HPLC Calibration Parameters are provided here as a guide to check the “High-Performance Liquid Chromatography (HPLC)” instrument performance.
Calibration is the most important aspect to meet the Good Manufacturing Practices (GMP / cGMP) and Good Laboratory Practices (GLP) requirements.
HPLC Calibration vs HPLC Performance Verification
The definition and the difference between HPLC Calibration and HPLC Performance Verification are given below.
HPLC Calibration
HPLC calibration is the process of verifying and adjusting the performance of a High-Performance Liquid Chromatography (HPLC) system to ensure accurate and reliable analysis. It involves measuring the accuracy and precision of the instrument by running standard solutions with known concentrations and comparing the results to the expected values. This process helps to detect and correct any deviations or errors in the instrument, thus ensuring the reliability and accuracy of the data generated by the HPLC system.
HPLC Performance verification
HPLC performance verification is the process of evaluating the overall performance of a High-Performance Liquid Chromatography (HPLC) system to ensure that it is operating within acceptable limits and is capable of producing accurate and reliable results. This involves verifying key performance parameters such as retention time, peak resolution, column efficiency, and detector linearity, among others. Performance verification is typically carried out using appropriate reference standards or test solutions and is an important part of routine quality control for HPLC systems to ensure that they continue to operate effectively over time.
List of HPLC Calibration Parameters
The following are some of the key parameters that are typically calibrated during the instrument calibration of a High-Performance Liquid Chromatography (HPLC) system:
HPLC Pump Calibration
- Flow rate accuracy
- Compositional accuracy
- Flow rate stability
HPLC Auto injector Calibration
- Injection volume accuracy
- Lubrication of the Injection
- Injection accuracy
- The Injection carryover
- Check the VIAL position
HPLC Detector Calibration
UV / PDA Detector Calibration
- UV Detector Noise & Drift
- UV/PDA Detector linearity
- HPLC Calibration Wavelength accuracy
RI Detector Calibration
- Detector Noise & Drift
- RI Detector linearity
Temperature
- Column oven temperature
- The temperature of the sample cooler
Software verification
Software files
HPLC calibration interview questions
Flow rate: This is the rate at which the mobile phase moves through the column and is usually measured in milliliters per minute (mL/min).
Injection volume: This is the volume of the sample that is injected into the HPLC system and is usually measured in microliters (µL).
Column temperature: This is the temperature of the column and is usually measured in degrees Celsius (°C).
Detector sensitivity: This is the sensitivity of the detector used to measure the signal produced by the analyte and is usually measured in millivolts per unit of concentration (mV/µg/mL).
Retention time: This is the time that it takes for an analyte to pass through the column and be detected by the detector.
Mobile phase composition: This is the composition of the solvent mixture used as the mobile phase and can be adjusted to optimize separation performance.
System pressure: This is the pressure of the mobile phase as it flows through the HPLC system and can be used to optimize the flow rate and separation performance.
Gradient program: This is the programmed change in the mobile phase composition over time and can be used to optimize separation performance.
Calibrating these parameters is essential to ensure accurate and reliable HPLC analysis and can help to identify and correct any issues with the HPLC system.
HPLC Pump Calibration
HPLC pump calibration is the process of verifying and adjusting the accuracy of the flow rate delivered by the HPLC pump. Calibration is typically carried out by measuring the volume of liquid delivered by the pump over a set period of time and comparing this to the expected value based on the pump speed and stroke volume. The following steps are typically involved in HPLC pump calibration:
Prepare a reference standard solution of known concentration.
Connect the HPLC pump to a flow meter or balance capable of measuring the volume of liquid delivered by the pump.
Set the pump speed to a known value and measure the volume of liquid delivered over a set period of time.
Calculate the actual flow rate delivered by the pump and compare this to the expected flow rate based on the pump speed and stroke volume.
Adjust the pump as necessary to correct any discrepancies and repeat the calibration process until the desired level of accuracy is achieved.
Document the results of the calibration and the adjustments made to the pump for future reference.
HPLC pump calibration is an important part of ensuring the accuracy and reliability of HPLC analysis and is typically carried out on a regular basis as part of routine instrument maintenance.
HPLC Auto injector Calibration
HPLC auto-injector calibration is the process of verifying and adjusting the accuracy of the volume of liquid injected by the auto-injector. Calibration is typically carried out by injecting a reference standard solution of known concentration and measuring the actual injected volume using a suitable measuring device. The following steps are typically involved in HPLC auto-injector calibration:
Prepare a reference standard solution of known concentration.
Connect the HPLC auto-injector to a suitable measuring device such as a balance or micropipette capable of measuring the volume of liquid injected by the injector.
Load the reference standard solution into the auto-injector and set the injector to the desired injection volume.
Inject the reference standard solution and measure the actual volume of liquid injected by the injector using the measuring device.
Calculate the actual injection volume delivered by the injector and compare this to the expected injection volume based on the injector settings.
Adjust the auto-injector as necessary to correct any discrepancies and repeat the calibration process until the desired level of accuracy is achieved.
Document the results of the calibration and the adjustments made to the auto-injector for future reference.
HPLC auto-injector calibration is an important part of ensuring the accuracy and reliability of HPLC analysis and is typically carried out on a regular basis as part of routine instrument maintenance.
HPLC UV Detector Calibration
HPLC UV detector calibration is the process of verifying and adjusting the accuracy of the detector’s response to ultraviolet light of a specific wavelength. Calibration is typically carried out by injecting a reference standard solution containing an analyte that absorbs UV light at a specific wavelength and measuring the detector response. The following steps are typically involved in HPLC UV detector calibration:
Prepare a reference standard solution of known concentration that contains an analyte that absorbs UV light at a specific wavelength.
Set the detector wavelength to the same wavelength as the analyte of interest.
Inject the reference standard solution and measure the detector response using a suitable measuring device such as a photodiode array detector.
Calculate the actual detector response and compare this to the expected response based on the known concentration of the analyte.
Adjust the UV detector as necessary to correct any discrepancies and repeat the calibration process until the desired level of accuracy is achieved.
Document the results of the calibration and the adjustments made to the detector for future reference.
HPLC UV detector calibration is an important part of ensuring the accuracy and reliability of HPLC analysis and is typically carried out on a regular basis as part of routine instrument maintenance.
HPLC RI Detector Calibration
HPLC refractive index (RI) detector calibration is the process of verifying and adjusting the sensitivity and baseline of the detector. Calibration is typically carried out by injecting a reference standard solution of a known refractive index and measuring the detector response. The following steps are typically involved in HPLC RI detector calibration:
Prepare a reference standard solution of known refractive index.
Inject the reference standard solution and measure the detector response using a suitable measuring device such as a refractometer or refractive index detector.
Calculate the actual detector response and compare this to the expected response based on the known refractive index of the solution.
Adjust the RI detector as necessary to correct any discrepancies and repeat the calibration process until the desired level of accuracy is achieved.
Document the results of the calibration and the adjustments made to the detector for future reference.
HPLC RI detector calibration is an important part of ensuring the accuracy and reliability of HPLC analysis, particularly for samples that do not have significant UV absorption. Calibration is typically carried out on a regular basis as part of routine instrument maintenance.
HPLC Temperature Calibration
HPLC temperature calibration is the process of verifying and adjusting the accuracy of the column temperature in the HPLC system. Calibration is typically carried out by measuring the temperature of the column using a temperature sensor or probe and comparing this to the expected temperature based on the set temperature of the HPLC system. The following steps are typically involved in HPLC temperature calibration:
Connect a temperature sensor or probe to the HPLC column.
Set the HPLC system to the desired temperature.
Allow the column to reach thermal equilibrium at the set temperature.
Measure the actual temperature of the column using the temperature sensor or probe.
Compare the measured temperature to the expected temperature based on the set temperature of the HPLC system.
Adjust the HPLC system as necessary to correct any discrepancies and repeat the calibration process until the desired level of accuracy is achieved.
Document the results of the calibration and the adjustments made to the HPLC system for future reference.
HPLC temperature calibration is an important part of ensuring the accuracy and reliability of HPLC analysis, particularly for temperature-sensitive compounds. Calibration is typically carried out on a regular basis as part of routine instrument maintenance.
HPLC Software verification
HPLC software verification is the process of ensuring that the software used to control and process data from the HPLC system is operating correctly and in compliance with applicable regulatory standards. This includes verifying that the software has been installed correctly, that it is functioning as intended, and that it is generating accurate and reliable data. The following steps are typically involved in HPLC software verification:
Verify that the software has been installed correctly and that all required components are present.
Check that the software is configured correctly, including instrument settings, method parameters, and data processing parameters.
Perform a system suitability test using a standard sample to verify that the software is generating accurate and reliable data.
Verify that the software is compliant with applicable regulatory standards, such as 21 CFR Part 11, if required.
Document the results of the software verification process and any issues that were identified.
HPLC software verification is an important part of ensuring the accuracy and reliability of HPLC analysis and is typically carried out on a regular basis as part of routine instrument maintenance. It is also typically required as part of the validation process for new or modified HPLC systems.
Never-before Pharma Articles and Pharmaceutical Guidelines only at PharmaeliX HPLC Lamp Energy Test Best Guidance 2023
HPLC Calibration Frequency
The frequency for HPLC calibration is usually every 6 Months (Half-yearly). However, the HPLC calibration frequency depends on the pharma company’s quality policy. The short frequency is always suggestible (3 Months/Quarterly Calibration).
HPLC Calibration Reference
For more information, refer to HPLC Calibration as per USP & HPLC Calibration as per EDQM.
Stay tuned here for the latest “HPLC Calibration Interview Questions” and much more. XRD Method Development Guidance 2023, Molar Extinction Coefficient Latest Guidance, Key Functions of Quality Control Department, Introducing HPLC Detectors Guidance
Trackbacks/Pingbacks