1.
Title:
Standard operating procedure for HPLC
2. Purpose:
To lay down the procedure for the operation of HPLC used
in the raw material, intermediates &
finished product analysis in FBL lab.
3. Scope:
This procedure shall be applicable for operation of HPLC
( Agilent ) in FBL Lab.
4. Responsibility: Analyst.
5.0
PROCEDURE:
5.1
OPERATING PROCEDURE:
5.2.1
Switch on instrument and system. Click the
‘Empower’ icon on desktop of computer. Enter user name and
password.
5.2.2
‘Empower pro’ displays. Click on ‘Browse project’
and select desire project. Browse project window will open.
5.2.3
New instrument method preparation. In Browse
project window click on ‘File’ ⟶new method⟶ click on ‘instrument method’ and make
changes as per your specification and save the instrument method.
5.2.4
New method set preparation. In Browse project
window click on ‘File’⟶new method⟶ click on ‘Method set’ and add update above
instrument method in method set.
5.2.5
Click on ‘Run sample’ icon in browse project and
select system FBL_HPLC_03 System. New window will appear. For conditioning the
column go on INSTRUMENT METHOD window select desire method then click on setup.
5.2.6
New sample set preparation Click on the File ⟶new
sample set method ⟶Empty prepare sequence as per specification and save it.
5.2.7
Start sample set by click on green Run icon.After
completion of sample set, process the chromatogram and take print in suitable
report template.
5.2.8
After completion analysis take entry.
5.2
CALIBRATION
PARAMETERS:
5.3.1
Pump flow rate accuracy
5.3.2
Sample compartment temperature accuracy
5.3.3
Column oven temperature accuracy
5.3.4
Injection volume accuracy
5.3.5
Auto Vial Injector Test
5.3.6
System
precision
5.3.7
Detector
linearity
5.3.8
Injector
linearity
5.3.9
Detector:
wavelength accuracy
5.3.10 Carry over Test
5.3.11 Gradient
proportioning value
5.3
PUMP
FLOW RATE ACCURACY:
5.4.1
REQUIREMENT:
5.4.1.1 Volumetric flasks, Class A, 10
ml and 25 ml
5.4.1.2 Stop watch
5.4.1.3 Distilled water
5.4.1.4 Column: X Bridge C18, 3.5 um, 4.6 x 50mm or equivalent.
5.4.2
PROCEDURE:
5.4.2.1 Flush the system with distilled
water.
5.4.2.2 Take the Class A of 10ml and 25
ml volumetric flasks and stopwatch.
5.4.2.3 When the flow and pressure have
stabilized, place the indicated flask under the outlet tube and start the
stopwatch immediately.
5.4.2.4 Stop the stopwatch when the lower
meniscus of water reaches up to mark of the volumetric flask.
5.4.2.5 Record the elapsed time (in
seconds) and pressure obtained.
5.4.2.6 Repeat above steps for the
following conditions indicated in the table below.
5.4.2.7 Calculate the flow rate for each
flow rate setting using the following equation:
Volume of flask
Calculated flow rate =
-----------------------------
Elapsed
time in seconds
Record the calculated flow rate in format
no. FBL/F/QC/081
|
Flow rate |
Volumetric
flask size |
Acceptable limits |
Remarks |
|
0.5 ml / min. |
10 ml |
0.469 – 0.531 |
With column |
|
1.0 ml / min. |
10 ml |
0.968 – 1.032 |
With column |
|
2.5 ml / min. |
25 ml |
2.470 – 2.530 |
Without column |
5.4
SAMPLE
COMPARTMENT TEMPERATURE ACCURACY:
5.5.1
REQUIREMENT:
5.5.1.1 Calibrated
Thermometer
5.5.2
PROCEDURE:
5.5.2.1 Place a thermometer in sample chamber,
Close the Chamber door.
5.5.2.2 Set temperature in INSTRUMENT
STATUS window. Right click on Sampler then click on method, and then enter
value 10°C, 25°C and 35°C. Record this set point value.
5.5.2.3 Allow
the sufficient time for the chamber temperature to reach and stabilize at the
set temperature.
5.5.2.4 Record the temperature observed
at the thermometer.
Acceptance criteria: observed temperature should be set
temperature ±2°C.
5.5
COLUMN
OVEN TEMPERATURE ACCURACY:
5.6.1
REQUIREMENT:
5.6.1.1 Calibrated
Thermometer.
5.6.2
PROCEDURE:
5.6.2.1 Place a calibrated thermometer
in Column oven. Close the chamber door.
5.6.2.2 Set temperature in INSTRUMENT
STATUS window. Right click on Column comp then click on method, and then enter
value 10°C, 25°C, 35°C and 50°C. Record this set point value.
5.6.2.3 Allow the sufficient time for
the column oven to reach and stabilize at the set temperature.
5.6.2.4 Record the temperature observed
at the calibrated thermometer.
Acceptance criteria: observed temperature should be set temperature ±2°C.
5.6
INJECTION
VOLUME ACCURACY:
5.7.1
REQUIREMENT:
5.7.1.1 Analytical
weighing balance
5.7.1.2 Pipettes
5.7.1.3 Distilled
water
5.7.1.4 Injection
vial
5.7.2
PROCEDURE:
5.7.2.1 Fill a three standard 2 ml vial
with 1.5 ml degassed distilled water.
5.7.2.2 Weigh all three vial on analytical weighing balance and note the
weight of vial before injection (W1).
.
Follow the instrument
parameters as per below table
|
1. |
Column |
X Bridge C18,
3.5 um, 4.6 x 50mm or equivalent. |
|
2. |
Mobile Phase |
Methanol (30):
Distilled water (70) |
|
3. |
Flow ml/min |
1.0 ml/min |
|
4. |
wavelength |
272 nm |
|
5. |
Run time (min) |
0.2 minutes |
|
6. |
Injection Volume |
50µL |
5.7.2.3 Run sample set of each vial in six
replicate injections.
5.7.2.4 After completion of run re-weigh
the vial and record the weight (W2).
5.7.2.5 Calculate the average volume of
water injected per Injection.
5.7.2.6 Calculation: [(W1-W2)/6] X 1000 = mg of water
withdrawn per injection = l per Injection Density of water at 25°C is
0.99707g/ml.
Acceptance
criteria: observed
injection volume should be set injection volume ±1µl or 50±1µl
5.7
AUTO
INJECTOR- VIAL TEST:
5.8.1 PROCEDURE:
5.8.1.1 Fill
and seal five vials with water and put randomly at different vial locations in
different location in the sample compartment.
5.8.1.2 Set
the system to draw the 5
µl from each vial.
5.8.1.3 Take
out each vial from the instrument and check for septum piercing.
5.8.1.4 Acceptance criteria: Should
be pierced.
Follow the instrument
parameters as per below table
|
1. |
Column |
X Bridge C18, 3.5 um, 4.6x 50mm or equivalent |
|
2. |
Mobile Phase |
Methanol (30): Water (70) |
|
3. |
Flow ml/min |
1.0 ml/min |
|
4. |
Wavelength |
272 nm |
|
5. |
Run time (min) |
0.2 minutes |
|
6. |
Injection Volume (water) |
5 µl |
5.8
SYSTEM
PRECISION:
5.9.1
REQUIREMENTS:
5.9.1.1 Analytical weighing balance
5.9.1.2 Distilled water
5.9.1.3 Caffeine AR grade.
5.9.1.4 Volumetric flask 100ml
5.9.2
Procedure:
5.9.2.1 Prepare the required standard
solution and set the chromatographic condition as mention below in the table.
5.9.2.2 Standard
preparation:
weigh accurately 100±2 mg caffeine AR grade in a 100 ml volumetric flask,
dissolve in 60 ml distilled water and make up to volume with water. Transfer 5
ml of above solution in another 100 ml volumetric flask and make up the volume
with distilled water (50 ppm).
Follow the instrument parameters as per below
table
|
1. |
Column |
X Bridge C18,
3.5 um, 4.6 x 50mm or equivalent. |
|
2. |
Mobile Phase |
Methanol (30):
Distilled water (70) |
|
3. |
Flow ml/min |
1.0 ml/min |
|
4. |
wavelength |
272 nm |
|
5. |
Run time (min) |
5 minutes |
|
6. |
Injection Volume |
20µL |
5.9.2.3 Run the sample set of six replicate
injections with 50 ppm caffeine solution
5.9.2.4 After completion of runs measure
peak retention time and area.
5.9.2.5 Calculate the % RSD for each and
record the observation as per current version of Format No. FBL/F/QC/081.
5.9.2.6 ACCEPTANCE CRITERIA:
|
%RSD for retention time |
NMT 1.0 % |
|
%RSD for Area |
NMT 2.0% |
5.9
DETECTOR
LINEARITY:
5.10.1 REQUIREMENTS:
5.10.1.1 Analytical weighing balance
5.10.1.2 Distilled water
5.10.1.3 Caffeine AR grade
5.10.1.4 Volumetric flask
5.10.1.5 Methanol (HPLC grade)
5.10.2 STANDARD
PREPARATION.
5.10.2.1 Solution
A: Weigh
accurately 100±2 mg caffeine AR grade in 100 ml volumetric flask dissolve in 25
ml distilled water and make up volume with same.
5.10.2.2 50
ppm: Pipette 5
ml of solution –A in 100 ml volumetric flask and make up volume with distilled
water.
5.10.2.3 25
ppm: Pipette
2.5 ml of solution –A in 100 ml volumetric flask and make up volume with distilled
water.
5.10.2.4 10
ppm: Pipette 10
ml of 50 ppm solution in 50 ml volumetric flask and make up Volume with
distilled water.
5.10.2.5 5ppm: Pipette 5 ml of 50 ppm solution in 50 ml
volumetric flask and make up volume with distilled water.
5.10.2.6 1ppm:
Pipette 1ml of
50 ppm solution in 50 ml volumetric flask and make up volume with distilled
water.
5.10.3 PROCEDURE:
Set the chromatographic
condition as mention below in the table.
|
1. |
Column |
X Bridge C18, 3.5 um, 4.6 x 50mm or equivalent. |
|
2. |
Mobile Phase |
Methanol (30):
Distilled water (70) |
|
3. |
Flow ml/min |
1.0 ml/min |
|
4. |
wavelength |
272 nm |
|
5. |
Run time (min) |
5 minutes |
|
6. |
Injection Volume |
20µL |
5.10.3.1 Inject the solutions of 1 ppm, 5
ppm, 10 ppm, 25 ppm and 50 ppm.
5.10.3.2 Enter run icon. Run will
automatically starts.
5.10.3.3 After the completion of each run
measure area of caffeine peak and record the observations as per current
version of format No. FBL/F/QC/081.
5.10.3.4 Calculate the co-relation
coefficient of the peak areas and print the same.
5.10.3.5 Calculate % RSD &
Co-relation coefficient.
5.10.3.6 Acceptance
criteria: Co-relation coefficient should be more than 0.99
5.10
INJECTOR
LINEARITY:
5.11.1 REQUIREMENT:
5.11.1.1 Analytical weighing balance
5.11.1.2 Distilled water
5.11.1.3 Caffeine
5.11.1.4 AR grade
5.11.2 PROCEDURE:
5.11.2.1 Set the chromatographic
condition as mentioned below.
|
1. |
Column |
X bridge c18, 3.5µm,4.6x50mm or
equivalent |
|
2. |
Mobile phase |
Methanol(30): Distilled water (70) |
|
3. |
Flow ml/min |
1.0 ml/min |
|
4. |
Wavelength |
272nm |
|
5. |
Run Time |
5 minutes |
5.11.2.2 Inject 25 ppm caffeine solution 10,
25, 50, 75, 100 µl and record the Chromatograms. Calculate co-relation
coefficient of the areas.
5.11.2.3 ACCEPTANCE CRITERIA: Correlation coefficient should
not be less than 0.99
5.11
DETECTOR
WAVELENGTH ACCURACY:
5.12.1
REQUIREMENTS:
5.12.1.1 Analytical weighing balance
5.12.1.2 Caffeine AR grade
5.12.1.3 Distilled water
5.12.1.4 Volumetric flask
5.12.1.5 Methanol (HPLC grade)
5.12.2 PREPARATION OF STANDARD
SOLUTION :
Weigh about 100±2 mg caffeine AR grade and transfer into
100mL volumetric flask, dissolve and dilute upto the mark with distilled water. Pipette out
5 ml of this solution into 100ml volumetric flask and dilute upto the mark with distilled water.
Chromatographic
Condition:
|
1. |
Column |
X Bridge C18, 3.5 um, 4.6x 50mm or equivalent |
|
2. |
Mobile Phase |
Methanol (30): Water (70) |
|
3. |
Flow ml/min |
1.0 ml/min |
|
4. |
Run time (min) |
5 minutes |
|
5. |
Injection Volume |
20 µl |
5.12.2.1
Ensure that, the
instrument is ready for calibration and Start-up procedure is followed. Ensure
that, the instrument is set according to the Chromatographic conditions.
5.12.2.2 Follow the
Instrument Operating procedure, Inject 20µL of Standard solution and the run system with 202nm to 208 nm , 269 to
275 nm and 242 to 248nm by increment of 1-nm wavelength and record the chromatograms.
5.12.2.3 Only Single
injection can be injected with one wavelength.
5.12.2.4 Extract the data
with 208nm to 212 nm, 242nm to 248 and 270 to 274 nm.
5.12.2.5 Take the print
out of the chromatograms and attach to the Calibration protocol.
5.12.2.6 Record the Area of the Caffeine peak in the
calibration protocol.
5.12.2.7 QC Executive
shall make entry in log book of the usage of the instrument as per Current version
of format No. FBL/F/QC/078.
5.12.2.8 QC Executive
shall make entry in log book of the usage of the column as per Current version
of format No. FBL/F/QC/088.
5.12.2.9 Compare the
result for its compliance against limit given in the Calibration protocol and
put the remark regarding the Calibration Status.
5.12.2.10 Prepare
Calibration Status Label and display on the instrument at the designated place.
5.12.2.11 In case of
non-compliance, inform to quality head and follow the Maintenance Program.
5.12.2.12 Acceptance Criteria:
Measured peak value should be two maxima at
wavelength 205± 2nm, 272 ±2nm and one minima at 245±2nm.
5.12
CARRY
OVER TEST:
5.13.1 PROCEDURE:
5.13.1.1
Set
the instrument parameters as mentioned in System Precision.
5.13.1.2
Make
injections of pre-blank solution.
Make three injection of 50ppm
Caffeine Solution.
5.13.1.3 Make two injections of post
Blank solution.
5.13.1.4 Take average of peaks area
observed at Caffeine RT for calculation.
CALCULATION:
(Avg. Area of post blank – Area of
pre-blank) x 100
%Carry over =
--------------------------------------------------------------
Mean Area of Caffeine Solution.
|
%Carry
over |
<
0.01 % |
Acceptance criteria:
5.13
GRADIENT
PROPORTIONING VALUE:
5.14.1 REQUIREMENT:
5.14.1.1 Acetone(HPLC
grade)
5.14.1.2 Distilled
water
5.14.2 PROCEDURE:
5.14.2.1 Connect
a PDA detector
5.14.2.2 Fill
the reservoir for the A and C Lines with Distilled water.
5.14.2.3 Fill
the reservoir for B and D lines with 0.4% Acetone in water.
5.14.2.4 Prime
the pump.
5.14.2.5 Run
the inline degasser for 10 minute or use appropriate sparge or sonication.
5.14.2.6 Create
an instrument method in software using the parameter listed in table.
5.14.2.7 Set the detector to 265 nm.
5.14.2.8 Save
the method with a name such as Gradient.
|
Time |
Flow |
%A |
%B |
%C |
%D |
|
1.00 |
2.0 |
50 |
0 |
50 |
0 |
|
1.01 |
2.0 |
40 |
10 |
40 |
10 |
|
4.00 |
2.0 |
40 |
10 |
40 |
10 |
|
4.01 |
2.0 |
30 |
20 |
30 |
20 |
|
7.00 |
2.0 |
30 |
20 |
30 |
20 |
|
7.01 |
2.0 |
20 |
30 |
20 |
30 |
|
10.00 |
2.0 |
20 |
30 |
20 |
30 |
|
10.01 |
2.0 |
10 |
40 |
10 |
40 |
|
13.00 |
2.0 |
10 |
40 |
10 |
40 |
|
13.01 |
2.0 |
0 |
50 |
0 |
50 |
|
16.00 |
2.0 |
0 |
50 |
0 |
50 |
|
21.00 |
2.0 |
50 |
0 |
50 |
0 |
|
26.00 |
2.0 |
50 |
0 |
50 |
0 |
5.14.2.9
Run single injection with 26 minute run time using
the above method.
5.14.2.10 Prepare
the sequence and run the sequence.
5.14.2.11 After completion of run bring the channel in to review
5.14.2.12 Process
the Chromatogram.
5.14.2.13 Five peak height observed in the chromatogram.
5.14.2.14 Calculate 10, 20, 30,
40% composition of B and D line
as per given formula.
Peak height at definite composition
x 50
% composition = ----------------------------------------------------------------------------
Peak height at highest composition (50%)
5.14.3 Acceptance criteria: % Composition ±1.
5.14
Calibration
Status Display:
After completion of calibration,
display the calibration status as per current version format: FBL/F/QC/453 (Refer SOP/QC/064).
5.15
SERVICING
AND PREVENTIVE MAINTENANCE:
5.16.1 In case of breakdown or problem
occurred while analysis inform to the service engineer By telephone or email
and affix the label as “OUT OF ORDER”.
6.0
ABBREVIATIONS:
Conc. : Concentration
QC :
Quality Control
QA :
Quality Assurance
°C :
Degree Celsius
ml : milliliter
SOP : Standard
Operating Procedure
µl : micro
liter
RSD :
Relative standard deviation
ppm : Part per million
NMT : Not more than
Doc. No.: Document number
CCIF :
Change control initiation form
7.0
REFERENCES:
7.1
Instrument user
manual
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