A spectrophotometer is a scientific instrument used in research and industrial laboratories for UV-Vis spectroscopy, measuring the energy of light at specific wavelengths. It is used in physics, molecular biology, chemistry, and biochemistry labs. Samples are measured in small containers called cuvettes, and extinction coefficients are used to calculate the concentration of compounds. Spectrophotometers are also used to measure the growth of bacterial cultures and the color changes in chemical and enzymatic reactions. They use a light source to shine a series of wavelengths through a monochromator and compare the intensity of the light passing through the sample with that passing through a reference compound. There are two types of UV-Vis spectrophotometers: single beam and dual beam.
A spectrophotometer is one of the scientific instruments commonly found in many research and industrial laboratories. Spectrophotometers are used for research in physics, molecular biology, chemistry and biochemistry laboratories. Typically, the name refers to ultraviolet-visible (UV-Vis) spectroscopy.
The energy of light depends on its wavelength, usually denoted as lambda. Although the electromagnetic spectrum spans a wide range of wavelengths, most laboratories can only measure a small fraction. UV-Vis spectroscopy measures between 200 and 400 nanometers (nm) for UV light measurements and up to about 750 nm in the visible spectrum.
For UV-Vis spectroscopy, samples are usually contained and measured in small containers called cuvettes. These can be plastic when used in the visible spectrum, but must be quartz or fused silica when used for UV measurements. There are some machines that can use glass test tubes.
Visible spectroscopy is often used industrially for colorimetry. Using this method, samples are measured at multiple wavelengths from 400-700 nm and their absorbance profiles compared to a standard. This technique is often used by fabric and ink manufacturers. Other commercial users of UV-Vis spectroscopy include forensic laboratories and printers.
In biological and chemical research, solutions are often quantified by measuring how well they absorb light at a particular wavelength. A value called the extinction coefficient is used to calculate the concentration of the compound. For example, molecular biology laboratories use spectrophotometers to measure the concentrations of DNA or RNA samples. Sometimes they have an advanced machine called a NanoDrop spectrophotometer that uses a fraction of the amount of sample than traditional spectrophotometers use.
For the quantification to be valid, the sample must comply with the Beer-Lambert law. This requires that the absorbance be directly proportional to the length of the cuvette path and the absorbance of the compound. Tables of extinction coefficients are available for many, but not all, compounds.
Many chemical and enzymatic reactions change color over time, and spectrophotometers are very useful for measuring these changes. For example, the polyphenol oxidase enzymes that cause fruit browning oxidize solutions of phenolic compounds, turning clear solutions into visibly colored solutions. These reactions can be tested by measuring the increase in absorbance as the color changes. Ideally, the rate of change will be linear and rates can be calculated from this data. A more advanced spectrophotometer will have a temperature controlled cuvette holder to run the reactions at a precise temperature ideal for the enzyme.
Microbiology and molecular biology laboratories often use a spectrophotometer to measure the growth of bacterial cultures. DNA cloning experiments are often performed in bacteria, and researchers need to measure the growth stage of the culture to know when to perform certain procedures. They measure absorbance, known as optical density (OD), on a spectrophotometer. You can tell by the OD whether the bacteria is actively dividing or starting to die.
Spectrophotometers use a light source to shine a series of wavelengths through a monochromator. This device then transmits a narrow band of light, and the spectrophotometer compares the intensity of the light passing through the sample with that passing through a reference compound. For example, if a compound is dissolved in ethanol, the reference would be ethanol. The result is displayed as the degree of absorbance of the difference between them. This indicates the absorbance of the sample compound.
The reason for this absorbance is that both ultraviolet and visible light have enough energy to excite chemicals to higher energy levels. This excitation results in a higher wavelength, which is visible when absorbance is plotted against wavelength. Different molecules or inorganic compounds absorb energy at different wavelengths. Those with the greatest absorption in the visible range are seen as colored by the human eye.
Solutions of compounds may be clear, but they absorb in the UV range. Such compounds usually have double bonds or aromatic rings. Sometimes there are one or more detectable peaks when the degree of absorption is plotted against wavelength. If so, this can help in the identification of some compounds by comparing the shape of the graph to that of known reference graphs.
There are two types of UV-Vis spectrophotometers, single beam and dual beam. These differ in how they measure the light intensity between the reference and test sample. Dual-beam machines measure the reference and test compound simultaneously, while single-beam machines measure before and after the addition of the test compound.
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