Light is a form of electromagnetic radiation, and like all waves, it has a specific wavelength. Molecules absorb light when its energy matches the energy required to excite their electrons to a higher state. Different molecules possess distinct arrangements of atoms and electrons, causing them to absorb light at particular wavelengths. The wavelength of 260 nanometers (nm) falls within the ultraviolet (UV) region of the electromagnetic spectrum and is particularly relevant in molecular biology due to its interaction with specific biological molecules.
Molecules That Absorb at 260 nm
The primary biological molecules that absorb light strongly at 260 nm are nucleic acids, specifically deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). This characteristic absorption is due to the presence of nitrogenous bases within their structure: adenine (A), guanine (G), cytosine (C), and thymine (T) in DNA, or uracil (U) in RNA. These bases contain ring structures with alternating single and double bonds, known as conjugated double bonds. This arrangement allows electrons to move freely within the ring systems, requiring a specific amount of energy, corresponding to 260 nm UV light, to be excited.
While proteins are another class of important biological molecules, their peak absorption of UV light typically occurs around 280 nm, mainly due to aromatic amino acids like tryptophan, tyrosine, and phenylalanine. This difference in peak absorption wavelengths allows scientists to distinguish between nucleic acids and proteins. The sugar-phosphate backbone of nucleic acids does not significantly contribute to the 260 nm absorption.
How 260 nm Absorption is Measured
Measuring light absorption at 260 nm typically involves a laboratory instrument called a spectrophotometer. This device works by directing a beam of light, often from a UV light source, through a sample solution. A detector then measures the amount of light that passes through the sample. The difference between the initial light intensity and the transmitted light intensity indicates how much light was absorbed by the molecules in the sample.
The relationship between the amount of light absorbed and the concentration of the absorbing substance is described by the Beer-Lambert Law. This law states that absorbance (A) is directly proportional to the concentration (c) of the substance and the path length (l) of the light through the sample. In simpler terms, a higher concentration of molecules in the sample will absorb more light, resulting in a higher absorbance reading. This principle allows scientists to quantify the amount of nucleic acids present in a solution.
Practical Applications in Science
The measurement of 260 nm absorption is a widely used technique in various scientific fields, particularly in molecular biology. One of its applications is the quantification of DNA and RNA in a sample. By measuring the absorbance at 260 nm, scientists can determine the concentration of nucleic acids, with an absorbance of 1.0 at 260 nm corresponding to approximately 50 micrograms per milliliter (µg/ml) for double-stranded DNA and 40 µg/ml for RNA.
Beyond simple quantification, 260 nm absorption is also used to assess the purity of nucleic acid samples. This is achieved by calculating ratios of absorbance at different wavelengths. The A260/A280 ratio helps indicate protein contamination, as proteins absorb light around 280 nm; a ratio of approximately 1.8 for DNA and 2.0 for RNA suggests a pure sample.
A lower A260/A280 ratio may indicate protein contamination, while a higher ratio can suggest RNA contamination in a DNA sample. Another important purity assessment uses the A260/A230 ratio, which helps detect contamination from various organic compounds and salts that absorb light around 230 nm. These contaminants include phenol, guanidine salts, and carbohydrates, often used in nucleic acid extraction. An ideal A260/A230 ratio for pure nucleic acids is generally between 2.0 and 2.2, with lower values indicating potential contamination. The presence of contaminants can lead to an overestimation of nucleic acid concentration, impacting downstream applications.