Calibration of HPLC

1.0    PURPOSE: 

1.1     To lay down the procedure for calibration of HPLC. 

2.0    SCOPE.

2.1    This SOP is applicable for the Calibration of HPLC’S in QC / IPQC Laboratories.

3.0    RESPONSIBILITY

3.1    Executive-QC

4.0    ACCOUNTABILITY

4.1     Asst. Manager-QC

5.0    PROCEDURE

5.1    Follow the respective SOP for operation and carry out the following tests for the calibration of a HPLC system as applicable.

5.1.1 FLOW RATE ACCURACY
5.1.2 GRADIENT COMPOSITION ACURACY
5.1.3 INJECTION VOLUME PRECISION
5.1.4 OVEN TEMPERATURE/SAMPLE CHILLER TEMPERATURE    
         ACCURACY
5.1.5 WAVELENGTH ACCURACY
5.1.6 PRECISION /CARRY OVER 
5.1.7 RESPONSE LINEARITY

5.2    FLOW RATE ACCURACY:
5.2.1 Flow rate accuracy shall be checked for 0.5 mL, 1.0mL and 2.0  mL/minute with 
      the following chromatographic conditions.
                Column: Restriction capillary
                Mobile phase: HPLC grade water
       5.2.1.1 Program the flow rate to 0.5 mL/minute and allow to stabilize.
        5.2.1.2 Take a clean and dried measuring cylinder of 10mL capacity   
                and weigh accurately(W1).
         5.2.1.3 Take a calibrated stop watch.
       5.2.1.4 Introduce the capillary outlet into the measuring cylinder and  
                start the stop-watch simultaneously.
        5.2.1.5 Stop the watch after collection of about 10 mL water in the   
                cylinder  and withdraw  the capillary outlet simultaneously. 
    Note the time elapsed as ‘t’. 
       5.2.1.6 Weigh the measuring cylinder containing water accurately 
                (W2). Calculate the weight of water collected (W).
        5.2.1.7 Calculate the volume (V) of water using the specific gravity of 
                water at  specified temperature  as given below. Calculate the   
                flow rate  as follows;
                Flow rate (mL/minute)= V/t
5.2.1.8    Repeat steps 5.2.1.1 to 5.2.1.7 and determine the flow rate accuracy for 1.0 mL/min and 2.0 mL/min.
5.2.1.9    Record the details as per Format No. xxxxx

                                                    TABLE 1

    VOLUME OF 1 g OF WATER AT VARIOUS TEMPERATURES

5.3    GRADIENT COMPOSITION ACCURACY

5.3.1    Determine the gradient composition accuracy using the following chromatographic conditions.

5.3.1.1    For Low pressure gradient system

Pre-Purge

Wavelength: 265 nm
Flow:         2.0 mL/min
                 Stop-time: 10.00
                 Solvent A: HPLC grade water.
                 Solvent B:0. 5% Acetone in HPLC grade water.
                 Timetable:

                  Time         Solvent B
                   0.01        100.0
                   8.0    100.0
                   8.01        0.0
                        

Gradient Run:                    
Wavelength: 265 nm
Solvent A: HPLC grade water.
Solvent B:0. 5% Acetone in HPLC grade water.
Flow:         2.0 mL/min
Stop-time: 25 + 10 minute’s pre-purge.
                                                 TABLE2

                                              TIME TABLE

5.3.1.1 Prior to test gradient composition perform a pre-purge.
        5.3.1.2 Perform the test in triplicate and Measure the average height of every step
        5.3.1.3 Re-scale the height and noise from absorbance to composition  (%B)  values.
        5.3.1.4 Calculate the composition accuracy for every step as the absolute    
            difference between the rescaled measured step height  and the corresponding
            gradient composition values.
      
ACCEPTANCE CRITERIA    : ± 1.500%     

5.3.1.2    For High pressure gradient system

Pre-Purge
Wavelength: 254 nm
Flow:         1.0 mL/min
Stop-time: 10.00
Solvent A: HPLC grade water.

Solvent B:0. 3% Acetone in HPLC grade water.

                 Timetable:

                  Time         Solvent B
                   0.01        10.0
                   10.0        10.0
                        
Gradient Run:
                
Wavelength: 254 nm
Solvent A: HPLC grade water.
Solvent B:0. 3% Acetone in HPLC grade water.
Flow:         1.0 mL/min
Stop-time: 50 min. 
        
                                                     TABLE3
                                                 TIME TABLE

5.3.1.1 Prior to test gradient composition perform a pre-purge.
        5.3.1.2 Perform the test in triplicate and Measure the average height of every step
        5.3.1.3 Re-scale the height and noise from absorbance to composition  (%B)  values.
        5.3.1.4 Calculate the composition accuracy for every step as the absolute    
            difference between the rescaled measured step height  and the corresponding
            gradient composition values.
       

ACCEPTANCE CRITERIA    : ± 1.0%

5.4    INJECTION VOLUME ACCURACY

5.4.1    Injection volume precision shall be determined for 10?L, 20 ?L, 50 ?L and 100?L Injections.
                    Chromatographic conditions:
                    Mobile phase: HPLC grade water.
                    Column:  Restriction capillary.
                    Run time: 1 min
                    Flow: 1 mL/min
    5.4.1.1 Fill four vials with HPLC grade water and cap them with  septum.
    5.4.1.2 Identify the vials and accurately weigh each of the vials  separately.
    
    5.4.1.3 Place the vials in the sample tray of the HPLC system and program an
                                         injection  sequence as follows.

                                                    TABLE3
   VIAL                           INJECTION VOLUME      REPETITIONS
   Vial1                                   10?L                           5
   Vial2                                   20?L                           5
   Vial3                                   50?L                           5
   Vial4                                   100?L                          5
   
    5.4.1.4 After completion of the injection cycle weigh the vials  accurately and 
          determine the average injection volume for each nominal volume using the 
          volume of water per gram at specified temperature as included  in table 1.
                                      Record the details as per Format No. xxxxx
  
                                      ACCEPTANCE CRITERION:  + 10.0% of programmed value.

Note  :  INJECTION VOLUME ACCURACY is applicable only to HPLC system 
  having autosampler / autoinjector. 

5.5    OVEN TEMPERATURE/SAMPLE CHILLER TEMPERATURE ACCURACY

5.5.1    Oven temperature accuracy shall be checked at 300C, 400C and 500C. 
5.5.2    Sample chiller accuracy shall be checked at the temperatures 100C, 150C and 200C.
5.5.3    Set the temperature of the oven /sample chiller to one of the above  
            mentioned values, insert the temperature probe  and allow to stabilize.  
            After stabilization monitor the temperature at intervals of 5 minutes and  
  note down three readings of the observed value. Repeat the procedure for the  other  nominal values and record the details as per Format No. xxxxx

    ACCEPTANCE CRITERION:  + 20C of programmed value.  

5.6    WAVELENGTH ACCURACY

5.6.1 Wavelength accuracy of UV detector 

       Chromatographic conditions:
       Sample: 20 ?g/mL caffeine in water.
       Run time: 7 min.
       Wavelength program:

5.6.1.1 Fill the flow cell with caffeine standard solution by injecting    
                directly  into  the detector inlet and start the program.
    5.6.1.2 Record the peak response at various wavelengths as per 
          Format Noxxxxx    
ACCEPTANCE CRITERION: Wavelength Maximum AT 273+ 1nm.

NOTE : If the system has no wavelength programming option, than perform the test by selecting wavelength manually for each injection.

5.6.1    Wavelength accuracy of PDA detector
5.6.1.1    Fill the flow cell with caffeine standard solution by injecting  Directly into the detector inlet and start the wavelength scan from 190 to 400nm.Record the details as per Format No. xxxxx       
ACCEPTANCE CRITERIA: Maxima at 205+ 1 nm and 273 + 1 nm. 

5.7    INJECTION PRECISION / CARRY OVER

5.7.1    Perform injection precision and carry over as follows.
 
    Chromatographic conditions;
    Column: Restriction Capillary
    Flow: 1.00 mL/min
    Run time: 1.00 min
    Mobile Phase: HPLC grade water.
    Injection volume: 20.0?L
    Wavelength:273 nm

        5.7.1.1Inject 20.0?L of mobile phase three times followed by six 20?Linj. of  
        Caffeine (20?g/mL) in water and again  inject one blank (mobile phase).

5.7.1.2 Calculate the injection precision for area as the % RSD of caffeine 
          standard peak responses.

5.7.1.3    Calculate the injection carry over for area as peak response in the end blank injection with the peak response from the third blank injection subtracted as % of the response of the sixth Caffeine injection. Record the calibration details as per Format No. xxxxx
        
        ACCEPTANCE CRITERIA:    Precision     : RSD NMT 2.0%
                            Carry over area: NMT 0.20%.

5.8    RESPONSE LINEARITY:

5.8.1    Determine the detector response linearity by injecting Caffeine standard solutions of concentrations 10?g/mL, 20?g/mL, 30?g/mL, 50?g/mL and 100?g/mL in water.
Chromatographic conditions:  Same as 5.7.1          
5.8.1.1 Inject Caffeine standard solutions of the above said concentrations. 
5.8.1.2 Record the chromatogram and take the peak areas of responses as per
            Format No. xxxxx
5.8.1.3    Plot a linearity graph of concentration Vs peak response areas and Record       
             the details as per Format No. xxxxx                
ACCEPTANCE CRITERION: Correlation Coefficient NLT 0.999
  

5.9    FREQUENCY:

5.9.1    Once in six months.    

Calibration of hplc, restriction capillary, specific gravity, separately, caffeine