Written by Yvette Mattley, PhD
Stray light limits the maximum absorbance level achievable with a given spectrometer. Once the stray light limit is reached, sample dilution or a smaller pathlength is required to measure more concentrated samples. In this application note, we show the impact of stray light on the maximum absorbance level and describe how optimizing the choice of light source can minimize stray light and increase the maximum absorbance measured.
The maximum absorbance level achievable with a given spectrometer is limited, in part, by stray light. Simply defined, stray light is undesired light of any wavelength that reaches the detector and includes light that reaches the detector from an unintended source. Stray light is caused by normal optical behaviors including scatter off the surface of the optical components or reflection from other surfaces in the spectrometer. Since the detector cannot distinguish light coming from the intended light path from an unintended source, stray light limits the maximum absorbance level that can be achieved by consuming a portion of the detector’s dynamic range.
Stray light manifests itself as an apparent deviation in the Beer-Lambert Law (relationship between absorbance and concentration). The effect is more obvious at higher analyte concentrations because the stray light component becomes a larger fraction of the total transmitted light. Stray light is observed in a plot of absorbance versus concentration as a flattening or roll-off in the spectrum as absorbance increases with higher analyte concentrations.
In this Application Note, we show the impact of stray light on the maximum absorbance level achievable with a spectrometer and describe how optimizing the light source can minimize stray light and increase the maximum absorbance measured.
Various concentrations of Salmon DNA (Sigma D-1626) were prepared in deionized water. DNA absorbance was measured in a 1 cm pathlength cuvette with an STS-UV and the deuterium lamp of a DH-2000-BAL balanced, deuterium and tungsten halogen light source. Measurements were made with both the deuterium and tungsten halogen lamps and with just the deuterium lamp to show the impact of out-of-band light on the maximum absorbance level achieved with the spectrometer.
The impact of the light source used for absorbance measurements is shown in the figure for DNA absorbance measured with the same spectrometer but different light sources. Using both the deuterium and tungsten halogen lamps increases the total stray light in the bench due to the addition of visible light outside of the region where DNA absorbs. This results in a lower maximum absorbance of ~1.7 versus ~2.1 for DNA measured with the same STS-UV and the DH-2000-BAL light source with only the deuterium lamp turned on. Stray light has the most profound impact to linearity. The linear range of the system drops from 1.2 AU when both lamps are used versus 1.6 AU when only the deuterium lamp is used.
As demonstrated by the dramatic impact on both maximum absorbance and absorbance linearity, simply eliminating the use of light outside the region of interest where the analytes absorb enables the measurement of more concentrated samples without the need for dilution. In the case of STS-UV, the careful choice of the light source for the measurement enables an incredible dynamic range of 0.005 AU to 2.0 AU with linear data up to about 1.6 AU in the UV region.
Stray light is always present in the total system used for absorbance measurements. It limits the maximum absorbance measurement that can be achieved, requiring sample dilution or smaller pathlength sampling cells with highly absorbing samples. As demonstrated by this data, one way to decrease stray light is to manage the light entering the spectrometer. Simply avoiding the use of light outside the wavelength range of interest lowers stray light in the spectrometer, enables a wider linear measurement range for a higher maximum absorbance measurement. While many of the typical causes of stray light are out of the user’s control, light source optimization is one option that has a significant impact on the absorbance measurements.