Reagents

• Urea standard
• MeOH (Methanol)
• Ehrlich's reagent (p-dimethylaminobenzaldehyde, DMAB)

Theory

Milk is a complete food, but its adulteration is a widespread issue, often done to increase bulk or mask the addition of water. One common adulterant is urea, added to milk to increase its non-protein nitrogen content and maintain the solid-not-fat (SNF) levels that are lowered when water is added. The consumption of milk adulterated with urea can lead to serious health issues, including gastrointestinal disorders and renal failure.

Principle of Urea Detection

Urea itself does not absorb UV light, making its direct detection by conventional methods difficult. Therefore, detection relies on a derivatization reaction. The method is based on the reaction of urea with Ehrlich's reagent (p-dimethylaminobenzaldehyde, DMAB) in an acidic medium. This reaction forms a yellow-colored complex, which can be quantitatively measured. A similar principle, utilizing the reaction of urea with DMAB to form a yellow complex measured at 425 nm, is used in standard colorimetric methods.

Application in HPTLC-Densitometry

High-Performance Thin Layer Chromatography (HPTLC) combined with densitometry is a powerful and efficient analytical technique for the quantification of urea. The procedure generally involves the following steps:

1. Sample Preparation: Milk samples often require protein precipitation, for example, using trichloroacetic acid (TCA), followed by filtration.

2. Chromatographic Separation: The prepared sample, along with standard urea solutions, is applied as bands onto an HPTLC plate (e.g., silica gel 60 F254). The plate is then developed in a suitable mobile phase to separate urea from other components.

3. Derivatization: After development, the plate is derivatized by spraying or dipping with Ehrlich's reagent (DMAB) and heated. This produces a stable, colored spot for urea, enabling its visualization and quantification.

4. Densitometric Scanning: The derivatized plate is scanned using a densitometer at the wavelength of maximum absorbance for the yellow urea-DMAB complex (e.g., 440 nm or 520 nm). The peak area or height corresponding to urea is then measured.

Validation and Linearity

For reliable quantification, the method is validated. A linear calibration curve is established by plotting the peak area against the concentration of standard urea applied (e.g., in the range of 100-700 ng per band). This curve allows for the accurate calculation of urea concentration in the milk sample. Method validation parameters like linearity (e.g., R² = 0.993), precision (relative standard deviation, RSD), and recovery (e.g., 98.45%) are evaluated to ensure the method's accuracy and reproducibility.