Calibration of HPLC: A Complete Guide

High Performance Liquid Chromatography (HPLC) is a widely used analytical instrument in the pharmaceutical industries. HPLC is used for the separation, identification and quantification of various chemical compounds. In order to obtain accurate and reliable results, it is important to calibrate the HPLC system. Calibration of the HPLC system involves a series of tests to ensure that the system is functioning correctly and that the results obtained are accurate and precise. In this article, we will discuss the calibration of HPLC as per Indian Pharmacopoeia commission in detail.

Calibration of HPLC involves a series of tests that are designed to ensure that the system is operating correctly and that the results obtained are accurate and precise as below:

1. Calibration of Pump

1.1 Flow Rate Accuracy:

• Remove the column and put all the channels inlets in reservoirs of HPLC grade water. Set the flow rate at 1.0 ml/min. using the following composition: Channel A, 25%; B, 25%; C, 25%; and D, 25%.
• Then Collect the HPLC grade water from column inlet into a dry 10.0 ml of calibrated volumetric flask and note down the time taken to fill the volumetric flask till the mark using a calibrated stopwatch. Perform the exercise in duplicate.
• Calculate the flow rate as follows:

Flow rate = Volume in ml /Time in minutes.

• Set the flow rate at 2.0 ml/min. and 3.0 ml/min. and perform the same exercise in duplicate.
• Acceptance Criteria: The flow rate should be within ±2.0% of the set value.

1.2 Flow Rate Consistency:

• Accurately weigh about 100 mg of Caffeine standard into a 100.0 ml volumetric flask. Dissolve in about 10 ml of methanol and make up the volume with mobile phase.
• Furthermore, dilute accordingly with mobile phase to get solution having concentration of 10 ppm.

Chromatographic conditions:

Column	Octadecylsilane (C-18) or Octylsilane (C-8) (250 mm x 4.6 mm x 5 µm)
Mobile phase	Methanol: Water (50 : 50)
Flow rate	1 ml/min.
Injection volume	20 µL
Detection	UV at 272 nm
Run time	10 minutes
Retention time of caffeine	About 5 minutes

• Inject 10 ppm caffeine solution six times and calculate %RSD of the retention time of caffeine as obtained from the chromatograms.

• Acceptance criteria: The %RSD of retention time of caffeine should not be more than 1.0%.

1.3 Compositional Accuracy (Gradient Profile)

• Remove the column from the system and replace with dead volume connector.
• Then Prepare a 0.25% v/v solution of acetone in water. Flush the channels at a flow rate of 1.0 ml/min. using the composition given below:

Time (min)	HPLC grade water (Channel A, B) (%)	0.25% v/v Acetone in water (Channel C, D) (%)
0	25 + 25	25 + 25
10	25 + 25	25 + 25
12	50 + 50	0 + 0
20	50 + 50	0 + 0

• Check the compositional accuracy of the HPLC system with the conditions given below:

Flow rate	1 ml/min.
Detection	UV at 254 nm
Run time	30 minutes
Injection delay	15 minutes

Time (min.)	HPLC grade water (Channel A) (%)	0.25% v/v Acetone in water (Channel C) (%)
0	100	0
4	100	0
6	80	20
10	80	20
12	60	40
16	60	40
18	20	80
22	20	80
24	0	100
28	0	100
30	100	0

• Run the gradient using channel combination A and C and repeat the same gradient using channel combination B and D.
• Then Inject 0 µL or minimum volume of HPLC grade water and record the gradient profile.
• Print the overlay plot of gradient profile of A/C and B/D. Chromatogram is attached for reference.

• Acceptance Criteria: The gradient profile of A/C and B/D should overlay with each other with difference in absorbance should not more than 0.01 AU and the difference in time should not more than 20 seconds. Chromatogram is attached for reference.

1.4 Delay Volume of the System

• Review the gradient profile performed under – Compositional accuracy (Gradient profile). Note the time in minutes taken for the actual first change in absorbance (Lift of the baseline).
• The delay volume of the system can be calculated in terms of ml by subtracting 4 minutes from the actual time in minutes taken for change in absorbance.
• Acceptance criteria: The delay volume of the system should be not more than 1.0 ml. Chromatogram is attached for reference.

2. Calibration of Autosampler

2.1 Injection Volume Accuracy

• Purge the instrument with HPLC grade water.
• Fill the HPLC vial with HPLC grade water and close with a cap. Weigh this vial and record weight in gram (W1). Water is used in auto injection volume accuracy test because its density is 0.9982 g/ml at 20°C and 0.9970 g/ml at 25°C. This introduces less than 0.3% error when volume is assumed equal to weight.
• Program HPLC system with a flow rate of 1.0 ml/min. with water and run time of 1 minute.
• Inject 20 µL from HPLC Vial and repeat it for 10 times from the same HPLC vial.
• After completion of 10 injections, remove the vial and weigh again (W2).
• Calculate the average volume (in µL) injected per injection using formula: Average Volume = [(W1-W2)/10] x1000= µl/injection
• Acceptance Criteria: Average volume of injection (µl/injection) should be 20 µl±0.4 µl.

2.2 Injection Volume Precision

• Flush the HPLC system with HPLC grade water for about half an hour.

Chromatographic conditions:

• Accurately weigh about 100 mg of caffeine in a 100 ml volumetric flask. Dissolve in 10 ml of methanol and make up the volume with mobile phase. Further dilute accordingly with mobile phase to get solution having concentration of 10 ppm.
• Inject this solution six times and calculate %RSD for the area obtained in the chromatograms.
• Acceptance criteria: %RSD for the area should not be more than 1.0%.

2.3 Injection volume linearity

• Inject 10 ppm caffeine solution with duplicate injection of 5 µl, 10 µl, 20 µl, 50 µl and 100 µl.
• From the data obtained, plot a graph of mean area count of duplicate injections on y-axis versus injection volume on the x-axis and calculate the value of R-square.
• Acceptance criteria: R-square should be not less than 0.9990.

2.4 Autosampler Temperature Accuracy

• Set the sample compartment temperature at 40º.
• After about 10 minutes record the observed temperature using a calibrated probe with digital thermometer.
• Repeat the same at 30°, 15°, 10° and 5°.
• Acceptance Criteria: The observed temperature should be within ±2ºC of the set temperature.

3. Calibration of Column Oven

• Set the column oven temperature at 60º.
• After about 10 minutes, record the temperature using a calibrated probe with a digital thermometer.
• Repeat the same at 50°, 30°, 20° and 10°.
• Acceptance Criteria: The observed temperature should be within ±2ºC of the set temperature.

4. Calibration of Detector

Set up the HPLC system using chromatographic conditions mention below:

Column                                    : Octadecylsilane (C-18) or Octylsilane (C-8) (250 mm x 4.6 mm x 5 µm)

Mobile phase                           : Methanol : Water (50 : 50)

Flow rate                                  : 1 ml/min.

Injection volume                       : 10 µL

Detection                                 : UV at 272 nm

Run time                                  : 10 minutes

Retention time of caffeine       : About 5 minutes

4.1 Detector Linearity

• Accurately weigh about 100.0 mg of caffeine in a 100 ml volumetric flask. Dissolve in 10 ml of methanol and make up the volume with mobile phase.
• Further dilute accordingly with mobile phase to get solution having concentration of about 0.001, 0.01 and 0.10 mg/ml (1.0, 10.0 and 100.0 µg/ml).
• Inject in duplicate 10 µl of 0.001, 0.01 and 0.10 mg/ml solutions of caffeine prepared above.
• From the data obtained plot a graph of mean area counts of duplicate injection on y- axis versus concentration (µg/ml) on x-axis and calculate the value of R-square.
• Acceptance Criteria: R-square should not be less than 0.9990.

4.2 Wavelength Accuracy

For Photo Diode Array Detectors (PDA)

• Set detector wavelength at 200 nm to 400 nm.
• Inject 20 µl of 0.01 mg/ml solution of caffeine.
• Record spectrum and report maxima and minima.

• 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.

For Variable Wavelength Detectors

• Create 32 acquisition program with same parameters except changing wavelength 269 to 278, 239 to 249 and 200 to 210.
• Inject 20 µL of 0.01 mg per mL solution of Caffeine.
• Record spectrum and report maxima and minima.

• 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.

FAQs

References:

1. https://www.ipc.gov.in/

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