Spectrophotometry – Basic Instrumentation

Objective:

To understand the basic principle of spectrophotometry and the working of a UV-Vis spectrophotometer by measuring absorbance of a colored solution at a specific wavelength.

 Principle:

Spectrophotometry is based on the principle that molecules absorb light at specific wavelengths. The amount of light absorbed is proportional to the concentration of the solution (Beer-Lambert Law).

Beer-Lambert Law:
$A=εc lWhere: A = Absorbance , ε = Molar absorptivity (L·mol⁻¹·cm⁻¹), c = Concentration (mol·L⁻¹), l = Path length (cm) $

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·         UV-Visible Spectrophotometer

·         Cuvettes (quartz or plastic, depending on wavelength)

·         Distilled water

·         Colored solution (potassium permanganate/cobalt chloride/food dye)

·         Pipettes and volumetric flasks

·         Tissue paper

 Procedure:

1.      Switch on the spectrophotometer. Select the wavelength suitable for the solution.

2.      Fill a cuvette with distilled water, place it in the sample holder, and set the instrument to zero absorbance.

3.      Rinse the cuvette with the test solution and fill it three-fourths full. Wipe the cuvette outside with tissue.

4.      Insert the sample cuvette into the sample compartment.

5.      Record the absorbance reading.

6.      Repeat with multiple concentrations of the standard solution to observe the relationship between concentration and absorbance.

 

Observation Table:

S.No	Concentration (mg/mL)	Absorbance at λmax

 Fig: Diagram of the elements in a single beam UV-Visible spectrometer.

(https://jascoinc.com/learning-center/theory/spectroscopy/uv-vis-spectroscopy/instrumentation/)

 

Result:

A graph of absorbance vs concentration resulted in a straight line, confirming Beer-Lambert law. With increase in the concentration of solution, the absorbance was found to increase.

 

Conclusion:

The experiment demonstrates the use of a spectrophotometer to determine the absorbance of solutions and the linearity between concentration and absorbance.

 

Precautions:

·         Always handle cuvettes with clean hands/gloves to avoid smudges.

·         Use the same cuvette for blank and test samples when possible.

·         Ensure the cuvettes are properly aligned in the sample holder.

·         Avoid bubbles in the cuvette as they can distort readings.

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