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Calibration

Calibration of HPLC

Calibration of HPLC

  

  1. Calibration of Pumps:-

1.1 Flow Accuracy and Flow precision

 Observation table for Accuracy of Flow rate

Sr. No. Set flow rate in

mL/ min

 Time require to collect 10mL volume Observed flow rate in

mL/ min

 Acceptance criteria

 
1 2 3 Average
1 0.5           0.49-0.51
2 1.0           0.98-1.02
3 1.5           1.47-1.53
4 2.0           1.96-2.04
5 5.0           4.90-5.10

 

Observation Table for Precision of flow rate

Sr. No. Set flow rate in

mL/ min

 Observed flow rate in mL/min %RSD Acceptance criteria

 
1 2 3 Average
1 0.5           ≤ 2.0%
2 1.0           ≤ 2.0%
3 1.5           ≤ 2.0%
4 2.0           ≤ 2.0%
5 5.0           ≤ 2.0%

 

 

  • 1.2 Gradient Accuracy:-

  Observation table for Channel A and B

Eluent Composition Hx Calculated    Bx (%) Acceptance Criteria
20% 19.4-20.6
40% 38.8-41.2
60% 58.2-61.8
80% 77.6-82.4
100% 97.0-103.0

 

Height at 100% composition of B (H100) = __________

Observation table for Channel C and D

Eluent Composition Hx Calculated    Dx (%) Acceptance Criteria
20% 19.4-20.6
40% 38.8-41.2
60% 58.2-61.8
80% 77.6-82.4
100% 97.0-103.0

 

Height at 100% composition of D (H100) = __________

 

Calculation:

The actually obtained mobile phase composition can be calculated from the Hx values by the following formula.

 

BX or DX (%) = HX (mm)/H100 (mm)————— x (100)

= _____________________

Where:

X         =          for the programmed step height (20, 40, 60 and 80).

HX          =          Height of the respective plateaus in mm of B or D

DX       =          the actual obtained mobile phase composition.

H100       =          Height at 100% composition of B or D.

 

  1. Calibration of Auto Injector:-

 

  • Vial Test

 

Sr No Vial Position/ Location Observation
1   Septum pierced/Not pierced
2   Septum pierced/Not pierced
3   Septum pierced/Not pierced
4   Septum pierced/Not pierced
5   Septum pierced/Not pierced

  

  • Volume accuracy of injection:-

 

Instrument/Equipments/Standards used:

Balance No
Balance calibration due date
Injection Volume 20µl
Number of Injections 10
Density of Water at 200C (d)

 

  

Observation Table:

Initial weight of Vial + Water(W1) in gram  

 
 Weight of Vial + Water after 10 Injection(W2) in gram  

 
W = (W1-W2) (in gram)  

 
mL of water injected = W/d  in mL  

 
mL of water injected in 10 injection = W/d *1000 µl=A  

 
µl per injection=A/10  

 

  

Acceptance Criteria:

The amount of water drawn by the auto injector per injection should be in between 19.0 µl and 21.0µl (i.e., ± 1.0 µl of the set value)

 

  • Injector Linearity:-

Instrument/Equipments/Standards used:

Balance No
Balance calibration due date
Standard used
Standard weight
Standard Lot number
HPLC Column  details

 

 Preparation of Standard Solution (20 ppm Caffeine):

  1. Weighed ______ mg of Caffeine in 100 mL volumetric flask, dissolved and diluted up to the mark with Mobile Phase.
  2. Further dilute 10mL of this solution to 100mL with Mobile Phase.

                                                        

Observation Table

 

Sr. No Injection Volume Area
1 5 µl  
2 10 µl  
3 20 µl  
4 50 µl  
5 100 µl  
        Correlation coefficient(r2)

(Acceptance criteria  r2 ≥ 0.999)

  

  

  • Injector Precision-

Instrument/Equipments/Standards used:

Balance No
Balance calibration due date
Standard used
Standard weight
Standard Lot number
HPLC Column  details

  

Preparation of Standard Solution (20 ppm Caffeine):

  1. Weighed ______ mg of Caffeine in 100 mL volumetric flask, dissolved and diluted up to the mark with Mobile Phase.
  2. Further dilute 10mL of this solution to 100mL with Mobile Phase.

                       

                                    Observation Table

 

Sr. No. Area
1  

 
2  

 
3  

 
4  

 
5  

 
6  

 
Average Area  

 
RSD (%)  

 

            Acceptance Criteria: RSD NMT 2.0 %             

 

 

  • Carryover Test :-

Instrument/Equipments/Standards used:

Balance No
Balance calibration due date
Standard used
Standard weight
Standard Lot number
HPLC Column  details

 

Preparation of Standard Solution (500 ppm Caffeine):

  1. Weighed ______ mg of Caffeine in 10 mL volumetric flask, dissolved and diluted up to the mark with Mobile Phase.

                       

                             Observation Table

 

Name Area % Carryover in Blank
Blank-1 NA
Sample NA
Blank-2
Sample NA
Blank-3
Sample NA
Blank    

                         Acceptance Criteria: NMT 0.02 %  

 

(Area response of blank-2) – (Area response of blank-1)

Carryover % =   ——————————————————— X 100

Average peak area of caffeine obtained from                                                    standard solution

Carryover % =   ———————————————————

 

  1. Calibration of Detector:-

 

  • Wave Length Accuracy:-

Instrument/Equipments/Standards used:

 

Preparation of Standard Solution (20 ppm Caffeine):

  1. Weighed ______ mg of Caffeine in 100 mL volumetric flask, dissolved and diluted up to the mark with Mobile Phase.
  2. Further diluted 10mL of this solution to 100mL with Mobile Phase.

 

Observation Table for wavelength 205 nm

Wavelength nm Response Wavelength Maxima/ Maximum response at nm Acceptance Criteria
Area Height
201      

 

 205nm ± 2 nm
202
203
204
205    
206    
207    
208    
209    

 

Observation Table for wavelength 245 nm

Wavelength nm Response Wavelength minima/ Minimum response at nm Acceptance Criteria
Area Height
241      

 

 245nm ± 2 nm
242
243
244
245    
246    
247    
248    
249    

                                                  

                                 Observation Table for wavelength 273 nm

Wavelength nm Response Wavelength Maxima/ Maximum response at nm Acceptance Criteria
Area Height
269      

 

 273nm ± 2 nm
270
271
272
273    
274    
275    
276    
277    

 

 

  • Detector Linearity:-

Instrument/Equipments/Standards used:

 

Preparation of Standard Solution (0.5mg/mL Caffeine):

Weighed ______ mg of Caffeine in 50 mL volumetric flask, dissolved and diluted up to the mark with Mobile Phase.

  • 0.25mg/mL standard Solution:

Diluted 1mL of stock solution to 20mL with mobile phase

  • 0.5mg/mL standard Solution:

Diluted 2mL of stock solution to 20mL with mobile phase

  • 100mg/mL standard Solution:

Diluted 4mL of stock solution to 20mL with mobile phase

  • 125mg/mL standard Solution:

Diluted 5mL of stock solution to 20mL with mobile phase

  • 250mg/mL standard Solution:

Diluted 10mL of stock solution to 20mL with mobile phase

  • 500mg/mL standard Solution:

Use stock solution

 Observation Table:

Theoretical Conc.

(mg/ ml)

 Actual Conc.

(mg/ ml)

 Area of injection 1 Area of injection 2 Mean Area Correlation Coefficient
0.025
0.050
0.100
0.125
0.250
0.500

 

     Acceptance Criteria:  Correlation coefficient ≥ 0.999

 

  • Lamp Energy:-

 

Acceptance Criteria and results for DAD:

 

Description Acceptance criteria Results Status Pass/Fail
Lowest intensity in range 190 nm to 220 nm Not less than 2000 cts.    
Lowest intensity in range 221 nm to 350 nm Not less than 5000 cts    
Lowest intensity in range 351 nm to 500 nm Not less than 2000 cts    
Lowest intensity in range 501 nm to 950 nm Not less than 4000 cts    
Highest intensity in range 190 nm to 350 nm Not more than 450000 cts    
Highest intensity in range 700 nm to 950 nm Not more than 300000 cts    
Highest intensity for D2 alpha line Not more than 1200000 cts    

 

 Acceptance Criteria and results for VWD:

 

Description Acceptance criteria Results Status Pass/Fail
Highest intensity More than 10000 cts    
Lowest  intensity More than 200 cts    
Average  intensity More than 5000 cts    

 

  1. Calibration of column Compartment:-

 

Observation Table for Right side Compartment

Set Temperature (in °C) Displayed Temperature on instrument

( in° C)

 Observed Temperature on thermometer

( in° C)

 Difference between set temp and observed temp on thermometer ( in° C)
25.0
40.0
60.0
80.0

 

Observation Table for Left side Compartment

 

Set Temperature (in °C) Displayed Temperature on instrument

( in° C)

 Observed Temperature on thermometer

( in° C)

 Difference between set temp and observed temp on thermometer ( in° C)
25.0
40.0
60.0
80.0

 

Acceptance Criteria:

All the observed thermometer temperature values are within ± 2°C of set value.

 

  1. Calibration of Autosampler Temperature:-

 

Observation Table

 

Set Temperature (in °C) Displayed Temperature on instrument

( in° C)

 Observed Temperature on thermometer

( in° C)

 Difference between set temp and observed temp on thermometer ( in° C)
25.0
20.0
10.0
4.0

 

Acceptance Criteria:

All the observed thermometer temperature values are within ± 2°C of set value.

 

Remark: System complies/does not comply with respect to above test.

 

 

Performed By:                                                         Checked By:

 

                Date:                                                                     Date:

 

 

 

 

 

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