Health and environmental issues have driven an almost hysterical rush to judgment over hexavalent chromium analysis in leather and tanning process liquors.

Unfortunately, the methodology for determining hexavalent chromium has had to evolve as the fierce controversy develops.

Each of the four or five common analytical methods for hexavalent chromium have been tried and tested but only the diphenylcarbazide colourimetric method has proven even close to satisfactory for easy, accurate and dependable results.

Although many of the better descriptions of the diphenylcarbazide procedure caution about analytical interferences, they typically only mention a few metals that can interfere but are rarely encountered in sufficient amounts to matter, such as hexavalent molybdenum, vanadium or mercury.

And there may be statements about destroying organic compounds by digestion but these assume total chromium determination rather than just hexavalent values which will be changed by digestion, with acid digestion likely to destroy (reduce) hexavalent chromium and alkaline digestions likely to create (oxidise trivalent chromium) hexavalent chromium.

However, many current techniques involve only extraction of the chromium and do not include digestions which destroy organic substances.

Consequently a serious new group of potential interfering substances is often present in these adapted determinations of hexavalent chromium.

Indeed, diphenylcarbazide has long been used as an indicator for alkaloids and other organic substances so interactions with organics were documented even before the indicator was ever used for chrome analysis.

A quick check of some common organic substances reveals that several common organic compounds may well give bright pink colours in the hexavalent chrome procedure, falsely leading the analyst to record values for chrome that really have nothing to do with chrome content.

In fact, the very first revelation by Bartless and James that chromium was oxidised in normal soils may be explainable in terms of organic interference rather than actual chrome oxidation.

Source: IULTCS proceedings

The following abstract of the paper has been provided by Catherine A Money, CSIRO, with the comment that the paper should be considered by regulators and that CSIRO are planning to carry out their own trials. ‘The concern about chromium III in wastes has been largely based on literature reporting the use of diphenylcarbazide methods to conclude that chromium VI can be formed in the environment and be a risk.’

Organic acids and amines produce colour with diphenylcarbazide determinations

The diphenylcarbazide colourimetric test method for hexavalent chromium is widely used for determining hexavalent chromium in soils and leather. The method has not been rigorously validated but there has been research into some of its weaknesses and failures.

The use of diphenylcarbazide evolved over many decades. Originally the methods for hexavalent chromium were largely methods used on ashed materials, therefore organic substances were of no consequence. Recently the use of the method on extracts from leather, soil and other heavily organic substrates has become common place.

References in the old methods to interference from iron, vanadium and other metals have been interpreted to mean that those were the most logical and reasonable false positive agents of concern, when in fact, those were only the major interfering substances in ashed or mineral samples. Historically diphenylcarbazide was an indicator for organic compounds such as alkaloids before it was used for hexavalent chromium analyses.

Alkaloids are plant metabolic products. Plants and microbes produce a wide range of organic metabolic products. Tanners use a variety of organic substances in the production of leather in addition to the potential degradation products of oxidation and photolysis acting upon the proteins, fats, retans, and other organics used in the production of leather. Chromium can contribute a catalytic effect to such fat deterioration.

Perhaps the first and most critical determination of hexavalent chromium with diphenylcarbazide in an extract from an organic matrix was the soil analysis by Bartlett and James reported in 1979. Believing that the coloured complex produced by the diphenylcarbazide was proof that hexavalent chromium could form and exist in typical soils, the authors started an investigation that has continued with other researchers to this day.

Even though no significant validation of the actual chromium content of the extract was ever demonstrated, the concern for environmental risk resulted in stringent regulations. One peculiar feature of the work in this research was the strongly noted point that the positive hexavalent chromium value from the soil samples disappeared or was destroyed by drying the soil before analysis.

The analysis had to be performed on fresh, moist soil samples. These were typical soils rich in organic compounds with slightly acid pH values, conditions that do not fit with the general understanding of hexavalent chromium chemistry. Indeed, most research clearly documents that such conditions would rapidly and definitely reduce any hexavalent chromium to the trivalent oxidation state.

Experiments with fresh soil confirmed that diphenylcarbazide does produce a bright pink colour with many soil extracts, often indicating a chromium concentration greater than the total chromium content of the solution as determined by atomic absorption spectro-photometry.

It was concluded by the authors that the presence of volatile agents in the wet soil could have been the reason the coloured response disappeared when the soil was dried prior to analysis. An investigation into potential false positive hexavalent chromium determinations was undertaken. The volatile chemicals investigated were simple organic acids and amines.


The conditions of colour development with diphenylcarbazide solution were strongly acidic, similar to those used in hexavalent chromium test methods.

Starting with the short chain organic acids and progressing to the medium chain length acids, the response of the diphenylcarbazide produced stronger and more permanent colour. Where the acetic acid caused only an instantaneous flash of pink colour when the diphenylcarbazide solution was added to the water and acetic acid blank, propionic and butyric acids produced a brighter and longer lasting pink colour.

The simple organic acids with 6 to 8 carbons appeared to give a strong, stable pink colour when the diphenylcarbazide solution was added. This indicates a potential for creating false chromium analyses, and it is possible that a great many other simple plant and microbial acids might also be capable of giving a coloured reaction with this indicator.

Methyl and ethyl amines also produced a strong, permanent colour when added to the diphenylcarbazide indicator solution. The colour was more red than pink, but at low concentrations might be difficult to distinguish. Other common analytical amines did not produce colour with the indicator under the test conditions.


Organic acids and amines react with diphenylcarbazide to produce a colour that could be wrongly interpreted as hexavalent chromium under the usual analytical conditions of the hexavalent chromium determination.

These compounds would not have been a concern in ashed samples, but the modern practice of extracting organic matrices such as soil and leather could lead to the introduction of these organic compounds into the analytical solutions giving false positives.

Bartlett and James may have extracted organic acids from plants and microbes from soils in their 1979 experiments.

Volatile organic acids that evaporated when the samples were heated during drying could easily account for the unexpected disappearance of the positive diphenylcarbazide result.

In the current dilemma of hexavalent chromium in leather extracts, the condition is strongly linked to fat oxidation in the processing or holding of the leather. One of the primary products of fat deterioration is short chain fatty acids.

The diphenylcarbazide test method for hexavalent chromium has been shown to give false positives and any results based on this method must be questioned.