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Terminalia chebula: a novel natural product colorimetric sensor for Fe2+ and Fe3+ ions


Natural product like Terminalia chebula as Fe2+ and Fe3+ ions sensor was not reported in the literature till now. Herein, we first reported Terminalia chebula (T. chebula), a natural product used in Ayurveda, as a highly sensitive, simple, and cost-effective colorimetric sensor for the detection of Fe2+ and Fe3+ ions. Terminalia chebula showed a selective colorimetric sensing ability for iron (2+/3+) by changing color from green and pale yellow to blue, having limit of detection level of 43.7 μM and 60.8 μM for Fe2+ and Fe3+ ions, respectively. The concentration-dependent colorimetric determination of iron (2+/3+) was carried out, and the color change to distinguish between different concentrations was excellent. Using High Performance Liquid Chromatography, the fraction having sensing ability was isolated and purified. From the mass spectra of the purified fraction, it was concluded that, the major component responsible for the sensing ability was tri-O-galloyl-β-D-glucose. This chemosensor could be used to detect and quantify Fe2+ and Fe3+ in water samples, which is particularly a useful tool.


Detection and estimation of transition metal ions are extremely important in chemical, biological, and environmental sciences. Iron is one of the most important transition metal ions in living organisms; it is a key transition metal ion in the electron transport process, oxygen transport, and storage in higher organisms. Also, it plays an important role in different enzymes like catalase, peroxidase, superoxide dismutase, etc. by controlling reactive oxygen species generation (You et al. 2015; de Silva 2000; Jiang and Guo 2004). Therefore, regulation and maintenance of iron level in our body is very crucial; because both deficiency and excessive iron uptake can cause a number of disorders like anemia, hemochromatosis, etc. It was reported that iron shortage can lead to heart failure and diabetes (Tkaczyszyn et al. 2018; Madhu et al. 2017). On the other hand, excessive iron can cause disruption of blood vessels, inducing bloody vomitus/stools, liver and kidney failure, and even cause death (; World Health Organization (WHO) recommended maximum allowed level of iron in drinking water is 0.3 mg/L, at this concentration drinking water sometimes acquires a reddish-brown color ( In water, iron is mostly found in two forms: soluble ferrous iron (Fe2+) and less soluble ferric iron (Fe3+) ( In the northern parts of West Bengal, especially, in Alipurduar, Jalpaiguri, Cooch Behar, and Darjeeling districts, ground water is heavily contaminated with iron, which is one of the major contaminants of domestic and agricultural water resources (Rajmohan and Prathapar 2016). Iron can also be found in surface water due to natural deposits, industrial wastes, iron ore refining, and corrosion of iron-containing metal cans. As a result, a cost-effective, simple, highly sensitive, and selective iron chemosensor for households would be beneficial for commoners.

Many analytical techniques, including atomic absorption spectroscopy (AAS), atomic fluorescence spectroscopy (AFS), inductively coupled plasma-mass spectroscopy (ICP-MS), electrochemical methods, and others, are being employed for the detection and quantitative estimation of iron (Welna et al. 2011; Hu 2011; Alberti et al. 2020). All of these methods are costly, time-consuming, and labor-intensive. These methods necessitate a well-trained analyst, pre-treatment of water samples, and in some cases, a specialized laboratory facility as well. Colorimetric detection using ion sensors, on the other hand, can be used to monitor target ions even with the naked eye. As a result, ion sensors have gained a lot of interest when it comes to detecting metal ions such as Fe2+ and Fe3+. Still, the iron sensors are not very easily accessible to the common people. Commercially available ion sensors are costly and chemically synthesized compounds are used as sensing materials, which may cause hazards to the environment if not disposed properly. That is why most of the ion sensors are not available for the general public. And previously, some natural products have been utilized in sensing applications (Pinyou et al. 2010; Saithongdee et al. 2014; Ballesteros et al. 2021). So, there are previous reports of natural products being utilized as a sensor.

In this article, a natural product has been used to solve the problems. We have used Terminalia chebula (T. chebula), which is a traditional medicine belonging to the genus Terminalia, family Combretaceae, and is mostly found in south east Asia (Kumar 2020). The mature dried fruit of T. chebula is an important Indian herb with homeostatic, antitussive, laxative, diuretic, and cardiotonic properties, widely used extensively in the indigenous system of medicine (ayurvedic) (Bag et al. 2013; Upadhyay et al. 2014; Afshari et al. 2016). Terminalia chebula has been reported to contain gallic acid, ellagic acid, tannic acid, ethyl gallate, chebulic acid, chebulagic acid, corilagin, mannitol, ascorbic acid, and other compounds (Grover and Bala 1992; Sheng et al. 2018; Chang and Lin 2012). Terminalia chebula, contains 32% tannin, which can bind to iron very efficiently (Bag et al. 2013; Fu and Chen 2019; South and Miller 1998; Sarkar et al. 2012). In this regard, T. chebula can be evaluated to be an excellent eco-friendly, easily available, and inexpensive colorimetric sensor for the detection of iron in the aqueous phase with a user-friendly method.


Preparation of Terminalia chebula extract

Dried ripen fruits of T. chebula were collected from the local market and authenticated by a professor from the Botany Department, Cooch Behar Panchanan Barma University (CBPBU). Appropriate T. chebula fruits (those which are well ripened, not moldy or rotten and free from any kind of insect attack) were selected, then seeds were removed, grounded to coarse powder using a domestic grinder. The powder thus obtained was stored in vacuum desiccators for subsequent use.

At first 1.5 gm of T. chebula powder was weighed and, taken in a conical flask containing 100 mL of double distilled water. The mix was kept in the conical flask for 24 h at room temperature and filtered. The filtrate thus obtained was preserved in the refrigerator for further use.


The T. chebula raw extract was initially column chromatographed using RP-HPLC (reverse phase high performance liquid chromatography) [Shimadzu SPD-20A UV detector and Shimadzu LC-20AR series pumping system (Shimadzu Corporation, Kyoto, Japan) with Eclipse XDB-C18 (reverse phase) and C18 monochromatic column, respectively], then the active ingredient was identified by treatment of Fe3+ and Fe2+ solutions to different collected fractions from HPLC. The active ingredient of the raw extract for iron sensing was further purified by preparative RP-HPLC with MeOH as mobile phase (isocratic system) on a C18 column (25 × 250 mm), at UV detector: 292 nm, flow rate: 1.0 ml/min with a runtime of 20 min. Subsequently, the purity of the isolated product was checked using the analytical column SB C18-column (ZORBAX SB-C18, 4.6 × 250 mm, 5 μm, UV detector: 292 nm, flow rate: 1.1 ml/min, tR = 2.5 min). Then the chemical structure of the isolated fraction was characterized and confirmed by FAB-MS spectroscopy (Fast Atom Bombardment Mass Spectrometry) (the Korea Basic Science Institute at Daegu, S. Korea) and was found to be m/z 637.9 [M + H]+, and calculated m/z 636.1 for C27H24O18.

UV–Vis titration measurements of Terminalia chebula extract with Fe2+ and Fe3+

For the UV–Vis titration, the prepared extract was diluted with double distilled water in a ratio of 1:250. The desired concentrations of FeSO4 and FeCl3 solutions were prepared for the colorimetric estimation. 2.4 mL of the prepared T. chebula extract was taken and 100–1000 µL of the metal ion solution (final concentration 1.44 × 10–5–1.44 × 10–4 M) was added to it. After mixing and keeping solutions for 5 min in the dark, UV–Vis spectra were measured at room temperature (You et al. 2015; Kang and Kim 2018).

Competitive experiments

For this experiment, the extract was diluted in the ratio of 5:250 and metal ion solutions of NiCl2, CoCl2, HgCl2, CdCl2, Al2(SO4)3, MnSO4, Cr2O3, LaCl3, MgCl2, CaCl2, NaCl, KCl were prepared in distilled water (0.004 M). 1 mL of each metal solution was taken in a test tube containing 1 mL 0.004 M of Fe2+ or Fe3+ solution, followed by addition of 1 mL of the extract. After mixing and keeping for 5 min in dark, the UV–visible spectra of each mixture were taken at room temperature (Gao et al. 2017).

Determination of Fe2+ and Fe3+ in water samples

UV–Vis spectra of water samples containing Fe2+ and Fe3+ were carried out by adding 1 mL of diluted (5:250) stock solution of T. chebula extract and 2 mL of sample solutions. Mixed homogenously, the solutions were allowed to stand for 5 min at room temperature before recording UV–visible spectra (You et al. 2015).

Colorimetric determination of iron concentration by naked eye

A colorimetric technique was utilized to determine the content of Fe2+ and Fe3+ in aqueous solution by the naked eye. Different concentrations of iron solutions (10, 50, and 100 ppm) were prepared, then 200 μL of T. chebula raw extract was added to each solution, to a final volume of 2.5 mL. Color changes were observed after 5 min of waiting. The same technique was performed using a paper strip, at first 200 μL T. chebula raw extract was added onto the strip and dried in the oven. Following that, Fe2+ and Fe3+ solution (50 μL) separately of various concentrations (10, 50, and 100 ppm) were added to the strips, dried and the colors for different concentrations of Fe2+ and Fe3+ were monitored.

Results and discussions

Characterization using HPLC and mass spectroscopy methods

To study the Fe2+ and Fe3+ active ingredient of T. chebula, water extract was purified using RP-HPLC. Initially, the extract was analyzed using preparative RP-HPLC and, out of all the fractions, only one fraction showed activity toward Fe2+ or Fe3+ ions. The single peak at 2.5 min was isolated and lyophilized [Fig. 1(inset)]. Subsequently, the mass spectrum of the isolated compound showed a m/z peak at 637.9 (M + H)+, indicating the presence of tri-O-galloyl-β-D-glucose as a major component responsible for the iron detection (Fig. 1) (; This conclusion is based on previous report that tri-O-galloyl-β-D-glucose is one of the major components of T. chebula. And the mass spectral data also supported this, as the m/z value of 637.9 (M + H)+ only matches with the tri-O-galloyl-β-D-glucose (Bag et al. 2013; Fu and Chen 2019; South and Miller 1998; Sarkar et al. 2012).

Fig. 1
figure 1

Mass spectra of the T. chebula water extract. (inset) The HPLC spectra of purified Fe2+ and Fe3+ active T. chebula

Sensing property toward Fe2+ and Fe3+

The selectivity of the sensor for the detection of Fe2+ and Fe3+ was investigated with different metal ions such as Co2+, Ni2+, Cr3+, Hg2+, Mg2+, Na+, K+, Ca2+, Al3+, Mn2+, La3+, Cd2+. The absorption spectra of the extract with different cations are shown in Fig. 2. Colors of iron with the extract changed instantly from faint green to deep blue and pale yellow to blue for Fe2+ and Fe3+ ions, respectively, and there were no detectable color changes of other metal ions with the extract (Cr3+ and Mg2+ showed very faint yellow color but in comparison to Fe3+ or Fe2+, it is negligible and did not interfere in iron detection). These findings suggested that T. chebula extract could be used as a colorimetric chemosensor for Fe2+ and Fe3+ ions with high selectivity in aqueous medium.

Fig. 2
figure 2

a The color changes of different metal ions in the (a) absence and (b) presence of T. chebula extract. c UV–Vis spectra, d bar graph showing the absorbance of different metal ions in the presence of T. chebula extract

UV–Vis titration was used to investigate the sensing characteristics of the extract with Fe2+ and Fe3+ (Fig. 3). An aqueous solution of T. chebula extract showed absorbance peaks at 215 and 274 nm. On successive addition of Fe2+ aliquots, the absorbance peak of T. chebula decreased and a new absorption peak appeared at 576 nm that increased with the gradual addition of ferrous ion (Fe+2) solution with an isosbestic point at 308 nm. In the presence of ferric ion (Fe+3), characteristic new absorption peak appeared at 576 nm, and that increased with the gradual addition of Fe+3 aliquots, Fe+3 with T. chebula extract exhibited three isosbestic points at 232 nm, 255 nm, and 289 nm. The absorption peaks at 576 nm (characteristics of Fe+2 and Fe+3 complex with T. chebula extract) have molar extinction coefficients of 6.2 × 102 M−1 cm−1 and 2.2 × 103 M−1 cm−1 respectively, and these values are too large for d-d transitions. Thus, the new peak could be attributed to a metal-to-ligand charge transfer (MLCT), which is responsible for the intense color of the solutions (You et al. 2015). By using 3σ/slope, the detection limit of Fe2+ and Fe3+ with T. chebula extract was determined and the values are 43.7 μM and 60.8 μM respectively (Fig. 4) (Kang and Kim 2018; Tsui et al. 2012). From Fig. 4, correlation coefficient (R) values were found to be 0.988292 and 0.91661, respectively, for Fe2+ and Fe3+, which indicates both variables move in the same direction. The P(T < = t) value for one-tail and two-tail was found to be 0.0163% and 0.0327% for Fe2+ and 0.000431% and 0.000863% for Fe3+, which are lower than 5%. The UV–Visible competitive experiments with Fe2+ and Fe3+ in the presence of other transition and common group metal ions were conducted to examine the selectivity for Fe2+ and Fe3+ as shown in Additional file 1: Fig. S1. The detection of both Fe2+ and Fe3+ with the naked eye was unaffected in presence of competing metal ions. In this experiment, the total concentration of the metal ions (iron and other competitive ions) was maintained constant in each test tube, the color intensity in this experiment decreased in the presence of competing ions. In the absence of other metal ions, just iron was present in its entirety. However, when other metals are present, the concentration of iron was only 50% of the total ion concentration. This is what caused the color intensity to change. These findings suggest that the extract may function as a specific chromogenic chemosensor for iron ions. We have conducted experiments to monitor the effect of pH on the iron sensing properties of T. chebula extract over a wide range of pH from 2 to 10. The absorbance at 576 nm increased considerably with increasing pH from 2 to 10, as shown in Additional file 1: Fig. S2 (Ahmed et al. 2020), which can be explained by the presence of acidic hydrogen in tri-O-galloyl-β-D-glucose. As with the increase in pH value, the hydrogen abstraction from the phenolic -OH group will enhance the concentration of the deprotonated form of tri-O-galloyl-β-D-glucose, which will increase the rate of complexation with Fe2+ and Fe3+, resulting an increase in the absorbance values. The iron sensing effect of T. chebula in the presence of Fe2+ and Fe3+ ions were tested in the temperature range of 20–60 °C. Interestingly, the absorbance at 576 nm increases with increasing temperature (Additional file 1: Fig. S2) which may be due to the kinetic effect. The rate of complex formation increases with increasing temperature. In order to check the applicability of T. chebula in extract for the detection of Fe2+ and Fe3+ in unknown water samples, calibration curves for both Fe2+ and Fe3+ ions were drawn. In Additional file 1: Fig. S3 the calibration curve showed a straight line with the suitable recoveries and permissible R.S.D. (Relative Standard Deviation) values (Table 1). The colorimetric assessment of iron concentration using only the naked eye produced excellent results. The color shift between the solutions can be seen very clearly in Fig. 5, and different levels of iron can be easily distinguished using the reported color which may be considered one of the major findings for iron sensing by the naked eye, in both solutions and paper strips (Whatman 41 filter paper strips) (Additional file 1: Fig. S4), thus, this will aid the common people in determining the iron content. Though naked eye detection is not a confirmatory result, yet it can give a qualitative idea. Previously, similar method of detection for iron and also other metals had been reported in the literature (Suresh et al. 2010; Wei et al. 2011; Sun et al. 2020), which proved that naked eye detection is also becoming a major tool for metal ion detection. Therefore, the naked eye detection can be treated as one of the secondary methods. These results warranted that T. chebula could be applied as a detector for iron in real water samples.

Fig. 3
figure 3

UV–Vis spectral changes of T. chebula extract titrated with (a) Fe2+ and (c) Fe3+. b and d are the enlarged part of the graph (500–700 nm range) of Fe2+ and Fe3+ respectively

Fig. 4
figure 4

The detection limit (via 3σ/slope) of T. chebula extract with (a) Fe2+ and (b) Fe3+ (fitting up to x = 0.000101(M), y = 0.09785 for Fe3+) on the basis of UV–Vis titrations. σ means the average of the standard deviations

Table 1 Determination of Fe2+.and Fe3+ in water samples
Fig. 5
figure 5

Colorimetric response of different concentrations of Fe2+ and Fe3+


Terminalia chebula may be the simplest, most selective, efficient, and least expensive chemosensor for Fe2+ and Fe3+ in aqueous conditions, with excellent selectivity for both cations over relevant competing metal ions. The presence of iron was detected by the sensor changing color from a very light green (Fe2+) and pale yellow (Fe3+) to a blue color with a LOD limit of 43.7 μM and 60.8 μM towards Fe2+ and Fe3+, respectively. Furthermore, the sensor might be utilized to measure iron in real-world water samples. Most importantly, the concentration of iron can be measured quickly and simply by comparing it with the reported color, which is a significant result and very valuable for the layperson. As a result of the findings presented here, a novel approach for selective recognition of the most abundant transition metal ions (Fe2+ and Fe3+) in the presence of various common metal ions has been developed. Most importantly, this method can be used for the quantitative estimation of Fe2+ and Fe3+ in water samples and may be accessible to the masses for routine iron detection. Though we cannot quantify Fe2+ and Fe3+ in a mixture. But this method is novel in determining both the ground water and surface water iron content individually, as mostly in ground water iron present as Fe2+ state, while in surface water iron present as Fe3+ (Gülay et al. 2018). The process of turning this analytical approach into a practical and easy-to-use procedure is in progress. The mechanism responsible for this color change as well as the influence of other metal ions of the periodic table on the T. chebula extract will be studied further in the future.

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The authors are thankful to Department of Chemistry, Cooch Behar Panchanan Barma University for providing the necessary support.


This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1F1A1062951). This work was supported by the Soonchunhyang University Research Fund.

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SS performed all the experiments; TJ performed the Mass spectra and HPLC; SS written the manuscript; GB, DD and JI reviewed and edited the manuscript; GB, DD and JI supervised the entire project. All authors read and approved the final manuscript.

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Correspondence to Jungkyun Im, Dilip Debnath or Goutam Biswas.

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Additional file 1

. Figure S1: The colour changes Fe2+ in presence of different metal ions in the (a) absence and (b) presence of T. chebula extract; The colour changes Fe3+ in presence of different metal ions in the (c) absence and (d) presence of T. chebula extract; Bar graph showing the absorbance of T. chebula extract with (e) Fe2+ and (f) Fe3+ in presence of various metal ions. Figure S2: Absorbance of T. chebula extract with Fe2+.and Fe3+ at different (a) pH (b) temperature. Figure S3: The calibration curve (at 576 nm) of T. chebula extract upon the addition of (a) Fe2+.and (b) Fe3+. [Fe2+] and [Fe3+] = 2.29×10-5 – 2.29×10-3 M. Figure S4: Colorimetric response of Fe3+ in paper.

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Sen, S., Singh, T., Im, J. et al. Terminalia chebula: a novel natural product colorimetric sensor for Fe2+ and Fe3+ ions. J Anal Sci Technol 13, 39 (2022).

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