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A cuvette (French: cuvette = "little vessel") is a small, straight-sided tube-shaped container with a circular or square cross section. It is sealed at one end, and made of an optically-clear material such as plastic, glass, or fused quartz (for UV light). Cuvettes are designed to hold samples for spectroscopic experiments in which a beam of light is passed through the sample within the cuvette in order to measure the absorbance (also called optical density), transmittance, fluorescence intensity, fluorescence polarization (also called fluorescence anisotropy), or fluorescence lifetime of the substance in the cuvette. This measurement is performed using a spectrophotometer.
- 1 Overview
- 2 History
- 3 Types of Cuvette
- 4 Cuvette Calibration
- 5 Standard Technique
- 6 Additional images
- 7 See also
- 8 External links
- 9 References
Traditional ultraviolet–visible spectroscopy or fluorescence spectroscopy typically use samples that are liquid - either a liquid substance or a substance dissolved in a solvent. The liquid sample is placed in a cuvette, and the cuvette is then placed in a spectrophotometer for testing. The cuvette can be made of any material that is transparent in the range of wavelengths being tested. Historically, reusable quartz cuvettes were required for measurements in the ultraviolet range, since glass and most plastic absorb light in this wavelength range, creating interference. Now, disposable plastic cuvettes manufactured using specialized ultraviolet-transparent plastics are available. Glass, plastic (traditional or ultraviolet-transparent), and quartz cuvettes are all suitable for measurements made at higher wavelengths (e.g. in the visible spectrum).
The size of the cuvette determines how long the light path is able to travel through the sample. For many applications, a 1 cm cuvette is used. Cuvettes typically have two parallel sides that are transparent so that the spectrophotometer light can pass through, but some tests can be performed by reflection and therefore only need a single transparent side. For fluorescence, two more parallel sides, perpendicular to the ones used for the spectrophotometer light, are needed for the excitation light. Cuvettes are typically 10 mm thick, allowing light to easily pass through. The thickness and sizes of cuvette affects the calculation of absorbance value. Some cuvettes have a glass or plastic cap for use with hazardous solutions, but others do not.
In 1934, James Franklin Hyde created a combined silica cell, which was free from other extraneous elements, as a liquefying technique of other glass products. In the 1950s, Starna Ltd. idealized the method to completely melt an optical segment of glass by using heat without deformation of its shape. This innovation has altered the production of inert cuvettes without any thermosetting resin. Before the rectangular cuvette was created, researchers used standard test tubes for laboratory work. As innovation motivated changes in technique, cuvettes were constructed to have focal points over ordinary test tubes.
Types of Cuvette
Different UV ranges can be analyzed using different types of cuvette. The smallest units are capable of holding 70 µL, while the largest can test samples of 2.5 mL or more. Some cuvettes will be clear only on opposite sides, so that a single beam of light can pass through them. Cuvettes used in fluorescence spectroscopy must be clear on all four sides, as fluorescence is measured at a right-angle to the beam path to limit contributions from the beam itself. Some cuvettes, known as tandem cuvettes, have a glass barrier medium that extends two-thirds of the way up in the middle of the container, so that measurements can be taken with two solutions separated, and again when they are mixed. Typically, cuvettes are 10 mm (0.39 in) in width, to allow for easy calculations of coefficients of absorption. To measure the sample, the transparent side must be placed toward the light in the spectrophotometer. For accurate measurement, these testing tubes should be clean and scratch-free. Cuvettes to be used in circular dichroism experiments should never be mechanically stressed, as the stress will induce birefringence in the quartz and affect the measurements made. There are several different types of cuvettes commonly used; each type has different usable wavelengths at which its transparency exceeds 80%:
Disposable plastic cuvette
Disposable plastic cuvettes are often used in fast spectroscopic assays, where high speed is more important than high accuracy. Plastic cuvettes, with a wavelength from 380 to 780 nm (visible spectrum), are easily expendable and thus disposed of once used, preventing contamination risk from reusing cuvettes. Owing to its low cost of manufacture, it is relatively low in price. Plastic cuvettes are accessible for UV measuring. Disposable cuvettes can be used in some laboratories where the beam light is not high enough to affect the absorption tolerance and consistency of the value.
Optical Glass has an optimal wavelength range of 340 - 2,500 nm. Glass cuvettes are typically for use in the wavelength range of visible light, whereas fused quartz tends to be used for ultraviolet (UV) applications.
Optical glass, or more technically, Optical Crown Glass, is manufactured from alkali-lime silicates containing around 10% potassium oxide. It typically has a refractive index of ≈1.52.
Quartz cells provide more durability compared to plastic and glass. Quartz excels at transmitting UV light, and can be used for wavelengths ranging 190-2500 nm.
Fused quartz cuvette
Fused silica cuvette
ES quartz has a usable wavelength range of 190 to 2,000 nm, and a matching tolerance of 1% at 220 nm. ES quartz is composed of a suprasil giving a marginally higher quality of quartz over the UV cuvette. High transmission in the low nano-meter range is available for the ES quartz cuvette.
Infrared quartz cuvette
IR quartz has a usable wavelength range of 220 to 3,500 nm, and a matching tolerance of 1% at 2,730 nm. The IR quartz cuvette is more resistive to chemical solution than other types designed for fluorescence measurements. The path-lengths are available at 5 mm up until 40 mm with 1.25 mm thickness.
Sapphire cuvettes are the most expensive, though provide the most durable, scratch-resistant, and transmissible material. The transmission extends from UV light to mid-infrared, ranging from 250 to 5,000 nm. Sapphire can withstand the extreme natural condition of some sample solutions and variances in temperature.
Cuvette calibration has a great effect on the absorbance measurement. Many variables need to be considered to ensure precision and accuracy. The spectrophotometric measurement is sensitive to the change in volume. The calibration cuvette helps scientists to convert the volume data from relative value units to the true volume with less uncertainty. The cuvette adjustment is important for laboratory technicians who require a precise estimation of the solution volume. In the laboratory, sometimes a round glass cuvette is utilized. The adjusted cuvette that has been aligned and sold in the market is expensive in comparison with the cost of normal glass tubes. The glass cell costs approximately 30 times more than an ordinary glass tube. In turn, the cuvettes do the most appropriate sort of spectrophotometer, but some researches need the great number of cuvette and it can be costly. A high quality glass tube can give similar results as a cuvette. However, the manually calibrated cuvette has less consistency. The adjustment and position of the tube affect the reading value of the absorbance as the light goes through the sample controlled by the width of the cuvette. The distance traveled by light and the concentration of solution influence the estimation of absorbance. The appropriate wavelength and type of cuvette is the main component for measuring the correct absorbance with manually calibrating the cuvette.
Stress factors in a cuvette reduces the expected measurement and possibly causes errors. Errors in the spectrophotometer such as light distribution can occur when light hits a scratched cuvette. A rubber or plastic rack protects the cuvette from accidentally hitting and being scratched by the machine casing. The type of solvent used, temperature, and the wavelength of the light affect the measurement at different levels of light transmitted through the cuvette.
Low-lint gauze or tissue paper is used to wipe clean the outer surface of the cuvette before usage. Fingerprints or droplets of water disrupt the reflection of light rays during the measurement. In the situation where the pasture pipette contains air during solution transfer, bubbles may form inside the cuvette reducing the purity of a solution and cause light beams to be more scattered. The finger-clad finger method is used to remove bubbles. The solution contained in the cuvette should be high enough to be in the path of the light source.
Mild detergent and ethanol rinsing with tap water is used to wipe off the outer layer of the cuvette. Acid and alkaline are not used due to their corrosive effects on glass, and acetone is avoided when dealing with plastic cuvettes. Wiping the container dry with a soft, low-lint cloth avoids scratching the surface of the cuvette. In case the cuvette needs incubation for various experiments, care must be taken to avoid raising the temperature above permissible levels for the material.
|Wikimedia Commons has media related to Cuvettes.|
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