UV-Vis Frequently Asked Questions - Cuvettes
What is a typical sample?
The majority of samples employed in UV/Vis spectroscopy are solutions placed into a cuvette for measurement. Cuvettes are small rectangular glass or quartz containers. They are often designed so that the light beam travels a distance of 1 cm through the contents, but the path length can vary from 1 or 2 mm all the way up to 10 cm. The sample cell contains a solution of the substance you are testing, usually very dilute. Long path cuvettes are normally used to measure very dilute solutions. Beer’s Law tells us that the longer the path length of sample, the greater the sensitivity of analysis.
The solvent is chosen so that it doesn’t absorb any significant amount of light in the wavelength range of interest. Most cuvettes are designed to fit into a 1 cm square holder in the instrument.
In high performance UV/Vis/NIR instruments it is common to measure other non-solution sample types. With the proper diffuse reflectance accessory, such as an integrating sphere, solid samples, both transparent and opaque, can be measured. With specular reflection accessories samples such as mirrors and optical coatings can be measured.
How should cuvettes be cleaned?
This is a common question without a fixed answer. In fact, it is hard to find any specific published methods. Why are there no specified methods?
The reason is because of the diverse variety of samples that are analyzed. Due to the wide variety of analytical samples, a wide variety of corresponding cleaning methods are required. However, since describing all methods is not realistic, it does not help solve the problem. Therefore, a few of the common cleaning methods are described below. These methods can be roughly categorized into few groups. One group uses water as the solvent and the other uses organic solvent.
1) Using Water as a Solvent - After cleaning with purified water, clean with ethanol and store dry. However, for more severe contamination, soak cells in a commercial cleaning solution made specifically for cleaning cells (for about 10 minutes at 30 to 50 °C). Then clean the cell with distilled water and soak them in a dilute solution of nitric acid and a small amount of hydrogen peroxide (for about 30 minutes). Finally, rinse the cell with distilled water and store cells dry.
2) Using Organic Solvents - After cleaning with the organic solvent being used, clean with ethanol or acetone and then clean using the same method as described for the aqueous solution above.
3) For stubborn contamination - the cell may be scrubbed lightly with a cotton swab. Avoid using alkaline cleaning solutions that can dissolve glass or ultrasonic cleaning devices that can damage the cell.
What is meant by cell or solvent cut-off?
The material of the cell must have no absorption at the measurement wavelength. Two materials often used for cells are glass and quartz. Polystyrene (PS) and polymethylmethacrylate (PMMA) are mainly used for disposable cells. The indicates the wavelengths for which the different types of cells can be used. In this way, the measurable wavelength range varies
according to the cell material. The figure shows the transmission spectra obtained for different types of cells using air as the object of measurement. It can be seen that there is no absorption by the cells in the measurement wavelength ranges given in the table.
In addition to measurement wavelength, there is also the issue of chemical resistance. Except for use with strongly alkaline solutions, glass and quartz have extremely good chemical resistance. With resin cells, however, the chemical resistance varies with the material, so care is required when selecting the cell used for measurement. Another point to be noted is that there are small differences in the optical path lengths of individual disposable cells, which are generally discarded after use. This may lead to errors in quantitative values.
As glass is opaque in the ultraviolet region, glass cells offer a measurement range of approximately 320 nm to 2500 nm, which is narrower than quartz cells. At just several hundred dollars each, however, they are a convenient and cheaper option than quartz cells if measurements are not required in the short wavelength region below about 320 nm. These cells are often marked "G".
To eliminate variations in the optical path length from cell to cell, a series of measurements is performed using the same cell and rinsing the cell between measurements when glass or quartz cells are used. Disposable cells are made of plastic and can be discarded after use. Unlike the glass and quartz cells described above, a different disposable cell is used for each sample. This makes it difficult to acquire accurate data because variations in the optical path length readily occur from cell to cell. Disposable cells are used when high measurement accuracy is not required. They are useful for the measurement of biological samples where it is difficult to wash the sample out of the cell. As disposable cells are made of plastic, they are unable to measure samples that attack plastics, such as organic solvents. They are also opaque in the ultraviolet and near-infrared regions; as a result, measurements are restricted to the visible light region.
What are “long path” cuvettes and why are they used? (sample too dilute)
Cells with an optical path length of 10 mm are used in many different types of solution analyses. If the sample concentration is low, however, it may not be possible to obtain enough absorbance with a cell of this size. Although concentrating such samples allows the use of 10-mm cells, concentration is difficult in cases where the sample vaporizes or undergoes a chemical change during the concentration process. In such cases, measurement using a "long-path cell" is effective. There are long-path cells with optical path lengths of 20 mm, 50 mm, and 100 mm. The absorbance increases in proportion to the optical path length of the cell. The figure here shows the results obtained by analyzing a 10-mg/L potassium permanganate solution with a 10-mm cell and a 100-mm cell. It can be seen that the absorbance of the 100-mm cell is ten times that of the 10-mm cell.
A well-known application of analysis using a long-path cell is the turbidity measurement of water. 50-mm and 100-mm cells are often used to analyze samples with a low turbidity.
What are “short path” cuvettes and why are they used? (sample too concentrated)
In the case of high-concentration samples, diluting the sample allows measurement with a 10-mm cell. There are samples, however, that cannot be diluted easily. For example, due to interaction with the solvent, diluting a sample may cause a change in the absorbance (i.e., a shift in the peak wavelengths).
In cases where the absorbance is high, and dilution is difficult, measurement using a "short-path cell" is effective. There are short-path cells with optical path lengths of 1 mm, 2 mm, and 5 mm. The figure shows the results obtained by analyzing toluene with a 1-mm cell and a 10-mm cell. It can be seen that there is much less absorption saturation (i.e., 0 % transmittance) with the 1-mm cell than there is with the 10-mm cell.
A well-known application of measurement using a short-path cell is solution analysis in the near-infrared region. If a 10-mm cell is used for measurement in the near-infrared region, saturation often occurs due to absorption by the solvent, making it impossible to ascertain the absorption of the sample. A short-path cell is used to prevent absorption saturation due to the solvent.
With low- and high-concentration samples, the optical path length of the cell is selected in accordance with the size of the absorbance or transmittance.