Saturday 7 October 2017

Calibration of HPLC

Posted by mypharmasolution.blogspot.com on October 07, 2017



















PROCEDURE :
PRECAUTIONS
  Ensure that the plug is connected to the mains.
  Check and ensure that the instrument is clean and calibration is valid.
Ensure that the pump, column, detector & processors are properly connected.
Set mobile phase and waste solution bottle in place.
Ensure that insert the end of solvent tube into solvent bottle.
Ensure that HPLC is connected with UPS and have battery back up.


CALIBRATION:
Procedure: To perform the calibration of instrument as per procedure given below follow  the operating instructions from above procedure.


PERFORMANCE OF PUMP FOR CONSTANT FLOW RATE:
Start the pump with ON/OFF key and set the flow rate of pump 1.0 ml/min.
Make sure that there is no air bubble in suction filter as well as in tubing carrying mobile

Take 10 ml cylinder of class A. Put the cylinder under the outlet of the capillary andstart.
The stop- watch of pump at the same time.
Collect the volume for flow rate set on pump.
Calculate  the flow rate & report it.
Calibrate all four pumps i.e., Pump A, Pump B, Pump C and Pump D as per the procedure given above.


WAVELENGTH ACCURACY TEST FOR UV DETECTOR:
  CHROMATOGRAPHIC CONDITIONS:
    The chromatographic conditions are as follows;
Column                            : Octadecylsilyl silica gel C18(250 mm × 4.6 µm)
Column temperature         : 250C
Flow rate                         : 1.0 ml/ min
Mobile phase                   : Methanol (70) : water (30)
Injection Volume              : 20 µl
Prepare the 20 ppm of caffeine in HPLC water.
Inject the preparation at 269, 270, 271, 272, 273,274, 275, 276 and 277 nm.
Same time check that the maximum area should be detected at the wavelength 272 nm ± 2.0 nm.
Confirm and record the results.



LINEARITY OF DETECTOR RESPONSE:
Chromatographic conditions: The chromatographic conditions are as follows;
Column                            : Octadecylsilyl silica gel C18(250 mm × 4.6 µm)
Column temperature        : 250C
Flow rate                         : 1.0 ml/min.
Mobile phase                  : Methanol (70) : HPLC water (30)
Wavelength                     : 272 nm
Injection volume              : 20 µl
Then prepare the level solution of 20, 30, 40, 50 and 60 ppm with the help of the dilution of stock solution.
Inject the each solution three times; calculate the mean area RSD%.


Plot the graph of concentration against area for the linearity of detector response.
Calculate the correlation coefficient and correlation coefficient should not be less than 0.998.
Remark the performance of detector in terms of linearity is satisfactory or not & note the results.



CALIBRATION OF INJECTOR & LOOP:
Chromatographic conditions:  The chromatographic conditions are as follows;
Column                            : Octadecylsilyl silica gel C18(250 mm × 4.6 µm)
Column temperature      : 250C
Flow rate                         : 1.0 ml/min.
Mobile phase                  : Methanol (70) : water (30)
Wavelength                     : 272 nm
Injection volume              : 10 µl; 20 µl; 50 µl  and 100 µl
Prepare the 20 ppm of caffeine in HPLC water and inject the triplicates of  10 µl, 20 µl, 50  µl  and  100 µl of sample preparation.
Check that the areas is increased proportionally with injection volume.



CALIBRATION FOR GRADIENT PROGRAM TEST:
High pressure gradient elute delivery of 2 mobile phases (HPLC water and 10 ppm caffeine solution) is performed, the absorbance produced by various set concentrations is measured and the measurements are used to calculate the actual concentrations.
First give washable warm HPLC water to HPLC system up to minimum 1 hour.
  Before gradient program test is carried out, Parameters set are as below.
Pump A - C Receiver               : 500 ml HPLC water.
Pump B - D Receiver               : 500 ml 10 ppm caffeine
Pumping mode                        : Gradient system ( A, B, C, D )

Flow Rate                               : 1 ml/min
UV Detector                            : 272 nm

Temperature                           : 250C
Stop Time                               : 35 minutes


GRADIENT PROGRAME TEST TIME FOR THERMO HPLC:

FLOW
1.00 ml/minute
STOP TIME
35.00 minutes
SOLVENT A/C
HPLC water
SOLVENT B/D
10 ppm caffeine in HPLC water

Time
Solvent A
Solvent B
Solvent C
Solvent D
5.00
100
0
100
0
5.01
90
10
90
10
10.00
90
10
90
10
10.01
50
50
50
50
15.00
50
50
50
50
15.01
10
90
10
90
20.00
10
90
10
90
20.01
0
100
0
100
25.00
0
100
0
100
25.01
100
0
100
0
35.00
Controller
Stop
Controller
Stop
Wavelength
272 nm
Temperature
250C


Calculation of Gradient Composition for Accuracy=  Gradient step area of height        X 100

                                                                   Gradient step 100 area of height


COLUMN OVEN TEMPERATURE:
Place a digital temperature thermometer probe inside he column heater.
Ensure that the thermometer probe does not contact any inside surface of oven.
Close the column oven door properly.
Set each of the three temperature 300C, 400C and 600C.
Take sufficient time to the oven temperature to reach and stabilize at set temperature.
Record the observation from digital thermometer indicator.


CARRY OVER TEST:
Sample preparation :
Weigh accurately about 100 mg caffeine WS in 100 ml volumetric flask.
Dissolve and dilute the volume with HPLC water.
Further dilute the 10 ml of this solution to 100 ml with water.


Acceptance criteria: NMT 0.01%