HPLC calibration questions and answers

1. Pump Calibration

Q1. What is the acceptance criterion for flow rate accuracy?
A1. The flow rate should be within ±2.0% of the set value.

Q2. How is flow rate accuracy measured in HPLC?
A2. By collecting HPLC grade water in a calibrated volumetric flask and recording the time to fill a known volume using a stopwatch. Flow rate = Volume (mL) / Time (min).

Q3. What is the acceptance criterion for flow rate consistency?
A3. The %RSD of retention time of caffeine should be NMT 1.0%.

Q4. How is flow rate consistency evaluated?
A4. By injecting 10 ppm caffeine solution six times and calculating the %RSD of the retention times.

Q5. What is meant by compositional accuracy?
A5. It refers to the accuracy of gradient composition delivery. Measured using 0.25% acetone in water and recording absorbance changes.

Q6. What is the delay volume limit for the pump system?
A6. The delay volume should be NMT 1.0 mL.


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2. Autosampler Calibration

Q7. What is the acceptable range for injection volume accuracy?
A7. The average volume for 10 injections should be 20 ± 0.4 µL.

Q8. How is injection volume accuracy determined?
A8. By weighing the HPLC vial before and after 10 injections and using the difference to calculate average injection volume.

Q9. What is the limit for injection volume precision?
A9. %RSD should not be more than 1.0%.

Q10. How is injection volume linearity evaluated?
A10. By injecting 5, 10, 20, 50, and 100 µL and plotting the peak area vs. volume. R-square should be NLT 0.9990.

Q11. What is the acceptance criterion for autosampler temperature?
A11. The measured temperature should be within ±2°C of the set temperature.


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3. Column Oven Calibration

Q12. What are the required set temperatures for column oven calibration?
A12. 60°C, 50°C, 30°C, 20°C, and 10°C.

Q13. What is the acceptance limit for column oven temperature accuracy?
A13. The measured temperature should be within ±2°C of the set temperature.

Q14. How is the temperature measured?
A14. Using a calibrated probe with a digital thermometer.


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4. Detector Calibration

Q15. How is wavelength accuracy evaluated?
A15. Using known UV maxima and minima, such as Maxima 273 ± 2 nm, Maxima 205 ± 2 nm, and Minima 245 ± 2 nm.

Q16. What is the acceptance criterion for detector linearity?
A16. R-square (correlation coefficient) should be NLT 0.9990.

Q17. What is the typical injection volume used for detector calibration?
A17. 10 µL.

Q18. What is the standard mobile phase composition for calibration?
A18. Methanol:Water (50:50 v/v).