HPLC Calibration

HPLC Calibration | Complete Guide
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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.

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HPLC Calibration | Complete Guide

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Gradient Composition (Acceptance Criteria: ± 1% of set composition):

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