The dyes “Gentian Violet” and “Eosin,” among others, commonly used in high school biology experiments, are classified as alkaline dyes. Despite their classification, they are actually acidic in nature, with a pH below 7. The classification of dyes is based on their chemical properties, and they are categorized as alkaline dyes, acid dyes, or complex dyes. Alkaline dyes refer to dyes in which the colored cation combines with the dye target, while acid dyes refer to dyes in which the colored anion combines with the target. Complex dyes are a mixture of acid and alkaline dyes.
To aid in understanding, the classification of alkaline (or acidic) dyes is not determined by the pH value of the dye solution. Instead, it is based on the charge carried by the dye molecule’s auxiliary color groups after they ionize. Dyes with positively charged auxiliary color groups are classified as alkaline dyes (also known as cationic dyes), while dyes with negatively charged auxiliary color groups are classified as acid dyes (also known as anionic dyes).
When used as dyes, two conditions must be met by the dye:
- It must have color, and the colored portion is referred to as the chromophore or dye chromophore.
- It must have an affinity for the tissue being dyed, and the part responsible for the affinity is called the auxochrome.
The auxochrome increases the polarity of the dye molecule, facilitates the interaction between the dye and the tissue, and produces the desired staining effect.
In terms of classification based on dyeing objects:
- Alkaline dyes can stain acidic substances because acidic substances can release H+ ions, thereby acquiring a negative charge. The colored cations in the alkaline dye solution are attracted to the negatively charged acidic substances, resulting in a firm staining effect.
- Acid dyes can stain alkaline substances because alkaline substances can release OH- ions, thereby acquiring a positive charge. The colored anions in the acid dye solution are attracted to the positively charged alkaline substances, resulting in a firm staining effect.
When targeting proteins for staining, the choice of dye depends on the pH of the solution. Proteins contain amino and carboxyl groups. In an acidic solution with a pH lower than the protein’s isoelectric point, the protein carries a positive charge and is easily stained by acid dyes. In an alkaline solution with a pH higher than the protein’s isoelectric point, the protein carries a negative charge and is easily stained by alkaline dyes.
