Dr Pramod Kumar Pandey, BSc (Hons), MSc, PhD, is the Founder of PharmaGuru.co and an Analytical Research Expert with 31+ years of experience in the pharmaceutical industry. He is recognized for driving analytical innovation, research excellence, and regulatory-compliant quality advancements across leading Indian and global pharmaceutical companies. He can be contacted at admin@pharmaguru.co
Learn UPLC calibration step-by-step procedures, key parameters, frequency, delay volume, detector and pump calibration, quaternary gradient testing, and FAQs.
How to Perform UPLC Calibration: Parameters, Frequency, Procedure, Case Study & 11 FAQs
UFLC VsUPLC Vs HPLC
UPLC Calibration Overview
UPLC calibration is a critical quality activity that ensures the accuracy, precision, linearity, and reliability of analytical results. Calibration verifies that all critical components—such as pump, injector, detector, autosampler, gradient system, column oven, and system pressure—are functioning within predefined acceptance criteria. Regular calibration ensures consistent performance, reproducible retention times, and regulatory compliance for pharmaceutical and analytical laboratories.
Inject 1 µl of 20 µg/ml Uracil from 5 vial positions.
Acceptance: RSD ≤ 1%
5. Gradient Calibration (Quaternary System)
Disconnect the UPLC column and connect the union in place of the column. Flush all the channels first with water and then with methanol. Put the channel A1 in solvent B and channel B1 in solvent A. Perform the gradient calibration as described in the gradient table:
Replace the column with a union. Flush channels with water, then methanol.
Set channels as follows: A1 → Solvent B, B1 → Solvent A.
Inject methanol and record chromatogram (two peaks expected).
Solvent A: = 5.6 mg/litre of Propyl paraben in methanol
Solvent B = Methanol
Flow rate: = 0.5ml/minute
Wavelength = 254 nm
Run time: 3 minutes
Time
Flow (ml/minute)
%A1
%B1
Curve
0
2.0
100
0
–
0.5
2.0
50
50
11
1
2.0
100
0
11
1.75
2.0
50
50
11
2,25
2.0
100
0
11
3
2.0
100
0
11
Gradient table
Inject µl of methanol and record the chromatogram. There should be only two peaks.
Delay Volume Calculation:
Delay volume = Flow rate x (Start time of the first peak – Flow rate)
Acceptance: ≤ 0.1 ml
6. Column Oven Calibration
Set three temperatures (e.g., 30°C, 50°C, 70°C).
Measure actual using a calibrated thermometer. Acceptance: ±2°C
7. System Pressure Check
Measure baseline pressure at specified flow rate with mobile phase.
Acceptance: Within manufacturer’s recommended range
Case Study: UPLC Carryover Detection
A lab detected unexpected peaks in blank injections after high-concentration Uracil injections.
Corrective action: Increased wash cycles and adjusted wash solvent composition.
Result: Carryover reduced from 0.025% to 0.003% (acceptance ≤0.01%).
Expert Tips:
Ensure degassing of solvents to avoid pump cavitation.
Validate autosampler needle wash to prevent cross-contamination.
Include routine drift/noise checks for UV/PDA detectors.
Document all calibration data for GMP compliance.
Frequently Asked Questions (FAQs) on UPLC Calibration
How many parameters are performed in UPLC calibration?
Six parameters are performed in UPLC calibration: Flow rate accuracy, Injector precision calibration, Wavelength accuracy, Detector linearity test, Vial location calibration and Gradient calibration.
Which chemical is used in UPLC calibration?
Uracil and Propyl paraben are used in UPLC calibration
How often should UPLC calibration be performed?
UPLC calibration should be performed every 6 months ± 5 days or immediately after any major system service, pump replacement, column change, or method modification. Intermediate checks may also be required based on instrument usage or regulatory requirements.
What is the acceptable flow rate deviation for pump calibration?
The measured flow rate should be within ±2% of the set flow rate. Any deviation beyond this limit requires corrective action, such as pump maintenance or recalibration.
How is injector precision evaluated?
Injector precision is assessed by injecting the same standard solution multiple times (typically 6 injections) and recording the peak area. The %RSD of the area responses should be ≤1% to confirm injector precision.
What standards are required for UPLC calibration?
Common standards include:
Uracil standard – for UV/PDA detector calibration and wavelength accuracy
Propyl paraben standard – for gradient calibration and delay volume determination All standards must have a valid Certificate of Analysis (COA).
How is detector wavelength accuracy checked?
Wavelength accuracy is checked by injecting Uracil solution across a range of wavelengths (e.g., 250–268 nm) and confirming the absorbance maxima. For UV/PDA detectors, the maxima should be 258 nm ± 2 nm.
What is delay volume, and why is it important?
Delay volume is the time or volume required for the mobile phase to travel from the pump to the detector. It is critical for accurate gradient formation, retention time reproducibility, and peak identification, especially in quaternary systems. Acceptance: ≤0.1 ml.
How do you perform gradient calibration for a quaternary system?
Confirm peak formation, start time, and gradient delivery as per the gradient table. Acceptance: Peaks should form correctly, and delay volumes must be within limits.
How is autosampler vial position calibration done?
Answer: Inject a standard solution from vials placed in different positions of the autosampler. Measure the peak area for each vial. The RSD of the area response should be ≤1%, confirming consistent injection from all vial positions.
What are typical acceptance criteria for detector noise and drift?
Noise: ≤60 µAU for UV detectors; ≤80 µAU for PDA detectors
Drift: ≤10 mAU/hr for UV detectors; ≤10 mAU/hr for PDA detectors These parameters ensure baseline stability and accurate quantification.
How to troubleshoot carryover in UPLC?
Carryover occurs when the analyte remains in the injection system after a run. Common remedies:
Increase needle wash cycles
Optimise wash solvent composition
Check autosampler and injector seals
Validate carryover with blank injections Acceptance: ≤0.01% of the previous sample peak area.
Why is column oven calibration important?
Column oven calibration ensures the set temperature matches the actual column temperature, which directly affects retention time, resolution, and reproducibility. Typically, the measured temperature should be ±2°C of the set value.
