Color surrounds us in every aspect of life, each chemical compound interacts with 
light across the electromagnetic spectrum by absorbing, transmitting, or reflecting it 
at different wavelengths. When light travels through a solution, a portion of it is 
absorbed. Spectrophotometry enables both qualitative and quantitative analysis, 
where higher concentrations of a substance lead to increased light absorption and 
reduced light transmission.
Spectrophotometry is a method used in Biochemistry/Clinical Chemistry Analyzers to 
measure the concentration of biomolecules by analyzing how light interacts with a 
sample. It relies on the principles of light absorption at specific wavelengths and is 
used to quantify compounds such as proteins, glucose, cholesterol, and enzymes.
Spectrophotometry operates on the Beer-Lambert Law, which states: A=ε⋅c⋅l
For reliable measurements, the absorbance range should ideally fall between 0.1 and 
1.0 where the Beer-Lambert law holds best. For very low absorbance (<0.05) noise 
can become significant affecting precision.

Elements of Spectrophotometry

Functions of each element/How spectrophotometry works:
1. Sample and Reagent Mixing: A small volume of the sample (e.g., blood, urine) is 
mixed with a reagent. The reagent reacts chemically with the target biomolecule to 
produce a measurable product (e.g., a colored compound).
2. Light Source: Produces light across a broad spectrum (e.g., UV, visible, or infrared 
light). Common sources include tungsten lamps (near infrared and visible light, 400-
760 nm) or deuterium lamps (UV light, 200-900 nm).
3. Monochromator/ Wavelength Selection: Selects specific wavelengths of light using
glass, prisms, or diffraction gratings. Ensures that only light of a particular wavelength 
reaches the sample. Filters absorbs light of unwanted wavelength and allow only 
monochromatic light to pass through. For example: Glucose (via glucose method)-
505 nm, Proteins (via Bradford assay)- 595 nm.
4. Interaction with the sample: Cuvettes are made of materials like glass, quartz, or 
plastic, depending on the wavelength being analyzed. Holds the sample, usually in a 
transparent container called a cuvette. Cuvette path length- standard: 1 cm (longer 
path lengths increase absorbance).
The light passes through the sample solution in a cuvette or micro-well. Some of the 
light is absorbed by the product in the sample and the rest passes through.
5. Detection: A photodetector measures the intensity of the transmitted light (I) 
compared to the initial intensity (I₀). These detectors are made up of light sensitive 
material such as selenium. Common detectors include photodiodes or 
photomultiplier tubes. 
 Formula: A=−log (I/ I₀)
6. Data analysis/Output: The spectrophotometer provides a readout of absorbance
or transmittance, which is proportional to the concentration of the substance.

Spectrophotometry types:
 UV-Vis Spectrophotometry- Wavelength range for ultraviolet (190–400 nm) 
and visible light (400–700 nm). Common applications- protein concentration 
(e.g., Bradford assay), DNA quantification, and enzyme activity.
For absorbance 0.0 to 2.0 (in some cases up to 3.0, depending on the 
sensitivity of the detector and the linearity of the system).
 Infrared (IR) Spectrophotometry- Wavelength range for near-infrared (NIR) 
light (700 nm–2500 nm) and mid-infrared (MIR) light (2500 nm–25000 nm). 
Common applications- identifying functional groups in organic compounds.
Absorbance values like UV-Vis often reported as transmittance (%T) instead of 
absorbance.
 Atomic Absorption Spectrophotometry- Wavelength range is 190–900 nm 
(depending on the element being analyzed). Calculate the absorption of light 
by free atoms in a gaseous state. Common applications- detecting metal ions 
in solutions.

Application of Spectrophotometry:
 Quantitative Analysis
 DNA/RNA Measurement 
 Enzyme Kinetics
 Quality Control
 Environmental Testing

Conclusion: Spectrophotometry continues to be a versatile and powerful tool in 
science and industry, enabling precise and reliable analysis across a wide range of 
applications. Its ability to measure and quantify substances with accuracy and ease has 
revolutionized fields such as biochemistry, medicine, and environmental science. As 
technology advances, spectrophotometry's integration with automated systems and 
complementary techniques ensures its relevance in addressing complex challenges. 
Whether in research, diagnostics, or quality control, spectrophotometry remains a 
cornerstone of progress, empowering discoveries and solutions that shape the future