Terahertz Vibrational Modes of Sodium Magnesium Chlorophyllin and Chlorophyll in Plant Leaves

doi:10.21203/rs.3.rs-2072935/v1

Download PDF

Research Article

Terahertz Vibrational Modes of Sodium Magnesium Chlorophyllin and Chlorophyll in Plant Leaves

https://doi.org/10.21203/rs.3.rs-2072935/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 27 Jan, 2023

Read the published version in Journal of Infrared, Millimeter, and Terahertz Waves →

You are reading this latest preprint version

The low-frequency (terahertz) vibrational spectroscopy of two chlorophyll species, Chl-𝑎 and one of its magnesium derivatives (Chl-Mg-Na), has been investigated experimentally. The combination of terahertz time-domain spectroscopy and Fourier transform infrared spectroscopy has enabled a broad frequency range to be covered (0.2 to 18 THz). For Chl-Mg-Na, the terahertz spectra show clear and well-marked features at 1.44, 1.64, and 1.83 THz dominated by intermolecular interactions. The frequency dependent refractive index and absorption coefficient of Chl-Mg-Na were determined using the Fit@TDS software. Below 1.0 THz, a refractive index of 2.09 was measured. In order to acquire further understanding of the observed vibrational modes, a detailed study of the temperature dependence of the line positions of the lowest modes in Chl-Mg-Na was performed. As the temperature is increased from 88 K to 298 K, the feature at 1.83 THz experiences a notable red shift of frequency and line shape broadening, whereas the feature at 1.44 THz shows little change. These results suggest that the 1.83 THz feature is dominated by intermolecular motions occurring over the crystalline unit cell of the Chl-Mg-Na molecular crystal. Finally, terahertz time-domain was used to acquire the spectra of an ornamental plant bearing yellow-green variegated leaves (ivy, Aureomarginata variety), the yellow sectors having lower chlorophyll content compared to green sectors. In dehydrated green tissue, the chlorophyll molecules showed well-marked intermolecular vibrational modes at 1.85 THz, indicating that chlorophyll molecules are prone to pack with an ordered molecular arrangement. These results demonstrate the potential application of THz spectroscopy in agricultural sciences.

Terahertz spectroscopy

chlorophyll derivatives

molecular crystal

chemical recognition

bio-sensing

life sciences

Chlorophyll is an important photosynthetic pigment in plants, playing an essential role in light harvesting and conversion processes. Chlorophyll and its derivatives have long found wide application as food colorants and therapeutic agents. Moreover, the demand for chlorophyll derivatives has recently increased for their application as efficient catalysts [1], functional materials in molecular electronics [2–4] and as photoactive materials for new types of solid-state biosolar cells[5, 6]. The chemical structures of natural chlorophylls and their derivatives strongly influence their absorption and emission spectra. It is therefore expected that all aspects of their spectra in the ultraviolet-visible and infrared regions have been thoroughly studied by spectroscopic techniques. However, while much is known about the optical properties of chlorophyll in these frequency ranges, this is not quite the case for the terahertz (THz) region. This region of the electromagnetic spectrum, between 3 and 333 cm^− 1, has a greater sensitivity towards intermolecular interactions compared to other techniques [7–15] The intermolecular vibrations observed in the crystalline phase have frequencies much lower (typically, less than 200 cm^− 1) than intramolecular vibrations. However, in some cases, where molecular vibrations of unusually low frequency are present, the separation is impossible and the low-frequency vibrational modes result from a mixture of intermolecular and intramolecular motions. Since the low-frequency vibrations strongly depend on the intermolecular interactions, each molecular crystal has a unique spectral fingerprint at THz frequencies.

The rapid development of THz time-domain spectroscopy (THz-TDS) as a highly versatile analytical tool for the characterisation of materials has drawn considerable attention over the past two decades. Typical THz-TDS instruments can cover the frequency range of approximately 0.1–4 THz (3–133 cm^− 1) [8]. In chemistry and pharmaceutical sectors, THz-TDS gives access to new means for identification, detection, characterization and quantification of a substance [7]. Therefore THz spectroscopy that reveals the global structure of the material is a promising tool for the study of the weak intermolecular interactions found in chlorophyll molecular crystals.

Although some articles have reported investigation of the vibrational modes of chlorophylls and chlorophyll derivatives [16–18] using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy, they merely presented some intramolecular vibrational properties of the molecules for frequency ranges larger than 5 THz (167 cm^− 1). Only a few experimental and theoretical investigations in the THz frequency range are known regarding the natural chlorophylls [19–22] or chlorophyll derivatives. In addition, the extension of such investigations to chlorophyll in plant tissues would offer a rich but challenging sector of study. When plants are exposed to abiotic stresses, they undergo complex molecular changes such as chlorophyll contents and ratios. An attractive feature of THz probing of chlorophyll in plants is i) its non-invasive character which allows the measurement on leaves during long periods of time, ii) the relatively low scattering losses due to long wavelengths compared to the leaf roughness [23], iii) the possibility to probe all the chlorophyll located in the different layers of the leaf. Surprisingly, to the best of our knowledge, only a few reports are available of attempts to discover chlorophyll-𝑎 (Chl-𝑎) fingerprint in plant leaves at THz frequencies [21, 24]. Chlorophyll is a group of fat-soluble magnesium porphyrin complex which is widely found in green sectors of plants. Sodium magnesium chlorophyllin (Chl-Mg-Na) is a derivative of chlorophyll which displays additional advantages properties, including good water solubility, excellent photosensitivity, and thermal- and photo-stability. The molecular structures are shown in Fig. 1. The structure of Chl-𝑎 consists of a porphyrin ring, whose four nitrogen atoms surround a central magnesium atom, several other functional groups and a hydrocarbon tail (Fig. 1a). The structure of Chl-Mg-Na has the same unsymmetrical porphyrin ring as Chl-𝑎 without the long flexible hydrocarbon chain (Fig. 1b). Chl-Mg-Na is thus more stable to long-term irradiation and exhibits characteristic blue and red absorption bands that are very similar to those of Chl-𝑎. Investigating the low-frequency vibrations of chlorophyll derivatives will be helpful for not only completing the data library of the THz properties of materials, but also for a better understanding of the THz properties of the natural chlorophylls, chlorophyll derivatives, and of more complex structures related to chlorophyll [25].

In this work, we report on our THz and far-infrared spectroscopic study of Chl-𝑎 and Chl-Mg-Na. Intrinsic optical characteristics of Chl-Mg-Na in the THz frequency range were extracted with the Fit@TDS software [26], and the frequency shift and amplitude dependence of the THz vibrational modes with temperature have been examined. Overall, our results obtained for green and yellow sectors of leaves of variegated plants (ivy, Aureomarginata variety) demonstrate the possibility of chlorophyll detection during different hydration states.

2.1 Chl-𝑎 and chlorophyll derivative samples preparation

Chl-𝑎 powder purchased from Sigma-Aldrich was stored in the dark at -25◦C before preparing pellets. Chl-Mg-Na powder was purchased from Catalyons and according to the manufacturer, 0.5% magnesium stearate was added as a lubricant. Nevertheless, the powder was used without any further purification. The chlorophyll derivative samples used for mass spectroscopy, absorption spectra in the visible range and for surface-enhanced Raman spectroscopy (SERS) were dispersed in water or methanol at appropriate mass ratios. For SERS measurements, colloidal silver nanoparticles (AgNPs) were added to the solutions. The samples used for THz-TDS and FTIR measurements were prepared by mixing the polycrystalline Chl-𝑎 or Chl-Mg-Na and high-density polyethylene (PE) powders. The mixtures were then pressed with an applied pressure of 5 tons for three minutes. The resulting pellets have a diameter of 13 mm. The Chl-𝑎-PE pellet has a small amount of chlorophyll: less than 5 mg in 100 mg PE. In contrast, having a large amount of Chl-Mg-Na powder, a fairly large number of Chl-Mg-Na-PE pellets were prepared, with different concentrations and thicknesses. These pellets, necessary for measuring the THz transmission in a suitable frequency range, contained from 10 to 50 weight-percent (wt-%) of Chl-Mg-Na within the PE matrix and have thicknesses between 50 µm and 1.2 mm. Moreover, as the chlorophyll derivative powders compact well, pellets containing only Chl-Mg-Na were prepared. Pellets containing from 0.5 to 2.0 wt-% of magnesium stearate within the PE matrix were also prepared. Table 1 summarizes the different pellet fabrication conditions.

Table 1

Weight of pellets containing different relative amounts of PE and Chl-𝑎, of PE and Chl-Mg-Na, of PE and magnesium stearate, and of PE and dehydrated ivy tissues. The mixture composition is given in weight-percent (wt-%).
Pellet No.	Mixture composition	Pellet Mass (mg)	Substance Quantity (mg)	Measurements
1	~ 4.0 wt-% Chl-a	115.0	4.6	FTIR &THz-TDS at 298 K
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16	100 wt-% Chl-Mg-Na 100 wt-% Chl-Mg-Na 100 wt-% Chl-Mg-Na 100 wt-% Chl-Mg-Na 100 wt-% Chl-Mg-Na 100 wt-% Chl-Mg-Na 50.0. wt-% Chl-Mg-Na 50.0 wt-% Chl-Mg-Na 50.0 wt-% Chl-Mg-Na 30.0 wt-% Chl-Mg-Na 30.0 wt-% Chl-Mg-Na 42.5 wt-% Chl-Mg-Na 25.7 wt-% Chl-Mg-Na 25.7 wt-% Chl-Mg-Na 17.2 wt-% Chl-Mg-Na	202.8 179.6 97.9 72.6 77.8 60.0 302.6 202.0 60.1 116.8 83.2 72.6 61.0 37.3 79.2	202.8 179.6 97.9 72.6 77.8 60.0 151.3 101.0 30.0 35.0 25.0 30.9 15.7 9.6 13.6	THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K THz-TDS vs temperature THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K FTIR at 298 K FTIR at 298 K FTIR at 298 K FTIR at 298 K
17 18 19 20	100 wt-% PE 100 wt-% PE 0.5 wt-% magnesium stearate 2.0 wt-% magnesium stearate	116.8 98.7 300.0 300.0	116.8 98.7 1.5 6.0	THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K THz-TDS at 298 K
20 21	50.0 wt-% dehydrated ivy green tissue 50.0 wt-% dehydrated ivy yellow tissue	200.0 185.0	100.0 92.5	THz-TDS at 298 K & 88 K THz-TDS at 298 K & 88 K

2.2 Plant Material

To investigate the intermolecular vibrational properties of chlorophyll molecules in plant tissues, well developed leaves of the variegated ivy were examined (Aureomarginata variety). For the Aureomarginata variety, leaves show a green centre, a yellow margin and intermediate pale green sectors. For THz-TDS transmission experiments, the yellow and green sectors of mature and fresh leaves were separated by cutting and used to prepare yellow or green powders. The powders were both mixed with PE and compressed into 13 mm diameter pellets by applying a constant pressure of 5 tons for three minutes.

2.3 Mass Spectroscopy

Electrospray ionization mass spectroscopy analysis (ESI-MS) and MS/MS experiments were performed on the Chl-Mg-Na product, the mass spectrometer operating in the positive mode. The MS-spectra were acquired by full range covering the scale m/z 100–1000. For the fragmentation study, MS/MS experiments were performed by deploying collision-induced dissociation with the normalized collision energy set between 10 and 50 eV.

2.4 Surface-Enhanced Raman spectroscopy

Complementary information about intramolecular vibrations of Chl-Mg-Na was investigated by SERS detection. For SERS spectra acquisition, the excitation photon wavelength (532 nm) was close in wavelength to the Q_y band [27] (see the Supporting Information), the spectral window recorded between 200 and 1600 cm^− 1, and each Raman and SERS spectrum was an average of 3 successive scans. Chl-Mg-Na was found to be readily soluble in water, but only slightly soluble in methanol. As a result, the absorption of Chl-Mg-Na recorded by a UV-Vis-NIR spectrometer showed that the Q_y band shifted slightly from 653 nm for the aqueous solution (1 mg/mL solution diluted to 60 µg/mL in H₂O) to 648 nm for the methanol suspension (0.5 mg/mL in MeOH further diluted to 25 µg/mL in MeOH)) (Supporting Information). In the Raman and SERS study, three solutions were analysed. Solution 1 (S1) was prepared by adding 1 mL of MeOH to 0.54 mg of Chl-Mg-Na. Solution 2 (S2) was prepared by mixing 200 µL of AgNPs (40 nm citrate-capped AgNPs at 0.03 nM) with 200 µL of S1. Solution 3 (S3) was prepared by adding 100 µL a 0.1 M aqueous solution of NaCl to S2. Addition of NaCl to AgNPs promotes aggregation of the nanoparticles, a key role to the signal enhancements observed in SERS. The laser power (incident laser power was 40 mW) was set at 2 mW for S1 and S2, 4 mW for S3 and an average scan time of 0.5 s for all solutions.

2.5 Terahertz spectroscopy

Low-frequency vibrational modes of Chl-𝑎 and Chl-Mg-Na were collected in transmission configuration with THz-TDS and Fourier transform infrared (FTIR) spectrometers, to cover the spectral absorption range from 0.2 THz to 18 THz.

FTIR Spectroscopy experimental setup. FTIR spectroscopy is most useful in high frequency range of the THz spectra and has low signal/noise ratio at lower frequencies. Measurements were performed with a Brucker IFS 66v/S spectrometer that accesses the THz range using a liquid helium cooled bolometer. The transmission was measured in vacuum over the frequency range (0.5–18 THz) with a spectral resolution of 0.5 cm^− 1 (0.015 THz). FTIR spectra were acquired with at least 100 scans. The FTIR spectra were normalized using the empty aperture as a reference. The absorbance spectra was calculated and had a background correction using the Vancouver algorithm [28] with a 5th order polynomial fit (Supporting Information). FTIR spectroscopy can obtain amplitude information only.

THz-TDS experimental setup. THz-TDS has the advantages of detecting the amplitude and phase of coherently generated THz radiation, and provides a better signal to noise ratio than FTIR spectroscopy at frequencies below 3 THz (100 cm^− 1). THz-TDS and FTIR can thus complement each other for the study of intramolecular and intermolecular vibrational modes. The THz-TDS spectra were acquired using a commercial TeraPulse 4000 instrument (TeraView Ltd, Cambridge, UK) across the spectral range of 0.1–3.5 THz. To minimize the contribution of the water vapour to the sample spectrum, the sample chamber was either purged continuously with dry nitrogen or evacuated throughout the measurements. For a given sample, the empty aperture was used as a reference and the raw absorbance calculated. In addition, the peaks associated to the low-frequency vibrational modes were treated as the spectra of interest. Then, to eliminate the varying background mainly caused by the presence of amorphous, disordered regions in the samples, the raw absorbance calculation was followed by a background correction using the Vancouver algorithm with a 5th order polynomial fit. Variable temperature were carried out using a liquid nitrogen cooled cryostat. The spectra were acquired while cooling over a temperature range, from 298 to 88 K. At each temperature, the samples were left to equilibrate for approximately 10 min before starting measurements.

3.1 FTIR and SERS spectra

Our initial efforts to explore intermolecular vibrations have focused on polycrystalline Chl-𝑎, for which intramolecular vibrations at frequencies above 5 THz (167 cm⁻¹) have already been explored with solutions, suspensions [17] and solid films [18]. Our measured far-infrared absorbance spectra of the Chl-𝑎-PE pellet containing a low amount of Chl-a (pellet 1, around 4.0 wt-%) are presented in Fig. 2a, after normalization and background correction with the Vancouver algorithm. The results from the FTIR experiments plotted between 4.5 and 11 THz (150 and 367 cm⁻¹) show that the main far-infrared spectral features of Chl-𝑎 molecules have three strong characteristic absorption bands centred at 6.01 THz (200 cm⁻¹), 8.83 THz (295 cm⁻¹), and 9.24 THz (308 cm⁻¹). These peak positions, reported in Table 2, agree well with the spectra presented in Refs. 17 and 18. Far-infrared studies have shown that aggregation of chlorophyll induces a supplementary 300–312 cm⁻¹ absorption band. The features above 200 cm⁻¹ primarily correspond to intramolecular bonds in Chl-𝑎. Many of the features have been assigned [16–18] and relate mainly to skeletal deformations of the porphyrin section of the Chl-𝑎 molecule, with strong contributions from Mg-N or Mg-O bending and stretching modes. A similar description may be applied to the far-infrared and THz spectra of Chl-Mg-Na but no comparison could be made using previous studies from the literature. Far-infrared absorbance spectra were obtained using different concentrations of Chl-Mg-Na powder in the PE matrix (pellets 13 to 16, Table 1) and are reported in Fig. 2b. Compared to the far-infrared spectra of Chl-𝑎 in Fig. 2a, a number of well-resolved and narrower absorption peaks are seen in the range 3–18 THz (100–600 cm⁻¹).

The higher Chl-Mg-Na concentration (pellet 13, blue curve) led to saturation of the strong absorption band centred at 5.32 THz (177 cm^− 1) in the far-infrared spectrum but resulted in more pronounced peaks in the THz range (Fig. 2b). These peaks will be discussed in more detail in the paragraph on THz-TDS spectra results. In addition to the major peaks reported in Table 2, many weaker features appeared, which leads to peak broadening, and formation of shoulders. Complementary Raman and SERS experiments were also carried out. While the intensity of many bands typically changes in both SERS and FTIR spectra, SERS combines the specificity of vibrational Raman spectroscopy with the increased sensitivity provided by plasmon assisted scattering, induced by colloidal AgNPs [29]. Further enhancement of the SERS signal can be gained by adding salt to colloidal AgNPs, this causes slight aggregation of the nanoparticles and leads to the formation of SERS ‘hotspots’ [30]. Figure 3 presents Raman spectrum of Chl-Mg-Na in solution and SERS spectra in presence of AgNPs. The peaks in this frequency range (200–1600 cm^− 1) are governed by the intra-molecular vibrations of the sample. Below 400 cm^− 1, the Chl-Mg-Na solution exhibits a broad feature in the 290–310 cm^− 1 region (8-69-9.29THz) observed in FTIR spectra and arising from aggregation. The comparison of the SERS absorption peaks of Chl-Mg-Na with both SERS [31] and resonant Raman spectra of Chl-𝑎 [32] reveals that the SERS spectrum of Chl-Mg-Na is almost similar to that of Chl-𝑎. Most of the frequency difference are less than 10 cm⁻¹. In the intramolecular frequency range, the SERS spectra of the natural Chl-𝑎 and of Chl-Mg-Na being qualitatively similar suggests that the features observed at 302 and 340 cm⁻¹ in the Chl-Mg-Na spectrum can be assigned to MgN₄ intramolecular vibrations.

Table 2

Frequency position of THz and far-infrared absorption features of Chl-𝑎 and of Chl-Mg-Na at room temperature (in THz and in cm⁻¹). Data from other reports are also included^{*,♦,♠,♣,♥}.
Chl-𝑎					Chl-Mg-Na
Chl-𝑎 THz-TDS & FTIR (This work)	Chl-𝑎♦ THz-TDS	Chl-𝑎♠ THz-TDS	Chl-𝑎♣ FTIR	Chl-𝑎♥ FTIR	Chl-Mg-Na THz-TDS (This work)	Chl-Mg-Na FTIR (This work)	Chl-Mg-Na SERS (This work)
1.45 (48.4) s 1.56 (52.0) w 1.73 (57.7) vs 1.86 (62.0) s 3.72 (124) m 4.83 (161) m 6.01 (200) vs 7.05 (235) m 7.85 (262) m 8.83 (295) vs 9.24 (308) vs 10.56 (352) m	1.64 (54.7) s 1.72 (57.4) vs 1.86 (62.0) vs 1.98 (66.0) s	1.42 (47.4) s 1.68 (56.0) sb 2.45 (81.7) m	5.46 (182) w 5.97 (199) mb 7.85 (262) w 9.08 (303) vs 10.40 (347) m 11.39 (380) w 11.93 (398) s 13.97 (466) m 14.27 (476) w	5.70 (190) mb 6.24 (208) sb 6.48 (216) m 7.31 (244) w 8.24 (275) m 9.11 (304) mb 11.33 (378) vs 13.01 (434) s 13.76 (459) m	1.442 (48.1) s 1.535 (51.2) vw 1.637 (54.6) m 1.823 (61.3) vs 1.96 (65.4) m 2.03 (67.7) vw 2.24 (74.6) w 2.57 (85.8) m 2.84 (94.5) m	1.83 (60.8) vs 2.24 (74.6) w 2.57 (85.8) s 2.84 (94.5) m 3.44 (114) s 5.32 (177) vsb 5.78 (193) vsb 6.47 (216) w 7.10 (237) m 7.83 (261) s 8.43 (281) vs 8.82 (294) vs 9.39 (313) m 10.36 (345) m 10.96 (365) s 12.01 (400) s 12.14 (405) s 12.54 (418) w 13.22 (441) m 14.17 (472) vs 15.02 (501) wb 15.67 (522) s 16.13 (538) s 16.56 (552) s 17.50 (583) m	8.69–9.29 (290–310) m 10.19 (340) m 10.85 (362) m 11.09–11.69 (370–390) mb 12.62 (421) s 13.64 (455) m 14.24 (475) w 15.02 (501) m 16.37 (546) m 17.50 (583) m
*Entries: s, strong; m, medium; w, weak; b, broad; v, very. ^♦Data from Wagner [21], ^♠Data from Qu [20], ^♣Data from Fujiwara [17], ^♥Data from Tajmir-Riahi [18].

3.2 THz-TDS Spectra of Chl-Mg-Na and Chl-𝑎

Figure 4a shows the raw absorbance data for three pellets of Chl-Mg-Na (pellets 8–10) and one pellet of PE (pellet 18). The weak oscillations at low frequencies for the Chl-Mg-Na pellets are artefacts caused by multiple reflections of the probe beam through the sample, while well-resolved and sharp spectral features are observed above 1.3 THz. In addition, the observed background absorption may indicate the presence of amorphous regions in the Chl-Mg-Na samples. Due to the lack of long-range symmetry all sharp spectral features disappear, but absorption remains significant due to the random orientation of intermolecular bonds and scattering losses. The background corrected spectra obtained using the Vancouver algorithm with a 5th order polynomial fit method for the four pellets are shown in Fig. 4b. For a clearer representation the data have been offset vertically. For the higher Chl-Mg-Na quantity (pellets 8 and 9), three well-resolved features were obtained at 1.44, 1.64, and 1.83 THz (Fig. 4b). An enlarged view of the features observed for Chl-Mg-Na above 1.3 THz is shown in Fig. 4c, and for comparison, the THz-TDS spectra of Chl-𝑎 is displayed in the inset of Fig. 2a.

While some similarities with the Chl-𝑎 spectra are observed, the strong features observed in Chl-𝑎 around 1.45 THz, 1.73 THz and 1.86 THz are slightly shifted towards lower frequencies for Chl-Mg-Na (0.01 THz, 0.09 THz and 0.03 THz, respectively). For the two polycrystalline species, these spectral features, as in many molecular crystals, may be explained as originating from mixed intermolecular and intramolecular vibrations. For the pellets with high quantity of Chl-Mg-Na, the feature around 1.96 THz was difficult to identify, as a direct consequence of limited dynamic range of the experiment, the sample signal reached the noise floor level of the experiment.

To acquire robust absorption spectra above 1.9 THz, smaller amounts of Chl-Mg-Na were therefore required. The absorption features at low frequencies (pellets 10 and 12) significantly decrease in intensity, but the sample signal reached the noise floor level of the experiment at higher frequencies [8]. Thus the features at higher frequencies (1.96, 2.24, 2.57 and 2.84 THz) can be clearly observed. In addition, the inset in Fig. 4c shows that THz-TDS spectra and FTIR spectra are in good agreement, and demonstrate the reproducibility and complementarity of both techniques in this study. It was aforementioned that the Chl-Mg-Na powder includes a small percentage of magnesium stearate (< 0.5 wt-%) for lubricating purposes. In order to determine the effects magnesium stearate can have on the current study, pellets 19 and 20 were prepared with 0.5 and 2 wt-% of magnesium stearate in PE respectively (Table 1). As expected, no absorption peaks were observed in the THz frequency range (Supporting Information), due to either the low concentration or to the amorphous nature of the magnesium stearate.

The relatively strong and well-resolved features observed for Chl-Mg-Na samples present an opportunity for quantitative analysis. Therefore, we have investigated the response of the stronger features (1.44, 1.64, and 1.83 THz) with change of chlorophyll quantity. The frequency positions of the three peaks were determined by a multipeak-fit using Lorentzian function (Inset of Fig. 5) with amplitude, full-width half-maxima, and offset as parameters. Figure 5 presents the plot of the absorbance (after background subtraction) as a function of quantity of chlorophyll in the pellet. This was done at all three frequencies with a fitted line crossing zero referring to zero amount. As anticipated, the background corrected absorbance increases linearly with Chl-Mg-Na quantity in the pellet. The results clearly suggest that the feature at 1.83 THz can be used for quantification of chlorophyll in polycrystalline form.

3.3 Refractive Index and Absorption Coefficient of Chl-Mg-Na material extracted from THz-TDS data

To extract precise and reliable intrinsic optical parameters from the Chl-Mg-Na material (Supporting Information), we made use of the open-source time-fitting software called Fit@TDS and developed by Peretti et al. [26]. The software enables a user to compare the recorded temporal pulse with the modelled one using an optimization approach. We performed the THz-TDS measurements in a timing window of 500 ps with steps of 20 fs for optimal frequency resolution. In order to adequately fit the data and retrieve the refractive index, the absorption coefficient, the frequency, the width and the oscillator strength of the features observed in the THz range, we followed the methodology used in Ref. 26. First, the Fabry-Pérot oscillations were retrieved by fitting the transmission data of a pure Chl-Mg-Na pellet (pellet 6) using the simplest model. The resulting residual error obtained for each pellet exhibited broad, high frequency losses, hence we added the strong oscillator observed around 5.5 THz in the far-infrared measurements (Fig. 2b). To take into account the well-marked features in the absorption spectrum, we added the four oscillators associated with the features at 1.44, 1.64, 1.83, and 1.96 THz (Table 3). The experimental time-trace of the pellet recording in the time-domain and the resulting fitted time-trace are illustrated in Fig. 6a. The corresponding spectra in the frequency-domain are displayed in Fig. 6b. The fitted values of the real part of the refractive index \(\varvec{n}\left(\varvec{\omega }\right)\) and of the absorption coefficient \(\left(\varvec{\omega }\right)\) are shown in Figs. 6c and 6d, respectively. All absorption peaks are accompanied by significant change of the refractive index near the feature. Below 1.0 THz, the Chl-Mg-Na material has a constant refractive index \(\varvec{n}\left(\varvec{\omega }\right)\) of approximately 2.09. The absorption coefficient \(\left(\varvec{\omega }\right)\) remains < 150 cm^− 1 below 2.5 THz. Table 3 lists the resulting fit parameters of the features, extracted from the pure Chl-Mg-Na pellet. It should be noted that the thickness extracted from the fit in the time-domain (408 µm), is in good agreement with the digital micrometer measurement (426 µm).

Table 3

Optimized parameters for the first four absorption features of the 77.8-mg pure Chl-Mg-Na sample (pellet 6).
Fitted thickness: 408 µm, Permittivity at very high frequency: 3.833
Oscillator	Oscillator Frequency [THz]	Oscillator Linewidth [THz]	Oscillator strength
1 2 3 4	1.449 1.659 1.836 1.963	84.3 10^− 3 85.3 10^− 3 87.9 10^− 3 81.2 10^− 3	1.79 10^− 3 0.50 10^− 3 7.04 10^− 3 1.50 10^− 3

3.4 Temperature dependence of the low frequency vibrational features for Chl-Mg-Na

Observing the temperature dependence of the low-frequency vibrational modes provides additional information and can play a decisive role in the assignment of the features. At 0 K, the motions of atoms around the equilibrium minima are well described by a harmonic potential. As temperature increases, a comparison of the experimental and calculated vibrational frequencies for most of the molecular crystals indicates the presence of pronounced anharmonicity in the intermolecular interactions [10, 11]. In addition to anharmonicity, temperature-dependent structural changes can also have a large effect on vibrational features. Thus, as the temperature is increased, the THz features experience notable line shape broadening and shifts in frequencies. Investigating the temperature changes of the low-frequency vibrational features of chlorophyll is critical for future simulations and assignments of the modes in chlorophyll THz spectra at room temperature. Figure 7a shows the background corrected absorbance spectra of a pure Chl-Mg-Na pellet from 88 K to 298 K (pellet 7). The low-temperature spectra have significantly sharper, well-defined features.

This thermal effect is particularly pronounced for the peak centred at 1.83 THz (Fig. 7b). The amplitude, frequency position, and full-width half-maxima of the four well-resolved features at 1.44, 1.64, 1.83, and 1.96 THz were determined by a multipeak fit using a Lorentzian function and these parameters were plotted against temperature in Figs. 8a, 8b, and 8c, respectively. As the temperature is increased (\(\varvec{T}\)= 212 K), the changes in amplitude of the strong absorption peak centred at 1.83 THz is of 17%. Its red shift is pronounced with a value of 0.030 THz (1.0 cm^− 1), while the changes in full-width half-maxima is of 0.02 THz (0.50 cm^− 1). It should be noted that the effect would have been more marked when using THz spectra recorded at the lowest possible temperature to reduce the anharmonic effects. Interestingly, the spectral features at 1.44 THz does not shift significantly and does not display a large temperature dependence in terms of amplitude and full-width half-maxima. This implies that anharmonicity and thermally induced structural changes only have a weak contribution in the cases of this vibrational feature. Further work to study the behaviour of the vibrational modes of Chl-Mg-Na without studying the temperature dependence has been done. This was carried out by introducing a small amount of solvent to the material to change the molecular environment and alter the intermolecular vibrational modes [33]. In a previous paper [34], we observed the changes of the THz features of Chl-Mg-Na pellets by changing the degree of hydration. The small amount of water in the Chl-Mg-Na polycrystals was easily removed under vacuum and as a consequence, as the sample dried the intensity of the 1.83 THz feature decreased and its position shifted to lower frequencies. After 6 h of dehydration, the 1.83 THz feature shifted by 0.014 THz (0.47 cm^− 1) towards lower frequencies. In contrast, no drastic change was observed for the 1.44 THz feature. Additional experimental details are provided in the Supporting Information. The results are consistent with the temperature dependence suggesting that the stronger feature at 1.83 THz is dominated by intermolecular vibrations in the Chl-Mg-Na polycrystals.

3.5 THz-TDS Spectra of chlorophyll in plant leaves

Estimating the concentration of leaf pigments using spectroscopy techniques has always been one of the most significant issues in plant sciences [35]. However, the overriding question concerning our study is: can we detect the presence of chlorophyll directly in a plant organ using THz spectroscopy? To address this issue, we have chosen to compare the THz absorption between green and non-green tissues of the leaves in variegated plants. Green sectors of pigment-related variegated plants contain high quantities of chlorophyll, while non-green sectors have little or no chlorophyll [36–38]. Here, leaves of variegated ivy shown in the inset of Fig. 9a were investigated. THz absorption spectra from pellets of dehydrated yellow and green tissues were measured. The first step of the experiments consisted to press dehydrated green and yellow tissues into pellets (20 and 21, respectively). This allowed to measure the chlorophyll absorption over a thickness greater than the thickness of a leaf and the formation of uniform, planar interfaces. THz-TDS measurements were performed at 298 K and 88 K (Fig. 9). The 298 K spectrum of the green pellet contains a very broad feature with a central frequency of 1.86 THz (62.0 cm^-1) (Fig. 9a), while the 88 K spectrum shown in the same figure has a significantly sharper peak that is more well-defined and with a central frequency of 1.87 THz (62.4 cm^-1). The frequency value obtained at 298 K remains very close to the one observed in our previous studies with isolated Chl-𝑎. The yellow pellet showed a very weak feature at low temperature with a central frequency of 1.86 THz (Fig. 9b). This indicates that the chlorophyll content in the yellow sectors of a variegated variety is low [36, 37]. It should be noted that plants also contain Chl-b that can contribute to the diversity of recorded spectra. Thus, for a more comprehensive analysis, the investigation of low-frequency vibrations in Chl-b would be important for future studies. In the work presented herein, the low-frequency vibration around 1.86 THz suggests that weak non-covalent interactions between chlorophyll molecules can form during the dehydration process. The self-aggregation (and crystal packing) is dense enough to show well-marked intermolecular vibrational modes.

In this paper, THz-TDS was used to obtain the absorption properties of two chlorophyll polycrystalline powders; Chl-𝑎 and ChI-Mg-Na, and for chlorophyll directly measured from plant leaves. For the Chl-𝑎 polycrystalline powder, the THz-TDS data are in agreement with the few results presented in the literature, with sharp features at 1.45, 1.73, and 1.86 THz, observed below 3 THz. Regarding the Chl-Mg-Na powder, for which this frequency range has not previously been explored, well-resolved spectral features with a slight shift in frequency (compared to Chl-𝑎) were identified experimentally at 1.44, 1.64, 1.83, 2.57, and 2.84 THz. Additional work showed that for the frequency range 3-18 THz, the Chl-Mg-Na strong features positions extracted from the FTIR measurements are in agreement with the peaks positions observed in the SERS spectra for Chl-Mg-Na in solution. This comparison shows that the detection of intramolecular vibrations in this chlorophyll derivative was not affected by the medium of the test, and was observed at similar frequencies in solution or in solid state. This is valuable information that can be useful during FTIR investigations of chlorophyll in fresh or dehydrated plants.

The temperature-dependent THz absorption spectra for Chl-Mg-Na provided additional information that will play a decisive role in the future assignment of the features. The temperature dependence reveals the presence of pronounced anharmonicity for the 1.83 THz feature and suggests that this feature is dominated by intermolecular vibrational modes. However, it is crucial that simulation methods are performed to facilitate the interpretation of experimental results shown here, and identify the vibrational motions in the molecular crystal structures. Supplementing these experimental data with accurate simulations will provide a detailed description of the low frequency vibrational spectra, including origin of the different modes, their frequencies and intensities. For variegated plants used in this work, a clear absorption feature was observed from dehydrated green sectors of leaves. The peaks in the fingerprint region were observed to decrease in intensity with decreasing chlorophyll concentration: green sectors gave more intense peaks compared with yellow sectors of the leaves, which contain little to no chlorophyll. These findings are very promising as first steps towards quantification of chlorophyll in plants. Finally, the absorption properties of chlorophyll derivatives using THz-TDS reported here, notably the sharp and well-resolved features of chlorophyll observed in leaves (1.87 THz at 88 K), adds a new entry to the ever-growing list of applications for THz in the agro-environmental field, a field which is becoming increasing important for the future of sustainability on our planet.

Acknowledgements

This project was supported by the Montpellier University of Excellence I-Site MUSE (PRIME@MUSE), by the Contrat de Plan Etat-Région (CPER) - Région Occitanie “PlantEnvi”, and has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N^o 964203 (FET-Open LINkS project).The authors thank Catalyons Laboratories for providing chlorophyll derivative powders.

Ethical Approval

The work and the drafting of this paper follow the ethical and professional principles of the authors and other external people involved. The research does not involve human participants.

Consent to Participate

No external participation is foreseen for this work.

Consent to Publish

If the research is accepted for publication, I consent to publish this work in the Journal of Infrared, Millimeter, and Terahertz Waves.

Authors Contributions

D.C. and N.D. conceived the idea and secured funding for this research. All authors designed the experiments. Y.M. prepared some of the samples. D.C., N.D., E.O., E.B. performed all of the experiments and measurements. D.C., N.D., E.O., E.B., and C.B. performed analysis. All authors interpreted the results. D.C., E.O., and E.B. wrote the main manuscript and prepared the figures. The manuscript was written through contributions of all authors: D.C., E.O., E.B., Y.M., C.B., D.N., K.F., J.T., N.D.. All authors reviewed the manuscript.

Funding

The Montpellier University of Excellence I-Site MUSE (PRIME@MUSE),

The Contrat de Plan Etat-Région (CPER) - Région Occitanie “PlantEnvi”

Funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N^o 964203 (FET-Open LINkS project)

Competing Interests

The authors have no competing interests.

Availability of data and materials

The datasets generated for this study are available from the corresponding authors on reasonable request.

B. Gajewska, S. Raccio, K. J. Rodriguez and N. Bruns, Polymer Chemistry, 10, 125−135 (2019).
S. Y. Chen, Y. Y. Lu, F. Y. Shih, P. Ho, Y. F. Chen, C. Chen, Y. T. Chen and W. H. Wang, Carbon, 63, 23-29 (2013).
Y. Yu, Y. Zhang, L. Jin, Z. Chen, Y. Li, Q. Li, M. Cao, Y. Che, H. Dai, J. Yang and J. Yao, Org. Electron., 65, 381–385 (2019).
B. Yang, Y. Lu, D. H. Jiang, Z. C. Li, Y. Zeng, S. Zhang, Y. Ye, Z. Liu, Q. Q. Ou, Y. Wang, S. L. Dai, Y. P. Yi and J. Huang, Adv. Mater., 32, 2001227 (2020).
S. Erten-Ela, K. Ocakoglu, A. Tarnowska, O. Vakuliuk and D. T Gryko, Dyes and Pigments, 114, 129-137 (2015).
S. Duan, Q. Zhou, A. Li, X. Wang, S. Sasaki and H. Tamiaki, Sol. RRL, 4: 2000162 (2020).
J. A. Zeitler, P. F. Taday, D. A. Newnham, M. Pepper, K. C. Gordon and T. Rades, J. Pharm. Pharmacol., 59, 209-23 (2007).
P. Jepsen, D. Cooke and M. Koch, Laser Photonics Rev., 5: 124-166 (2011).
F. Zhang, H.-W. Wang, K. Tominaga and M. Hayashi, J. Phys. Chem. A, 119, 3008−3022 (2015).
M. T. Ruggiero and J. A. Zeitler, J. Phys. Chem. B, 120, 1733–11739 (2016).
M. T. Ruggiero, J. A. Zeitler and A. Erba, Chem. Commun., 53, 3781-3784 (2017).
J. Neu, H. Nikonow and C. A. Schmuttenmaer, J. Phys. Chem. A, 122, 28, 5978-5982 (2018).
P. Bawuah and J. A. Zeitler, TrAC Trends Anal. Chem., 139, 116272 (2021).
K. Siegrist, C. R. Bucher, I. Mandelbaum, A. R. Hight Walker, R. Balu, S. K. Gregurick, and D. F. Plusquellic, J. Am. Chem. Soc., 128, 5764-5775 (2006).
D. Lee, H. Cheon, S. Jeong and J. Son, Sci Rep, 10, 10271 (2020).
L. J. Boucher, H. H. Strain and J. J Katz, J. Am. Chem. Soc., 88, 7, 1341–1346 (1966).
M. Fujiwara, H. Hayashi and M. Tasumi, Croatica Chemica Ac-ta, 61, 435-446 (1988).
H. A. Tajmir-Riahi, G. Wang and R. M. Leblanc, Photochemis-try and Photobiology, 54, 265-271 (1991).
H. Chen and L. Wang, J. Appl. Phys. 102, 074701 (2007).
Y. Qu, H. Chen, X. Qin, L. Wang, L. Li and T. Kuang, Sci China C Life Sci., 50, 350-355 (2007).
C. Wagner, C. Peveling-Oberhag, C. Heinen, F. Fritzsche, A. Hommes, D. Nüßler and H. Essen, IEEE, 36th International Conference on Infrared, Millimeter, and Terahertz Waves, IRMMW-THz 2011, Houston, Texas, USA (2011).
L. Jiang, J. Yu, C. Li, Y. Xu, B. Jin and Y. Liu, IEEE, 40th International Conference on Infrared, Millimeter, and Terahertz Waves, IRMMW-THz 2015, Hong Kong, China (2015).
Y. Abautret, D. Coquillat, M. Zerrad, X. Buet, R. Bendoula, G. Soriano, N. Brouilly, D. Héran, B. Grèzes-Besset, F. Chazallet and C. Amra, Opt Express 2020, 28, 35018-35037 (2020).
Liu Yi-Ke, Liu Yu-Tong, Xu Xiang-Dong, Yan Wei, Ma Miao, Zhu Hong-Zhao, Ma Chun-Qian, Zou Rui-Jiao Ding Lian and Luo Meng-Jia, Acta Physica Sinica, 2015, 64, 068701.
S. Matsubara and H. Tamiaki, Applied Nano Materials, 2020, 3, 1841-1847.
R. Peretti, S. Mitryukovskiy, K. Froberger, M. Mebarki, S. Eliet, M. Vanwolleghem and J. F. Lampin, IEEE Transactions on Terahertz Science and Technology, 2019, 9, 136-149.
Y. L. Wang, Z. K. Zhou, X. N. Peng, L. Zhou, Z. H. Hao and Q. Q. Wang, Chin. Phys. Lett., 2013, 30, 098702.
J. Zhao, H. Lui, D. I. McLean and H. Zeng, Appl. Spectrosc., 2007, 61, 1225-32.
D. Graham, K. Faulds and W. E. Smith, Chem. Commun., 2006, 4363-4371.
K. Faulds, R. E. Littleford, D. Graham, G. Dent and W. E. Smith, Anal. Chem., 2004, 76, 592-598.
L. L. Thomas, J. H. Kim and T. M. Cotton, J. Am. Chem. Soc., 1990, 112, 25, 9378–9386
C. Zhou, J. R. Diers and D. F. Bocian, J. Phys. Chem. B, 1997, 101, 9635-9644.
N. Nagai, R. Kumazawa and R. Fukasawa, Chem. Phys. Lett., 2005, 413, 495-500.
D. Coquillat, E. O'Connor, E. V. Brouillet, Y. Meriguet, C. Bray, D. J. Nelson, K. Faulds, J. Torres and N. Dyakonova, Proc. SPIE 2021, 11827, Terahertz Emitters, Receivers, and Applications XII, 118270F.
J. B. Féret, A. A. Gitelson, S. D. Noble and S. Jacquemoud, Remote Sensing of Environment, 2017, 193, 204-215.
G. Manenti and G. Tedesco, Caryologia, 1977, 30, 163-176.
S. H. Pao, J. W. Liu, J. Y. Yang, P. Chesson and C. R. Sheue, Taiwania, 2020, 65, 74-80.
Q. Zhang, J. Huang, P. Zhou, M. Hao and M. Zhang, Plants, 2021, 10, 552.

No competing interests reported.

SupplementaryJInfraredMilliTerahzWaves.docx

Download PDF

Journal Publication

published 27 Jan, 2023

Read the published version in Journal of Infrared, Millimeter, and Terahertz Waves →

Editorial decision: Major revision
07 Dec, 2022
Reviews received at journal
18 Oct, 2022
Reviewers agreed at journal
28 Sep, 2022
Reviewers invited by journal
22 Sep, 2022
Editor assigned by journal
22 Sep, 2022
Submission checks completed at journal
19 Sep, 2022
First submitted to journal
16 Sep, 2022

You are reading this latest preprint version

Terahertz Vibrational Modes of Sodium Magnesium Chlorophyllin and Chlorophyll in Plant Leaves

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Experimental

2.1 Chl-𝑎 and chlorophyll derivative samples preparation

2.2 Plant Material

2.3 Mass Spectroscopy

2.4 Surface-Enhanced Raman spectroscopy

2.5 Terahertz spectroscopy

3 Results And Discussion

3.1 FTIR and SERS spectra

3.2 THz-TDS Spectra of Chl-Mg-Na and Chl-𝑎

3.3 Refractive Index and Absorption Coefficient of Chl-Mg-Na material extracted from THz-TDS data

3.4 Temperature dependence of the low frequency vibrational features for Chl-Mg-Na

3.5 THz-TDS Spectra of chlorophyll in plant leaves

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1