Many dyes and stains are sold with a figure such as "dye content ≥85%" on the label. This does not mean that 15% of the powder is impurity. The figure is the share of the powder that is coloured compound. Most of the rest is water and inorganic salt from manufacture, which the official specifications for colours permit within set limits.

What the figure measures

The clearest definition is in the official specifications for food colours, because they set a minimum dye content. The EU specification for erythrosine (E 127), for example, requires a "content not less than 87 % total colouring matters, calculated as the anhydrous sodium salt".^[1]

Dye content is therefore the percentage by weight of the powder that is coloured compound, calculated as the salt form in which the dye is sold. It is measured on each lot. JECFA, the joint FAO/WHO expert committee on food additives, describes two general methods:^[2]

• Spectrophotometry. The absorbance of a solution of the powder is compared with the absorptivity of a reference standard.
• Titration with titanium(III) chloride. The dye is titrated with this reducing agent, and the amount of titrant used gives the amount of dye.

Some dyes are assayed by gravimetry instead. The JECFA method for erythrosine, for example, dries a precipitate to constant weight and weighs it.^[3]

Both general methods also count compounds other than the main dye. For spectrophotometry, JECFA states that "all colours present in the sample that absorb in the same region as that of the main colour will contribute".^[2] The titration method assumes that isomers and subsidiary colouring matters have the same titanium(III) chloride equivalent as the main dye.^[2]

Dye content counts the main dye together with related coloured compounds. It is therefore not the same as the chemical purity of the main dye.

What the rest of the powder is

The same specifications state what the rest of the powder is. For erythrosine, the EU text lists "water, sodium chloride and/or sodium sulphate as the principal uncoloured components".^[1] The JECFA specification for fast green FCF lists the same three components, and allows up to 15% of the powder to be water, chloride and sulfate together.^[3]

Table 1 gives the official minimum dye content of five colours that are also sold as stains. These specifications are for colours used in food, drugs and cosmetics, not for microscopy stains. They are given here because they state the composition that the authorities accept for these dyes.

The US specification for eosin Y shows the difference between dye content and purity. The total colour must be at least 90%, but the main compound, the disodium salt of tetrabromofluorescein, needs to be only 72%. Up to 25% of the powder may be tribromofluoresceins, which have one bromine atom fewer than the main compound.^[4]

Not all of these other components are accidental. Conn's book Biological Stains (1925), published by the Commission on Standardization of Biological Stains, states that some impurities are accidental and that others are added intentionally, so that dyers can obtain the shade they want.^[5] The Biological Stain Commission states that almost all dye powders contain colourless substances such as sodium chloride and dextrin, and that many contain more than one coloured component.^[6]

Two common stains are examples:

• Crystal violet (gentian violet) is mainly hexamethyl-pararosaniline, with the penta- and tetramethyl compounds as impurities. Unreacted reagents, such as Michler's ketone, may also be present.^[7]
• Basic fuchsin is a mixture. The Commission states that pararosaniline is one of its four components, and reports that most samples submitted to it in recent years contain only pararosaniline.^[6]

The rest of the powder is mostly water and inorganic salt. Part of the colour can come from compounds related to the main dye. The official specifications accept both, within stated limits.

What a Biological Stain Commission certificate adds

The Biological Stain Commission (BSC) tests samples of dye powders that manufacturers and vendors submit to it. Its current table lists 64 certified stains.^[6] A certificate means three things:

• The lot passed an assay and a staining test. In the Commission's words, "only those passing our assay limits and working satisfactorily in specified staining procedures are Certified". The assay covers dye content and composition.^[6]
• It applies to one lot of powder. The label shows the lot number with the BSC certification code. The Commission "certifies dye powders, not staining solutions".^[6]
• For some dyes, it states the measured dye content. For example, each BSC certificate for an alcian blue lot states the assayed dye content of the powder.^[6]

The Commission also publishes minimum dye contents for some dyes. Basic fuchsin must reach 80%, a figure the Commission has reduced from 88%. Pararosaniline must reach 88%, thionine 85% and azure B 80%. Thionine acetate, by contrast, usually has a dye content close to 99%.^[6]

Dye content can also differ between older and current batches. The Commission reports that the alcian blue used in published methods usually contained less than half as much dye as some batches sold today.^[6] With such a batch, a method that gives a weight of alcian blue can therefore give more than twice the intended amount of dye.

A BSC certificate applies to one lot of powder. It confirms that the lot passed the assay limits and worked in specified staining methods.

How to weigh a dye for a solution

Conn's 1925 book gives an example. It compares two samples of methylene blue with dye contents of 90% and 55%, and states that solutions made at 1 g per 100 ml from the two samples "must differ greatly in their strength".^[5] The calculation is:

• 1 g of the first sample contains 1 × 0.90 = 0.90 g of dye.
• 1 g of the second sample contains 1 × 0.55 = 0.55 g of dye.
• 0.55 ÷ 0.90 = 0.61, so the second solution has 61% of the dye of the first.

The book concludes that solutions can be made identical only "on the basis of the weight of actual dye present in the stain used". The Commission on Standardization therefore issued its certification "only to batches of stain on which the total dye content is stated".^[5] Salt also affects saturated solutions: a dye high in sodium chloride gives a saturated solution with considerably less dye.^[5]

The same correction applies to absorbance measurements. Conn's book states that the height of the absorption curve "varies directly with the amount of dye present".^[5] A molar absorptivity calculated from the weight of powder without this correction is therefore too low by the factor of the dye content: about 15% too low for a powder at 85%.

To keep the amount of dye the same between lots, divide the weight of dye you need by the dye content of the lot.

Hematoxylin: a special case

Hematoxylin, the nuclear stain of H&E, is almost colourless and is not itself the dye. The dye is hematein, which forms when hematoxylin is oxidised.^[8][9]

Older formulations let air oxidise the solution, which takes 4 to 10 weeks. Most current formulations add a chemical oxidant, usually sodium iodate, at 0.10 to 0.20 g per gram of hematoxylin.^[8]

The strength of a hematoxylin solution depends on how much of the hematoxylin has been oxidised to hematein. For this reason, recipes state the amount of oxidant per gram of hematoxylin.

Our dyes and their dye content

The eleven dyes in Table 2 are labelled with their dye content, as a minimum for each lot.

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