5.4 KiB
| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| Ultraviolet–visible spectroscopy | 3/5 | https://en.wikipedia.org/wiki/Ultraviolet–visible_spectroscopy | reference | science, encyclopedia | 2026-05-05T10:06:41.378064+00:00 | kb-cron |
Stray light in a UV spectrophotometer is any light that reaches its detector that is not of the wavelength selected by the monochromator. This can be caused, for instance, by scattering of light within the instrument, or by reflections from optical surfaces. Stray light can cause significant errors in absorbance measurements, especially at high absorbances, because the stray light will be added to the signal detected by the detector, even though it is not part of the actually selected wavelength. The result is that the measured and reported absorbance will be lower than the actual absorbance of the sample. The stray light is an important factor, as it determines the purity of the light used for the analysis. The most important factor affecting it is the stray light level of the monochromator. Typically a detector used in a UV–VIS spectrophotometer is broadband; it responds to all the light that reaches it. If a significant amount of the light passed through the sample contains wavelengths that have much lower extinction coefficients than the nominal one, the instrument will report an incorrectly low absorbance. Any instrument will reach a point where an increase in sample concentration will not result in an increase in the reported absorbance, because the detector is simply responding to the stray light. In practice the concentration of the sample or the optical path length must be adjusted to place the unknown absorbance within a range that is valid for the instrument. Sometimes an empirical calibration function is developed, using known concentrations of the sample, to allow measurements into the region where the instrument is becoming non-linear. As a rough guide, an instrument with a single monochromator would typically have a stray light level corresponding to about 3 Absorbance Units (AU), which would make measurements above about 2 AU problematic. A more complex instrument with a double monochromator would have a stray light level corresponding to about 6 AU, which would therefore allow measuring a much wider absorbance range.
==== Deviations from the Beer–Lambert law ==== At sufficiently high concentrations, the absorption bands will saturate and show absorption flattening. The absorption peak appears to flatten because close to 100% of the light is already being absorbed. The concentration at which this occurs depends on the particular compound being measured. One test that can be used to test for this effect is to vary the path length of the measurement. In the Beer–Lambert law, varying concentration and path length has an equivalent effect—diluting a solution by a factor of 10 has the same effect as shortening the path length by a factor of 10. If cells of different path lengths are available, testing if this relationship holds true is one way to judge if absorption flattening is occurring. Solutions that are not homogeneous can show deviations from the Beer–Lambert law because of the phenomenon of absorption flattening. This can happen, for instance, where the absorbing substance is located within suspended particles. The deviations will be most noticeable under conditions of low concentration and high absorbance. The last reference describes a way to correct for this deviation. Some solutions, like copper(II) chloride in water, change visually at a certain concentration because of changed conditions around the colored ion (the divalent copper ion). For copper(II) chloride it means a shift from blue to green, which would mean that monochromatic measurements would deviate from the Beer–Lambert law.
==== Measurement uncertainty sources ==== The above factors contribute to the measurement uncertainty of the results obtained with UV–Vis spectrophotometry. If UV–Vis spectrophotometry is used in quantitative chemical analysis then the results are additionally affected by uncertainty sources arising from the nature of the compounds and/or solutions that are measured. These include spectral interferences caused by absorption band overlap, fading of the color of the absorbing species (caused by decomposition or reaction) and possible composition mismatch between the sample and the calibration solution.
== Ultraviolet–visible spectrophotometer ==
The instrument used in ultraviolet–visible spectroscopy is called a UV–Vis spectrophotometer. It measures the intensity of light after passing through a sample (
I
{\displaystyle I}
), and compares it to the intensity of light before it passes through the sample (
I
o
{\displaystyle I_{o}}
). The ratio
I
/
I
o
{\displaystyle I/I_{o}}
is called the transmittance, and is usually expressed as a percentage (%T). The absorbance,
A
{\displaystyle A}
, is based on the transmittance:
A
=
−
log
(
%
T
/
100
%
)
{\displaystyle A=-\log(\%T/100\%)}