HPLC
Calibration Procedure :
Know the procedure to calibrate the High
Performance Liquid Chromatography ( HPLC) including leakage test, flow rate,
reproducibility and linearity, lamp energy and pump pressure drop in
Pharmaceutical Quality Control.
Check HPLC chromatography (Pump) for
the following:
- Checkpoint: Leakage test (By Pressure Drop).
- Flow rate calibration
CHECKPOINT:
LEAKAGE TEST (BY PRESSURE DROP):
1. Ensure that, the instrument is
ready for calibration and Start-up procedure is followed.
2. Place inlet tubing of the Pump into
the Water HPLC grade through a suction filter.
3. Allow the mobile phase to flow for
about 5 min.
4. Block Pump outlet with the block
screw.
5. The pressure rises and on crossing
the 300 bar, “ERROR P-MAX” appears on the display window. Note the time. Press
“CE” key and observe the pressure drop for 5 min.
6. After 5 min., record the pressure
in the Calibration Log.
7. Make entry of the HPLC column usage
in the Column Usage Log Register.
8. Make entry of the user into the
Instrument Usage Log Register.
9. Compare the result for its
compliance with the limit given in the Calibration Log and put the remark
regarding the HPLC chromatography Calibration Status.
10. In case of non-compliance, follow the
Maintenance Program.
FLOW
RATE CALIBRATION
1. Ensure that, the instrument is
ready for calibration and Start-up procedure is followed.
2. Ensure that, the Pump is passing
the “Leakage Test (By Pressure Drop)”.
3. Keep the Drain tube in such a way
that the mobile phase (Water) drops falls into 10 ml clean, dry volumetric
flask without touching the walls of the flask and start immediately the
stopwatch when the first drop falls into the flask.
4. Wait till the collected mobile
phase reaches 10 ml mark of the volumetric flask and Stop the stopwatch.
5. Record the time required to collect
the 10 ml mobile phase in the calibration log.
6. Repeat the procedure for 1.0 ml,
1.5 ml and 2.0 ml/ min. flow rates.
7. Repeat step 3 to 6 but using
methanol HPLC grade as mobile phase instead of water.
8. Compare the results for its
compliance with limits given in the Calibration Log and put the remark
regarding the Calibration Status.
9. Make entry of the usage of the
instrument and HPLC column in the Instrument Usage Log Register and Column
Usage Log Register respectively.
10. Prepare Calibration Status Label
and display on the instrument at the designated place.
11. In case of non-compliance, follow
the Maintenance Program
REPRODUCIBILITY
AND LINEARITY OF INJECTION VOLUME
Solution
Mixer Preparation
1. Take clean and dry 50 ml volumetric
flask.
2. Pipette out 1.0 ml of Benzene and
Toluene into the clean and dry 50 ml volumetric flask.
3. Make up the volume to 50 ml with
Methanol and mix well
Chromatographic
Condition
HPLC Column: ODS C18, (25 cm x 4.6 mm
ID, 5 μm)
Mobile Phase: Methanol : Water (70 :
30)
Flow Rate: 1.0 ml/min.
Wavelength: 254 nm
Injection Volume: 20 μl
Calibration
:
1. Ensure that, the instrument is
ready for calibration and Start-up procedure is followed.
2. Ensure that, the instrument is set
according to the Chromatographic conditions.
3. Follow the Instrument Operating
procedure, Inject 10 μl in triplicate and record the chromatograms.
4. Repeat the injection of the above
solution by injecting 15, 20, 25, 30 μl in triplicate.
5. Take the print out of the
chromatograms and attach to the Calibration Log.
6. Record the Area and Retention times
of the of the Benzene and Toluene peaks in the Calibration Log.
7. Make entry of the usage of the
instrument and HPLC column in the Instrument Usage Log Register and Column
Usage Log Register respectively.
8. Plot the curve for the area
corresponding to Benzene to Toluene peaks v/s injection volume, Find out the
RSD (reproducibility) and record in the Calibration Log.
9. Find out the Correlation
coefficient “r2” for each peak at five levels and record in the Calibration
Log.
10. Compare the result for its
compliance with the limit given in the Calibration Log and put the remark
regarding the Calibration Status.
11. Prepare Calibration Status Label
and display on the instrument at the designated place.
12. In case of non-compliance, follow
the Maintenance Program.
CHECKPOINT:
D2 LAMP ENERGY CHECK (DETECTOR)
1. Ensure that, the instrument is
ready for calibration and Start-up procedure is followed.
2. On the Detector’s display window,
some values at the functions “l(nm)”, “abs(AU)”, “range(AUFS)” and “lamp”
appears.
3. On the display, previously set
value blinks at “l(nm)” function, enter the wavelength to 254 nm by pressing
numeric keys.
4. Press “Func Back” key and select
“lamp” functions, enter 1 to select D2 lamp.
5. Further press “Func Back” key till
“REF EN ” appears.
6. Record the Reference Energy of the
D2 Lamp at 254 nm in the Calibration Log.
7 Compare the result for its
compliance with the limit given in the Calibration Log and put the remark
regarding the Calibration Status.
8. In case of non-compliance, follow
the Maintenance Program.
LINEARITY
OF DETECTOR RESPONSE :
Solution
Mixer Preparation:
1. Take three clean and dry 50 ml
volumetric flasks
2. Pipette out ml of Benzene and
Toluene as per the following table into the clean, dry 50 ml volumetric flask
separately.
3. Make up the volume to 50 ml with
Methanol and mix well.
Solution (Level)
|
ml of Benzene to be taken
|
Ml of Toluene to be taken
|
To be Diluted with Methanol to
|
1
|
0.5
|
1.0
|
50 ml
|
2
|
1.0
|
1.0
|
50 ml
|
3
|
1.5
|
1.0
|
50 ml
|
Chromatographic
Condition
1.
Set
each of the equipment to the following parameters as per the Equipment’s
Parameter Setup Procedure.
HPLC
Column: ODS C18, (25 cm x 4.6 mm ID, 5 m)
Mobile
Phase: Methanol : Water (70 : 30)
Flow
Rate: 1.0 ml/min.
Wavelength:
254 nm
Injection
Volume: 20 μl
Calibration
1.
Ensure that, the instrument is ready for calibration and Start-up procedure is
followed.
2.
Ensure that, the instrument is set according to the Chromatographic conditions.
3.
Follow the Instrument Operating procedure, Inject each of the Solutions in
triplicate and record the chromatograms.
4.
Take the print out of the chromatograms and attach to the Calibration Log.
5.
Record the Area and Retention times of the of the Benzene and Toluene peaks in
the Calibration Log.
6.
Make entry of the usage of the instrument and HPLC column in the Instrument
Usage Log Register and Column Usage Log Register respectively.
7.
Calculate the Area Ratio of Benzene to Toluene, Find out the Mean value of the
ratios and record in the Calibration Log.
8.
Find out the Correlation coefficient “r2” from the Mean area ratio values of
the three levels.and record in the Calibration Log.
8.
Plot the curve for area ratio corresponding to Benzene to Toluene peaks v/s
concentration, Find out the RSD (reproducibility) and record in theCalibration
Log.
9.
Compare the result for its compliance against limit given in the Calibration
Log and put the remark regarding the Calibration Status.
10.
Prepare Calibration Status Label and display on the instrument at the designated
place.
11.
In case of non-compliance, follow the Maintenance Program.
CALIBRATION
OF HPLC ( Liquid Chromatography ) PUMP
Model No. : Make :
Model No. : Make :
Instrument
Code No.
a.
Checkpoint : Leakage Check Test (Pressure Drop)
|
|||||||
P. Max to be set
|
P. Max Set
|
Start Time
|
Stop Time
|
Total Time
|
Pressure Observed
|
Remark
|
|
300 bar
|
|||||||
Flow Rate Calibration :
|
|||||||
Flow Rate (ml / min.)
|
Time required to collect 10 ml mobile phase
|
Limit
|
Remarks
|
||||
Theoretical
(in sec.)
|
Actual with water
|
Actual with Methanol
|
|||||
0.5 ml
|
1200
|
1194 – 1206
|
|||||
1.0 ml
|
600
|
594 – 606
|
|||||
1.5 ml
|
450
|
443 – 457
|
|||||
2.0 ml
|
300
|
294 - 306
|
|||||
Calibration
Status : Satisfactory / Not Satisfactory
Next
Calibration Due :
Calibrated
By : Checked By :
Date
: Date :
CALIBRATION OF INJECTOR
Model:
Make:
Instrument Code No.:
a. Preparation
of solvent mixture :
Taken
_____ ml (1.0 ml) of the ________ (Benzene) and ________ (Toluene) in to a
_____ ml (50 ml) clean, dry volumetric flask, make up the volume with methanol,
mixed well.
b. Chromatographic
Condition :
Parameters
|
Test Condition
|
Applied Condition
|
Mobile Phase
|
Methanol: Water
(70: 30)
|
|
HPLC Column
|
ODS
C18, (25 cm x 4.6 mm ID, 5 μm)
|
|
Flow Rate
|
1.0 ml / min.
|
|
Wavelength
|
254 nm
|
Injection volume
|
Injection -1
|
Injection -2
|
Injection -3
|
Mean
|
%RSD:(Not more than 2.0%)
|
||||
Benzene
|
Toluene
|
Benzene
|
Toluene
|
Benzene
|
Toluene
|
||||
10 ml
|
RT
|
||||||||
Area
|
|||||||||
15 ml
|
RT
|
||||||||
Area
|
|||||||||
20 ml
|
RT
|
||||||||
Area
|
|||||||||
25 ml
|
RT
|
||||||||
Area
|
|||||||||
30 ml
|
RT
|
||||||||
Area
|
|||||||||
Coefficient of co-relations : r2 : _____________ (Limit :
NLT : 0.999)
Calibration Status : Satisfactory / Not
Satisfactory
Next Calibration Due :
Calibrated By:
Checked By :
Date: Date
:
CALIBRATION OF DETECTOR
Model
: Make :
Instrument Code No. :
D2 LAMP
ENERGY CHECK :
|
||||||
Wavelength to be set
|
Wavelength Set
|
Reference Energy Observed
|
Limit
|
|||
254 nm
|
Not less than 200
|
|||||
LINEARITY OF DETECTOR RESPONSE :
|
||||||
a. Solution
Preparation :
|
||||||
Solution
|
ml
of Benzene
|
ml
of Toluene
|
in Methanol
|
|||
(Level)
|
To be taken
|
Taken
|
To
be taken
|
Taken
|
To
be diluted
|
Diluted to
|
1
|
0.5 ml
|
1.0 ml
|
50 ml
|
|||
2
|
1.0 ml
|
1.0 ml
|
50 ml
|
|||
3
|
1.5 ml
|
1.0 ml
|
50 ml
|
|||
Mobile Phase Preparation: Taken ______ ml of
___________ (Methanol HPLC grade) in to a _______ ml clean, dry volumetric
flask, added ____________ ml of___________(Water HPLC grade), mixed well,
allowed to cool to room temperature. Filtered through 0.45/0.22 m membrane
filter, degassed for _____ min. by vacuum / sonication.
Parameters
|
Test Condition
|
Applied Condition
|
Mobile Phase
|
Methanol:
Water ( 70: 30)
|
|
HPLC Column
|
ODS
C18, (25 cm x 4.6 mm ID, 5 μm)
|
|
Flow Rate
|
1.0 ml / min.
|
|
Wavelength
|
254 nm
|
|
Range
|
1.0 AUFS
|
|
Injection Volume
|
20
μl
|
Solution(Level)
|
Benzene Area
|
Toluene Area
|
Area Ratio(Benzene/Toluene)
|
||
RT
|
Area
|
RT
|
Area
|
||
1
|
|||||
Mean :
|
|||||
% RSD :
|
|||||
2
|
|||||
Mean :
|
|||||
% RSD :
|
|||||
3
|
|||||
Mean :
|
|||||
% RSD :
|
|||||
Linearity of Ratio : Correlation
Co-efficient r2 :
|
Limit
: % RSD of the Retention Time : Not more than 2.0%
Linearity
of Ratio : Correlation Co-efficient r2 : Not less than 0.999
|
Calibration Status : Satisfactory / Not
Satisfactory
Next Calibration Due :
Calibrated By :
Checked By :
Date :
Date :
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