Validation and Calibration of HPLC, HPTLC, and GC

Calibration of HPLC, HPTLC and GC.

3. High-Performance Liquid Chromatography (HPLC) is an analytical
technique used to separate a mixture of compound in analytical
chemistry and biochemistry with the purpose of identifying,
quantifying or purifying the individual components of the mixture.
Principle:
HPLC is based on the principle of adsorption as well as partition,
depending on the nature of stationary phase, if stationary phase is
solid principle is based on adsorption and if stationary phase is liquid it
is partition.

4. Instrument Part
Pump AutosamplerDetector Column
Injection carryover
Injection volume
accuracy
Injection volume
linearity
Injection volume
precision
Flow rate
Pressure test
Gradient flow
measurment
Wavelength
accuracy
Detector
linearity
Column oven
temperature

5. Procedure:
❖Prime all the solvent line with water and set the flow rate to 1 mL/min.
❖Wait for 15 min to stabilize the system and ensure that the pressure is stable.
❖Insert the outlet tubing into 10 mL volumetric flask and start the stopwatch
simultaneously.
❖Stop the stopwatch when the water reaches the mark.
❖Record the time and similarly check for 2 mL/min and 3 mL/min.
Acceptance criteria: The flow rate should be within ± 2% of the set value.
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6. Procedure:
❖Purge the system for each flow line with water as mobile phase.
❖Remove the tubing from reservoir to column inlet.
❖Plug the outlet of the pump using a dead nut.
❖Set the flow rate 0.1 mL/min.
❖Observe the pressure until it reaches its maximum limit.
❖If pressure reaches to maximum limit, it implies that good check valve and no leakage
in pumping system.
❖If not than poor check valves or leakage in pumping system.
Acceptance criteria: Maximum pressure limit should be as per company qualification.

7. Acceptance criteria:
❖Wavelength maxima found should be between 273±2 nm.
❖Wavelength maxima found should be between 205±2 nm.
❖Wavelength minima found should be between 245±2 nm.
Chromatographic condition:
❖Column : C-18 or C-8
❖Mobile phase – Methanol:Water (50:50)
❖Flow rate : 1 mL/min
❖Run time : 10 min
Procedure:
❖Set detector wavelength from 200 nm to 400 nm.
❖Inject 20 µL of 0.01 mg/mL solution of caffeine in methanol.
❖Record the spectrum and report maxima and minima.

8. Procedure:
❖Weigh accurately 100 mg of caffeine and dissolve in 100 mL of methanol.
❖From this solution prepare the solution of concentration 1 µg/mL, 10 µg/mL, 100 µg/mL.
❖Inject the solution in duplicate and record the data.
❖Plot a calibration curve of the mean area of duplicate injection vs the concentration
and calculate the value of correlation coefficient.
Acceptance criteria: The R
2
should be not less than 0.999

9. Procedure:
❖Prepare a sample of 4µg/mL of reference caffeine with methanol.
❖Mobile phase is Water:ACN in the ratio of 85:15.
❖Run HPLC for 30 min with mobile phase to get saturated.
❖Inject 20 µL methanol as blank for baseline.
❖After getting the baseline inject the sample in duplicate.
❖Again inject 20 µL methanol as blank and observe for any peak.
Acceptance criteria: The % carryover should not more than 0.2%
% Carryover =
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10. Procedure:
❖Purge the instrument with HPLC grade water.
❖Fill the vial with HPLC grade water and close the cap and weigh the vial.
❖Program HPLC system with flow rate 1 mL/min.
❖Inject 20 µL from vial and repeat it for 10 times.
❖After 10 injection weigh the vial again.
Acceptance criteria: The average volume should be 20 µL ± 0.4 µL
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11. Procedure:
❖Prepare a 10 µg/mL sample solution of caffeine by mixing it in methanol.
❖In duplicate inject 5 µL, 10 µL, 20 µL, 50 µL and 100 µL of sample solution.
❖Observe the peak and plot a calibration curve of area under curve vs concentration and
calculate the value of correlation coefficient.
Acceptance criteria: The R
2
should be not less than 0.999

12. Procedure:
❖Flush the HPLC system with HPLC grade water for about half an hour.
❖Prepare a 10 µg/mL solution of caffeine in methanol.
❖Inject this solution six times and calculate %RSD for the area obtained of the
chromatograms.
Acceptance criteria: The %RSD for the area should not be more than 1%.

Chromatographic condition:
❖Column : C-18 or C-8
❖Mobile phase – Methanol:Water (50:50)
❖Flow rate : 1 mL/min
❖Injection volume : 20 µL
❖Run time : 10 min

13. Procedure:
❖Carry out calibration for the temperature point at 15°C, 30°C and 60°C using digital
temperature indicator.
❖ Set the temperature of oven at 30°C and keep the temperature probe in the center of
column and allow stabilizing the set temperature for at least 15-20 min.
❖Check the temperature displayed on the screen and compare it with the temperature
indicator.
Acceptance criteria: The observed temperature should be within ± 2°C of the set
temperature.

14. High-Performance Thin Liquid Chromatography (HPTLC) is a sophisticated form of
TLC that enhance the resolution, sensitivity and reproducibility of traditional TLC. It
is used for separating, identifying and quantifying components in a mixture.
Principle:
Principle of HPTLC is similar to that of TLC i.e. adsorption chromatography. The
mobile phase flows through capillary action and the component move according to
their affinity towards adsorbent.

16. Chromatographic condition:
❖Stationary phase – Silica gel 60F254 plate
❖Mobile phase – Toulene:Acetone – 4:1 v/v
❖Sample volume - 5µL
❖Scan wavelength – 273 nm
Procedure:
❖Accurately weigh and transfer about 5mg of caffeine into 100 mL volumetric flask.
❖Dissolve and dilute upto the mark with methanol to prepare 50 µg/mL solution.
❖Apply 6 bands of this solution into the TLC plate using sample applicator.
❖Allow the TLC plate for development in the mobile phase.
❖Scan the plate in scanner and find out the area of each band.
Acceptance criteria: The %RSD of these area should not be more than 2.0%

17. Procedure:
❖The same TLC plate can be used to study reproducibility of
scanner.
❖The plate is scanned 12 times under same condition.
❖The area of any one band is measured for 12 times and the %RSD is
calculated.
Acceptance criteria: The %RSD of these area should not be more
than 2.0%

18. Chromatographic condition:
❖Stationary phase – Silica gel 60F254 plate
❖Mobile phase – Toulene:Acetone – 4:1 v/v
❖Sample volume - 5µL
❖Scan wavelength – 273 nm
Procedure:
❖Prepare 50 µg/mL solution of caffeine by dissolving 5 mg of caffeine in 100 mL of
methanol.
❖Apply 2 µL, 3 µL, 4 µL, 5 µL and 6 µL band of prepared caffeine solution using sample
applicator.
❖Allow the plate to develop in mobile phase.
❖Scan the TLC plate in scanner and measure the area of each concentration.
❖Plot the calibration curve of area vs concentration and calculate the value of correlation
coefficient.
Acceptance criteria: The R
2
should be not less than 0.999

19. Gas chromatography is a analytical separation
technique used to analyze volatile substance in the gas
phase. The component of sample are dissolved in a
solvent and vaporized in order to separate the analyte
by distributing the sample between two phases:
Stationary phase and mobile phase. The mobile phase is
a chemically inert gas that serves to carry the molecule
of the analyte through the heated column.
Principle:
The principle of separation in GC is partition. The
component which has more affinity towards stationary
phase travel slower and elute later. The component
which less affinity towards stationary phase travels
faster and elute out first.

21. Procedure:
❖Set the column oven temperature for at least 3
points from 40 – 300°C.
❖After 10 min, record the observed temperature using
a calibrated temperature probe.
❖Ensure whether the temperature inside the oven is
the same as that set temperature.
Acceptance criteria: The observed temperature should be within ±2°C of the set
temperature

22. Procedure:
❖Connect the digital flow meter to the detector outlet port.
❖Set the carrier gas flow and wait till it reaches the set flow.
❖Note the observed flow in duplicate.
❖Repeat the procedure for hydrogen and air flow.
Acceptance criteria: The tolerance limit is ±5% of set flow.
Parameter Set Flow Rate
(mL/min)
Tolerance Limit
(mL/min)
Hydrogen Gas 40 38 - 42
45 43.75 – 47.25
Air Flow 400 380 – 420
450 437.50 – 472.50
Nitrogen Gas 2 1.9 – 2.10
5 4.75 – 5.25
10 9.5 – 10.5

23. Procedure:
❖Inject six replicate injection of the standard mixture of 0.3 mg/mL and calculate the area
ratio of pentadecane and hexadecane to that of the tetradecane.
❖Determine the relative standard deviation of the ratio of the area count of the peaks.
Acceptance criteria: %RSD of the ratio of area count of the peak corresponding
to n-pentadecane and n-hexadecane to that of n-tetradecane
should not more than 2%
Standard Sample Preparation:
❖Weigh accurately 50 mg each of n-tetradecane, n-pentadecane and n-hexadecane in a 50 ml
volumetric flask and make-up the volume upto the mark with n-hexane.
❖From above solutions prepare 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, and 0.5
mg/mL solutions by dilution 1 mL, 2 mL, 3 mL, 4 mL and 5 mL in 10 mL volumetric flask each.

24. Procedure:
❖Inject 1 µL of all the five standards 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, and
0.5 mg/mL respectively in duplicate.
❖From the data obtained, plot regression curve with concentration on x-axis and mean
response of standard on y-axis and calculate the value of correlation coefficient.
Acceptance criteria: The correlation coefficient should not be less than 0.999

25. 1.Indian Pharmacopoeia Commission. (2021). Calibration of HPLC [Guidance
Document IPC/GD/05]. https://www.ipc.gov.in/images/GD-05-
Calibration_of_HPLC.pdf
2.Indian Pharmacopoeia Commission. (2021). Calibration of Gas Chromatograph
[Guidance Document IPC/GD/06]. https://www.ipc.gov.in/images/GD-06-
Calibration_of_GC.pdf