UHPLC · Calibration procedure
Detector wavelength accuracy
Confirms the UHPLC UV / PDA detector reads the correct wavelength, using the built-in holmium oxide filter or a caffeine solution.
PURPOSE
Why this is checked
A wavelength error reduces sensitivity and changes peak areas, especially for methods that measure on the slope of an absorption band. PDA peak-purity checks also depend on an accurate wavelength scale.
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
- Detector's built-in holmium oxide filter (bands near 361, 418 and 536 nm; use the reference values from the filter certificate or manufacturer)
- or caffeine solution in water (maxima near 205 and 273 nm)
BEFORE YOU START
Prerequisites
- Warm up the lamp fully (often 30–60 minutes)
- Flush the flow cell with water and make sure it is free of bubbles
PROCEDURE
Step by step
- 1Run the detector's built-in wavelength verification with the holmium oxide filter, or fill the cell with caffeine solution.
- 2Record the wavelength of maximum absorbance for each band.
- 3Compare each observed maximum with the reference value.
- 4Print or save the detector's verification report with the raw data.
RECORD
Example observation table
| Reference (nm) | Observed (nm) | Difference (nm) |
|---|---|---|
| 360.8 (Ho) | 361.1 | +0.3 |
| 418.5 (Ho) | 418.2 | −0.3 |
| 536.4 (Ho) | 536.9 | +0.5 |
| 273 (caffeine) | 273 | 0 |
CALCULATION
How to calculate the result
Difference = Observed maximum − Reference maximum
- Reference 536.4 nm, observed 536.9 nm
- Difference = 536.9 − 536.4 = +0.5 nm (within ±1 nm)
ACCEPTANCE CRITERIA
Pass if
- Each observed maximum typically within ±1 nm (PDA) or ±2 nm (variable-wavelength detector) of the reference value, as per your SOP and the manufacturer's specification.
IF IT FAILS
What to do
- Check lamp energy and hours; replace the lamp if low.
- Run the manufacturer's wavelength recalibration and repeat the check.
- Label the detector out of calibration until it passes.
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.