Chlorophyll as a simple, inexpensive and environment-friendly colorimetric indicator for NO₂ gas

INTRODUCTION

Nitrogen dioxide (NO₂) is one of the prevalent oxide species of nitrogen in the atmosphere collectively known as NO_x. It is characterized by a reddish-brown color with a pungent irritating odor. This pollutant gas is produced from combustion of fossil fuels and industrial processes. It is also a component of photochemical smog and responsible for production of acid rain. Its accumulation poses a threat to the environment because NO₂ is highly reactive, corrosive and oxidative, causing discoloration and degradation of plant species.¹ Its presence in the troposphere can also lead to various health issues. At low concentrations, NO₂ can induce cough, nasal and eye irritations. At higher concentrations, it can lead to serious respiratory infections.² These environmental and health concerns due to extensive circulation of NO₂/NO_x in the atmosphere necessitates development of novel approaches for its detection and monitoring.

In the last decade, many studies have been devoted to selectively monitor the levels of NO₂ in the atmosphere. These include metallic oxides,^3–6 organic semiconductors,^7–9 metal complexes,^10,11 and cage-like molecules.^12–17 These approaches however require expensive materials and lengthy procedures for its synthesis. Here, we report a very simple and cheap approach towards sensing of NO₂ gas by using a common pigment naturally occurring in plants: chlorophyll.

Chlorophyll is a metalloporphyrin pigment responsible for the green color of plants. It plays a major role in photosynthesis. However, chlorophyll is greatly affected by pollutant gases. Chapados and co-workers have observed that chlorophyll reacts with gases such as SO₂, H₂S, NO and NO₂,¹⁸ while Guidi et al noted that it can interact with ozone.¹⁹ Bevilaqua and co-workers also suggested that chlorophyll can potentially bind CO₂ gas based on density functional calculations.²⁰ These studies provide possibilities for chlorophyll as gas sensor. However, application of this natural pigment for naked-eye detection of pollutant gases has not been explored.

EXPERIMENTAL

All reagents and chemicals used were analytical grade and purchased from commercial sources. Solvents were used without any purification.

Extraction of chlorophyll

100 g of cogon grass (Imperata cylindrica) was grinded and macerated for 3-5 min. in ethyl acetate. The crude sample was filtered, placed in a shaker for 5 min_. then left overnight at room temperature. The extract was rotavaped to a final volume of 200 mL and carefully decanted into an evaporated dish to exclude any undissolved matter. The extract was then heated to dryness in a water bath at 40°C. The residue was purified using column chromatography (30 g of silica gel with ethyl acetate). The first eluate which was yellow in color was discarded. Methanol was then used to elute the purified extract. The eluate was air dried, resulting to a dark green solid product (0.0395 g). The green product was dissolved with CH₂Cl₂ and used throughout the experiment.

Production of gases

Sulfur dioxide (SO₂) was prepared by adding 10 drops of 2 M HCl to 0.1 g NaHSO₃ in a test tube. Nitrogen dioxide (NO₂) was prepared by reacting a small piece of copper metal with concentrated HNO₃. Carbon dioxide (CO₂) was produced by reacting 0.1 g CaCO₃ with 10 drops of 5M HCl. The gases were introduced into 3.0 mL chlorophyll solutions via a 5-mL syringe.

Titration of chlorophyll solution

NO₂-CH₂Cl₂ solution was prepared by carefully bubbling NO₂ gas to dry CH₂Cl₂. Concentration of NO₂ was determined according to published procedure.²¹ 0-900 mL of NO₂-CH₂Cl₂ solution was used in the titration.

Gas detection using test strips

Filter paper strips (5 cm x 1.5 cm) were soaked in a 5-ml chlorophyll-CH₂Cl₂ solution until dark green strips were formed. Strips were air dried, transferred in a vial and covered. Gas was introduced into each vial using a syringe.

Spectral Analysis

Spectral scans of untreated and gas-treated chlorophyll solutions in CH₂Cl₂ were recorded on a Shimadzu 1700 spectrometer from 300-800 nm. Infrared spectra were recorded on a KBr pellet and analyzed using a Perkin-Elmer 1600 series Fourier Transform Spectrophotometer.

RESULTS AND DISCUSSION

Chlorophyll, a green pigment in plants, was investigated for its potential use as colorimetric indicator for NO₂. Chlorophyll was extracted from cogon grass using ethyl acetate, purified using column chromatography, and then dried. The CH₂Cl₂ solution of chlorophyll was placed in a sealed vial and bubbled with NO₂ gas. This resulted to a drastic color change of the chlorophyll solution from green to yellowish-brown (Fig. 1). Other gases such as CO₂ and SO₂ did not exhibit any color change. Previous studies have shown that CO₂ has no effect on the structure of chlorophyll, while SO₂ gas does not react with chlorophyll in the absence of water.^18,22

Fig. 1 Visual detection of gas samples in solution. A) chlorophyll solution in CH₂Cl₂, B) chlorophyll solution after exposure to CO₂, C) chlorophyll solution after exposure to NO₂, D) chlorophyll solution after exposure to SO₂.

 

Fig1: Visual detection of gas samples in solution. A) chlorophyll solution in CH2Cl2, B) chlorophyll solution after exposure to CO2, C) chlorophyll solution after exposure to NO2, D) chlorophyll solution after exposure to SO2. Click here to View Figure

 

Visible spectra of chlorophyll exposed to NO₂ show prominent bands that are distinct from the bands observed in chlorophyll alone (Fig. 2A). Absorption bands with λ_max at 414 nm and 666 nm shifted to 424 nm and 679 nm, respectively. The band around 470-480 nm also disappeared upon addition of NO₂. These spectral changes are consistent with a study conducted by Chapados et al. showing the effect of NO₂ gas on chlorophyll a multilayer_.¹⁸

 ^{Fig.2 (A) UV-Vis spectra of chlorophyll bubbled with NO₂ gas. (B) UV-Vis titration of chlorophyll with NO₂-CH₂Cl₂ solution. Insert: Plot of change in absorbance at 414 nm versus NO₂ concentration.}

 

Fig 2A: UV-Vis spectra of chlorophyll bubbled with NO2 gas. (B) UV-Vis titration of chlorophyll with NO2-CH2Cl2 solution. Insert: Plot of change in absorbance at 414 nm versus NO2 concentration. Click here to View Figure

 

Fig2 A: UV-Vis spectra of chlorophyll bubbled with NO2 gas. (B) UV-Vis titration of chlorophyll with NO2-CH2Cl2 solution. Insert: Plot of change in absorbance at 414 nm versus NO2 concentration. Click here to View Figure

 

The same spectral change was recorded when chlorophyll was titrated slowly with NO₂-CH₂Cl₂ solution (Fig. 2B). It was also noted that the chlorophyll solution was able to detect NO₂ with concentrations greater than 2 mM in CH₂Cl₂ solution (Fig. 2B insert). Similar titration experiments have been conducted by Rudkevich et al. on calixarenes with NO₂^–CH₂Cl₂ solution to estimate limit of detection for NO_2.¹⁶

Additionally, bubbling oxygen gas into the NO₂-treated chlorophyll solution did not change the spectra. This suggests that there is a strong interaction or irreversible reaction that took place between chlorophyll and NO₂ gas. Thin layer chromatography of the product formed from the reaction of chlorophyll and NO₂ suggests a complete transformation of chlorophyll into a new compound, possibly a nitrated product. This was confirmed by FTIR spectroscopy where distinct bands were detected at 1555 cm^-1 and 1372 cm^-1 characteristic of an –NO₂ group (Fig. 3). Another band at 1282 cm^-1 which was not present at the spectrum of chlorophyll solution may be due to formation of an N-NO₂ group.²³ This result suggests that nitration occurred when chlorophyll was exposed to NO₂. Nitration reactions are known to occur in porphyrin complexes caused by NO₂ gas.²⁴

 

Fig3: Portion of FTIR spectra of chlorophyll before and after NO2 exposure.  Click here to View Figure

 

Interaction between gases and chlorophyll was also studied in solid state using test strips. Figure 4 shows green coloration of filter paper after soaking it in chlorophyll solution. When the dried test strip was exposed to NO₂, the color turned from green to yellow-brown, similar to the color change observed in solution. No color change was noted when test strips were exposed to CO₂ and SO₂. This observation clearly indicates the selectivity of chlorophyll towards NO₂ gas. The study also demonstrates as proof-of-principle the potential use of chlorophyll-based sensors for naked-eye detection of NO₂ gas.

 

Fig4: Gas detection using test strips. A) Test strip with chlorophyll alone, B) Test strip after exposure to CO2, C) Test strip after exposure to NO2, D) Test strip after exposure SO2. Click here to View Figure

 

CONCLUSION

In conclusion, a simple and cheap colorimetric indicator for NO₂ gas based on chlorophyll was developed. This pigment can be obtained easily from plant materials and be used either in solution or as test strips.

Moreover, the method presented here is inexpensive since gases can be produced from common laboratory reagents, and the set-up requires minimal use of equipment. Thus, this method can be performed in the classroom to demonstrate not only the sensitivity of chlorophyll towards NO₂ but also the destructive effect of NO₂ gas to chlorophyll pigment of plants. Further work will be directed towards improving the detection limit and applying it as NO₂ sensor for environmental monitoring.

ACKNOWLEDGMENT

Financial support was provided by the Creative Research and Scholarship Program of the University of the Philippines System.

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