradient table as per below table for pumps A and B.    

1.15.8 Running the Gradient proportionate Valve test:                                                                                                

Time	A                        Methanol	B  0.5% Acetone in Methanol
0.01	100	0
4.01	90	10
8.01	100	0
12.01	75	25
16.01	100	0
20.01	50	50
24.01	100	0
28.01	25	75
32.01	100	0
36.01	0	100
40.01	100	0
44.0	100	0

.          

 

 

 

 

 

 

 

1.15.9    Purge all the pumps with a flow rate of 2 to 5 ml/min for about 2 to 5 minutes.

1.15.10  Set the total flow rate at 2.0ml/min and wait until the baseline is stable.

1.15.11  Set the gradient profile for Pumps A and B.

1.15.12  Record the height of peaks.

1.15.13 Consider height at B conc. At 100% 100 calculate the other peaks' heights.

1.15.14 For Quaternary systems, repeat the study for other channels of C and D.

1.15.15  Consider height at D conc. At 100% 100 calculate the peak heights.

    Calculation:

                                          Height of B/D peak at different concentrations

 Height % of Peak B/D = ----------------------------------------------------------  X100

                                         Height of full-scale peak (100% B/D peak height)

 

1.15.16 Acceptance criteria: The difference in the proportion of B or D concentration should not vary by more than ± 1.5% for the set concentration.

1.15.17 then select the Setup Detector signal option enter the required values and click OK.

1.15.18 to her that if already method already was saved we have to open it, and if it any necessary to modify the method we can change it.

1.15.19 now run the sample, the sampler will pick the sample after the sample is picked the needle is washed from the additional vial kept for purposes
5.20 Check the flow on running and we can get the result.

1.15.21 here select printer and press OK. Again select the Report/print option. The report is printed and filed.

1.16.0   Signal noise and Drift:

1.16.1   Fix the union as a column.

             Mobile phase: Water.

             Flow rate        : 1.0 ml/min

             Wavelength    : 254 nm

             Run Time       : 24 min

             Start Time      : 03 min, End Time: 20 min

1.16.2    Run the chromatogram and record the data.

1.16.3    Evaluation of the noise and drift between 3 min to 20 min.

1.16.4    Determine the noise and drift using software.

1.16.5    Acceptance criteria: Should be less than 40µV for noise and Should be less than 500µV/Hz 8.3µV/min.                     

2.0     CARE AND MAINTENANCE:

2.1 Ensure the details are documented in the column usage log after the analysis & washing activity are completed

2.2   Use 85:15 Acetonitrile and water for the plunger wash.

2.3   Use mobile phase/diluent vial for syringe needle wash.

2.4 Verify the performance of the system as per the schedule/after a major breakdown of the instrument.

2.5    Replace the inlet filters based on their performance.

2.6    Enter the breakdown details & spares utilized in the Instrument malfunction record. 

* All mobile phase solutions shall be prepared as per the analysis requirement. However mobile phase solutions stored beyond 48 hrs shall not be used. And document the details in the mobile phase and mobile phase log

REFERENCE:

*  Instrument Manual of  HPLC

REVISION HISTORY

Version	Effective	Summary of Revision
0.0	DD/MM/YYYY	New SOP

 

END O F THE DOCUMENT

                                     

                                     Prepared By           Checked By             Approved By

        Sign & Date

        Name & Dept    QA Executive         QC Manage               QA Manager.   

#HPLC #Calibration, #GPV #Performance