UV-Vis · Calibration procedure
Stray light
Measures how much unwanted light (light outside the selected band) reaches the detector, using a solution that blocks essentially all light at the test wavelength, such as potassium chloride at 198 nm.
PURPOSE
Why this is checked
Stray light makes high absorbances read too low and bends calibration curves at the top end. It matters most at low UV wavelengths, where the lamp is weak and many assays are measured.
Frequency
Typically every 6 months (some labs quarterly), and after lamp replacement, major repair or relocation, as defined in your calibration SOP.
Learn how the UV-Vis worksYOU WILL NEED
Standards & tools
- Potassium chloride solution, 12 g/L in water (test near 198 nm)
- Sodium iodide solution, 10 g/L in water (test near 220 nm), if your SOP includes it
- Acetone or sodium nitrite cut-off solutions for longer wavelengths, if your SOP includes them
- Sealed certified cut-off cells may be used instead of freshly prepared solutions
BEFORE YOU START
Prerequisites
- Lamps fully warmed up; quartz cuvettes clean
- Water used for the blank is of high purity and freshly drawn
PROCEDURE
Step by step
- 1Fill one 10 mm quartz cuvette with water as the blank and zero the instrument at 198 nm.
- 2Fill a second cuvette with the potassium chloride solution.
- 3Measure the absorbance at 198 nm (or scan about 195–205 nm and take the reading at the specified wavelength).
- 4Repeat with any other cut-off solutions in your SOP at their test wavelengths.
- 5If your chapter uses the paired path-length approach (sample in a 10 mm cell measured against the same solution in a 5 mm cell), follow that procedure instead.
RECORD
Example observation table
| Solution | Wavelength (nm) | Absorbance | Equivalent stray light (%T) |
|---|---|---|---|
| KCl 12 g/L | 198 | 2.45 | 0.355 |
| NaI 10 g/L | 220 | 3.12 | 0.076 |
CALCULATION
How to calculate the result
Stray light (%T) ≈ 10^(−A) × 100, where A is the absorbance measured for the cut-off solution
- KCl 12 g/L at 198 nm reads A = 2.45
- Stray light ≈ 10^(−2.45) × 100 = 0.355 %T
- A ≥ 2.0 (stray light below 1 %T) passes the typical limit
ACCEPTANCE CRITERIA
Pass if
- Potassium chloride at 198 nm: absorbance typically not less than 2.0 against water in a 10 mm cell.
- Other cut-off solutions and the paired path-length approach: as per the current chapter and your SOP (limits differ between USP and Ph. Eur.).
IF IT FAILS
What to do
- Check the cuvettes, the water blank and the solution preparation; repeat once if an error is documented.
- Check the deuterium lamp energy, which falls with age and makes stray light worse in the low UV.
- Have the optics cleaned or the monochromator serviced; do not use the instrument for low-UV work until it passes.
MORE
Other UV-Vis 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.