In a spectrophotometer, stray light is light that passes by the sample and falls directly on the detector. This can lead to incorrect measurement results. Stray light may be caused by scattering or diffraction, by poor optical alignment, the use of incorrect or damaged cuvettes, incorrectly fitted sampling accessories or damaged seals around a light-tight sample chamber.
Stray light is problematic, as it reduces the range of measurable absorbance and impairs the linearity between concentration and absorbance. Cut-off filters (filters with a strictly defined spectrum) are required to check the device for stray light.
The following liquid filters are due to their very sharp spectrum excellent for qualifying the stray light of the spectrophotometer according to the regulations of the pharmacopoeias. The procedure is the same for all stray light filters.
In the European Pharmacopoeia (chapter 2.2.25) the checking of stray light is described as follows:
Stray light is determined at an appropriate wavelength using suitable filters or solutions.
The acceptance criterion depends on the filters used, for example:
|Filter / Solution||Absorbance at wavelength|
|Potassium chloride (12 g/L)||≥ 2,0 Abs at198 nm|
|Sodium iodide (10 g/L)||≥ 3,0 Abs at 220 nm|
|Potassium iodide (10 g/L)||≥ 3,0 Abs at 250 nm|
|Sodium nitrite (50 g/L)||≥ 3,0 Abs at 340 and 370 nm|
When checking for stray light, the stray light filter used is measured against the reference filter filled with pure water. The measured absorbance value at the recommended wavelength must meet the respective acceptance criterion.
Hellma Analytics stray light filters do not allow light to pass through them below a certain wavelength (cut-off wavelength). Any transmittance values displayed in the cut-off wavelength range therefore represent stray light. According to the Ph. Eur method, the measurement is made against the reference filter which is filled with water.
In USP <857> (update Dec. 2022), two possible procedures are described for checking for stray light:
Here the stray light filter with a path length of 10 mm is measured against the reference filter (filled with the same solution) with a path length of 5 mm. The stray light value can now be calculated from the absorbance maximum obtained, using the following formula:
Sλ = 0.25 x 10-2∆A
The following acceptance criteria apply:
∆A ≥ 0.7 Abs and Sλ ≤ 0.01
∆A = observed maximum absorbance
Sλ= stray light value calculated at wavelength λ
The measurement indicated on the calibration certificate for the wavelength at the peak maximum refers only to the measurement taken with the UV-Vis-NIR spectrophotometer shown on the calibration certificate. This wavelength is instrument-dependent due to the different optical components installed and the resulting differences in performance, it is not applicable to other UV-Vis-NIR spectrophotometers. The indicated measurement of the wavelength at the peak maximum is not suitable for checking the wavelength scale.
The analysts can measure the stray light filter with a path length of 10 mm against the reference filter filled with pure water (10 mm path length), with the exception of acetone, which is measured against air. The measured absorbance value per filter should be greater than 2.0 at the recommended wavelength listed in the table:
|Filter / Solution |
used for Procedure A or B
|Spectral Range||Recommended Wavelength |
for Procedure B
|Potassium chloride (12 g/L)||190 - 210 nm||198 nm|
|Sodium iodide (10 g/L)||210 - 270 nm||220 nm|
|Potassium iodide (10 g/L)||210 - 270 nm||220 nm|
|Acetone||250 - 330 nm||300 nm|
|Sodium nitrite (50 g/L)||300 - 400 nm||340 nm|