HPLC · Calibration procedure
Detector wavelength accuracy
Confirms the UV detector reads the correct wavelength, using a compound with known absorbance maxima or the built-in holmium oxide filter.
PURPOSE
Why this is checked
If the detector wavelength is off, sensitivity drops and results change, especially for methods measured on the slope of an absorbance band.
Frequency
Typically every 6 months, and after major repair or relocation, as defined in your calibration SOP.
Learn how the HPLC worksYOU WILL NEED
Standards & tools
- Caffeine solution in water (maxima near 205 nm and 273 nm)
- or the detector's built-in holmium oxide filter (lines near 361, 418 and 536 nm)
BEFORE YOU START
Prerequisites
- Allow the lamp to warm up fully
- Flush the flow cell with water
PROCEDURE
Step by step
- 1Fill the flow cell with the caffeine solution (or start the built-in holmium oxide test).
- 2Scan or step the wavelength across each maximum (e.g. 265–281 nm in 1 nm steps).
- 3Record the wavelength giving the highest absorbance for each maximum.
- 4Compare the observed maxima with the reference values.
RECORD
Example observation table
| Reference maximum (nm) | Observed maximum (nm) | Difference (nm) |
|---|---|---|
| 205 | 206 | +1 |
| 273 | 273 | 0 |
CALCULATION
How to calculate the result
Difference = Observed maximum − Reference maximum
- Reference 273 nm, observed 273 nm
- Difference = 0 nm (within ±2 nm)
ACCEPTANCE CRITERIA
Pass if
- Observed maxima within ±2 nm of the reference value (tighter limits may apply to PDA detectors).
IF IT FAILS
What to do
- Check lamp energy and age; replace the lamp if low.
- Recalibrate the wavelength using the manufacturer's procedure.
- Do not use the detector until it passes.
MORE
Other HPLC 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.