UHPLC · Calibration procedure
Injector precision
Checks that the UHPLC autosampler injects the same small volume every time, using replicate injections of a caffeine solution.
PURPOSE
Why this is checked
UHPLC injections are small (often 1–2 µL), so any air bubble, worn seal or partial blockage gives a large relative error. Injector precision underpins every quantitative result and system suitability %RSD.
Frequency
Typically every 6 months, and after major repair or relocation, as defined in your calibration SOP.
Learn how the UHPLC worksYOU WILL NEED
Standards & tools
- Caffeine reference solution (e.g. about 20–50 µg/mL in water)
- Short UHPLC C18 column (e.g. 50 × 2.1 mm, sub-2 µm)
- HPLC-grade water and methanol
BEFORE YOU START
Prerequisites
- Equilibrate with a suitable mobile phase (e.g. water : methanol 80 : 20) until the baseline is stable
- Set the detector to 273 nm and a data rate suited to the narrow peak
- Prime the needle wash and make sure the sample syringe is free of air
PROCEDURE
Step by step
- 1Inject the caffeine solution six times at the routine volume (e.g. 1 µL).
- 2Integrate each peak with the same processing method.
- 3Calculate the mean, standard deviation and %RSD of the peak areas.
- 4If your SOP requires, repeat at a second volume (e.g. 5 µL) and inject a blank after the highest standard to check carry-over.
RECORD
Example observation table
| Injection | Volume (µL) | Peak area |
|---|---|---|
| 1 | 1.0 | 285,412 |
| 2 | 1.0 | 286,031 |
| 3 | 1.0 | 284,977 |
| 4 | 1.0 | 285,760 |
| 5 | 1.0 | 286,288 |
| 6 | 1.0 | 285,104 |
CALCULATION
How to calculate the result
%RSD = (Standard deviation ÷ Mean) × 100
- Mean area = 285,595
- Standard deviation = 520
- %RSD = 520 ÷ 285,595 × 100 = 0.18%
ACCEPTANCE CRITERIA
Pass if
- %RSD of peak area typically not more than 1.0% (n = 6) at the routine volume; follow the manufacturer's specification for very small volumes and your SOP.
IF IT FAILS
What to do
- Check the syringe, needle, needle seat and rotor seal for wear or blockage.
- Purge the syringe and wash lines to remove air.
- Service the autosampler and repeat the test.
- Assess the impact on results since the last passing calibration.
MORE
Other UHPLC calibrations
In LimsPulse · Calibration Management
LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.