1.0
PURPOSE:
1.1
2.0
SCOPE.
2.1
3.0
Executive-QC
ACCOUNTABILITY
4.1
5.0
This SOP is applicable for the Calibration of HPLC’S in QC / IPQC
Laboratories.
RESPONSIBILITY
3.1
4.0
To lay down the procedure for calibration of HPLC.
Asst. Manager-QC
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
S.No TEMPERATURE( 0C)
VOLUME (mL)
1
20
1.0027
2
22
1.0033
3
24
1.0037
4
26
1.0044
5
28
1.0047
6
30
1.0053
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
0.01
8.0
8.01
Gradient Run:
Solvent B
100.0
100.0
0.0
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
TIME
1.00
1.01
5.00
5.01
8.00
8.01
11.00
11.01
14.00
14.01
17.00
17.01
20.00
20.01
25.00
SOLVENT B (%)
0.0
100.0
100.0
90.0
90.0
50.0
50.0
6.0
6.0
5.0
5.0
50.0
50.0
0.0
0.0
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
0.01
10.0
Solvent B
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
TIME
0.01
10.00
10.01
20.00
20.01
30.00
30.01
40.00
40.01
50.00
SOLVENT B (%)
10.0
10.0
50.0
50.0
90.0
90.0
100.0
100.0
0.0
0.0
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.
VIAL
Vial1
Vial2
Vial3
Vial4
TABLE3
INJECTION VOLUME
10L
20L
50L
100L
REPETITIONS
5
5
5
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:
TIME
0.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
7.0
WAVELENGTH
350
268
269
270
271
272
273
274
275
276
277
350
STOP
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:
5.8
RESPONSE LINEARITY:
Precision
: RSD NMT 2.0%
Carry over area: NMT 0.20%.
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.
END OF DOCUMENT