Calibration of HPLC is essential for reliable quantitative analysis in pharmaceutical laboratories. This introduction covers HPLC calibration, calibration curves, system suitability, linearity, accuracy, precision, sensitivity, limit of detection (LOD) and limit of quantification (LOQ), retention time, resolution, and detector response. B.Pharm students will learn practical steps for standard preparation, instrument performance checks, use of internal and external standards, regression and weighting, and troubleshooting chromatographic issues. Emphasis is on method validation, routine verification, and regulatory expectations to ensure accurate drug assay results and data integrity in quality control. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the primary purpose of calibrating an HPLC system?
- To increase column lifetime
- To ensure accurate, precise quantitative measurements
- To reduce solvent consumption
- To sterilize the mobile phase
Correct Answer: To ensure accurate, precise quantitative measurements
Q2. Which of the following best describes a calibration curve in HPLC?
- A plot of retention time versus column length
- A plot of detector response versus analyte concentration
- A list of mobile phase compositions
- A plot of pump pressure versus flow rate
Correct Answer: A plot of detector response versus analyte concentration
Q3. Which parameter indicates the ability of two peaks to be separated in HPLC?
- Plate count (N)
- Retention factor (k’)
- Resolution (Rs)
- Tailing factor (Tf)
Correct Answer: Resolution (Rs)
Q4. What is the typical minimum number of calibration standards recommended for an HPLC calibration curve during validation?
- 1 standard
- 3 standards
- 5 to 6 standards
- 10 standards
Correct Answer: 5 to 6 standards
Q5. In calibration, what does linearity assess?
- The column packing quality
- The proportionality between response and concentration over a range
- The mobile phase pH stability
- The detector’s wavelength accuracy
Correct Answer: The proportionality between response and concentration over a range
Q6. Which statistical parameter from a regression line indicates goodness of fit for a calibration curve?
- Relative retention
- Correlation coefficient (r or r²)
- Column efficiency
- Tailing factor
Correct Answer: Correlation coefficient (r or r²)
Q7. What is the most common method to calculate the limit of detection (LOD) in HPLC?
- 3.3 × (standard deviation of response) / slope
- 10 × (standard deviation of response) / slope
- Area under blank peak
- Retention time variability
Correct Answer: 3.3 × (standard deviation of response) / slope
Q8. Which system suitability parameter is directly related to column efficiency?
- Resolution (Rs)
- Theoretical plates (N)
- Retention factor (k’)
- Signal-to-noise ratio
Correct Answer: Theoretical plates (N)
Q9. When using an internal standard for calibration, why is it used?
- To change the mobile phase polarity
- To correct for injection and sample preparation variability
- To flush the column between runs
- To increase column temperature
Correct Answer: To correct for injection and sample preparation variability
Q10. What is the effect of peak tailing on quantitative calibration?
- Improves slope of calibration curve
- Causes integration errors and inaccurate peak area measurement
- Reduces detector noise
- Decreases retention time uniformly
Correct Answer: Causes integration errors and inaccurate peak area measurement
Q11. Which weighting factor is commonly used in linear regression for HPLC calibration when heteroscedasticity is present?
- 1 (no weighting)
- 1/x or 1/x²
- log(x)
- sqrt(x)
Correct Answer: 1/x or 1/x²
Q12. What does %RSD of calibration standards assess?
- Peak shape only
- Precision of repeated measurements
- Column dead volume
- Detector wavelength drift
Correct Answer: Precision of repeated measurements
Q13. Which practice helps maintain calibration validity between full calibrations?
- Changing column tubing daily
- Running quality control (QC) samples and calibration verification standards
- Using different detectors for each run
- Increasing mobile phase pH by 3 units
Correct Answer: Running quality control (QC) samples and calibration verification standards
Q14. What is the purpose of a blank injection during calibration?
- To prime the pump
- To check for carryover and baseline contamination
- To equilibrate column temperature
- To calibrate the autosampler volume
Correct Answer: To check for carryover and baseline contamination
Q15. Which detector response behavior requires nonlinear calibration or transformation?
- Strict proportionality across all concentrations
- Detector saturation at high concentrations
- Stable baseline at low concentrations
- Constant retention time
Correct Answer: Detector saturation at high concentrations
Q16. How is limit of quantification (LOQ) commonly estimated?
- LOD × 0.5
- 10 × (standard deviation of response) / slope
- Retention time plus 2 minutes
- Using mobile phase conductivity
Correct Answer: 10 × (standard deviation of response) / slope
Q17. Which of the following affects detector response and thus calibration accuracy in UV-Vis HPLC detectors?
- Column particle size only
- Wavelength selection and lamp stability
- Injector needle length
- Ambient light in the lab
Correct Answer: Wavelength selection and lamp stability
Q18. During calibration, a non-zero intercept in the regression line may indicate:
- Perfect linearity
- Systematic baseline offset or blank contamination
- That slope equals zero
- Column void volume is zero
Correct Answer: Systematic baseline offset or blank contamination
Q19. What is the recommended action if a standard near the top of the calibration range shows nonlinearity?
- Exclude it and reduce the calibration range or dilute the sample
- Increase the column temperature
- Decrease the mobile phase flow rate
- Run the sample without standards
Correct Answer: Exclude it and reduce the calibration range or dilute the sample
Q20. Why is it important to prepare calibration standards in the same matrix as samples when possible?
- To change detector type
- To account for matrix effects and recovery differences
- To extend column life
- To ensure identical retention times
Correct Answer: To account for matrix effects and recovery differences
Q21. Which parameter is checked to assess peak symmetry during system suitability?
- Retention factor
- Tailing factor
- Mobile phase pH
- System pressure
Correct Answer: Tailing factor
Q22. What is a common cause of drift in calibration slope over time?
- Consistent mobile phase composition
- Detector lamp aging or fouling
- Stable column temperature
- Accurate standard weights
Correct Answer: Detector lamp aging or fouling
Q23. When validating an HPLC method, calibration must be demonstrated across:
- Only the lowest concentration
- The expected working concentration range of the assay
- Only the highest concentration
- Random concentrations unrelated to dosage
Correct Answer: The expected working concentration range of the assay
Q24. What is the impact of improper injection volume on calibration?
- No impact if column is preconditioned
- Introduces proportional errors in measured peak areas
- Only affects retention time
- Improves linearity automatically
Correct Answer: Introduces proportional errors in measured peak areas
Q25. Which quality metric is commonly plotted on control charts to monitor calibration stability?
- Mobile phase color
- Calibration standard response or QC % recovery
- Column serial number
- Date of last column wash
Correct Answer: Calibration standard response or QC % recovery
Q26. What role does method robustness testing play in HPLC calibration?
- To determine detector brand
- To assess how small deliberate changes affect calibration and quantitation
- To eliminate the need for standards
- To shorten run time only
Correct Answer: To assess how small deliberate changes affect calibration and quantitation
Q27. For gradient HPLC methods, calibration may require additional attention because:
- Detector cannot measure gradients
- Gradient reproducibility and equilibration can affect response and retention
- Columns are not compatible with gradients
- Solvent viscosity is irrelevant
Correct Answer: Gradient reproducibility and equilibration can affect response and retention
Q28. Which is the best practice for long-term calibration documentation?
- Keep only the most recent calibration report
- Maintain calibration records, trend analyses, and corrective actions
- Discard failed calibration records immediately
- Only note the calibration date without details
Correct Answer: Maintain calibration records, trend analyses, and corrective actions
Q29. Which factor is NOT typically part of system suitability criteria for calibration?
- Theoretical plates (N)
- Resolution (Rs)
- Injector needle brand
- Percent relative standard deviation (%RSD) for replicate injections
Correct Answer: Injector needle brand
Q30. What is the advantage of using a calibration verification (CCV) standard during routine runs?
- It cleans the column
- It confirms that calibration remains valid during sample analysis
- It increases mobile phase pH
- It doubles the run speed
Correct Answer: It confirms that calibration remains valid during sample analysis
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
Mail- Sachin@pharmacyfreak.com