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Dye Classification by Color Index

Dyes are generally defined along the lines of being colored, aromatic compounds that can ionize. They are thus able to interact with oppositely charged tissue constituents.

The Color Index uses this as a classification and naming system. Each dye is named according to the pattern:

acid + base color + number

Acid Dyes

Dyes that are negatively charged and are used to bind to positively charged tissue components, are termed acid dyes.

These dyes are thereby specifically identified as acid dyes of the stated color, and whose primary mechanism of staining is by ionic bonding. Note that this is a functional and color classification. It contains no chemical information, nor does it imply that dyes with similar names but unique numbers are in any way related. It should also be noted that the classification refers to the primary mechanism of staining. Other mechanisms may also be possible.

The groups that are responsible for the ionizing capability are the auxochromes. Acid dyes have hydroxyl, carboxyl or sulphonic groups as their auxochromes, and consequently have an overall negative charge. In other words, the colored part of the molecule is the anion. Although the molecular charge is often shown on a specific atom in structural formulas, it is the whole molecule that is charged.

An example of a dye with hydroxyl groups as the auxochrome is martius yellow (acid yellow 24).

The carboxyl group is also negatively charged, but is usually found in conjunction with other groups rather than as the sole auxochrome. This is illustrated in the dye eosin Y (acid red 87), which has a hydroxyl group as well.

The final negatively charged auxochrome is the sulphonic group. This is also found in conjunction with other auxochromes. An example is biebrich scarlet (acid red 66), which also has a hydroxyl group. The sulphonic group frequently has the effect of causing an otherwise positively charged dye to have an overall negative charge, as demonstrated by acid fuchsin (acid violet 19), which is sulphonated pararosanilin (basic red 9).

There are numerous other examples.

Basic Dyes

Dyes which are positively charged, and are used to bind to negatively charged tissue components, are termed basic dyes.

These dyes are thereby specifically identified as basic dyes of the stated color, and whose primary mechanism of staining is by ionic bonding. Note that this is a functional and color classification. It contains no chemical information, nor does it imply that dyes with similar names but unique numbers are in any way related. It should also be noted that the classification refers to the primary mechanism of staining. Other mechanisms may also be possible.

Basic dyes have amino groups, or alkylamino groups, as their auxochromes, and consequently have an overall positive charge. In other words, the colored part of the molecule is the cation. Although the molecular charge is often shown on a specific atom in structural formulae, it is the whole molecule that is charged.

An example of a dye with amino groups as the auxochrome is pararosanilin (basic red 9), and with alkylamino groups is methylene blue (basic blue 9).

There are numerous other examples.

Direct Dyes

Although the definition of dyes infers that ionic interaction with oppositely charged tissue constituents is the norm, there are exceptions. Some dyes are used in the textile industry to dye cotton without using a mordant. An older name for dyeing without a mordant is direct dyeing, and the dyes are termed direct dyes or, sometimes, direct cotton dyes.

These dyes are thereby specifically identified as dyes of the stated color, and whose primary mechanism is staining without a mordant. Note that this is a functional and color classification. It contains no chemical information, nor does it imply that dyes with similar names but unique numbers are in any way related. It should also be noted that the classification refers to the primary mechanism of staining. Other mechanisms may also be possible.

Direct dyes are usually negatively charged. In other words, the colored part of the molecule is the anion. Although the molecular charge is often shown on a specific atom in structural formulae, it is the whole molecule that is charged.

It has been shown that direct dyes can stain cotton by hydrogen bonding. This mechanism is also the basis for their value in histological staining and explains why some are able to demonstrate amyloid very selectively. However, they are also able to stain positively charged tissue components by ionic interaction in much the same way as acid dyes. They may be viewed as a special subgroup of acid dyes, many being azo dyes.

The most commonly used direct dye is probably congo red (direct red 28), which is used for demonstrating amyloid. Erie garnet (direct red 10) can be used in a rapid frozen section staining method, and sirius red F3B (direct red 80) can be used to stain collagen ionically in a modified Van Gieson, as well as for staining amyloid by hydrogen bonding.

There are numerous other examples.

Mordant Dyes

Although the definition of dyes infers that ionic interaction with oppositely charged tissue constituents is the norm, there are exceptions. Some dyes require the presence of a metal to properly develop their color or staining selectivity. These are termed mordant dyes.

These dyes are thereby specifically identified as dyes of the stated color, and whose primary staining mechanism requires the presence of metal atoms. Note that this is a functional and color classification. It contains no chemical information, neither does it imply that dyes with similar names but unique numbers are in any way related. It should also be noted that the classification refers to the primary mechanism of staining. Other mechanisms may also be possible.

The most commonly used mordant dyes have hydroxyl and carboxyl groups and are negatively charged, i.e. anionic. It is convenient to view these as a specialized subgroup of acid dyes. Some other mordant dyes may possess amino groups, and are cationic overall. Despite this, they must still have hydroxyl or carboxyl groups, since lake formation requires it. Mordant dyes can usually stain by ionic interaction in the same way as other ionizable dyes. The color is often pale, sometimes so pale that the results have no value.

It is often noted that when a mordant dye forms a lake with a metal, there is a strong colour change. This is because metals have low energy atoms. The incorporation of these low energy atoms into the delocalised electron system of the dye causes a bathochromic shift in the absorption. It is this delocalised electron system which is fundamentally responsible for color in dyes. Since different metal atoms have differing energy levels, the colour of the lakes may also differ.

The most commonly used mordant dye is undoubtedly hematein (natural black 1), whose status as a natural product supercedes its mode of dyeing, apparently. Others are chromoxane cyanine R (mordant blue 3) and celestin blue B (mordant blue 14), both used as substitutes for alum hematoxylin but with a ferric salt as the mordant. Alizarin red S (mordant red 3) is valuable for the demonstration of calcium, particularly in embryo skeletons.

There are numerous other examples.

Natural Dyes

Coloring materials have been used for many thousands of years by man. Leather, cloth, food, pottery and housing have all been modified in this way. The two old ways were to cover with a pigment (painting), or to color the whole mass (dyeing). Pigments for painting were usually made from ground up cultured rocks and minerals, and the dyes were obtained from animals and plants. Today, many of the traditional dye sources are rarely, if ever, used (onion skins, for instance). However, some of our most common dyes are still derived from natural sources. These are termed natural dyes.

These dyes are thereby specifically identified as dyes of the stated color, and may still be derived from animals or plants. Note that this is a classification based on the dye’s source and color. It contains no chemical information, nor does it imply that dyes with similar names but unique numbers are in any way related. It gives no information about the mechanism by which staining occurs.

Natural dyes are often negatively charged. Positively charged natural dyes do exist, but are not common. In other words, the colored part of the molecule is usually the anion. Although the molecular charge is often shown on a specific atom in structural formulae, it is the whole molecule that is charged. Many, but by no means all, natural dyes require the use of a mordant.

The use of dyes is very ancient. Kermes (natural red 3) is identified in the bible book of Exodus, where references are made to scarlet colored linen. Similar dyes are carmine (natural red 4) and lac (natural red 25). These three dyes are close chemical relatives, obtained from insects of the genus Coccus. All require a mordant.

The most commonly used natural dye is undoubtedly hematein (natural black 1), obtained from the heartwood of a tree. This dye also requires a mordant.

Saffron (natural yellow 6) is obtained from the stigmata of Crocus sativus, and is used without a mordant, staining as an acid dye. Although its use is very ancient, it is more common now as a coloring and spice for food than for dyeing, due to its expense.

Solvent Dyes

Solvent dyes differ from other dyes, in that they color by dissolving in the target, which is invariably a lipid or non-polar solvent.

These dyes are thereby specifically identified as dyes of the stated color, and whose primary mechanism of staining is by dissolving in the target. Note that this is a functional and color classification. It contains no chemical information, neither does it imply that dyes with similar names but unique numbers are in any way related. It should also be noted that the classification refers to the primary mechanism of staining. Other mechanisms may also be possible, but are rare.

As a general principle, solvent dyes do not ionize. Many are azo dyes which have undergone some molecular rearrangement and lost the ability to ionize. In the process, they gained the ability to dissolve in non-polar materials such as triglycerides. They are commonly used to stain such materials in sections. They are frequently called lysochrome dyes. The prefix lyso means dissolve, and chrome means color.

Sudan III (solvent red 23), sudan IV (solvent red 24) and oil red O (solvent red 27) are commonly used for demonstrating fat in sections. Sudan black B (solvent black 3) is also very effective, but can also stain ionically under some circumstances.

References

  1. Edward Gurr, (1971)
    Synthetic dyes in biology, medicine and chemistry
    Academic Press, London, England.