Eppendorf North America
Product Manager, PCR, Detection, Cell Technology and Cell Imaging Consumables
A spectrophotometer is perfectly suited to measure how much a substance of interest absorbs or transmits a specific wavelength of light, thus yielding information on concentration and purity. The spectrophotometer in a typical research lab has a standard-sized chamber in which a cuvette is placed, allowing a certain wavelength of light to traverse a defined distance through the sample, called the path length. The sample type, volume requirements, concentration levels and measurements being made should influence your decision when selecting the appropriate cuvette for your samples and applications.
Here are three options to consider when selecting a cuvette:
- A macro cuvette requires a measurement volume of more than 1.5 ml to 2 ml.
- A standard cuvette typically requires a measurement volume between 50 ml and 2 ml.
- A microvolume adapter requires samples in a single microliter range, typically between 2 ml and 10 ml.
It’s also important to make sure the cuvette selected is transparent to the wavelengths that will be used to measure the samples of interest. Glass and polystyrene are typically transparent to visible light but absorb in the UV spectrum. Therefore, glass and polystyrene are suitable for colorimetric protein assays or measuring the density of a bacterial culture, but they cannot be used for determining the concentration and purity of nucleic acids in the UV range at 230, 260 and 280 nm. Quartz and other UV-compatible plastics, on the other hand, are generally transparent in both the visible light and UV range and are appropriate choices when measuring samples in the UV-light spectrum.
There are advantages and disadvantages to both a cuvette and a microvolume adapter. Here are some tips to guide you on selecting the appropriate vessel for accurately and reliably measuring your samples.
Cuvettes
Spectrophotometers take measurements at one of two standard optical-beam path heights (the distance from the bottom of the cuvette)—either 8.5 or 15 mm—depending on the instrument. Macro cuvettes are generally straight walled and will work in most spectrophotometers. On the other hand, standard cuvettes have the same exterior footprint, but their interior is typically tapered to restrict the amount of sample material required. Regardless, the standard cuvette is designed to measure through the sample at a specific path height in the chamber. It is essential to confirm that the cuvette you select to measure your sample with will work with the path height of your instrument. Here are some important features of cuvettes to consider:
- When measuring samples that are normally found at low concentrations—such as RNA, single-stranded DNA and oligonucleotides, for example—you want a long enough path length for the readings to be within the linear measuring range of the instrument. The standard requirement is 10 mm, although some cuvettes currently on the market offer an optional path length. The Eppendorf UVette®, for example, can be rotated 90 degrees to allow for a reading at a 2 mm path length.
- If you plan to recover your sample after a measurement, it’s best to use a sterile, disposable plastic cuvette. Cuvettes can be purchased in bulk, but certain applications may demand individually wrapped, sterile cuvettes that are certified protein-free or nuclease-free.
- The concentration of a bacterial culture is typically determined by measuring the absorbance of a sample at a wavelength of 600 nm, commonly called the “OD600.” The larger sample volume of a macro cuvette allows for heterogeneous samples such as this to provide a more reliable reading.
Microvolume adapters
A microvolume adapter is a cuvette-shaped measuring cell designed to fit into a cuvette chamber, and it requires a minimal sample volume—as little as 1 µl and as much as 10 µl, depending on the adapter. The sample is sandwiched between two optically clear quartz plates held a short, fixed distance apart. In the case of the Eppendorf µCuvette G1.0, the fixed distance is 1.0 mm, which allows for a short path length through the sample. Besides the obvious feature of using smaller amounts of sometimes precious material, microvolume adapters are also reusable; they require only a wipe with lint-free tissue to clean the adapter before reading the next sample. Consider these additional features of microvolume adapters:
- It’s simpler and quicker to measure a small volume across a short path length than to carry out multiple steps to dilute a sample. Also, because there are fewer pipetting steps, the readings are likely to be more accurate.
- Microvolume adapters have a shorter path length than a cuvette. Readings with a microvolume adapter are more reliable and accurate when testing highly concentrated nucleic acids and/or proteins vs. more dilute sample preparations.
- Microvolume adapters are reusable, eliminating the need to continually replenish your supply of consumable cuvettes. It is possible to recover precious undiluted samples for certain downstream applications, such as those in which sterility is not required.
A standard spectrophotometer can accommodate a variety of different cuvettes and even a microvolume adapter. The selection of the appropriate cuvette or microvolume adapter is influenced by the sample type, volume used, path length and transparency of the cuvette material, providing scientists with the flexibility to reliably and accurately perform their experiments. So review the options we have covered, and then set the wavelength and path length and start measuring away!
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