HPLC
Calibration SOP
1.0 OBJECTIVE:
To
lay down the calibration procedure of High Performance Liquid Chromatography (HPLC) for reliable and accurate
results.
2.0 SCOPE:
This
shall be applicable to the installed High Performance Liquid Chromatography (HPLC) of Quality Control Instrument
Laboratory.
3.0 RESPONSIBILITY:
3.1
Technical
Assistant/ Helper.
3.2
Q.C Analyst.
4.0 ACCOUNTABILITY:
4.1
Manager-Quality
Control.
5.0 MATERIAL AND
EQUIPMENT:
5.1
High Performance Liquid Chromatography (HPLC).
5.2
Stopwatch.
5.3
Purified water.
5.4
Methanol.
5.5
Toluene.
5.6
Glassware.
6.0 PROCEDURE:
6.1
Make sure that the
instrument is clean.
6.2
Switch on the HPLC
apparatus and operate it according to operating procedure.
6.3
Calibrate the apparatus
according to the instructions for the reliable and accurate results.
6.4
Calibration
of pressure drop (Leakage test):
6.4.1
Ensure that,
the instrument is ready for calibration and Start-up procedure is followed.
6.4.2
Place inlet tubing
of the Pump in to the Water HPLC grade through suction filter.
6.4.3
Allow mobile phase
to flow for about 5 min.
6.4.4
Block Pump outlet
with the block screw.
6.4.5
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.
6.4.6
After 5 min., record
the pressure in Calibration Log.
6.4.7
Make entry of the
column usage in the Column Usage Log register.
6.4.8
Make entry of the
usage into the Instrument Usage Log Register.
6.4.9
Compare the result
for its compliance against limit given in the Calibration Log and put the
remarks regarding the HPLC chromatography Calibration status.
6.4.10
In case of non-compliance,
follow the Maintenance Program.
6.5
Calibration
of flow rate:
6.5.1
Ensure that, the
instrument is ready for calibration and Start-up procedure is followed.
6.5.2
Ensure that, the
Pump is passing the “Leakage Test (By Pressure Drop)”.
6.5.3
Keep the Drain
tube in such a way that the mobile
phase (Water) drops falls into 10.0ml clean, dry
volumetric flask without touching the walls of the flask and start immediately
the stopwatch when first drop falls into the flask.
6.5.4
Wait till the
collected mobile phase reaches 10 ml mark of the volumetric flask and Stop the
stopwatch.
6.5.5
Record the time
required to collect the 10 ml mobile phase in calibration log.
6.5.6
Repeat the
procedure for 1.0ml, 1.5ml and 2.0ml/ min. flow rates.
6.5.7
Repeat the step 6.5.3
to 6.5.6 but using methanol HPLC grade as mobile phase instead of water.
6.5.8
Make entry of the
column usage in the Column Usage Log register.
6.5.9
Make entry of the
usage into the Instrument Usage Log Register.
6.5.10
Compare the result
for its compliance against limit given in the Calibration Log and put the
remarks regarding the HPLC chromatography Calibration status.
6.5.11
Prepare the
calibration status Label and display on the instrument at the designated place.
6.5.12
In case of non-compliance,
follow the Maintenance Program.
6.6
Calibration
of gradient proportionate valve:
6.6.1
Install union in
place of column and flush solvent lines (A & B) at the flow rate of
2.0ml/min with purified water.
6.6.2
Prepare the mobile
phase.
6.6.3
Prepare 0.3%
acetone with HPLC grade purified water.
6.6.4
Fill reservoir A
with 100% HPLC grade purified water and reservoir B with 0.3% acetone in HPLC
grade purified water as mobile phase.
6.6.5
Enter the
following time set up.
|
Time
|
Function
|
Value
|
|
0.01
|
B
conc.
|
10
|
|
10.00
|
B
conc.
|
10
|
|
10.01
|
B
conc.
|
50
|
|
20.00
|
B
conc.
|
50
|
|
20.01
|
B
conc.
|
90
|
|
30.00
|
B
conc.
|
90
|
|
30.01
|
B
conc.
|
100
|
|
40.00
|
B
conc.
|
100
|
|
40.01
|
B
conc.
|
0
|
|
50.00
|
B
conc.
|
0
|
6.6.6
Use detector at
wavelength of 254nm.
6.6.7
Record the print out of gradient valve test as
per Annexure-1.
6.6.8
The gradient valve
test shall be accepted if actual concentration with ±1% of set concentration.
6.6.9
Make entry of the
column usage in the Column Usage Log register.
6.6.10
Make entry of the
usage into the Instrument Usage Log Register.
6.6.11
Compare the result
for its compliance against limit given in the Calibration Log and put the
remarks regarding the HPLC chromatography Calibration status.
6.6.12
Prepare the
calibration status Label and display on the instrument at the designated place.
6.6.13
In case of non-compliance,
follow the Maintenance Program.
6.7
Calibration
of injector (to check reproducibility and linearity of the injection volume):
6.7.1
Standard
preparation:
6.7.1.1
Take clean and dry
50.0ml volumetric flask.
6.7.1.2
Pipette out 1.0ml
of Benzene and Toluene in to the clean and dry 50.0ml volumetric flask.
6.7.1.3
Make up the volume
to 50.0ml with Methanol and mix well.
6.7.2
Chromatographic
condition:
6.7.2.1
Column:
ODS C18, (25 cm x 4.6 mm ID, 5 µm).
6.7.2.2
Mobile
Phase: Methanol: Water (70:30).
6.7.2.3
Flow
Rate: 1.0 ml/min.
6.7.2.4
Wavelength:
254 nm.
6.7.2.5
Injection
Volume: 20 µl.
6.7.3
Calibration:
6.7.3.1
Ensure that, the
instrument is ready for calibration and Start-up procedure is followed.
6.7.3.2
Ensure that, the
instrument is set according to the Chromatographic conditions.
6.7.3.3
Follow the
Instrument Operating procedure i.e. SOP,
Inject 10µl in triplicate and record the chromatograms.
6.7.3.4
Repeat the
injection of above solution by injecting 15, 20, 25, 30 µl in triplicate.
6.7.3.5
Take the print out
of the chromatograms and attach to the Calibration Log.
6.7.3.6
Record the Area
and Retention times of the of the Benzene and Toluene peaks in the Calibration
Log.
6.7.3.7
Make entry of the
column usage in the Column Usage Log register.
6.7.3.8
Make entry of the
usage into the Instrument Usage Log Register.
6.7.3.9
Plot the curve for
area corresponding to Benzene to Toluene peaks v/s injection volume, Find out
the RSD (reproducibility) and record in the Calibration Log.
6.7.3.10
Find out the
Correlation coefficient “r2” for each peak at five levels and record
in the Calibration Log.
6.7.3.11
Compare the result
for its compliance against limit given in the Calibration Log and put the
remarks regarding the HPLC chromatography Calibration status.
6.7.3.12
Prepare the
calibration status Label and display on the instrument at the designated place.
6.7.3.13
In case of
non-compliance, follow the Maintenance Program.
6.8
Calibration
of detector (D2 lamp energy check):
6.8.1
Ensure that, the
instrument is ready for calibration and start-up the procedure as followed.
6.8.2
On the detector’s
display window, some values at the functions “l (nm)”, “abs (AU)”, “range
(AUFS)” and “lamp” appears.
6.8.3
On the display,
previously set value blinks at “l (nm)” functions, enter the wavelength to
254nm by pressing numeric keys.
6.8.4
Press “Func Back”
key and select “lamp” functions, enter 1 to select D2 lamp.
6.8.5
Further press
“Func Back” key till “REF EN” appears.
6.8.6
Record the
reference energy of the D2 lamp at 254nm in the Calibration Log.
6.8.7
Compare the result
for its compliance against limit given in the Calibration Log and put the
remarks regarding the HPLC chromatography Calibration status.
6.8.8
In case of non-compliance,
follow the Maintenance Program.
6.8.9
Standard preparation:
6.8.9.1
Take clean and dry
50.0ml volumetric flask.
6.8.9.2
Pipette out ml of
Benzene and Toluene as per the following table in to the clean and dry 50.0ml
volumetric flask, separately.
6.8.9.3
Make up the volume
to 50.0ml 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
|
50.0ml
|
|
2
|
1.0
|
1.0
|
50.0ml
|
|
3
|
1.5
|
1.0
|
50.0ml
|
6.8.9.4
Chromatographic
conditions:
6.8.9.4.1
Set each of the equipment
to the following parameters as per the equipment’s parameter set up procedure:
6.8.9.4.1.1 Column: ODS C-18,
(25cm×4.6mm ID, 5m)
6.8.9.4.1.2 Mobile phase: Methanol:
Purified water (70:30).
6.8.9.4.1.3 Flow rate: 1.0ml/min.
6.8.9.4.1.4 Wavelength: 254nm.
6.8.9.4.1.5 Injection volume: 20μl.
6.8.9.5
Calibration:
6.8.9.5.1
Ensure that, the
instrument is ready for calibration and Start-up procedure is followed.
6.8.9.5.2
Ensure that, the
instrument is set according to the Chromatographic conditions.
6.8.9.5.3
Follow the
instrument operating procedure, inject each of the solutions in triplicate and
record the chromatogram.
6.8.9.5.4
Take the print out
of the chromatograms and attach to the Calibration Log.
6.8.9.5.5
Record the Area
and retention times of the Benzene and toluene peaks in the Calibration Log.
6.8.9.5.6
Make entry of the
column usage in the Column Usage Log register.
6.8.9.5.7
Make entry of the
usage into the Instrument Usage Log Register.
6.8.9.5.8
Calculate the area
ratio of the benzene to toluene, find out the mean value of the ratios and
record in the Calibration Log.
6.8.9.5.9
Find out the
correlation co-efficient “r2” from the mean area ratio values of the
three levels and record in the Calibration Log.
6.8.9.5.10 Plot
the curve for area ratio corresponding to benzene to toluene peaks v/v
concentration. Find out the RSD (reproducibility) and record in the Calibration
Log.
6.8.9.5.11 Compare
the result for its compliance against limit given in the Calibration Log and
put the remark regarding the Calibration Status.
6.8.9.5.12 Prepare
calibration status label and display on the instrument at the designated place.
6.8.9.5.13 In
case of non-compliance, follow the maintenance program.
6.9
Frequency
of calibration:
6.9.1
Pump:
Once
in three months and after each maintenance job.
6.9.2
Gradient:
Once
in six months and after each maintenance job.
6.9.3
Injector:
Once
in six months and after each maintenance job.
6.9.4
Detector:
Once
in three months and after each maintenance job.
6.10
After calibration
status label on the instrument containing “Calibrated on”, “Due on” and
“Signature”.
6.11
Report to QC head,
if any discrepancy observed during calibration or operating the instrument and
affix ‘Under Maintenance’ label on the instrument.
|
UNDER MAINTENANCE
Equipment:
___________________________________________
Since:_________________________________________________
Signature:
______________________
|
7.0 REVISION LOG:
|
Revision No.
|
Effective Date
|
Reason
|
|
00
|
|
New
SOP
|
8.0 REFERENCES:
8.1 Not Applicable.
9.0 ANNEXURES:
Annexure 1:
Calibration of HPLC apparatus.
Annexure:
1
Calibration
record of HPLC apparatus
|
Calibration
of HPLC apparatus
Observation Chart
1. Calibration
of pressure drop (Leakage test):
2. Calibration
of flow rate:
3. Calibration
of gradient proportionate valve:
Value=
Height of B conc.___
Height of a 100% conc.
4. Calibration
of injector:
Co-efficient
of co-relations r2: _______________
(Limit: NLT 0.999).
5. Calibration
of detector:
|
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10.0
ABBREVIATIONS:
|
Abbreviation
|
Expanded Form
|
|
SOP
|
Standard operating
procedure
|
|
&
|
And
|
|
No.
|
Number
|
|
Ltd.
|
Limited
|
|
Q.C
|
Quality control
|
|
Q.A
|
Quality assurance
|
|
QCEB
|
Quality control
equipment calibration
|
|
F
|
Format
|
|
Sr.#
|
Serial number
|
|
g
|
Grams
|
|
%
|
Percentage
|
|
oC
|
Degree centigrade
|
|
mg
|
Milligram
|
|
SD
|
Standard deviation
|
|
NMT
|
Not more than
|
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