Spectrophotometry

Spectrophotometry is built on the principle of light and its interaction with matter. Light is a form of electromagnetic radiation that travels in waves. 

When light encounters a material, several things can happen. The light can be reflected off the surface, transmitted through the material, or absorbed by the material. Absorption occurs when the energy of the light matches the energy needed to move electrons within the molecules of the material to a higher energy level. Different substances absorb light at different wavelengths, depending on their molecular structure.

In spectrophotometry, we focus on measuring the absorption of light by a substance. The amount of light absorbed at a particular wavelength can tell us a lot about the substance’s concentration and its identity. This measurement is based on the Beer-Lambert Law.

The Beer-Lambert Law is a fundamental equation in spectrophotometry that relates the absorbance of light by a substance to its concentration. The law is mathematically expressed as

A = εˑcˑl

Where:

– A represents absorbance

– ε (epsilon) is molar absorptivity, a constant value specific to each substance

– c signifies concentration

– l denotes path length

According to this law, absorbance is directly proportional to both the concentration of the absorbing substance and the path length through which the light passes. This relationship allows scientists to determine the concentration of an unknown sample by measuring its absorbance and comparing it to a standard.

The device used in spectroscopy is called a spectrophotometer. It is an instrument used to measure the intensity of light absorbed by a sample at specific wavelengths, allowing for the analysis of the sample’s concentration and composition. It consists of several key components.

Light Source

The light source is the first component in a spectrophotometer. It provides the light that will pass through the sample. Different types of light sources are used depending on the range of wavelengths needed for the analysis. For instance, a tungsten lamp is commonly used for visible light spectrophotometry because it produces a broad spectrum of light in the visible region (400–700 nm). For ultraviolet (UV) light, a deuterium lamp is often used, as it emits a continuous spectrum in the UV region (190–400 nm).

Monochromator

The light must be filtered to select the specific wavelength that will interact with the sample. It is where the monochromator comes into play. The monochromator uses a prism or diffraction grating to separate the light into its component wavelengths, much like a prism creates a rainbow from sunlight. By adjusting the monochromator, a single wavelength of light is isolated and directed toward the sample. This ability to select specific wavelengths is crucial because different substances absorb light at different wavelengths, and precise measurement requires focusing on the wavelength where absorption occurs.

Sample Holder (Cuvette)

The sample holder, or cuvette, is where the sample being analyzed is placed. Cuvettes are typically made of materials like quartz or plastic, which are transparent to the wavelength of light being used. The choice of material is important because it must not absorb light at the wavelengths of interest, as this would interfere with the measurement. Cuvettes also come in different path lengths, usually 1 cm, which is important for applying the Beer-Lambert Law in quantifying the absorbance.

Detector

After the light passes through the sample, the remaining light is measured by the detector. The detector converts the light into an electrical signal, which is then used to calculate the absorbance of the sample. Common types of detectors include photodiodes and photomultiplier tubes, which are highly sensitive to small changes in light intensity. The accuracy of the detector is critical because it determines how precisely the absorbance can be measured.

• Pharmaceutical Industry: Used to determine the concentration of active pharmaceutical ingredients (APIs) and assess the stability and purity of drugs.
• Clinical Diagnostics: Helps in analyzing blood and urine samples to measure levels of various compounds, such as glucose, proteins, and hormones.
• Food and Beverage Industry: Analyzes color and concentration of additives, preservatives, and nutrients in food products.
• Forensic Science: Identifies and quantifies substances in forensic samples, such as drugs or toxins, by measuring their absorbance characteristics.
• Material Science: Assesses the optical properties of materials, including their absorbance and transmission characteristics, for applications in coating and polymer analysis.

Problem: A solution of a dye is prepared, and its absorbance is measured using a spectrophotometer at a wavelength of 450 nm. The absorbance of the solution is found to be 0.65. The path length of the cuvette used in the measurement is 1 cm. The molar absorptivity of the dye at this wavelength is 1.25 × 10² L/mol·cm. Calculate the concentration of the dye in the solution.

Solution:

We use Beer-Lambert Law, which is expressed as:

A=ε⋅c⋅l

Given:

Absorbance, A = 0.65

Molar absorptivity, ε=1.25 x 10² L/molˑcm

Path length, l = 1 cm

Rearrange the equation

c = A/ε⋅l

Substituting

c = 0.65/(1.25 x 10² L/molˑcm x 1 cm) = 0.0052 mol/L

The concentration of the dye in the solution is 0.0052 mol/L.