1.0 Objective:
To provide written procedure for calibration of High Performance
Liquid Chromatography system.
2.0 Scope:
This SOP covers calibration
of all High Performance Liquid Chromatography system
3.0 Responsibility:
Jr.
Research Officer, Research Officer: Responsible for operation, calibration and
maintenance of the instrument as per procedure.
Head of Department: Responsible
for calibration and maintenance, timely as per schedule.
QA Officer/QA Manager: Review the records and governing the document.
4.0 Procedure:
4.1 Ensure
that the instrument is visibly clean and free from dust.
4.2 CALIBRATION OF PUMP:
4.2.1 Keep water as mobile phase at a flow
rate of 0.5 ml, 1.0 ml, 1.5 ml and 2.0 ml.
4.2.2 Collect the eluent in a tare-measuring
cylinder for 5 minutes and 10 minutes for the above flow rate.
4.2.3 Weigh the measuring cylinder and note
down the weight. Check the observed weight against the expected weight. (1 ml
of water = 0.99602 g at 25° C).
4.2.4 Record the details in HPLC calibration
format as given in Annexure - 1.
4.2.5 Frequency: Quarterly.
4.3 CALIBRATION OF DETECTOR:
4.3.1
System Suitability check (RSD
%):
4.3.1.1
Methanol filter, degas and use
as mobile phase. Set the wavelength at 258 nm.
4.3.1.2
Separately prepare 10 µg / ml
w/v solution of Uracil in methanol.
4.3.1.3
Fix a column. Run the
instrument at a flow rate of 1.0 ml /minute.
4.3.1.4
Inject 20 microlitres of the
10µg / ml w/v solution in 6 replicate.
4.3.1.5
Record the chromatogram and
check the response on peak due to Uracil.
4.3.1.6
Calculate the RSD. It should be
less than 2.0 %.
4.3.1.7
Record results in calibration
format Annexure-1
4.3.1.8
Frequency: Quarterly.
4.3.2 Linearity:
4.3.2.1 Methanol filter, degas and use as mobile phase. Set the
wavelength at 258 nm.
4.3.2.2 Separately prepare 5, 10, 15, 20 and 25
µg / ml w/v solution of Uracil in methanol.
4.3.2.3 Fix a column. Run the instrument at a
flow rate of 1.0 ml /minute.
4.3.2.4
Inject 20 microlitres each of the five different concentrations.
4.3.2.5 Record the chromatograms and responses.
Check the linearity of detector by plotting a graph of area verses
concentration. The correlation coefficient should not less than 0.99.
4.3.2.6 Record the details in HPLC calibration
format as given in Annexure - 1.
4.3.2.7 Frequency: Quarterly.
4.3.3 Wavelength accuracy:
4.3.3.1 Methanol
filter, degas and use as mobile phase.
4.3.3.2 Prepare 10 µg / ml w/v solutions of
Uracil in methanol.
4.3.3.3 Fix a column. Run the instrument at a
flow rate of 1.0 ml/minute.
4.3.3.4 Inject 20 µl of sample at 254, 255, 256,
257, 258, 259, 260, 261, 262 nm.
4.3.3.5 Record the chromatograms and responses. It should give the
area maxima at 258 ± 2 nm.
4.3.3.6 Record the details in HPLC calibration
format as given in Annexure - 1.
4.3.3.7 Frequency:
Quarterly.
4.4 CALIBRATION OF INJECTOR (FOR AUTO
SAMPLER):
4.4.1 Methanol filter, degas and use as
mobile phase.
4.4.2 Prepare 10 µg / ml w/v solutions of
Uracil in methanol.
4.4.3 Inject 5, 10, 15, 20, 25 microlitres of
the sample separately into the system at 258 nm.
4.4.4
Record the chromatograms and responses by plotting a graph of area
verses
4.4.5 Concentration and check the linearity. The
correlation coefficient should not less than 0.99.
4.4.6 Record the details in HPLC calibration
format as given in Annexure - 1.
4.4.7 Frequency: Quarterly.
4.5 INJECTOR CARRY OVER TEST :
4.5.1 Methanol filter, degas and use as mobile
phase. Set the wavelength at 258 nm.
4.5.2 Prepare 25 µg / ml w/v solutions of
Uracil in methanol.
4.5.3 Fix a column. Run the instrument at a
flow rate of 1.0 ml/minute.
4.5.4 Inject 20 microlitres of Blank , 25
µg / ml w/v solutions of Uracil and Blank Solution.
4.6 CALIBRATION COLUMN OVEN
TEMPERATURE:
4.6.1 Set the column oven temperature 15°, 25°, 30°,
40°, 50°, 60°.
4.6.2 Record the display temperature and probe
temperature observed on HTC thermometer.
4.6.3 Acceptance
criteria for temperature: ± 2 °C of the set value.
4.6.4 Frequency:
Quarterly.
4.7 CALIBRATION SAMPLE COOLER TEMPERATURE
CALIBRATION:
4.7.1 Set
the sample cooler temperature 4°, 15°, 25°, 30°, 40°.
4.7.2 Record the display temperature and
probe temperature observed on HTC thermometer.
4.7.3 Acceptance criteria for temperature: ± 2 °C of the set
value.
4.7.4 Frequency: Quarterly.
6.0 Documentation:
6.1 Annexure – 1 HPLC Calibration record format XXX/FRM/000
7.0 History of Revision:
Revision No.
|
Effective
Date
|
Revision
details
|
Reason for
revision
|
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
CALIBRATION RECORD
|
Page No:
|
|
Date of calibration :
|
Next due date for calibration :
|
|
Instrument
ID No. :
|
Instrument
Serial No. :
|
|
Make & Model
No. :
|
||
1.0
Calibration of Pump:
Time
|
5
Minute
|
10
Minute
|
|||||||
Flow rate
ml / minute
|
Observed
Weight in
Gm
|
Theoratical Weight
in gm
|
Variation
In Gm
|
Variation
in %
(Limit :
± 0.1 %)
|
Observed
Weight in
Gm
|
Theoratical Weight
in gm
|
Variation
In Gm
|
Variation
in %
(Limit :
± 0.1 %)
|
|
Pump
A
|
0.5 ml
|
2.4900
|
4.9801
|
||||||
1.0 ml
|
4.9801
|
9.9602
|
|||||||
1.5 ml
|
7.4702
|
14.9403
|
|||||||
2.0 ml
|
9.9602
|
19.9204
|
|||||||
Pump
B
|
0.5 ml
|
2.4900
|
4.9801
|
||||||
1.0 ml
|
4.9801
|
9.9602
|
|||||||
1.5 ml
|
7.4702
|
14.9403
|
|||||||
2.0 ml
|
9.9602
|
19.9204
|
|||||||
Pump
C
|
0.5 ml
|
2.4900
|
4.9801
|
||||||
1.0 ml
|
4.9801
|
9.9602
|
|||||||
1.5 ml
|
7.4702
|
14.9403
|
|||||||
2.0 ml
|
9.9602
|
19.9204
|
|||||||
Pump
D
|
0.5 ml
|
2.4900
|
4.9801
|
||||||
1.0 ml
|
4.9801
|
9.9602
|
|||||||
1.5 ml
|
7.4702
|
14.9403
|
|||||||
2.0 ml
|
9.9602
|
19.9204
|
|||||||
Remarks: Satisfactory / Not satisfactory
Calibrated by :
Checked by :
2.0 System
Suitability Check (RSD):
Weight of Uracil :
Mobile
phase : Methanol as mobile phase.
Flow
rate : 1.0
ml / minute
Detector
: 258 nm
Diluting
solution : Methanol
Preparation
of Solution : Weigh accurately 25 mg of Uracil in 50 ml volumetric flask and
make volume with methanol (Solution A). Pipette 5 ml above solution A and make
volume 50 ml with Methanol (Solution B). Pipette 2 ml solution B in 10 ml volumetric flask and
make volume with methanol (10ppm).
Injection No.
|
Area of 10ppm Uracil
|
Injection
1
|
|
Injection
2
|
|
Injection
3
|
|
Injection
4
|
|
Injection
5
|
|
Injection
6
|
|
Average
|
|
SD
|
|
%RSD
Limit : NMT 2.0%
|
Calibrated
by : Checked by
:
3.0
Linearity of Detector Calibration :
Weight of Uracil :
Mobile
phase : Methanol as mobile phase.
Flow
rate : 1.0
ml / minute
Detector
: 258 nm
Diluting
solution : Methanol
Preparation
of Solution : Weigh accurately 25 mg of Uracil in 50 ml volumetric flask and
make volume with methanol (Solution A). Pipette 5 ml above solution A and make
volume 50 ml with Methanol (Solution B). Pipette 1 ml, 2 ml, 3 ml, 4 ml and 5
ml solution B in five different 10 ml volumetric flask and make volume with
methanol. They will be indicated 5, 10, 15, 20 and 25 µg/ml solution.
Sr. No.
|
Concentration of Uracil
Solution in µg / ml
|
Area
|
Correlation
Coefficient (Limit: NLT 0.99)
|
Remarks
|
||||
Inj
1
|
Inj 2
|
Inj 3
|
Average
|
|||||
1
|
5
|
|||||||
2
|
10
|
|||||||
3
|
15
|
|||||||
4
|
20
|
|||||||
5
|
25
|
|||||||
Remarks: Satisfactory / Not satisfactory
Calibrated by :
Checked by :
4.0
Wave length accuracy of Detector Calibration:
Mobile
phase : Methanol as mobile phase.
Flow
rate : 1.0
ml / minute
Diluting solution : Methanol
Use 10 µg/ml solution for injection
Sr. No.
|
Wave length in nm
|
Area
|
Remarks
|
1
|
254
|
||
2
|
255
|
||
3
|
256
|
||
4
|
257
|
||
5
|
258
|
||
6
|
259
|
||
7
|
260
|
||
8
|
261
|
||
9
|
262
|
||
Limit
: 258 ± 2 nm
|
|||
Status
/ Remarks : Satisfactory / Not satisfactory
Calibrated by :
Checked by :
5.0
Injector carry over test :
Weight of Uracil :
Mobile
phase : Methanol as mobile phase.
Flow
rate : 1.0
ml / minute
Detector
: 258 nm
Diluting
solution : Methanol
Preparation
of Solution : Weigh accurately 25 mg of Uracil in 50 ml volumetric flask and
make volume with methanol (Solution A). Pipette 5 ml above solution A and make
volume 50 ml with Methanol (Solution B). Pipette 5 ml solution B in 10 ml
volumetric flask and make volume with methanol. It will be indicated 25 µg/ml solution.
Sr. No.
|
Blank Injection Area
|
Sample Injection Area
|
Blank Injection Area
|
1
|
Area
of blank Injection
Calculation
= ------------------------------- X
100
Area of sample
Injection
Observation:
_____________ Limit: Not more than 0.2%
Status
/ Remarks : Satisfactory
/ Not satisfactory
Calibrated by : Checked
by :
SOP No. : XXX/SOP/000-00 Format No. : XXX/FRM/000-00
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