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Absorbance Repeatability with the Square One Cuvette Holder
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Cuvette-based absorbance measurements are only as good as the system used for the measurements. Baseline drift and other spectral changes attributed to varying ambient conditions can be addressed by taking frequent background and reference measurements. Cuvette-to-cuvette variability is overcome using matched cuvettes. Measurement acquisition parameters are set to provide the highest possible signal to noise ratio. So why does your baseline drop below zero when you switch out samples? Why are your absorbance values negative? Why is your standard curve so poorly correlated? The answers may exist in a component of your system you probably don’t even think about – your cuvette holder.
Accurate absorbance data requires a well-engineered cuvette holder that enables users to remove and replace cuvettes repeatably (Figure 1).
Figure 1: Even when best practices are observed, less than ideal equipment can affect experiment results. The Square One cuvette holder is designed to minimize errors in absorbance measurements than can result from poorly fitted cuvettes.
Even the most expensive, matched set of quartz cuvettes will not provide accurate data if the cuvettes are not placed consistently into the cuvette holder. Cuvettes should fit snugly in the holder, so they can be removed and replaced without causing a change in spectral data. Baselines that shift below zero and negative absorbance values are immediate warning signs of problems. These issues call the data into question and erode confidence in the results. How can you trust any of the data you just collected when these events happen?
You worked hard to create your samples and design your experiment. Don’t waste all that valuable time and effort on a cuvette holder that undermines your ability to make consistent, repeatable measurements. The results from even the most precisely prepared samples are only as accurate as the system used to measure them. Don’t let the cuvette holder sabotage your work. Use the Square One cuvette holder because great data comes from reliable equipment and thoughtful use.
Sometimes, cuvette holders simply don’t “feel” right. Have you ever placed your cuvette into the cuvette holder and questioned the wobbly fit of your cuvette? How repeatable is the spectral data when the cuvette isn’t held securely? Does your standard curve look as inaccurate as the curve in Figure 2?
Figure 2: Example of a standard curve with poor correlation between concentration and absorbance values. Are differences in absorbance due to protein concentration, errors in sample preparation, or issues with repeatable placement of cuvettes in the holder?
With Square One, cuvettes are held snugly in place without damaging the delicate cuvette optical surfaces. Inserting the cuvette into the cuvette holder provides the user with tactile feedback that the cuvette is secure.
Square One provides repeatable data with both quartz (Figure 3) and plastic disposable cuvettes (Figure 4). To test this, we performed absorbance by removing and replacing the cuvette in the cuvette holder 10 times. As demonstrated, the resulting spectra overlay with great agreement, whether quartz or plastic cuvettes are used.
Figure 3: Example of spectral repeatability with Square One when a quartz cuvette containing diluted green food dye is removed and replaced in the cuvette holder 10 times. The spectra overlay almost perfectly, with an average absorbance of 0.47 AU at 410 nm with standard deviation of just 0.0007 AU.
Figure 4: Example of spectral repeatability with Square One when a disposable cuvette containing a slightly more concentrated green food dye is removed and replaced in the cuvette holder 10 times. Even with disposable cuvettes, the spectra overlay almost perfectly, with an average absorbance of 0.47 AU at 410 nm with standard deviation of just 0.0008 AU.
The Square One cuvette holder accepts 1 cm pathlength cuvettes. Included with each holder are three collimating lenses with fiber optic couplings, an integrated mirror to improve sensitivity in fluorescence measurements, a fully integrated cover to block ambient light, and two filter holders that accept both 12.5 and 25 mm diameter filters. When used with Ocean Insight spectrometers and light sources, Square One is ideal for absorbance, transmission, fluorescence, scattering or any combination of these optical phenomena.
With optimized equipment in place including the Square One cuvette holder, you can turn your attention to other criteria related to accurate absorbance measurements. Here are some tips: