Choosing the Suitable Volume for Measuring You Samples
A spectrophotometer is ideal for determining how much a material absorbs or transmits a certain wavelength of light, resulting in concentration and purity information. In a normal research lab, a spectrophotometer includes a standard-sized chamber in which a cuvette is inserted, allowing a certain wavelength of light to travel a set distance through the sample, known as the path length. When choosing the right cuvette for your samples and applications, consider the sample type, volume needs, concentration levels, and measurements to be taken.
When choosing a cuvette, there are three choices to consider:
- A macro cuvette necessitates a measuring capacity of 3.5 to 35 ml.
- A conventional cuvette has a measuring capacity of 3.5 mL.
- A microvolume adapter requires samples that are in the single microliter range, usually between 2ul to 3.5mL.
It’s also essential to ensure the cuvette you choose is transparent to the wavelengths you’ll be using to test your samples. Although glass and polystyrene are generally transparent to visible light, they absorb UV light. Glass and polystyrene are therefore appropriate for colorimetric protein assays or detecting the density of a bacterial culture, but not for detecting the concentration and purity of nucleic acids in the UV range at 230, 260, and 280 nm. Quartz and other UV-compatible polymers, on the other hand, are usually transparent in both visible and ultraviolet light, making them ideal for measuring UV-light samples.
Both a cuvette and a microvolume adaptor have benefits and disadvantages. Here are some ideas to help you choose the right vessel for measuring your samples accurately and consistently.
Depending on the instrument, spectrophotometers take measurements at one of three optical-beam light heights (the distance from the bottom of the cuvette, also know as Z dimension or light height): 8.5, 15 or 20 mm. Most spectrophotometers will accept macro cuvettes since they have a straight wall. Micro cuvettes, on the other hand, have the same outer footprint as standard cuvettes, but their interiors are generally tapered to limit the quantity of sample material necessary. The standard cuvette, on the other hand, is intended to measure through the sample at a particular light height in the chamber. It’s critical to double-check that the cuvette Z dimension you use to measure your sample is compatible with your instrument’s light height. Consider the following key characteristics of cuvettes:
- When measuring materials with low concentrations, such as RNA, single-stranded DNA, and oligonucleotides, you’ll need a long enough path length to keep the values within the instrument’s linear measurement range. Although some cuvettes now on the market allow an alternate path length, the standard size is 10 mm. Some dual path length cuvettes may be turned 90 degrees for a reading at a another path length.
- It’s recommended to use a sterile, disposable plastic cuvette if you want to retrieve your sample after a measurement. Although bulk cuvettes are available, some applications may need individually wrapped, sterile cuvettes that are certified protein-free or nuclease-free.
- The absorbance of a sample at a wavelength of 600 nm, usually referred to as the “OD600,” is used to estimate the concentration of a bacterial culture. A macro cuvette’s greater sample capacity allows heterogeneous samples like these to produce a more accurate readout.
A standard spectrophotometer may hold a variety of cuvettes, as well as a microvolume adaptor. The sample type, volume utilized, path length, and transparency of the cuvette material all impact the cuvette volume used, giving scientists the freedom to execute their experiments consistently and correctly. So go over the choices we discussed, select the wavelength and path length, and start measuring!