A Perspective on Indigo: An Iconic Colorant

Abstract

This perspective sets out to raise awareness about the chemical and photophysical properties of indigo, a highly distinguished colorant with an extraordinary history. Indigo, like many other dyes, was first extracted from plants at an inordinately low yield and at great ecological expense. Such was its popularity that indigo was among the first natural colorants to be synthesized in a laboratory before refinement and cost reduction resulted in its economical industrial-scale production. The color of indigo is highly characteristic but difficult to describe, since it falls at the blue/violet interface. It is a small, planar molecule with an exceptionally high degree of π-electron conjugation that pushes the absorption maximum to above 600 nm. Its structure helps explain the high level of photostability enjoyed by indigo, while recent spectroscopic studies have added to our understanding of the longevity of this emblematic colorant. The reversible formation of leuco-indigo increases the ways in which indigo can be used to add color to objects while helping to circumvent the effects of attack by free radicals. It is stressed that the journal Colorants would welcome submissions that describe the chemistry and/or spectroscopy of other representative colorants.

1. Introduction

This is the second report in a series of Editorial Perspectives introducing research topics of direct relevance to the journal Colorants. This style allows for personal observations in addition to hard science. It was sometime last year, when waiting for a much-delayed train and with no light reading matter at hand, that I started to think about landmark colorants. My personal preference is for natural materials, and, because of a lifetime spent researching artificial photosynthesis, I have always been attracted to the chromophores developed by plants and algae. These are often macrocycles, or linear versions thereof, designed and subsequently refined for specific tasks associated with the conversion of sunlight to chemical fuel. Over a few billion years, these chromophores have been systematically optimized for particular tasks and for their ability to act co-operatively with other essential ingredients, which, together, make up the complex energy-storing machinery employed by nature. These are functional materials. There is, however, a separate part of the natural world that manufactures beautiful colors for ascetic reasons. It is into this second domain that we now delve.

Color plays an important, possibly critical, role in establishing our understanding of the environment around us [1]. Color can modulate our moods, expand our horizons and stimulate our senses. The essence of color is provided by colorants: sometimes a single substance but more often a blend of substances that can produce a wide dispersion of colors. With imagination and experience, color carries hidden messages; for example, red means danger, green means security and the appearance of safrin yellow means spring is on the way, but when the vine leaves turn purple, we know the summer is coming to an end. More insightful is the observation noted by Oscar Wilde in his novel The Portrait of Dorian Gray: “Never trust a woman who wears mauve. It always means they have a history”. Not all such generalizations hold true, however. For example, redheads, like myself (and Queen Elizabeth I of England), having high levels of the reddish pigment pheomelanin, are expected to possess fiery temperaments and high levels of pain tolerance. Neither of these features applies to me!

Coloration, however, is not a simple property, and individuals do not always agree on a color. In any case, this will change with time and with immediate surroundings, especially exposure to sunlight; colors disappear entirely under sodium lighting. Knowing that particular colors can be obtained by mixing primary pigments is not always reassuring. Indeed, upon leaving secondary school to work in the laboratory of a local paint manufacturer, I was given the task of matching colors by eye. This is not so difficult when dealing with small quantities but can be quite daunting when handling a few thousand liters of paint in the presence of a critical audience. Nowadays, there is a machine to perform this job!

Simple inquisitiveness dictates that we give a name to the underlying colorant responsible for generating the color of an object. Often, this relates to a plant or a flower. The next step is to extract the colorant and characterize the color by spectroscopic means. A chemist would not stop there but would set out to identify the chemical structure before synthesizing a sample in the laboratory. Natural products are often deceptively simple in terms of their structure, but their synthesis can be extremely demanding; look at the effort involved in R. B. Woodward’s amazing synthesis of chlorophyll-a, which required 46 stages to obtain the target compound [2,3]. I was fortunate to meet Woodward on several occasions when he visited the Royal Institution in London. I was also privileged to collaborate with Ray Bonnett, one of the co-authors of the original publications and a great advocate for porphyrin synthesis. Gerhard Closs, also an original co-author, gave me advice on designing biomimetic systems for artificial photosynthesis. Apart from satisfying scientific curiosity, the successful synthesis of natural colorants can be extremely lucrative. This was a great driving force for Perkin in the mid-nineteenth century, who was responsible for the large-scale preparation of an inordinately wide range of chemical dyes and pigments, starting with mauve in 1856.

Not all colorants leave good memorials. For example, copper arsenite was a favorite substance used to develop a pea-green hue. During the Victorian period, it was heavily used to print fabrics and wallpaper and to enhance artists’ palettes. This compound, known as Scheele’s Green, was unbelievably popular in the mid-nineteenth century to such an extent that emerging concerns about its possible toxicity were largely ignored. The result was the untimely death of countless workers in the dye industry exposed to unacceptably high levels of arsenic. The story goes that Napoleon Bonaparte was poisoned by the British during his imprisonment on the distant island of St. Helena. Based on the reported symptoms, David E. H. Jones, a visiting scholar at Newcastle University and inventor of the perpetual motion machine that can be seen at the Royal Society in London, considered that perhaps Napoleon died because of accidental arsenic poisoning. David was fortunate to obtain an authentic sample of the wallpaper used in Napoleon’s rooms on St. Helena, and, from spectroscopic analysis, found that it did indeed contain high levels of Scheele’s Green. It was further confirmed that Napoleon had complained repeatedly that his rooms were damp. This is important because mold will grow on damp wallpaper, and this mold will release volatile arsenic trimethyl, which will be ingested by the inhabitants of the rooms. This is a nice detective story that was published in Nature (D. Jones and K. Ledingham, 1982, 299, 626) and New Scientist (D. Jones, 14 1982, 101). Following his death in 2017, I helped remove many dangerous chemicals from the house in Newcastle that David used as both laboratory and living quarters, but fortunately, I did not find arsenic.

One colorant that has enjoyed a long and distinguished antiquity is indigo. Indeed, the ancient Britons painted themselves with indigo, extracted from woad, as a way to frighten their enemies, although it did not help much with the Roman invaders. The modern Britons apply indigo in the form of tattoos for much the same reason. Originally, indigo was extracted from plants by an extremely laborious, noxious and wasteful process. The yield was minimal, and the toxic waste was prodigious, but the vivid blue color was deemed to be worth the phenomenal effort. In many parts of the world, notably Europe, Japan and China, indigo became the color of the working population. Napoleon’s Grande Armèe, for example, worn indigo-dyed uniforms, while blue jeans have brought indigo to the forefront of the modern world. The universalization of indigo owes much to the successful synthesis of “pure” material on an industrial scale. This was attempted first by van Baeyer and Drewsen, using procedures developed by Perkin, in 1880, although success was only at the laboratory scale.

The color of indigo, which sits somewhere between blue and violet in the electromagnetic spectrum, is instantly recognized; its hex code is #4B0082 and its RBG code is (75, 0, 130). A lot has been written about the history of indigo but much less is known, at least to the general public, about the substance itself. How is it made? What is its chemical structure? What makes the distinguishing color? Why is it so stable? These are questions we can try to answer in this Editorial Perspective. We are not attempting here to present an exhaustive review of the historical development of this spectacular colorant, but rather, we set out to highlight certain properties of an ever-enduring colorant. Perhaps not too surprisingly, there are still aspects of its photochemistry that remain relatively obscure.

2. Structure and Synthesis

The usual form of indigo, for which the IUPAC name is 2-(3-oxo-2,3-dihydro-1H-indol-2-ylidene)-2,3-dihydro-1H-indol-3-one, is a powder with a distinctive blue/violet coloration. It is a small, highly symmetrical molecule that adopts the trans geometry as the most stable species (Figure 1). The structure is planar, with two intramolecular 6-membered hydrogen bonds and a high degree of π-electron conjugation. The latter helps to push the absorption maximum toward lower energy, this appearing at around 610 nm with a slight dependence on the nature of the solvent. In addition, the hydrogen bonding motif between adjacent carbonyl and imino groups helps to stabilize the planar geometry, but also this is a crucial feature in terms of achieving unusually high levels of photostability, as will be discussed below. Indigo has limited solubility in common organic solvents and does not dissolve in water. The aromatic rings, however, are open to synthetic modification to give a range of derivatives with improved solubility and with less tendency toward aggregation, including water-soluble variants.

An important derivative is the leuco form of indigo, often referred to as white indigo. This substance is formed by the two-electron reduction of indigo, and, as the name suggests, this is associated with a loss of blue coloration. Of great interest to the dye industry is the observation that the aerial oxidation of leuco-indigo restores the characteristic blue/violet color. Of further interest is the realization that leuco-indigo dissolves in water at a neutral pH to give a slightly yellow solution. Reduction can be achieved most conveniently by electrochemical means or by treatment with alkaline sodium dithionite solution. The chemical structure of leuco-indigo is drawn in Figure 2 and shows the loss of π-electron conjugation inherent to indigo. The molecular structure is no longer planar, with the two rings adopting an almost orthogonal geometry, and the hydrogen bonding pattern is lost.

The extraction of indigo from natural plants is no longer economically or ecologically viable, and furthermore, the demand for the dye far exceeds what could be obtained by extraction procedures. Fortunately, several procedures are available for its large-scale synthesis under fairly mild conditions. The original aldol condensation methodology introduced by van Baeyer and Drewsen in 1880 was the first laboratory protocol for the synthesis of indigo, but it was unsuitable for expansion to an industrial scale. The reaction used 2-nitrobenzaldehyde and acetone as starting materials, but was expensive, wasteful in terms of solvent and time-consuming. Subsequent procedures, notably that introduced by Heumann in 1890, started with aniline and set out to prepare indoxyl, which could be oxidized directly to indigo. Later, Heumann used anthranilic acid as the starting compound, which allowed lower temperatures to be used and significantly increased the yield of indoxyl. Further reductions in cost, including lowering the reaction temperature and shortening the reaction time, resulted in the preference for 2-chloroethanol as the starting material. This is readily converted to oxirane before reaction with aniline forms hydroxy-ethylaniline. The latter cyclizes in alkaline solution to give indoxyl in a good yield. This is now the preferred route to indigo [4].

3. Spectroscopy

Our knowledge of the photophysical properties of indigo and its derivatives owes everything to the investigations carried out by J. Sérgio Seixas de Melo at the University of Coimbra. Because of this work, there now exists a large database of information regarding the spectroscopy of indigo, in both the keto and leuco forms, and of several derivatives. For indigo in N,N-dimethylformamide (DMF), the absorption maximum occurs at 610 nm, where the molar absorption coefficient is ca. 23,000 M⁻¹ cm⁻¹ [5]. This is quite a long wavelength for such a small molecule and arises because of the extensive conjugation running throughout the whole molecule, with the central double bond playing a key role. Substitution at the different ring positions of indigo is likely to promote significant shifts in both the visible and near-UV bands. Sulfonation of the phenyl rings provides access to a water-soluble form, known as indigo carmine, which has an absorption maximum at 618 nm. Fluorescence has been described for indigo in DMF, although the corresponding quantum yield is very low. This is difficult to measure with real accuracy, and values in the region of 0.002 have been reported [6].

The low-fluorescence quantum yield found for indigo merits further investigation. The emission maximum in DMF solution occurs at 653 nm, amounting to a sizeable Stokes shift of 1080 cm⁻¹. This indicates a moderate structural change upon excitation. The fluorescence lifetime for indigo in DMF, measured by time-correlated single-photon counting, is reported to be 135 ps [6,7]. This is close to the temporal resolution of the instrument, and there might be faster decay components that are not made apparent by this technique. Indeed, transient absorption spectroscopic studies made with femtosecond time resolution point toward a complex excited-state landscape. The experimental results, supported by DFT calculations, have been interpreted in terms of very fast intramolecular proton transfer [7]. This is a common mechanism for compounds that have well-defined intramolecular hydrogen bonds in the ground state. Light-induced proton transfer can be extremely fast, although the mechanism is often multi-step and complex while the identity of key intermediates might be poorly defined. Regardless, the short excited-singlet state lifetime minimizes competing reactions, such as triplet state formation, and this leads to highly photostable colorants.

Early attempts to characterize the triplet-excited state of indigo did not give clear results [6,7]. The direct excitation of indigo in solution gives small transient absorption signals on the microsecond timescale, possibly associated with the formation of the triplet state, but nothing convincing. Triplet–triplet energy transfer under pulse radiolysis conditions have also given small absorption changes across the visible spectral range but without definite formation of the triplet state. These experiments were hampered by having to use low concentrations of indigo. Under such conditions, triplet energy transfer from the sensitizer to indigo is slow relative to the rate of decay of the triplet. Subsequent experiments were more successful, and clear evidence for formation of the indigo triplet state was presented [8]. Again, the pulse radiolysis technique was used to sensitize the transient population of the indigo triplet. The latter was found to absorb weakly in the region of 450–700 nm. By using a range of sensitizers having different triplet energies, it was possible to establish the triplet energy for indigo as being 1.04 eV. This corresponds to an energy gap of 0.91 eV between the excited singlet and triplet states [8]. This large energy gap restricts mixing between the excited states, leading to a slow rate of intersystem crossing.

Additional studies were carried out using photoacoustic calorimetry, a technique that measures the amount of heat liberated into a solution during a photophysical process. In the case of indigo in solution, one experiment measured the product of the triplet quantum yield and its excitation energy [8]. Taking the latter to be 1.04 eV, the quantum yield for triplet formation could be determined as being 0.0066. This confirms the ineffective intersystem crossing for indigo inferred from earlier experiments. There is no doubt that the combination of low triplet yield and low triplet energy is responsible for the high level of photostability enjoyed by indigo.

4. Stability and Removal

Indigo is a particularly stable colorant, which is good news for the consumer but, given its exceptionally high usage, amounts to some 70,000 tons of dye annually, potentially bad news for the environment. Indeed, the widespread employment of indigo dyeing technology has resulted in the build-up of possibly toxic by-products and sludge, which needs to be addressed. In part, the resistance of indigo to direct attack is related to the two intramolecular hydrogen bonds formed between the amino and carbonyl groups. These block the sites that would otherwise be susceptible to nucleophilic or electrophilic attack. Certain free radicals, notably the hydroperoxyl radical, can add to the central double bond under mild conditions. Subsequent bond cleavage forms isatin as the majority product. Other free radicals, such as superoxide anions, can react with indigo in solution, with subsequent loss of color. These reactions, however, tend to be relatively slow.

Indigo is also highly resistant to light-induced damage under exposure to sunlight, especially in the absence of molecular oxygen. This is not too surprising, given the fact that the excited-singlet state lifetime is extremely short because of the rapid intramolecular proton transfer alluded to above. There is little time for any competing reaction, and this includes intersystem crossing to the triplet state. The most common mechanism responsible for the photobleaching of a colorant involves a reaction between the triplet-excited state of the dye and molecular oxygen. The resultant singlet molecular oxygen, which is short-lived in solution, attacks the dye in a geminate step and forms highly reactive oxy-radicals, which can initiate a chain reaction. A key point for indigo is that the triplet state is formed in a very low yield. Moreover, the excitation energy of the triplet is too low to effectively sensitize the formation of singlet molecular oxygen, and, in any case, the ground state is not reactive toward this ubiquitous species. Quantitative studies reported by Sousa et al. [5] have indicated a quantum yield for the photobleaching of dye in aerated DMF in the region of 10⁻⁴. The major breakdown product is isatin (Figure 3), but the products considered typical of attack by singlet molecular oxygen were not found. It was also observed that the photobleaching of indigo carmine, a water-soluble form of indigo, in water was significantly slower.

The removal of the color from indigo-contaminated water supplies is easily realized using sodium dithionite as the predominant reducing agent, but this does not solve the pollution problem, In fact, this is exacerbated by the introduction of sulfate into the environment. The adsorption and photocatalytic degradation of the water-soluble form of indigo, indigo carmine, have been achieved in an aqueous suspension of titania as a photocatalyst under UV irradiation. Again, this is not a practical solution. The existing traditional bioremediation approach is a successful technique for treating effluents containing coloring agents; it is aimed at providing a cost-effective, chemical-free and environmentally compatible treatment to mitigate the negative effects of industrial pollution. Various studies have highlighted the enzymatic decolorization of indigo carmine using laccase obtained from different strains or oxidase-enzymes. With appropriate engineering, such technology can be carried out in a cost-effective manner under eco-friendly conditions [9].

A highly innovative approach toward the catalyzed removal of indigo was introduced by Sirbu et al. [10]. This system was designed on the understanding that indigo is reactive toward both hydroperoxyl radicals and superoxide anions in solution. This recognition led to the synthesis of a dual-component catalyst comprising a light-collecting macrocycle equipped with a redox-active terminal subunit. Under illumination with visible light, an intramolecular electron-transfer reaction competes effectively with fluorescence and forms a charge-transfer state. In competition to reverse charge transfer, the oxidized donor, being a butylated hydroxytoluene subunit, ejects a proton to form a neutral phenyloxyl radical. In turn, the initially formed π-radical anion of the colored macrocycle transfers an electron to an oxygen molecule, restoring the original macrocycle and yielding a crop of superoxide anions. The latter reacts with the available proton, forming hydroperoxyl radicals. Finally, the hydroperoxyl radical reacts with the phenyloxyl radical to form a stable alkyl hydroperoxide via a radical recombination step. This latter species functions as the catalyst for the thermal breakdown of indigo. One molecule of photocatalyst can bleach more than 40 molecules of indigo under visible light illumination [10].

5. Conclusions

Indigo is a popular colorant with a long and proud history, stretching back some 5000–6000 years. It was named as such because it reached Europe from the Indus Valley in India. It has advanced from being produced in minute quantities through the extraction from plants, such as woad, to being manufactured on a huge scale. The current annual synthetic production is estimated to be 70,000 tons. Many derivatives have been synthesized, including water-soluble species, and it is now available in a highly purified form. The parent compound has very low solubility in water, and this has helped establish its excellent wash fastness. Indigo is known to be resistant to photofading, and this high level of photostability can be traced to its inherent photophysical properties. For example, the triplet-excited state, which is usually (but not always) responsible for photodegradation, is formed in very low yield and possesses an unusually low excitation energy [8]. The latter ensures that the triplet state cannot enter into energetic photoreactions, such as hydrogen atom abstraction or electron transfer with the substrate. Just as important is the realization that the excited-singlet state is tied up in intramolecular proton transfer, this step being rapid and thereby highly competitive with all other deactivation routes. Light-induced proton transfer is highly reversible and might be considered the ultimate black hole for excited states.

The popular interest in indigo stems almost exclusively from its use in dyeing clothes; the nomadic people of the Sahara desert were called the “blue people” because of their preference for wearing indigo-dyed clothes. During the past decade or so, however, our attitude toward dyeing technologies has changed, and it has become necessary to radically reduce the environmental impacts of these processes. One outcome of this new attitude has been so-called Smart-Indigo™, which replaces the dithionite treatment with electrochemical reduction. Other procedures are seeking to eliminate the use of solvents in the dyeing process. Can indigo meet these challenges? On the scientific front, one of the major successes has been the establishment of a thorough understanding of the chemical and photochemical properties of indigo in terms of contemporary theory. This makes for a nice story. But indigo is not alone in this respect, and other colorants merit similar analytical treatment. Colorants would like to hear about such studies!

6. Note

Editorial Perspectives present personal but authoritative viewpoints on contemporary research topics of particular interest to the journal Colorants. They should present a brief overview of the subject, with any key references, and provide a forward-looking commentary on where a particular research area is heading and where new insight is needed. The articles reflect the authors’ own research interests and should highlight matters that fit nicely into the scope of the journal.