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Calibration Procedure:
Calibration
of Pump:
Check the Flow
Rate by collecting milli-Q water at the detector outlet for a specified time
period. Procedure:
-
Put the milli-Q
water in the channel A and connect through union between auto injector &
detector. Set 0.5 ml/min flow rate and collect the milli-Q water in 10 ml volumetric
flask and record the weight of water. Note the time required in minutes and
calculate the corresponding flow rate.
Repeat the
procedure for 1 ml/min, 2.0 ml/min & 2.5 ml/min
Acceptance criteria: ± 1.0 % of Flow rate.
Retention Time Reproducibility:
Set the HPLC parameters and prepare
the sample solution of caffeine as given
below:
HPLC Condition : Column C18 250 x 4.6 mm, 5µ.
Column Oven temperature :
40°C
Detector : 272 nm (for caffeine)
Injection
volume : 10 µl.
Mobile phase
: Methanol: water (70:30)
Flow rate :
1.0 ml/min.
Standard solution Preparations:
Stock Solution:
Weigh accurately
about 100 mg of caffeine transfer in 100 ml volumetric flask dissolve &
dilute to volume with mobile phase.
Prepare 40 ppm
solution from stock solution by diluting 2 ml of above solution to 50 ml with
mobile phase for retention time reproducibility. Calculate the RSD of the
retention time. It should not be more than 1.0 %.
Gradient Composition (Acceptance
Criteria: ± 1% of set composition):
Purge channel A
& B with Purified Water and channel C & D with 0.3 % Acetone in
purified water to remove the air bubble from the line. Set the Chromatographic
condition as mentioned below:
HPLC condition : Use union for test
Detector : 265 nm
Injection
volume :
0.1µl
Mobile
phase : Solvent A/B-water HPLC Grade,
Solvent C/D-
0.3% Acetone.
Temperature :
40°C
Gradient Program:
S. No.
|
Time
|
Solvent A/B
|
Solvent C/D
|
1.
|
0.01
|
100
|
0
|
2.
|
5.00
|
100
|
0
|
3.
|
5.01
|
90
|
10
|
4.
|
10.0
|
90
|
10
|
5.
|
10.01
|
50
|
50
|
6.
|
15.00
|
50
|
50
|
7.
|
15.01
|
10
|
90
|
8.
|
20.0
|
10
|
90
|
9.
|
20.01
|
0
|
100
|
10.
|
25.00
|
0
|
100
|
11.
|
25.01
|
100
|
0
|
12.
|
30.00
|
100
|
0
|
After setting all
HPLC conditions, start pump and record the chromatogram and check the mV
(absorbance) from the chromatogram and calculate the % of different composition
by using formula given below.
Calculation formula: - For Channel A and C
Abs at 10.0%
Actual
concentration of 10.0 % (c) = ------------------------
x 100
Abs at 100 %
Abs at 50.0%
Actual
concentration of 50.0 % (c) = ----------------------------- x 100
Abs at 100 %
Abs at 90.0%
Actual
concentration of 90.0 % (c) = ------------------------- x 100
Abs at 100 %
Note: - Above calculation shall be repeated for channel B & D.
Calibration
of injector: -
(A). Injection
precision (area reproducibility): (use solution 40 ppm of
caffeine solution.)
From RT reproducibility
Calculate the RSD of the area and it should not be more than 2.0 %.
(B). Injector
& UV Detector linearity: -
Inject the solution 40 ppm - 5 µl, 10 µl, 20 µl, 40 µl, 60 µl, 80 µl and 100 µl (one injection each).
Record the peak area. The peak area and injection volume is represented in a
graph plotted through software and determine the regression line. Calculate the
correlation coefficient for linearity. It should not less than 0.999.
(C). Carry over test:
(Use 300 ppm
caffeine standard solution and mobile phase as a blank) The peak area is
measured for 25 µl injection of mobile phase as a blank, followed by six 25 µl
injections of 300 ppm caffeine standard solution and after that one blank
injection again. The Injection carry over is calculated for area as the peak
response from the mobile phase injection with the comparison to average area of
six standard caffeine injection. The value should not more than 0.05 %.
(D) Injector accuracy: -
To Confirm the auto injector has withdrawn the proper
sample amount from vial:
Fill a sample
vial, full with degassed & filtered water and cap the vial with
septum.
Carefully keep the
vial on the analytical balance by using the forceps
Placed the vial in sample position 1 and
generate the sequence for 6 injections by putting the sample volume 10 µl. and
run the system.
Using the formula
calculate the average amount injected per injection.
Water withdraw per injection (W1-W2)
W3=-------------------
0.997x6
Where 6= total
number of injection.
Acceptance criteria: - ±2.0% of set injection volume
(i.e. 9.8 to 10.2µl).
Detector Performance:
- (UV Detector)
Wavelength accuracy: -
To verify the
wavelength accuracy using caffeine standard solution (40 ppm caffeine solution
in mobile phase) wavelength verification from 201 to 209 nm, 241 to 249 nm
& 269 to 277 nm.
Acceptance criteria: -
Maxima at 205 ± 2 nm.
Minima at 245 nm ± 2 nm.
Maxima at 273 nm ± 2 nm.
Temperature Accuracy: (Acceptance criteria: - ± 1.0° C
of set temperature)
Procedure: Set the oven
temperature at 25°C,
40°C& 60°C from system. Check
the oven temperature from display and note down temp. in the Calibration format.
Also verify the oven temperature by keeping the external pre-calibrated
thermometer in the oven and verify the same against the displayed temp. by the
system. Difference should be ± 1.0 °C from set temp.
Lamp Energy
a. Click On the
Lab Solution Icon.
b. Enter user Name
and password then Click on ‘Instrument ‘. Double click on Real time
Analysis Icon.
c. Click On the
‘Main’ from ‘Assistant Bar ‘and then click on system check icon.
d. Click on LC
icon set parameters.
e. Then click on
report out and finally click on RUN. Print out will receive.
Acceptance criteria: -
The lamp energy
should not less than 400.
Action to be
taken in case unsatisfactory results:
Attach Not to
be used / under maintenance label to the instrument.
