Accurate high absorbance measurements in UV-Vis spectrophotometers
UV-Vis spectrophotometers provide quantitative measurements by comparing the intensity of a beam of light transmitted through the sample to the intensity of a reference beam without the sample in place. The higher the concentration of the sample, the more light it absorbs so high concentration measurements correspond to high absorbance values. However, at high absorbance, very little light actually reaches the instrument detector and it can be very challenging for many commercially available UV-Vis spectrophotometers to make accurate, linear measurement of these very low signal levels. If the measurement of very low light levels is not linear, then the instrument's high absorbance value measurements will not be accurate and cannot be used for reliable quantitative measurements. One approach is to dilute the sample before measurement so that the signal levels are larger, but for many applications, including many biological samples, high density printer inks, technical paints and solid samples such as optical components, sample dilution is not an option and accurate direct measurement of absorbance is essential.
Absorbance and Linearity
According to the Beer-Lambert law, absorbance (Abs) is a linear function of the concentration of the sample. although the spectrophotometers actually measure the transmittance of light. For high absorbance measurements where only very small quantities of light reach the detector any instrumental effects that influence these low level measurements can have significant implications on the linearity of measurements. While most spectrophotometers specify a measurable absorbance range between 0 - 4 Abs, the quality of the instrument optics determine the real performance at low light levels. The most important considerations are the contributions from stray light and noise.
Stray Light
Stray light is any light arriving at the detector from the monochromator which is not directly from the measurement beam, and this influences the measured transmitted light as shown here:
Mid range instruments would have typical stray light figures of around 0.05%T while upper range instruments would have stray light contributions of around 0.015%T in single monochromator versions. However, research grade instruments featuring two monochromators offer the best linear absorbance range with stray light as low as 0.00025%T.
The tables below show the influences of stray light on absorbance linearity, using the relationship above.
For mid range instruments, errors due to stray light in the 3 Abs range are significant and considerably higher at 4 Abs, meaning that quantitative measurements should be limited to 2 Abs and below. For the single monochromator instrument, the errors at 3 Abs are smaller, meaning that it could be used for quantitative measurements up to that point, but not up to 4 Abs. However, in the double monochromator version the errors are negligible right up to 4 Abs.
Noise
Signal-to-noise is critical in determining the sensitivity of the spectrophotometer. Peak to peak noise within the detection system can affect the measurement accuracy of very small signal levels, i.e.at high absorbance. The better the optical design of the system, the better the energy throughput, which in turn gives very low noise measurements. Noise is either quoted as a peak-to peak value or an RMS value. RMS has a smoothing effect on the peak-to peak values, giving apparently lower noise values. However it is the peak-to peak noise which directly influences low signal level measurements, so peak-to-peak noise is the value that should always be considered. Typical examples of noise values are 0.0003A peak-to-peak at zero absorbance, but for top of the range instruments, a noise measurement of 0.0001A peak-to-peak can be achieved at zero absorbance and only 0.0005A peak-to-peak at 2 absorbance.
High absorbance UV-Vis Spectrophotometers from Lambda
Lambda Advanced Technology is one of the industry-leading suppliers of UV-Vis spectrophotometers for biochemistry, life sciences, and production applications. We supply a world-class range of Hitachi instruments, from the entry-level U-5100 to the research-grade U4150. If you would like to learn more about the high absorbance capabilities of the spectrophotometers that we have available at Lambda, simply contact a member of the team today.