The assay of vitamin A by colorimetry is a method used to quantify the amount of vitamin A in a sample based on its color reaction with specific reagents. The principle behind colorimetry is that vitamin A can form a colored complex when it reacts with certain chemicals, and the intensity of the color produced is proportional to the concentration of vitamin A in the sample.

Here’s a general outline of the procedure:

Principle:

Vitamin A (retinol) is oxidized to form a compound that produces a distinct color when it reacts with specific reagents, often such as antimony trichloride or ferric chloride in a chloroform solution. The color intensity of the resulting solution is then measured using a colorimeter or spectrophotometer at a specific wavelength.

Procedure:

  1. Sample Preparation:

    • The sample containing vitamin A (e.g., food, serum) is extracted with a suitable solvent like ethanol or hexane.
    • The sample is then purified to remove any interfering substances, ensuring only vitamin A or its derivatives are present.

  2. Reaction with Reagents:

    • The purified vitamin A is treated with antimony trichloride (SbCl₃) in a chloroform solution (Carr-Price reaction). Antimony trichloride reacts with vitamin A to form a blue color.
    • Alternatively, ferric chloride may be used in some methods, producing a different colored complex.

  3. Measurement of Color:

    • The solution's color intensity is measured using a colorimeter or spectrophotometer at a specific wavelength (usually between 620–680 nm, depending on the reagent).
    • The absorbance is proportional to the concentration of vitamin A in the sample.

  4. Calibration Curve:

    • A calibration curve is prepared using standard solutions of known concentrations of vitamin A.
    • The absorbance readings from the sample are compared with the calibration curve to determine the concentration of vitamin A.

Calculation:

Using the absorbance value obtained from the sample and the calibration curve, the amount of vitamin A in the sample is calculated.

Advantages:

  • Simple and quick.
  • Can be used for various types of samples (e.g., biological fluids, food products).

Limitations:

  • Vitamin A is sensitive to light and oxygen, so samples must be handled carefully.
  • Other substances that absorb at the same wavelength may interfere with the results, requiring careful sample preparation.

This method is widely used in pharmaceutical, nutritional, and clinical laboratories to ensure accurate quantification of vitamin A levels.