Spectrophotometers are instruments that measure how much light a sample absorbs or transmits. They operate by shining a beam of light through a sample and detecting the light that passes through or is absorbed. These measurements are important in various scientific fields, including biology and chemistry, for understanding the properties and concentrations of different substances. This article will clarify the primary units spectrophotometers measure and what these measurements reveal about a sample.
Understanding Wavelength
Wavelength is a fundamental property of light, representing the distance between successive crests of a wave. In spectrophotometry, wavelength determines the “color” or type of light being used for measurement. The standard unit for wavelength is nanometers (nm), with 1 nanometer equaling one billionth of a meter.
Selecting a specific wavelength is important because different substances absorb light most effectively at particular wavelengths. For example, a red solution absorbs green light strongly, and a blue solution absorbs orange light. Spectrophotometers can operate across the electromagnetic spectrum, including visible light (typically 400-780 nm), ultraviolet (UV) light (below 400 nm), and infrared (IR) light (above 780 nm).
The Concept of Absorbance
Absorbance is a measure of how much light a sample prevents from passing through. It is a dimensionless unit, often referred to as Optical Density (OD), particularly in biological applications. When a spectrophotometer measures absorbance, it compares the intensity of light before it enters the sample to the intensity of light that passes through the sample.
A direct relationship exists between a substance’s concentration in a solution and its absorbance. More concentrated solutions will absorb more light, resulting in a higher absorbance reading. This makes absorbance a preferred measurement for quantifying the amount of a substance in a solution. Although dimensionless, absorbance is often reported in “Absorbance Units” (AU) for practical purposes.
Measuring Transmittance
Transmittance measures the amount of light that successfully passes through a sample without being absorbed. This value is typically expressed as a percentage (%). If a sample transmits 100% of the light, it means no light was absorbed, similar to clear water. Conversely, a sample transmitting 0% of the light means all light was absorbed, like a very murky substance.
Transmittance has an inverse relationship with absorbance. As absorbance increases, transmittance decreases. For instance, a solution that highly absorbs light will have low transmittance, while a solution that absorbs little light will have high transmittance.
What These Measurements Tell Us
The units measured by spectrophotometers—wavelength, absorbance, and transmittance—provide meaningful information about a sample. By selecting a specific wavelength and measuring absorbance or transmittance, scientists can determine the concentration of various substances. For example, the concentration of DNA and RNA is often determined by measuring absorbance at 260 nm, while protein concentration is commonly assessed at 280 nm.
Spectrophotometers also monitor bacterial cell growth by measuring the turbidity, or cloudiness, of a bacterial culture, which relates to its absorbance. These measurements are used to assess the purity of a sample by analyzing absorbance ratios at different wavelengths. These quantitative data are essential for research, diagnostics, and quality control in numerous scientific and industrial applications.