UV-Visible Spectroscopy

UV-Visible Spectroscopy is employed to identify and quantify compounds present in a sample by utilizing the interaction between light and matter. Here are the key points discussed:

Principles of UV-Visible Spectroscopy

• Light in the form of waves is characterized by its wavelength and frequency.
• Wavelength (λ) is the distance between two consecutive crests.
• Frequency indicates how many crests pass a particular point per second. Higher frequency means more crests per second.
• Wavelength and frequency are inversely proportional to each other.

Electromagnetic Spectrum

• UV and visible light are parts of the electromagnetic spectrum.
• Different wavelengths correspond to different types of radiation within the spectrum.
• Longer wavelengths have lower frequencies and, consequently, less energy.
• For instance, green light has a wavelength of about 549 nm, while red light has about 650 nm.

Basics of Spectroscopy

• Spectroscopy measures the interaction of light with materials. The specific wavelengths absorbed by a material reveal the presence and concentration of compounds.
• The process involves passing light through a sample and measuring the intensity of light before and after the sample interaction.
• Differential absorption of specific wavelengths indicates the presence of certain compounds.

Instrumentation

• A light source emits both UV and visible light.
• A diffraction grating disperses the light into its component wavelengths.
• The sample interacts with specific wavelengths, absorbing some and letting others pass through.
• A detector then measures the intensity of light after passing through the sample compared to a reference.
• The difference in light intensity indicates the compound's concentration.

Quantitative Analysis

• Absorbance (A) is calculated using the formula A = log10(I0/I), where I0 is the intensity of light before interacting with the sample, and I is the intensity after.
• A graph plotting absorbance against concentration can determine the compound's quantity in the sample.

Beer-Lambert Law

• The absorption of light by a sample is directly proportional to its concentration and the length of the light path through the sample.
• This relationship allows for the quantitative analysis of the sample's components based on their absorbance values.

Applications

• Identifying the presence of specific compounds, such as nucleic acids, by their characteristic absorption peak.
• For example, nucleic acids show maximum absorbance at 260 nm, allowing for their identification and quantification.

In summary, UV-Visible Spectroscopy is a crucial technique in analytical chemistry for identifying and quantifying compounds through their interactions with light.