I.
PURPOSE :
To provide a calibration procedure for High Performance Liquid Chromatograph
(Waters HPLC).
II.
SCOPE :
This procedure is applicable for all the HPLCs (Waters) used in the
Quality Control department.
III.
RESPONSIBILITY :
It is the responsibility of the Quality Control personnel involved in the HPLCs to
follow the procedure and schedule as mentioned in this Standard Operating
Procedure.
IV.
V.
DOCUMENT REFERENCE :
SOPs
:
Core procedure, Calibration of Equipment
Forms
:
Calibration of HPLC(Waters) system
SCHEDULE :
The calibration of HPLC system shall be performed in the first week of every
third
month.
VI.
PROCEDURE :
A.
CALIBRATION OF PUMP:
1.
Accuracy of flow rate:
01.
Fill filtered water in the pump reservoir and purge the pump.
02.
Set the Flow rate of the pump to 1.0 ml/minute and ensure no air bubble is stuck
up
in the path.
03.
Take a clean 10 ml Class – A graduated cylinder.
04.
Note the time and immediately start collecting water into the 10 ml graduated
cylinder.
05.
After 9 minutes note the time and remove the 10 ml cylinder.
Acceptance Criteria : The volume of water collected should be 9.0 ml + 0.2.
2.
Gradient Accuracy check:
Chromatographic Conditions :
Column
:
Hypersil ODS 100 x 4.6 mm ID, 5  or equivalent
Flow rate
:
2.0 ml /min.
Detector wavelength :
254 nm
Injection volume
:
20 l
Flow rate
:
2mL/min
Runtime

:
30 minutes
obile Phase
by
obile Phase A or C :
Water generated by Milli-Q Apparatus
Mobile phase B or D :
0.3%v/v Acetone in filtered water generated by Milli-Q
Preparation of Mobile Phase B: Take 3 mL of acetone in 1000 mL of water generated
Milli-Q
Gradient program :
Procedure using Pump A and pump B:
Time (min)
0.01
2.00
2.01
8.00
8.01
13.00
13.01
18.00
18.01
23.00
23.01
23.01
Mobile phase B
percentage
0
0
10
10
50
50
90
90
100
100
0
0

Open the drain valves and purge the flow lines of each channel

Equilibrate the column with initial concentration with above mentioned
conditions
and wait until the baseline is stable

Adjust the baseline level to fit the full Scale of the integrator

Inject exactly 20 l of mobile phase A and start the time program for gradient
accuracy

test.
Determine the signal level at 0%(B Con),10%(B Con), 50%(B Con), 90%(B Con)
and 100%(B Con)

Calculate the actual B concentration level at 10%(B Con), 50%(B Con), 90%(B
Con)
Using 0% (B Con) and 100% (B Con)
Calculation:
Calculate the actual B concentration level at 10%
=
B concentration level at 10% - B concentration level at 0%
B concentration level at 100% - B concentration level at 0%
Similarly calculate the actual B concentration level at 50%(B Con) and 90%(B Con)
Acceptance Criteria :
At B concentration 10% level should be between 9.0 and 11.0
At B concentration 50% level should be between 49.0 and 51.0
At B concentration 90% level should be between 89.0 and 91.0
Procedure using Pump C and pump D:
Time (min)
0.01
2.00
2.01
8.00
8.01
13.00
13.01
18.00
18.01
23.00
23.01
23.01
Mobile phase D
percentage
0
0
10
10
50
50
90
90
100
100
0
0

Open the drain valves and purge the flow lines of each channel

Equilibrate the column with initial concentration with above mentioned
conditions
and wait until the baseline is stable

Adjust the baseline level to fit the full Scale of the integrator

Inject exactly 20 l of mobile phase A and start the time program for gradient
accuracy

test.
Determine the signal level at 0%(D Con),10%( D Con), 50%( D Con), 90%( D
Con) and 100%( D Con)

Calculate the actual D concentration level at 10%( D Con), 50%( D Con), 90%( D
Con)
Using 0% (D Con) and 100% (D Con)
Calculation:
Calculate the actual B concentration level at 10%
=
D concentration level at 10% - D concentration level at 0%
D concentration level at 100% - D concentration level at 0%
Similarly calculate the actual B concentration level at 50%( D Con) and 90%( D Con)
Acceptance Criteria:
At D concentration 10% level should be between 9.0 and 11.0
At D concentration 50% level should be between 49.0 and 51.0
At D concentration 90% level should be between 89.0 and 91.0

Calibration of PDA Detector:
1.
Wavelength Accuracy

Go to diagnostics and click on PDA calibration.

Screen will appear and feed the expose time as 44 seconds and press
start
run icon, followed a spectrum will appear.

After 1 minute stop the run and press ‘ Detect Lines and take out the print out.

Press Calibration accuracy and note down the variation at 486.0 nm and 656.1
nm.

Go for PDA diagnostics and click on ‘lamp’ and ‘wavelength’ icons and
press run.

Take the print outs of all the above data.
Acceptance Criteria : The variation should be within + 1 nm at the specified
wavelengths.
2.
Lamp Intensity test: Measure the lamp intensity from the spectrum obtained
from wavelength accuracy test at 220 nm and record the results
Acceptance Criteria : The lamp intensity at 220 nm should be greater than or equal to
5000.
C.Calibration of UV Detector:
1. Wavelength Calibration:

Switch on the UV detector and warm up at least 30 minutes

Set the Methanol flow 1 mL per minute and wash the detector cell for at least 30
minutes.

Press shift switch followed by calibrate switch and press enter to start the
calibration.

After completion of the calibration process a message will appear as follows.

‘Calibration successful, maximum deviation since last calibration = 0.2 nm.
(current deviation in nm).

Go for wavelength diagnostics and click on lamp and wavelength icons
and press run.
Acceptance Criteria : The variation should be within + 1 nm.
2. Lamp Energy test:

Set the Methanol flow 1 mL per minute and wash the detector cell for at least 30
minutes. Press shift botton followed by diagnostic botton twice and then select
Sample and reference energy mode and feed the 220 nm and press enter to
observe the lamp energy at reference cell.
Acceptance Criteria : The reference energy at 220 nm should be more than 20 .
D.
CALIBRATION OF PUMP, AUTOINJECTOR and DETECTOR
Chromatographic system :
Column
:
Hypersil ODS 100 x 4.6 mm ID, 5  or equivalent
Flow rate
:
1.5 ml /min.
Detector wavelength :
254 nm
Injection volume
:
20 l
Runtime
:
8 minutes

Preparation of Calibration Standard :
01.
Weigh accurately about 150 mg each of Dimethylphthalate, Diethylphthalate,
10 mg of Biphenyl and 30 mg of O-Terphenyl, transfer into a 100 ml
volumetric
flask. Dissolve and dilute to volume with methanol.
Procedure :
01.
Equilibrate the column for 30 minutes with mobile phase at a flow rate of 1.5
ml/min
(with the initial concentration in case of gradient
system).
02.
Inject exactly 20 l of methanol as blank in to the system and record
the chromatogram.
03.
Program the data processor to inhibit the integration of peaks due to blank.
04.
Inject 20 l of Isocratic Standard for 6 times and record the chromatograms.
05.
Calculate the %RSD for Retention Times observed from the replicate injections to
check the precision of the Pump flow rate.
06.
Calculate the %RSD for areas observed from the replicate injections to check the
precision of the Auto sampler and Detector.
Acceptance Criteria : % RSD calculated for Retention times should not be more than
1.5%
% RSD calculated for areas should not be more than 1.5%
Note :
01.
In case the equipment fails in calibration, call the service engineer for repairing
the instrument. The data pertaining to previous samples analysed (analysed after
the previous due of calibration) using the equipment / instrument shall be
reviewed and the review shall be documented. All the proceedings shall be in
accordance to the core procedure, “Calibration of Equipment”.
02.
After repair, Samples from N + 1 batches, where N is the total number of
batches
details shall be
since previous calibration shall be analysed again and the
documented.