extfieldsofstudy: Chemistry Medicine

text:
A Strategy for Simultaneous Isolation of Less Polar Ginsenosides, Including a Pair of New 20-Methoxyl Isomers, from Flower Buds of Panax ginseng

The present study was designed to simultaneously isolate the less polar ginsenosides from the flower buds of Panax ginseng (FBPG). Five ginsenosides, including a pair of new 20-methoxyl isomers, were extracted from FBPG and purified through a five-step integrated strategy, by combining ultrasonic extraction, Diaion Hp-20 macroporous resin column enrichment, solid phase extraction (SPE), reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and preparation, and nuclear magnetic resonance (NMR) analysis. The quantification of the five ginsenosides was also discussed by a developed method with validations within acceptable limits. Ginsenoside Rg5 showed content of about 1% in FBPG. The results indicated that FBPG might have many different ginsenosides with diverse chemical structures, and the less polar ginsenosides were also important to the quality control and standardization of FBPG.

Introduction
Panax ginseng (PG) C. A. Meyer, well-known as "renshen" in Chinese, is one of the most famous medicinal herbs and used widely as an herbal remedy and healthy food for thousands of years [1]. Ginsenosides are considered as the major active component of PG with multiple pharmacological activities [2][3][4]. Up to now, more than 100 naturally-occurring ginsenosides have been isolated from roots, leaves/stems, fruits, and/or flower buds of PG [5][6][7][8]. In traditional Chinese medicine, PG root is the most commonly used part [9][10][11][12]. Due to its significant tonic and restorative effects, more attention was paid to PG. In addition to PG root, such as leaves and stems, flower buds and fruits were also studied for their medicinal values [13][14][15]. Recently, the ginsenosides in the flower buds of Panax ginseng (FBPG) gradually attracted people's attention, and it was considered as a valuable functional food with medicinal potential. People commonly use it as tea for anti-fatigue and for enhancing immunity [16].
Due to the higher content of total ginsenosides in FBPG than the other parts of PG [17,18], to find new active compounds, further isolation and identification studies of constituents in FBPG were carried out. Some new and minor ginsenosides, such as ginsenoside Rd, floralginsenoside Ka, floralginsenoside Ta, ginsenoside Rk 3 , and ginsenoside Rs 4 , have been identified with extensive bioactivities, including gastroprotective effect [19], radical scavenging [20], and anti-tumor [14] and anti-inflammatory activity [21]. In order to extend the utility value of FBPG, it is necessary to systematically uncover its chemical basis. PG contains many complex and structurally similar ginsenosides. Thus, multidisciplinary methods are required to analyze and identify its genuine structures. With the development of liquid chromatography coupled to mass spectrometry (LC-MS) [22,23], and assisted by the proper sample pre-preparation methods, it has become relatively simple and efficient to study ginsenosides.
Based on our previous work on the isolation and determination of the isomeric compounds ginsenoside F 5 and F 3 from FBPG [24], in this study, a five-step integrated strategy, by combining ultrasonic extraction, Diaion Hp-20 macroporous resin column enrichment, solid phase extraction (SPE), reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and preparation, and NMR analysis, is presented ( Figure 1). We obtained five less polar ginsenosides, including a pair of new isomeric ginsenosides ( Figure 2). Moreover, the quantification of the five ginsenosides in FBPG was also involved. This research will provide a novel scientific method for comprehensive studies of various ginsenosides in FBPG and make full use of ginseng resources. systematically uncover its chemical basis. PG contains many complex and structurally similar ginsenosides. Thus, multidisciplinary methods are required to analyze and identify its genuine structures.
With the development of liquid chromatography coupled to mass spectrometry (LC-MS) [22,23], and assisted by the proper sample pre-preparation methods, it has become relatively simple and efficient to study ginsenosides. Based on our previous work on the isolation and determination of the isomeric compounds ginsenoside F5 and F3 from FBPG [24], in this study, a five-step integrated strategy, by combining ultrasonic extraction, Diaion Hp-20 macroporous resin column enrichment, solid phase extraction (SPE), reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and preparation, and NMR analysis, is presented ( Figure 1). We obtained five less polar ginsenosides, including a pair of new isomeric ginsenosides ( Figure 2). Moreover, the quantification of the five ginsenosides in FBPG was also involved. This research will provide a novel scientific method for comprehensive studies of various ginsenosides in FBPG and make full use of ginseng resources.    systematically uncover its chemical basis. PG contains many complex and structurally similar ginsenosides. Thus, multidisciplinary methods are required to analyze and identify its genuine structures.
With the development of liquid chromatography coupled to mass spectrometry (LC-MS) [22,23], and assisted by the proper sample pre-preparation methods, it has become relatively simple and efficient to study ginsenosides. Based on our previous work on the isolation and determination of the isomeric compounds ginsenoside F5 and F3 from FBPG [24], in this study, a five-step integrated strategy, by combining ultrasonic extraction, Diaion Hp-20 macroporous resin column enrichment, solid phase extraction (SPE), reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and preparation, and NMR analysis, is presented ( Figure 1). We obtained five less polar ginsenosides, including a pair of new isomeric ginsenosides ( Figure 2). Moreover, the quantification of the five ginsenosides in FBPG was also involved. This research will provide a novel scientific method for comprehensive studies of various ginsenosides in FBPG and make full use of ginseng resources.

Preparation of Crude Ginsenoside Extracts
The most appropriate and simple method for extracting less polar ginsenosides from the ginseng plant is ultrasound-assisted extraction [25,26]. Methanol was used as the solvent, and it was tested under the following conditions: a sample of 50.0 g, a solvent to solid ratio of 10 mL/g, a temperature of 30 • C, ultrasound power of 1000 W, an extraction time of 30 min, a particle size of 80-100 mesh, and it was extracted three times. The combined extract was concentrated under vacuum yielding 8.2 g (16.4%) of crude extract powder. To obtain the high yield of less polar ginsenoside from FBPG extracts, Diaion HP-20 macroporous resin was used to remove the polar ginsenosides by the elution with H 2 O and 60% aqueous methanol, followed by 80% aqueous methanol. The last fraction was rich in less polar ginsenosides ( Figure S1), which were used to further isolate and purify specific ginsenosides.

Optimization of Solid Phase Extraction Conditions
In order to simplify the procedure of the semi-preparative liquid chromatography separation, it is important to obtain a relatively uncomplicated sample. It might achieve good results of isolation under fewer steps. Based on the HPLC chromatogram ( Figure S1), a simple and efficient method was established for rapid purification of the crude ginsenoside extracts by SPE.
In this section, three different strategies were used to test the efficiency of the elution. Crude ginsenoside powder (102.5 mg) was dissolved in 2.0 mL methanol and was subjected to C18 Sep-Pak ® cartridge column chromatography with a stepwise gradient methanol-water. The first step was methanol-water (40:60, 65:35, 80:20, 90:10 v/v), the second step was methanol-water (68:32, 80:20, 90:10 v/v), and the third step was methanol-water (65:35, 80:20, 90:10 v/v) (6.0 mL solvent per step, performed three times). By comparing the peaks in terms of the five less polar ginsenosides in the chromatogram, the majority of five less polar ginsenosides absorbed by the SPE column was eluted by methanol-water (80:20) solution ( Figure 3B) after it was firstly eluted by methanol-water (65:35) solution ( Figure 3A), where complete desorption was conducted with an elution volume of 18 mL. We could prove that a good desorption was achieved by methanol-water (65:35, 80:20 v/v) since there was no target compound in the 90% aqueous methanol fraction ( Figure 3C). However, when the methanol-water (68:32, 80:20 v/v) was used as the elution, the target compounds were also eluted together with the polar ginsenosides by methanol-water (68:32 v/v) ( Figure S2). Therefore, a stepwise gradient methanol-water (65:35, 80:20, 90:10 v/v) was selected as the eluent in the SPE processing section for preparation of samples for the semi-preparative liquid chromatography separation.

Establishment of HPLC Isolation Conditions
The chromatogram in Figure 3B shows the isolated peaks after the SPE treatment, but a satisfactory separation was not obtained with a mobile phase composed of methanol-water, especially for compounds 1-3. Usually the ginsenosides could achieve a complete separation effect with acetonitrile-water as the mobile phase [27,28]. Using acetonitrile-water, we discuss the parameters of volume ratios, flow rate, and column temperature here.
The best separation was obtained with the system of acetonitrile/water (53:47 v/v), as shown in    Different flow rate could also affect the separation effect of ginsenoside compounds. With the mobile phase of acetonitrile/water (53:47 v/v), the flow rate of 0.5 mL/min ( Figure S4) was satisfactory, and the analytical time was less than 30 minutes. While the flow rate of 0.6 mL/min led to partial overlapping of the two new isomeric ginsenosides, ginsenoside M1 was also affected by the adjacent compounds ( Figure 4).
As for the influence of column temperature, the lower column temperature should be adjusted to meet the separation needs of these target compounds. As shown in Figure S5, the column temperature of 25 °C resulted in a poor separation effect, and when it was set at 15 °C ( Figure S4), the five ginsenosides were all separated in good condition.
According to the analytical method, the separation process was scaled up to preparative-scale chromatography to obtain a significant quantity of the five ginsenosides. Using the semi-preparative HPLC Daisogel C18 column (5 μm, 30 mm internal diameter (i.d.) × 250 mm) column, we obtained five pure specific ginsenosides with a mobile phase of acetonitrile/water (53:47 v/v) at a flow rate of 5.0 mL/min within 30 min. The typical chromatogram of the semi-HPLC method is illustrated in Figure S6.

Identification of the Isolated Ginsenosides
Compound 1 was obtained as a colorless amorphous powder. The molecular formula was deduced to be C43H74O13 on the basis of the protonated molecular ion peak in positive high resolution electrospray ionization mass spectrometry (HR-ESI-MS) at m/z 799.5195 ([M + H] + , calcd. 799.5208). Its IR spectrum displayed strong absorption bands at 1636 cm −1 , indicating an olefinic functional group, and at 3397 and 1080 cm −1 suggestive of hydroxyl and ether functions. The 1 H-NMR spectrum of 1 displayed signals due to eight tertiary methyls, an olefin, and two anomeric protons ( Table 1). The 1 H-and 13 C-NMR data of 1 were very similar to those of 20(S)-ginsenoside Rg3 [29], except for signals indicating the presence of an extra methoxyl group (δ H 3.27; δ C 48.8), suggesting that 1 was a methyl ether of 20(S)-ginsenoside Rg3. The methoxyl group could be located at C-20 based on the downfield shift of C-20 (δ 79.9) and the highfield shifts of C-17 (δ 50.0), C-21 (δ 21.2) and C-22 (δ 35.1), which appeared at δ 73.0 (C-20), δ 54.8 (C-17), δ 27.1 (C-21), and δ 35.9 (C-22) in 20(S)-ginsenoside Rg3 [29], respectively. This was further supported by the long-range correlation between the methoxyl signal at δ 3.27 (OCH3) and the carbon signal at δ 79.9 (C-20) in the HMBC spectrum. The detailed NMR data was assigned by the heteronuclear singular quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) spectrum (Table 1). Different flow rate could also affect the separation effect of ginsenoside compounds. With the mobile phase of acetonitrile/water (53:47 v/v), the flow rate of 0.5 mL/min ( Figure S4) was satisfactory, and the analytical time was less than 30 minutes. While the flow rate of 0.6 mL/min led to partial overlapping of the two new isomeric ginsenosides, ginsenoside M1 was also affected by the adjacent compounds ( Figure 4).
As for the influence of column temperature, the lower column temperature should be adjusted to meet the separation needs of these target compounds. As shown in Figure S5, the column temperature of 25 • C resulted in a poor separation effect, and when it was set at 15 • C ( Figure S4), the five ginsenosides were all separated in good condition.
According to the analytical method, the separation process was scaled up to preparative-scale chromatography to obtain a significant quantity of the five ginsenosides. Using the semi-preparative HPLC Daisogel C18 column (5 µm, 30 mm internal diameter (i.d.) × 250 mm) column, we obtained five pure specific ginsenosides with a mobile phase of acetonitrile/water (53:47 v/v) at a flow rate of 5.0 mL/min within 30 min. The typical chromatogram of the semi-HPLC method is illustrated in Figure S6.

Identification of the Isolated Ginsenosides
Compound 1 was obtained as a colorless amorphous powder. The molecular formula was deduced to be C 43 H 74 O 13 on the basis of the protonated molecular ion peak in positive high resolution electrospray ionization mass spectrometry (HR-ESI-MS) at m/z 799.5195 ([M + H] + , calcd. 799.5208). Its IR spectrum displayed strong absorption bands at 1636 cm −1 , indicating an olefinic functional group, and at 3397 and 1080 cm −1 suggestive of hydroxyl and ether functions. The 1 H-NMR spectrum of 1 displayed signals due to eight tertiary methyls, an olefin, and two anomeric protons ( Table 1). The 1 H-and 13 C-NMR data of 1 were very similar to those of 20(S)-ginsenoside Rg 3 [29], except for signals indicating the presence of an extra methoxyl group (δ H 3.27; δ C 48.  [29], respectively. This was further supported by the long-range correlation between the methoxyl signal at δ 3.27 (OCH 3 ) and the carbon signal at δ 79.9 (C-20) in the HMBC spectrum. The detailed NMR data was assigned by the heteronuclear singular quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) spectrum (Table 1).
Compound 2 was also obtained as a colorless amorphous powder. The molecular formula was deduced to be C 43 H 74 O 13 on the basis of the protonated molecular ion peak in positive HR-ESI-MS at m/z 799.5194 ([M + H] + , calcd. 799.5208). It had the same molecular weight as compound 1. Its IR spectrum showed identical features as that of 1. The 13 C-NMR spectrum gave 43 carbon signals, of which 12 were assigned to the sugar moiety and 31 to a triterpene aglycone moiety. The NMR data assignments were elucidated by the HSQC and HMBC spectrum. By comparing NMR data of 2 with those of 1, few differences were found due to the 13 C-NMR data of C-17, C-20, C-21, and C-22 (Table 1). Compounds 1 and 2 were a pair of isomers with different stereochemistry configuration at C-20.
The determination of the configuration at C-20 was based on the rotating frame overhauser effect spectroscopy (ROESY) experiment. In Compound 2 was also obtained as a colorless amorphous powder. The molecular formula was deduced to be C43H74O13 on the basis of the protonated molecular ion peak in positive HR-ESI-MS at m/z 799.5194 ([M + H] + , calcd. 799.5208). It had the same molecular weight as compound 1. Its IR spectrum showed identical features as that of 1. The 13 C-NMR spectrum gave 43 carbon signals, of which 12 were assigned to the sugar moiety and 31 to a triterpene aglycone moiety. The NMR data assignments were elucidated by the HSQC and HMBC spectrum. By comparing NMR data of 2 with those of 1, few differences were found due to the 13 C-NMR data of C-17, C-20, C-21, and C-22 (Table 1). Compounds 1 and 2 were a pair of isomers with different stereochemistry configuration at C-20.
The determination of the configuration at C-20 was based on the rotating frame overhauser effect spectroscopy (ROESY) experiment. In  The three known compounds, ginsenoside Rk1 (3), ginsenoside Rg5 (4), and ginsenoside M1 (5), were readily identified by comparison of their data with the literature [32,33]. To the best of our knowledge, they were the first scientific report from FBPG.  The three known compounds, ginsenoside Rk 1 (3), ginsenoside Rg 5 (4), and ginsenoside M1 (5), were readily identified by comparison of their data with the literature [32,33]. To the best of our knowledge, they were the first scientific report from FBPG. We could make a distinction between 20(S) and 20(R) by the 13 C-NMR data at C-17, C-20, C-21, and C-22. Sometimes C-20 did not show the difference [29,34], but the data at C-17, C-21, and C-22 are always different and could be used to distinguish 20(S) from 20(R). Its chemical shift differences are shown in Table 2.
We could also distinguish 20(S) from the 20(R) ginseoside isomer by the retention time in RP-HPLC. Usually, the retention time of the 20(S) isomer was shorter than the 20(R) isomer [34,35]. In this paper, our study validated the correct retention property of the isomers on the C18 column. Table 2. Chemical shift differences of C-17, C-20, C-21, and C-22 between 20(R) and 20(S) isomer ginsenosides (in C 5 D 5 N).

Ginsenoside
C Optical rotation for the two new isomers was analyzed in dimethyl sulfoxide (DMSO) and/or MeOH due to differences in solubility. Like the positive data of 20(S)-ginsenoside Rg 3 previously reported [34], we obtained a positive value in DMSO and MeOH for 20(S)-methoxyl-ginsenoside Rg 3 , and there was also a negative value reported for 20(S)-ginsenoside Rg 3 in DMSO [36]. However, according to the synthesis method of 20(S)-ginsenoside Rg 3 with the positive value in MeOH [37], we prove that, at least, 20(S)-methoxyl-ginsenoside Rg 3 shows positive optical rotation in MeOH. Since 20(R)-methoxyl-ginsenoside Rg 3 was not soluble in MeOH, just like 20(R)-ginsenoside Rg 3 , we could not observe its optical rotation in MeOH.
It was reported that similar C-20 methyl ether compound has been isolated from ginseng genus plants, such as Panax vietnamensis [38]. Thus, our findings further proved that this kind of ginsenoside was native to the plant. Together, they made up a wide variety of chemical structures of ginsenosides. Furthermore, the variations observed in the C-17 side-chain of ginsenosides were usually considered as artifacts, which were produced through the processed ginseng [8], but some ginsenosides have been also isolated from the natural plant, such as the stems and leaves [32]. This might indicate that the aerial part of PG, including flower buds, and stems and leaves, had more abundant compounds of structural changes than roots due to the long time in sunlight and high temperature. Thus, it is important to analyze these less polar ginsenosides in FBPG for quality control.

Method Validation of Quantification
All calibration curves were constructed by plotting the peak areas versus the concentration and showed good linear regression (R 2 > 0.9994) within the test ranges. The limits of detection (LODs) (signal-to-noise ratio, S/N = 3) and the limits of quantification (LOQs) (S/N = 10) for the five ginsenosides were less than 0.092 and 0.275 µg, respectively ( Table 3). The overall intra-and inter-day variations were within 0.35%-1.51% and 0.41%-1.54% for the five analytes ( Table 4). As shown in Table 5, the developed analytical method had an excellent accuracy with an overall recovery from 97.20% to 99.71% (n = 3) for the analytes. All of the above indicated that this high-performance liquid chromatography diode array detector (HPLC-DAD) method was precise, accurate, and sensitive enough for the simultaneous quantitative evaluation of the five ginsenosides in FBPG.

Quantitative Analysis of the Five Less Polar Ginsenosides in FBPG
Ten grams (dry weight) of FBPG sample was extracted in triplicate by ultrasound-assisted extraction with methanol (each 100 mL, w/v = 1:10). Then the extracts were treated as described in Section 2.1 by a Diaion HP-20 macroporous resin column and C18 cartridges column to prepare the sample for HPLC analysis. The residue from SPE was concentrated and dissolved in methanol with a 10 mL volumetric flask, then filtered through a 0.45 µm syringe filter. This was conducted with three parallel tests to obtain three solutions, and each solution was analyzed three times. The content of the five less polar ginsenosides in FBPG was shown in Table 6.

General
Ultrasound-assisted extraction was carried out in a SB-5200DTD ultrasonic device (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China). Open column chromatography was carried out using a Diaion HP-20 (Mitsubishi Chemical Corp., Fuji, Tokyo, Japan) as the stationary phase. C18 Sep-Pak ® cartridge (12 cc/2 g) columns were obtained from Waters (Milford, MA, USA). Double-distilled water was used in all experiments and samples for HPLC were filtered through a 0.45 µm membrane before injection. HPLC was carried out on an Agilent Series 1260 (Agilent Technologies, Palo Alto, CA, USA) liquid chromatography system, equipped with a vacuum degasser, a quaternary pump, a column oven, a 7125i Reodyne Model manual injector with a 20 µL loop, and a diode array detector. The semi-preparative HPLC was performed on a CXTH HPLC system (Chuang Xin Tong Heng Sci. Technol. Co. Ltd., Beijing, China) equipped with two P3050 pumps, a UV-3000 UV-VIS detector (Chuang Xin Tong Heng Sci. Technol. Co. Ltd., Beijing, China), and a 7125i Reodyne Model manual injector with a 5 mL loop. The IR spectra were recorded as KBr pellets on a Jasco 302-A spectrometer (Jasco, Tokyo, Japan). Optical rotation was recorded on a Jasco P-2000 polarimeter. NMR spectra were measured on a Bruker DRX500 spectrometer (Bruker S.A., Wissembourg, France) with tetramethyl silicane (TMS) as internal standard. HR-ESI-MS were measured on a 6550 Agilent iFunnel Q-TOF LC/MS (Agilent Technologies). The melting point was recorded on an X-4 microscopic melting point meter (uncorrected) (Maisiqi High-tech Co. Ltd., Beijing, China). All of the reagents were of analytical grade bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Plant
The FBPG were collected from Tonghua County (Jilin, China) in 2014 and identified by one of the authors, Professor Xiao-Jie Gong.

Sample Preparation
An ultrasonic extraction method was employed. In brief, the air-dried 1 kg particle size of 80-100 mesh powder of FBPG was ultrasonically extracted in 10 L of methanol on a water bath (1000 W) at 30 • C for 30 min, repeating the extraction operation two times. After removing the solvents under vacuum, the extract was dissolved in a small amount of water and then chromatographed on a Diaion HP-20 column eluted with H 2 O, 60% aqueous methanol, and 80% aqueous methanol. The last fraction with enriched less polar ginsenosides was collected for further processing by SPE. The elution was carried out with increased concentrations of methanol (65%, 80%, and 90%, 6 mL each, for three times) successively. The eluted fraction of 80% aqueous methanol was rich in our targeted ginsenosides. It was ready for the isolation of specific compounds and quantification.

HPLC Analysis and Preparation
The analytical HPLC conditions was selected as: Agilent Zorbax Eclipse XDB C-18 (

Acid Hydrolysis
A solution of compounds 1 and 2 (1 mg each) in 1 M HCl (1.0 mL) was stirred for 4 h at 80 • C to release the sugar moiety by a previously reported method [5]. After the extraction with EtOAc, the concentrated aqueous layer was analyzed by the following HPLC conditions: Kaseisorb LC NH2-60-5 column (5 µm; 4.6 mm i.d. × 250 mm, Tokyo Kasei Co. Ltd., Tokyo, Japan); optical rotation detection; and mobile phase of acetonitrile/water (75:25 v/v). Peaks of the hydrolysate were detected by comparison with retention time of authentic samples of D-glucose (12.3 min).

Validation
Six concentrations of compounds 1-5 solutions were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas against the concentration of each analyte. The diluted solutions of the analytes were further diluted with methanol to give a series of concentrations for determining the LODs and LOQs. The LOD and LOQ for individual ginsenosides in FBPG under present chromatographic conditions were determined on the basis of the responses at a S/N of 3 and 10, respectively. The mixed standard samples were chosen to determine intraand inter-day precision of the method and analyzed as described in Section 2.1.3. The intra-day precision was performed in triplicate on a single day. The inter-day precision was carried out on five different days. The concentrations were calculated from the corresponding calibration curve. Variations were expressed by the RSD. Accurate amounts of standard ginsenosides were added to an accurately-weighed portion of FBPG, and then extracted and analyzed as described above.

Conclusions
An efficient simultaneous purification method combining SPE chromatography with semi-preparative HPLC was established to isolate five less polar ginsenosides from FBPG in the present study. The purified ginsenosides included a pair of new 20-methoxyl isomers and three known compounds which were identified for the first time from FBPG. The new compounds were elucidated by NMR and MS analysis. Furthermore, a rapid and efficient RP-HPLC method was established for systematic quantitative analysis of the five less polar ginsenosides in FBPG. Ginsenoside Rg 5 was recognized as the relatively most abundant (approximate 1%) in FBPG, which could also be selected as a marker compound for quality control, except for the polar ginsenosides, such as ginsenoside Re and ginsenoside Rd. The developed RP-HPLC method demonstrated an effective analytical means for less polar ginsenosides in FBPG. The results could provide a new material chemistry basis for the further complete application of FBPG in functional food with medicinal properties.


β- O -4 type dilignol compounds and their iron complexes for modelling of iron binding to humic acids: synthesis, characterization, electrochemical studies and algal growth experiments

A series of β- O -4 type dilignol compounds and their iron(III) complexes, designed as model compounds for humic acids, were prepared and characterized by 1 H-NMR and 13 C-NMR spectroscopy, elemental analysis, EPR spectroscopy, IR spectroscopy, UV‒Vis spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Properties regarding iron binding, stability, liphopilicity and bioavailability for microorganisms have been evaluated with cyclic voltammetry, stability studies in water and seawater by means of UV‒Vis spectrophotometry and the algae growth assays with seawater algal species Chlorella salina and Prymnesium parvum. Both established ligands and their iron complexes undergo deprotonation processes in seawater wheares no changes in UV‒Vis spectra were observed in distilled water. The iron(III) complex formation constants, p K a values and liphopilicity of the dilignols were in the same range as for the analogous catechol coordination compound. Synthetized dilignols were prone to redox reactions under biological conditions similar to natural aquatic humic acids. Moreover, an increased iron bioavaiability was observed for the presented complexes compared to corresponding catechol complexes and comparable to the bioavaialability of iron bound in humic acid complexes recovered from Craggie Burn river. Those results confirm that β- O -4 type dilignol compounds are excellent model ligands for aquatic humic acids. molecular peaks of

Introduction
Iron chemistry in seawater and its bioavailability for microorganisms has been intensively investigated for a long time. [1][2][3][4] Several experiments have shown that iron is limiting the productivity of marine ecosystems in huge areas of the ocean [5][6][7][8][9] but only little is known about the chemical speciation of iron in seawater and its accessibility for phytoplankton. Iron is not only an important factor for growth of algae and other microorganisms but also selectively supporting the development and expansion of several species (with lower iron demand), which impacts the food chain within the whole ecosystem. 4,10 Analytical methods provide information about the concentration of dissolved and undissolved iron or iron solubility dependent on the presence of organic ligands. However; exact structures of bioavailable iron complexes and uptake mechanisms remain still unclear. There are several factors influencing iron bioavailability for microorganisms. Besides different uptake mechanisms; kinetics and iron requirements for each species, one pressing issue is the chemical form of bioavailable iron. It is known, that inorganic iron displays an extremely low solubility in seawater, which is the result of the low solubility of iron oxide (the general term 'iron oxide' describes various forms of iron oxide, oxyhydroxide and amorphous iron hydroxide). 11,12 Thus, almost all of dissolved iron in the ocean is bound to organic scaffolds. [13][14][15][16] Those ligands have a large range of different structures and origins. One group of them are siderophores, which are produced by bacteria particularly in order to complex iron(III). [17][18][19] Stability constants for iron complexes of these natural chelators are extremely high and special mechanisms have been developed by certain organisms to release the iron from formed complexes. 20 Another big group of important iron chelators are substances which originate from decomposition of organic material, known as humic substances.
Their main sources are rivers, especially derived from peat bog regions which are rich in aquatic humic substances (AHS). It has been shown, that AHS are greatly improving the bioavailability of iron in coastal waters. [21][22][23][24][25] Also algal studies on microalgae Chlorella salina and Diacronema lutheri in batch cultures support the assumption of the positive impact of AHS on iron supply. 26 Algal cultures treated with isolated AHS showed increased growth response compared to samples treated with iron(III) chloride and EDTA (ethylenediaminetetraacetate), a complexation agent used for commercial algal cultivation 27 ). Knowing the importance of those chelators on phytoplankton growth and vitality, which affect huge amount of other processes like for example the global CO 2 cycle, [28][29][30] it is of great interest to clarify the chemistry behind iron complexation, release and uptake as well as the structure of the complexes and ligands. Unfortunately, research on humic substances, especially AHS is very challenging due to their low concentration in seawater 31 and the structural complexity depending on origin, incorporated building blocks and formation processes. [32][33][34][35] In order to understand the chemistry of AHS regarding iron binding properties and the postulated release by photoreduction 36 the utilization of model systems represents a highly promising approach. In our previous studies, we synthesized simple iron complexes with different coordination motifs and ligand scaffolds and investigated them for their suitability as model compounds for humic acids iron complexes. 37 The model compounds were characterized and investigated by various analytical techniques (cyclic voltammetry, EPR, EXAFS, 36 UV-Vis spectroscopy etc.) in addition to algal batch culture studies on chlorophyte and haptophyte unicellular algae species. The results of the latter assays led to the conclusion, that catechol-based ligand systems are excellent scaffolds for modeling of iron-AHS complexes. Therefore catechol-derived ligands and complexes with structural characteristics and molecular weight in range of natural AHS were established. The main components of humic substances are lignin decomposition products, originating from support tissues in plants and guaiacylglycerol-β-guaiacyl ether represents a suitable model for lignin. 38,39 Binding studies of iron with guaiacylglycerol-β-guaiacyl ether precursors (coniferyl alcohol, sinapic acid, ferulic and coumaric acid) and dehydrogenation polymers of coniferyl alcohol have been already carried by Guillon et al. regarding their sorption properties in comparison to lignin. 40,41 Although guaiacylglycerol-β-guaiacyl ether seems to be a good model for AHS, formation of stable iron complexes is unlikely. For this reason, we decided to modify the β-O-4 backbone of guaiacylglycerolβ-guaiacyl ether by introduction of a free catecholic moiety.
In this work we report the synthesis of diastereomerically pure lignols designed as model compounds for AHS, bearing free catecholic groups and their respective iron complexes. The synthesized substances were characterized by elemental analysis, spectroscopic methods (IR, EPR, UV-Vis), NMR spectroscopy (in the case of the ligands), mass spectrometry (ESI-MS) and cyclic voltammetry. The The proton dissociation constants (pK a ), complex formation constants and lipophilicity (as distribution coefficients) were determined. The bioavailability of the respective iron coordination compounds was elucidated in two unicellular algal species, namely chlorophyta Chlorella salina and haptophyte Prymnesium parvum in batch cultures.

Results and discussion
Synthesis -general overview Scheme 1. Synthesis of β-O-4 type lignol compounds.
Diastereomerically pure guaiacylglycerol-β-guaiacyl ether 6a was synthesized according to previously published procedure. 42 Two novel β-O-4 type lignol compounds 6b,c were synthesized in a similar approach (Scheme 1, see SI). Compound 3 was prepared according to the procedure described by Nakatsubo et al. 43 using tert-buthyl chloroacetate. Aldehydes bearing free phenolic groups were protected with benzyl groups to avoid undesired side reactions (2a-c). Aldol condensation of 3 and 2a-c in the presence of lithium diisopropyl amide at -78°C led to diastereoisomeric mixture of 4a-c (1:1 ratio of erythro and threo, respectively). As described in literature, recrystallization of 4a from EtOAc gave rise to pure erythro diastereoisomer. For the compounds 4b and 4c recrystallization was not successful and column chromatography (n-hexane/EtOAc 5:1 to 1:1) was performed. Due to the very similar elution time, it was difficult to separate both diastereoisomers and only the erythro disatereoisomer was purely separated. The isolated compounds were reduced with lithium aluminum hydride and benzyl groups were cleaved by palladium-catalyzed hydrogenation yielding 6a-c. Formation of the desired dilignols was confirmed by NMR spectroscopy and elemental analysis.
The ESI-MS spectra of the ligands were measured in methanol or in acetonitrile. Overall the positively charged Na + adducts, with m/z values of 329 (6a and 6b), were detected. Lignols were isolated in low to moderate yields due to the crucial purification step including the separation of diastereoisomers.
Thus in this step the yield of the pure erythro diastareoisomers was between 7% and 20%. The Fe(III) complexes were synthesized by deprotonation of the ligand with potassium hydroxide and addition of iron(III) chloride in methanolic solution (see SI). The complexes 7b and 7c were isolated in low to moderate yields (7-27%) and characterized by standard analytical methods. We presume that similar to analogous Fe(III) catechol coordination compounds, dinuclear complexes with two lignols bridging two iron centers were obtained. The mass spectra of the complexes display, similar to isolated and characterized catechol complex, one negatively charged fragment with m/z value of 664, which can be assigned to Fe(lignol) 2 -. All detected molecular peaks of the complexes showed the expected isotopic iron pattern. Also elemental analysis, electrochemical studies and complex formation studies support our proposed structure.

Cyclic voltammetry
The electrochemical behavior of the ligands and complexes was studied in order to estimate the possibility of iron reduction under physiological conditions. The proposed mechanism of iron release in natural humic acid complexes includes the photoreduction of Fe(III) to Fe(II). 36 Fe(II) has low affinity to AHS, dissociates from the complex and can be uptaken by the microorganisms. Although Fe(II) undergoes oxidation in oxygenated seawater, the steady state concentration of Fe(II) remains higher due to this reduction process. 44 Thus redox potentials present important information regarding the bioavailability of iron and cyclic voltammetric measurements of 6b and 6c were performed in aqueous solution at various pH values in the absence and in the presence of iron(III) ions. Representative cyclic voltammograms are shown in Figure 1 for 6b and catechol as reference, and electrochemical data are presented in Table S1. A reversible (E 1/2 = 0.00 V) and a quasi-reversible process (E 1/2 = +0.82 V) can be observed at pH 3.7 in the case of 6b. The reversible process might belong to the one-electron transfer between the catechol and the semiquinone radical. 45 On the other hand mostly irreversible oxidation peaks appear at pH > 8.8, which is the pH range where the ligand 6b is present in its monodeprotonated (HL -) form ( Figure S4A). The current maximum of the cathodic peak at +0.65 V is decreasing with increasing pH and a novel oxidation peak appears at +0.50 V and becomes dominant at pH 9.89, but disappears at pH >12. In addition a new reduction peak is observed at pH ≥11 at +0.27 V. In the presence of half-equivalent iron(III) the position of the quasi-reversible peak pair is already shifted at pH 1.8 and E is somewhat lower (+0.33 vs. +0.24 V) ( Fig. 3b), although practically no complex formation takes place under these conditions ( Figure S4B), because peaks belong to the unbound iron are observed at a lower potential range (E 1/2 = -0.15 at pH 2.7, not shown here). The cathodic peak at +0.42 V is disappearing with increasing pH, while the oxidation peak still can be observed shifting from +0.76 to +0.42 V. The anodic peak is most probably related to the electrochemical oxidation of the ligand, which is mostly bound to the metal ion (e.g. 95% of the ligand is coordinated at pH 8.) In addition a novel cathodic peak is seen at ca. -0.1 V at pH > 9 where more and more tris-ligand species are assumed to be formed and the reduction process can be described to the iron(III) / iron(II) transition. Notably, only irreversible processes are observed at pH > 5. Overall, 6c behaves very similarly to 6b; however, formal potentials E 1/2 of the ligand are always lower by 170-180 mV (Table S1). At the same time in the presence of iron(III) ions the observed anodic and cathodic peak positions are just slightly different in the case of the studied ligands.
Comparing the recorded voltammograms of 6b, 6c to those of catechol at pH ~ 8 when iron(III) was added to the solution it can be noted that the oxidation peak of catechol (+0.64 V) appears at a similar potential as in the case of the other two ligands, while a cathodic peak with high intensity is seen at +0.42 V and another smaller reduction peak appears at lower potentials (+0.11 V). In the case of catechol, iron(III) is present in the bis complex predominantly at 1:2 metal-to-ligand ratio at pH 8 due to the formation of the higher stability complexes compared to ligands 6b and 6c (c.f. Figure S4B in the case of 6b) based on the determined stability constants (vide infra). The isolated Fe(III) complexes 7b,c of the respective dilignols ligands 6b,c were investigated for comparison. However, due to the lower aqueous solubility the experiments were performed in DMF ( Figure S1). Under these conditions the two irreversible reduction peaks with values -0.07 V and +0.52 V and an irreversible oxidation peak at +1.2 V were detected for 7b which can be assigned to ligand and iron reduction. Complex 7c showed three irreversible reduction peaks with the values +0.01 V, +0.29 V and +0.44 V and similar to 7b an oxidation peak at +1.2 V.
Overall, the observed reduction potentials of model ligands, complexes and in situ prepared iron(III) complexes are lying in the area accessible to photoreduction as well as for natural occurring reducing agents. 46 Those studies confirm that those dilignols can be seen as suitable model compounds for AHS regarding their redox activity.

EPR spectroscopy
EPR spectral analyses of the ligands 6a-c, revealed a g-factor of 2.0 similar to the free electron ( Figure 2). Thus, all ligands might represent organic free radicals. Santana-Casiano et al. showed that catechol-type structures can be readily oxidized in the presence of oxygen yielding stable radicals. 47 Stable radicals were also detected in humic acids. 48,49 The high stability of the free radicals can be explained by the delocalization of the electron over the phenol ring. The hyperfine splitting of the signal indicates an interaction between the unpaired electron and magnetic hydrogen nuclei. The line pattern of the EPR spectrum suggests that the electron is delocalized over the carbon atoms of the aromatic ring. The EPR spectrum of the ligand, 6a, differs from the other ligands exhibiting a g-factor of approximately 1.9. The methoxy group at the aromatic ring might participate in the delocalization of the electron, thereby inducing the appearance of additional signals. The two iron complexes, 7b and 7c, showed a distinct signal at g = 4.3 ( Figure 2). This isotropic-looking line is characteristic of

Proton dissociation processes and lipophilicity of the ligands
The proton dissociation constants of the studied ligands 6a-c were determined by pH-potentiometric and UV-Vis spectrophotometric titrations in aqueous solution ( Table 1). The first deprotonation process in all cases takes place at pH < 11.5, therefore pK 1 values could be determined accurately by the evaluation of the pH-potentiometric titration curves; on the other hand, these titrations were used to obtain the exact concentration of the ligands stock solutions and to check their stability in aqueous solution. Consecutive titrations showed that no ligand decomposition occurred in the pH range studied under an argon atmosphere and the stock solutions (pH ~1.8) were stable over 48 h.
The proton dissociation processes could be followed by UV-Vis spectrophotometric titrations as well, since the deprotonation of the hydroxyl functional groups is accompanied by characteristic spectral changes. However, the second step occurs only at pH > 11.5 and spectra had to be recorded at high The lipo-hydrophilic character (logD) of the ligands 6a-c was studied at pH 2.5 and 8.3 via the partitioning between n-octanol and water (Table 1, Figure S7A-B All results indicate that the methoxy derivative has a more lipophilic character compared to 6b,c, which have moderate hydrophilic character like the reference compound catechol. At the chosen slightly alkaline pH the ligands are partly deprotonated, which causes the increased hydrophilicity. We have made an attempt at determining the lipophilicity of the iron(III) complexes of the studied compounds, but a precipitate was formed and clogged between the two phases even at lower ligand concentration (100 M), which hindered the quantitative analysis. Comparing the spectra of the

Complex formation processes of ligands
The complex formation processes of the ligand 6b with iron(III) were studied primarily by pHpotentiometry in aqueous solution. However, the complexation was found to be sluggish, especially in the acidic pH-range meaning that the equilibrium was not reached within 10 min as also reported for catecholates. 50 Therefore UV-Vis spectrophotometry was applied to follow the complexation of 6a-c with iron(III) using longer waiting time in the pH range from 2 to 11 with the exclusion of air; and the process was found to be reversible under this condition. Spectra were recorded in the wavelength range 350 -1000 nm where mostly the strong metal-to-ligand charge transfer (CT) bands can be seen and the non-coordinated ligands do not absorb. A representative spectrum series for the iron(III) -6c (1:3) system is shown in Figure 3A, which shows characteristic changes upon increasing pH. The  max values of the main CT band were decreased with increasing pH ( Figure 3B) and a well-  Table 2, and Figure 3C in the case of 6c). In these complexes most probably the completely deprotonated ligands (L 2-) coordinate via an (O -,O -) donor set as it was reported for the reference compound, catechol, and for other catecholate derivatives. 50,53,54 The coordination of the monoprotonated ligand (HL -) in the metal complexes is very rare and observed only in compounds isolated from organic solvents; 55, 56 however, it was also suggested for the iron(III) mono-ligand complexes of catechol by R.C. Hider et al. 54 It is noteworthy that intramolecular redox processes between Fe(III)-catecholate and Fe(II)-semiquinone species are known to occur, mostly in the case of the mono complex. 45,54,57 In addition existence of dimeric species [Fe 2 L 4 ] 2of catechol in the pH range of the formation of the bis-complex was also suggested based on Mössbauer spectroscopy.
This species was suggested to contain two equivalent sites and a bridging hydroxyl group. 54 Spectrophotometry is not an adequate method to distinguish between the formation of [Fe 2 L 4 ] 2and [FeL 2 ]complexes with the same metal-to-ligand ratio. Therefore two kinds of models could be calculated for the studied systems using the titration data which consist of the formation of the mononuclear or the dinuclear complex, but the latter model gave somewhat poorer fits between the experimental and calculated absorbance values. The calculated molar absorbance spectra and  max values (  Since ligand 6a has lower water solubility compared to the other two studied compounds its complexation with iron(III) was studied via the ligand's bands at lower concentrations (~ 200 M).
The measured absorbance values in the absence and the presence of the metal ion (Fig. S3) were fairly similar suggesting the negligible formation of the iron(III) complexes under these conditions.
The coordination of the monodentate phenolate type ligand is very weak similarly to the case of the phenol itself. 58 Direct comparison of stability constants (Table 2) and concentration distribution curves calculated for the iron(III) complexes formed with 6b, 6c ( Figure S5A) and catechol ( Figure S5B) shows that the studied ligands form somewhat lower stability complexes than catechol. (Notably, direct comparison of the log values in this case is possible due to the similar stochiometry of the complexes formed and the similar pK a values of the ligands.) In these cases the predominant formation of the bis complexes is found at neutral and slightly basic pH values. However, the actual speciation at pH 8 depends on the actual metal-to-ligand ratio ( Figure 3D).
In order to compare the metal ion binding abilities of the studied ligands to each other and to catechol or to other iron chelators (such as the catecholate-containing siderophores) pM compounds (see S10-S12). The time dependent spectra of 7b and 6a-c in water at pH ~8 (see S13-S14), showed also an increase in the same signals as observed in seawater. The same behavior was observed for as time period of 21 days (duration of algal growth tests) and only ligand 6a showed an additional band after 14 days (see S15-S16). Those results confirm that the established compounds are sufficiently stable for algal growth experiments. In order to elucidate the bioavailability of the iron complexes 7b,c as well as the free ligands 6a-c to provide bioavailable iron, algal tests on C.
salina and P. parvum have been performed. Those two species were intentionally chosen due to their wide occurrence and different biology. C. salina is a representative for unicellular green algae. The species react very sensitive to iron limitation. It has been recently reported, that cultures cultivated without iron or chelator EDTA showed poor growth. 26,37,60 The second species P. parvum, is a unicellular haptophyte and this algae is able to grow under photoautotrophic and heterotropic conditions. The species is also less sensitive to iron deficiency and can adapt to several environmental limitations. Moreover, P. parvum is well known for its toxicity and is a cause of mass fish deaths in coastal waters. Investigating both species may help to understand the alterations in iron uptake and preferences of each algae species towards different iron sources. All algae experiments were carried out in batch cultures, using three different nutrition setups. As full medium, seawater with micronutrient and vitamin solution as well as solution of NaNO 3 , Na 2 SiO 3 , NaH 2 PO 4 as described by Guillard et al. 27 , were used (see Table S5)    Figure S17). Table S2 shows the end concentrations of the samples as well as the relation between the end-algae concentration of control samples to samples treated with model compounds from both experiments with C. salina.
Additionally to the maximal algae cell concentration, we also calculated the specific growth rate of algae for all tested compounds after 17 days of each experiment (which was the duration of the algal growth experiment for 7c (Table S3). Similar to the maximal algae cell concentrations, highest specific growth rates were observed for samples treated with 6b and 7b.
The model ligands were also tested on P. parvum for they ability to bind and supply iron ( Figure S18).
The culture of P. parvum was not as sensitive as C. salina to iron deficiency and we did not observe such pronounced effects on algal growth as for C. salina. In fact, the control with full medium  Table S4. Similar to the maximal algae cell concentrations, highest specific growth rates were observed for samples treated 6c.
Summarizing the algal studies results, some of our herein presented compounds can be seen as suitable models for humic acids regarding the iron transport and ability to supply microorganisms with iron. In all experiments, the algal growth in samples without iron was strongly inhibited. For C. salina typical growth pattern, known from iron enrichment experiments, 61,62 was observed. 63,64

Conclusions
In this work, the novel dilignols-based models for aquatic humic acids were synthesized and extensively characterized. In order to investigate the redox properties, cyclicvoltammetric measurements were carried out. All of the compounds can be possibly reduced under biological conditions, which is in accordance with the properties of AHS. Various UV-Vis studies were performed in order to elucidate iron(III) complex formation constants, pK a values of the ligands, lipopilicity and the stability of the compounds in aquatic systems, especially in artificial seawater to verify the potential of the developed model to supply marine microorganisms with iron. Regarding the stability of the compounds we observed changes in spectra of the recorded in seawater which were the result of the deprotonation of free catecholic hydroxy groups. Comparison of the pK 1 values of 6b,c to 6a revealed a significantly weaker acidity of 6a due to the electron donating effect of the methyl group, while 6b and 6c have pK a values, which are similar to catechol. The lipophilicity studies of the ligands showed that methoxy derivative 6a has more lipophilic character compared to the catecholate derivatives, which have moderate hydrophilic character due to the partial deprotonation at increased pH. Stability constants for the formation of the iron(III) complexes with model ligands were somewhat lower as for catechol whereas no data could be collected for 6a due to its very low affinity towards iron. At neutral and slightly basic pH values bis-complexes were predominant. The iron(III) binding ability of the studied ligands was found to be weaker as for the known siderophores. The ability to supply algae with iron was investigated in algal batch cultures. Ligands 6b,c moiety showed very good impact on the culture of C. salina and P. parvum comparable with natural AHS and significantly better as for catechol itself. Also experiments on C. salina with isolated iron complexes gave similar results. However, guaiacylglycerol-β-guaiacyl ether did not enhance the growth of C. salina. Those results suggest that catecholic moieties can play an important role by complexation and stabilization of inorganic iron by humic substances.


extfieldsofstudy: Engineering Medicine

text:
Raman Under Water – Nonlinear and Nearfield Approaches for Electrochemical Surface Science

Abstract Electrochemistry is re‐gaining attention among scientists because the complex interplay between electronic and chemical interfacial processes lies at the bottom of a broad range of important research disciplines like alternative energy conversion or green catalysis and synthesis. While rapid progress has been made in recent years regarding novel technological applications, the community increasingly recognizes that the understanding of the molecular processes that govern macroscopic device properties is still rather limited – which hinders a systematic and more complete exploration of novel material and functionality space. Here, we discuss advanced Raman spectroscopies as valuable analysis tools for electrochemists. The chemical nature of a material and its interaction with the environment is contained in the label‐free vibrational fingerprint over a broad energy range so that organic species, solid‐state materials, and hybrids thereof can be investigated alike. For surface studies, the inherently small Raman scattering cross sections can be overcome with advanced nonlinear or nearfield‐based approaches that provide signal enhancements between three and seven orders of magnitude, sufficient to detect few scatterers in nano‐confined spaces or adsorbate (sub)monolayers. Our article highlights how advanced Raman techniques with extreme chemical, spatial and temporal resolution constitute valuable alternative surface analysis tools and provide otherwise inaccessible information about complex interfacial (electro)chemical processes.

Electrochemistry is re-gaining attention among scientists because the complex interplay between electronic and chemical interfacial processes lies at the bottom of a broad range of important research disciplines like alternative energy conversion or green catalysis and synthesis. While rapid progress has been made in recent years regarding novel technological applications, the community increasingly recognizes that the understanding of the molecular processes that govern macroscopic device properties is still rather limited -which hinders a systematic and more complete exploration of novel material and functionality space. Here, we discuss advanced Raman spectroscopies as valuable analysis tools for electrochemists. The chemical nature of a material and its interaction with the environment is contained in the label-free vibrational fingerprint over a broad energy range so that organic species, solid-state materials, and hybrids thereof can be investigated alike. For surface studies, the inherently small Raman scattering cross sections can be overcome with advanced nonlinear or nearfield-based approaches that provide signal enhancements between three and seven orders of magnitude, sufficient to detect few scatterers in nanoconfined spaces or adsorbate (sub)monolayers. Our article highlights how advanced Raman techniques with extreme chemical, spatial and temporal resolution constitute valuable alternative surface analysis tools and provide otherwise inaccessible information about complex interfacial (electro) chemical processes.

Nanoscale Chemical Characterization of Electrified Interfaces
When we ask an electrochemist about the most important and exciting part of the electrochemical system they investigate, they are likely to answer "the electrochemical interface". Independent of the electrochemical processes under study, ubiquitously ranging from electrocatalysis, [1] to biological membrane behaviour, [2] or energy conversion, [3] to name a few key players, the central aspect common to all systems is the interface where the electrochemical action, namely, charge transfer and molecular conversion, takes place.
To tailor (electro)active interfaces for specific application needs, a fundamental understanding of the molecular processes that govern the macroscopic device behaviour is quintessential: [4] What are the chemical species involved, where are they located, how do they respond to the local environment? The ideal electrochemist's tool should thus provide Aside from the common electrochemist's workhorse, cyclic voltammetry, that provides a wealth of thermodynamic and kinetic information about interfacial properties and reactivity of electrodes ranging from mm to nm size, [5] a number of advanced analytical tools are available to increase our knowledge about nanoscale electrochemical processes. Nanoscale topographic imaging can be achieved with electrochemical scanning probe approaches, [6] even allowing video-rate imaging of electrochemical processes, [7] however, without chemical specificity. Scanning electrochemical microscopy [6,8] as well as scanning flow cell based tools [9] provide complementary insight into (electro)chemical conversion processes with high temporal and spatial resolution. Nonetheless, as the detection sensitivity of scanning chemical analyses is mostly limited to the ppm range, individual reactive surface sites are difficult to characterize. In situ x-ray scattering attracts increased attention among electrochemists to push chemical spatial resolution toward the molecular level. [10] While important insight has been obtained into, for example, water adsorption on electrode surfaces, [11] in situ x-ray experiments remain rather scarce and expensive because they are synchrotron based.
Detailed information about adsorbate interactions can also be obtained with vibrational spectroscopy. For example, infrared spectroscopy and its nonlinear analogue sum frequency generation spectroscopy specifically tailored for electrochemical surface investigation provide direct chemical insight into molecular behaviour under reaction conditions with micronscale resolution. [12][13][14][15][16] However, the accessible spectral range is typically limited on the low-energy side, and aqueous electrolyte poses an additional difficulty because strong water absorption may mask vibrational bands of the reactive adsorbate species of interest. Despite its inherent low scattering cross sections, Raman spectroscopy constitutes a valuable alternative for molecularscale surface analysis of electrochemical systems and processes. [17][18][19] Advanced nonlinear and nearfield Raman tools are being developed to obtain in situ chemical fingerprints of physico-chemical actions and reactions of molecules at complex electrified solid/liquid interfaces with ms temporal or sub-tennm spatial resolution. Examples presented in this review concentrate on energy and catalytic conversion processes as well as on the development of novel Raman-based tools for nanoscale analysis, including the molecular behaviour in nanoporous systems studied with nonlinear Raman spectroscopy and the successful development and implementation of a novel tip-enhanced Raman setup capable of working at solid/liquid interfaces of non-transparent substrates under potential control. Correlating known macroscopic device behaviour to molecular-level processes observed under in situ working conditions with advanced Raman approaches enables performance evaluation in an unprecedented detailed manner and paves the way for rational design of improved electrochemical systems.

Raman Spectroscopy Revisited
Vibrational Raman spectroscopy is based on the inelastic scattering of light where the energy difference between the incident and scattered photons (w inc -w sc ; Raman shift) corresponds to the energy difference between two vibrational states of a material under investigation ( Figure 1a). Excitation typically occurs from the ground state (n 0 ) into the first vibrational excited state (n 1 ) of the Raman-active modes of a particular substance (Stokes scattering). A broad energy range is easily accessible with Raman spectroscopy, covering low-energy modes between 50 and 300 cm À1 , such as solid-state phonons in metal oxides or metal-organic vibrations, molecule vibrations between 700 and 2000 cm À1 and CH/OH stretches around 3000 cm À1 and higher.
As vibrational energies depend on the atomic bonds involved, a Raman spectrum provides a unique chemical fingerprint of the sample. Furthermore, Raman spectroscopy provides direct quantitative information, because the scattering intensity is directly proportional to the number of scatterers. Note that Raman scattering cross sections are typically very low so that only about one in a million photons is inelastically scattered. As a result, Raman spectroscopy is conventionally employed to characterize bulk samples, such as powders or highly concentrated solutions, and second-long spectral integration times and mW excitation powers are typically required to obtain good signal-to-noise spectra. On the one hand, this can be of advantage for electrochemical applications as work-ing in aqueous electrolyte gives only weak, if any, Raman water signals. On the other hand, the investigation of (sub)monolayer adsorbates at electrochemical interfaces or in nano-confinement is a huge challenge, because the sensitivity is generally too low to detect few scatterers. How can we exploit the advantages of Raman spectroscopy to access local information of the molecules at electrochemical interfaces?

Monitoring Molecular Interactions and Diffusion in Nanopores with Nonlinear Raman Spectroscopy
One way to enhance the inherently weak Raman scattering from confined molecules is to enforce coherent scattering with short, intense laser pulses. In coherent anti-Stokes Raman spectroscopy (CARS), three photons of specified energies w 1,2,3 interact with the sample molecules in a way that a fourth photon at the anti-Stokes energy is scattered (Figure 1b).
Through the coherent signal emission and nonlinear nature of the wave-mixing approach, the CARS signal is enhanced by three to five orders of magnitude compared to conventional Raman scattering. For broadband CARS approaches covering a few hundred wavenumbers energy range, mathematical approaches to extract Raman-like quantitative spectra from the CARS spectra are readily available. [20,21] As a result, spectral integration times can be reduced compared to conventional Raman spectroscopy to tens of ms, rendering CARS a valuable novel tool for in situ quantitative monitoring of dynamic molecular processes on the nanoscale. Commonly, CARS is employed in the field of bio(medical) imaging [22,23] and has only recently been introduced to the field of materials science. In the following, we highlight the potential CARS holds for the study of important (electro)chemical processes occurring inside nanopores, such as the catalytic chemical conversion on zeolite particles and the interaction and diffusion of water inside fuelcell membranes.

Zeolite Nanopore Catalytic Reactivity
Huge efforts are being carried out to develop 'greener' liquidphase, low-temperature catalytic approaches, and molecularlevel insight into the spatio-temporal distribution of reactants and products of catalytic conversion processes in situ would greatly benefit the development of cost-efficient next-generation catalysts. To answer fundamental questions about initial chemical transformation steps and the location of the corresponding catalytically active sites under liquid-phase working conditions, new operando analytical approaches are sought. [24,25] Introducing CARS to the field of heterogeneous catalysis, the 3D distribution and geometric alignment of thiophene reactant in the nanopores of individual zeolite particles were mapped, giving unprecedented insight into the early activation stages of sulfur-removing catalysis processes. [27,28] The full power of CARS for catalysis research was demonstrated by mapping renewable bio-fuel conversion of glycol with alkenes on single catalyst particles with submicron 3D spatial chemical resolution. Figure 2a shows an overlay of the CAR band intensity of the reactant C=C double bond (red) and of the fluorescence intensity of the cations (green) in 2D maps recorded from an individual catalyst particle. After chemisorption of the alkene, reactant activation and formation of stabilized alkyl cations inside the zeolite pores can be watched to proceed with increasing reaction temperature from the rim toward the center of the zeolite, following the aluminum zoning naturally present in the catalyst. While conventionally, the ether product can only be detected off-line after conversion, CAR spectra allow the in situ quantification of ether species directly on the catalyst: Figure 2b depicts CAR spectra recorded immediately after reaction onset (t = 0 min) and after 90 min (t = 90 min) and the respective non-negative least squares fitting results. The yield of the ether product component (green) is seen to increase with time to about 50 %, about five times more than extracted from the catalyst after the reaction. In summary, the findings suggest that using small ZSM-5 particles without aluminum zoning and finding more efficient ways to extract the product from the catalyst could greatly improve this important liquid-phase catalytic reaction.

Water Transport in Nafion Fuel-cell Membranes
Proper water management in fuel-cell membranes is critical for the design of efficient devices, because the level of membrane humidity, specifically, the interactions between water molecules and membrane matrix, strongly affect the cell performance. [29,30] An in-depth understanding of the water and proton transport inside the membrane nanopores is quintessential for the development of novel materials with increased proton transport efficiencies for improved fuel cells.
The nanoscale chemical interactions in two differently processed Nafion membranes, N117 and N212, of identical chemical composition but distinct proton transport properties has been elucidated with CARS. [31,32] While the Nafion signatures of both N117 and N212 are identical, distinct spectral shapes of to a vibrational excited state (n 1 ) whose energy difference matches the one between incident and scattered photons (w inc -w sc , Raman shift). Raman spectra cover a broad energy range, including solid-state phonons or metal-R interactions, the molecular fingerprint region, and high-frequency modes of CH/OH. Bottom: To investigate a small amount of scatterers at interfaces or in confinement, the weak Raman scattering can be enhanced by nonlinear (coherent anti-Stokes Raman, CARS) or nearfield (surface-or tip-enhanced Raman, SERS/TERS) approaches. b) CARS is a four-wave mixing technique where the enhancement is mainly based on coherent scattering from the sample. For c) SERS and d) TERS, field enhancement is achieved through the excitation of surface plasmons at rough surfaces or in a tip-sample gap, respectively. the water OH stretch signature for the two membranes were observed (Figure 3a) that can be fitted with two types of water signatures (Figure 3b), namely bulk water (water molecules coordinated to other water molecules, blue) and undercoordinated non-bulk water (orange), located in the center of the ionic channels and at the interface between the hydrophobic channel walls and bulk water, respectively. [31] The relative higher amount of non-bulk water in N117 than in N212 can be explained by smaller nanopores in N117 that lead to more widely spaced SO 3 À head groups, more surface area, and thus to more non-bulk water compared to N212 (Figure 3c). The CARS results further suggest that non-bulk water molecules diffuse an order of magnitude faster (D nb % 16 10 À10 m 2 /s) than bulk-water molecules (D b % 10 À10 ) inside the membrane nanochannels, which could lie at the origin of the faster macroscopic water and proton transport observed in N117 compared to N212. [31] While CARS provides quantitative information about solid/ liquid chemical interactions, molecular adsorption geometry and reactivity of nano-confined species, it is important to remember that the coherent scattering response in fact still stems from the nanopore ensemble probed in the focal volume. As such, direct nanoscale sensitivity is still lacking with CARS. To obtain Raman responses from interfacial adsorbate monolayers, or even from individual scatterers, signal enhancement has to be achieved differently.

Hot-spotting Nanoscale Chemistry with Surface-enhanced Raman Spectroscopy
Surface-enhanced Raman spectroscopy (SERS) is based on the formation of interface-confined electromagnetic nearfields through plasmon excitation in nano-structured coinage (Cu, Ag, Au) metal surfaces (Figure 1c). Raman signal enhancements of three to seven or more orders of magnitude are routinely achieved for molecules located in close vicinity of Au, Ag or Cu nanometer-sized particles or structures or inside (sub-)nanometer gaps, i. e. at 'hot spots' where the nearfield is particularly strong, [33] rendering possible the trace analysis of small amounts of scatterers present in (sub)monolayer adsorbates, and even single-molecule detection. [34,35] The potential of SERS character- Figure 2. a) Combined CARS and 2-photon fluorescence images of reactant (red) and product (green) distribution monitored in situ on a single zeolite particle as the reaction progresses with temperature. b) The ether product formation can be followed in situ in the time-resolved CAR spectra with help of non-negative least squares fitting. After 90 min, 50 % of the reactant has been converted into product. Reproduced from Ref. [26] Figure 3. a) CAR spectra of hydrated N117 (green) and N212 (black) indicate chemical identity of the two Nafion membranes, but the presence of different water species. b) Fitting with bulk (blue) and non-bulk (orange) water spectra show that N117 contains more non-bulk water than N212 which can be explained by c) the different nanoscale chemical constitution of the membrane channels of N117 (smaller channels, less densely packed SO 3 À groups) compared to N112. Reproduced from Ref. [31] ization of electrochemical interfaces based on organic molecules adsorbed at metal surfaces has been repeatedly demonstrated. [36,37] In the following, examples for the specific strength of SERS regarding nanoscale chemical analysis of the electrosynthesis of novel metal-organic hybrid materials and of the (photothermal) degradation of quantum-dots (QDs) in sensitized solar cell devices are highlighted.

The Electrochemical Growth Mechanism of Cu-MOF
Metal-organic frameworks (MOFs) possess great potential for energy storage and conversion applications, [38,39] because their chemical and structural properties can be tuned through the intentional choice of metal ions and organic linkers. However, the lack of understanding of the nucleation stage and early growth process of this novel class of functional crystalline materials still hinders the development of tailored bottom-up MOF fabrication protocols. Specifically, electrochemical MOF synthesis offers extraordinary possibilities for precise size [41,42] and functionality [43] tailoring through potential control. In a systematic SERS study of varying synthesis conditions, it was recently clarified that the electrochemical synthesis of prototype CuBTC proceeds in a two-step oxidation mechanism at the Cu electrode surface. [40] The strong nearfield signal enhancement at the atomically rough Cu electrode rendered possible the observation of the SERS signatures of the thin Cu 2 O precursor layer and of few CuBTC crystals forming on the electrode surface ( Figure 4). Combined SEM and SERS results indicate that the common first reaction step is the oxidation of metallic Cu to Cu(I) oxide in the presence of H 2 O or O 2 . Cu 2 O is then further oxidized with the linker molecules to form Cu(II)BTC as octahedral crystals growing directly at the electrode surface. These findings generate interesting possibilities for the design of patterned MOF devices based on Cu 2 O pre-patterning of any imaginable substrate.

The SERS Signature of PbS Quantum Dot Oxidation
PbS QDs, amongst other small-bandgap semiconductor materials, are of prime interest for photovoltaic energy conversion, for example, in sensitized solar cells, reaching conversion efficiencies up to about 10 %. [44] However, fast device degradation still hinders commercialization of QD-sensitized solar cells. To understand the origin of device degradation, systematic analytical approaches to monitor the chemical state of the QD sensitizer in situ during fabrication and operation are needed.
SERS fingerprints of PbS QDs deposited on Au SERS substrates showed different stages of (photothermal) oxidative QD degradation. [45] Isolated PbS QDs of 5 to 10 nm diameter were fabricated wet-chemically on commercial nanostructured Au substrates following typical solar-cell fabrication protocols under inert-gas conditions in a glove box (Figure 5a, b). The SERS signatures from different sample batches indicate that the chemical state of the QD sensitizers strongly depends on the oxygen level in the preparation atmosphere as evident from the simultaneous relative decrease of PbS (196 cm À1 ) and increase of PbO (312 and 450 cm À1 ) Raman marker bands in the low-wavenumber region (Figure 5c-e). [45] Only samples prepared and tightly sealed-at lowest oxygen levels show longterm stability even at increased power densities up to ca. 2 10 18 W/m 2 . This work demonstrates how SER spectral markers allow the direct in situ visualization of the oxidative degrada-   [45] tion state of PbS sensitizers, and SERS can serve as a fast benchtop control for in situ monitoring of PbS QD oxidation during device fabrication.
These examples illustrate the great versatility of SERS for obtaining valuable in situ information about interfacial chemical conversion processes underlying photovoltaic applications or novel electrosynthesis approaches of advanced functional materials. The exact location and enhancement properties of the few active 'hot spots', however, still remains elusive, and the SERS response, in general, has to be considered an ensemble response from few scatterers in SERS-active locations. To watch the first nucleation steps of single MOF crystals or probe size-dependent optical properties of individual QDs, or even to record potential-dependent maps of particle reactivity, it would be advantageous to create a free-floating, movable 'hot spot' over a location of interest. In addition, such an external 'hot spot' would also broaden the applicability of SERS to oxide-based or other non coinage-metal electrodes.

(Electrified Solid/liquid Interfaces Probed with Tip-enhanced Raman Spectroscopy
One way to generate a substrate-independent 'hot spot' close to a sample interface is by means of tip-enhanced Raman spectroscopy (TERS; Figure 1d), where a coinage-metal scanning-probe tip is brought in close vicinity to the surface and acts as a nano-antenna to provide high field-enhancement at the location of interest. With TERS, similar Raman signal enhancement as with SERS can be achieved to probe, for example, molecular catalytic properties [46,47] and interactions [48] of a few scatterers underneath the tip at a chemical spatial resolution in the order of around 5 nm. [49,50] Due to the setup complexity, TERS experiments are commonly carried out in air. A few ultrahigh-vacuum setups exist that provide particularly stable working conditions and extreme spatial [51] or temporal resolution. [52] However, air and vacuum are only crude approximations for electrochemical systems and there is a great interest in developing TERS systems capable of operating at electrified solid/liquid interfaces.

A Versatile Side-illumination TERS for Solid/liquid Interfaces
The possibility to detect TER scattering at solid/liquid interfaces was first demonstrated by Zenobi and coworkers in 2009 [53] with a transmission configuration. To enable the use of opaque electrodes, a side-illumination configuration is desirable. Here, the main challenge lies in the beam aberrations that occur at the air/glass/water interfaces in the excitation laser path. [54] Recently, we have developed a versatile side-illumination solid/liquid TER spectroscope based on a home-built sample cell design (Figure 6a) that can be easily implemented in a commercial scanning tunneling microscope (STM). [55] By keep-ing the beam path in water a few mm short, focusing the laser onto the tip apex is as straightforward in water as in air.
As depicted in Figure 6b, a comparison of nearfield (i, iii) and farfield spectra (ii, iv) from electronically resonant dye molecules adsorbed on an opaque Au(111) crystal showed that similar Raman enhancement factors in the order of 10 5 can be reached for both in-air (top; i, ii) and in-water (middle; iii, iv) experiments. Importantly, the tip check (v) proved that the tip was clean and the TER spectra originated from dye molecules adsorbed at the single-crystal substrate. Furthermore, the high surface sensitivity of the solid/liquid TER approach was Figure 6. a) Side-illumination TERS setup design for work at solid/liquid interfaces. b) Resonant TER spectra of malachite green isothiocyanate (MGITC) at Au (111) recorded in air (i) and in water (iii). Respective far-field Raman spectra (ii, iv) and tip check (v). Nonresonant TER spectra of thiophenol at Au(111) in air (i) and in water (iii) and the respective far-field spectra (ii, iv). Reproduced from Ref. [55] confirmed by probing a monolayer of non-resonant thiophenol/Au(111) (Figure 6c) where the in-water experiment (bottom; iii, iv) shows a similar nearfield enhancement effect upon tip approach as the in-air experiment (top; i, ii). The difference in relative Raman enhancement between air-versus-water dye and thiophenol experiments was ascribed to the difference in the chosen STM parameters for each experiment. Both, tip-sample bias and tunneling current setpoint infl the tip-sample distance. As the TER scattering intensity is known to depend exponentially on the tip-sample gap size, [50] a relative decrease in bias voltage and a relative increase in tunneling current setpoint lead to a smaller gap and hence to a TER intensity increase. [56][57][58]

Electrochemical TERS
Adjusting the solid/liquid TERS apparatus for potential-dependent spectro-electrochemical (EC-TERS) experiments requires additional electrical contacting and control of the working electrode under investigation, thus rendering the setup even more complex and technically challenging. EC-TERS has been a long sought-after goal of the community, and finally in late 2015, the groups of Ren and of Van Duyne could first demonstrate its succesful implementation: Ren and coworkers observed potential-dependent changes in TER spectra due to adsorbate reorientation on Au(111), [59] followed shortly after by a report by the Van Duyne group who studied the redox behaviour of Nile Blue dye molecules with AFM-based EC-TERS. [60] These works demonstrate the impressive potential that EC-TERS holds for studying nanoscale electrochemical processes in molecular detail.
A few important points need to be considered for the successful implementation of EC-TERS. First, as for all electrochemical experiments, utmost care has to be taken to provide a clean working environment. Note that this is of particular importance in TERS because of the extremely high chemical and spatial resolution. Even few contaminant molecules present in the 'hot spot' under the tip apex can significantly contribute to the TER signal. Next, particularly when working in an STM-TERS configuration, the tip has to be insulated with a coating that prevents from Faradaic currents and does not fluoresce so as not to mask the TER signals with a strong background. We found that proper tip coating provides a much more stable 'hot spot' also under non-electrochemical solid/liquid conditions. [55] Furthermore, it is crucial that adsorption of probe molecules on the tip is prevented during the experiment, because the atomically rough tip surface itself can easily generate large SER signals that may be difficult to discriminate from the sample TER response. Bipotentiostatic control of substrate and tip can help to maintain the tip free from adsorbates; however, in STMbased EC-TERS, a rational choice of STM-parameters has to be made to avoid molecular movement between sample and tip, and to keep the gap size stable. Figure 7 shows potential-dependent TER spectra of a monolayer of physisorbed adenine on Au(111) in 0.01 M H 2 SO 4 from our laboratory (inset: cyclic voltammogram recorded in the EC-TERS cell showing the reconstruction peak in the double-layer region; scan rate 0.2 mV/s). With constant sample bias of 0.4 V, the characteristic adenine ring breathing (732 cm À1 ) and C=C/C=N stretching modes (1320 and 1460 cm À1 ) are observed at negative electrode potentials (À0.4 V vs Pt). When increasing the Au-electrode potential to 0.2 V vs Pt, the adenine TER signatures decrease while the broad spectral background remains largely unchanged, indicating that the molecules stay adsorbed at the surface and the enhancement is still active. This trend of increasing band intensity with decreasing electrode potential agrees well with recently published electrochemical surface-and shell-isolated particle enhanced Raman spectroscopy data. [61,62] Note that the spectral changes are reversible, i. e. when applying a negative potential again, the adenine EC-TER bands reappear.
The observed EC-TER signature of adenine/Au(111) can be ascribed to potential-induced molecular reorientation. The geometrical arrangement of adenine on Au single crystals has been extensively investigated with electrochemical infrared spectroscopy, EC-STM and cyclic voltammetry. [63,64] There is a general agreement that in acidic media and at low adenine concentrations, as the case for the EC-TERS data presented here, protonated adenine is chemisorbed at positive potentials, while at lower potentials, protonated adenine physisorbs. However the specific geometrical arrangement of the adenine molecules with respect to the electrode surface has remained controversial [63] As the Raman polarizability tensor of the ring breathing mode (732 cm À1 ) is highly anisotropic, [65,66] a reorientation of the adenine rings could lead to the observed TER intensity changes. While the preliminary results shown here are very promising for future applications of EC-TERS to the investigation of molecular behaviour at electrochemical interfaces, a more detailed interpretation of the EC-TERS data at hand -but also in general -will require further experimental and particularly also simulation efforts to deconvolute the effects of the metal electrode, electrolyte, molecular interactions, surface charge and tip on the signal.

Summary and Outlook
We have presented examples of how advanced nonlinear or nearfield Raman spectroscopies can greatly benefit the nanoscale analysis of molecules or nanoparticles adsorbed at electrochemical interfaces or in confinement. As summarized in Table 1, each Raman-based approach has its specific strengths and can be chosen based on the requirements of the system under investigation. Dynamic measurements of adsorbates inside nanoporous systems can be realized with nonlinear CARS, while solid/liquid TERS provides extreme chemical spatial resolution in the order of a few nm and precise interrogation of the sample region of interest. Both TERS and SERS can be operated under electrode potential control and thus are powerful spectro-electrochemical tools for the visualization of potential-dependent adsorbate reorientation or chemical conversion.
As an outlook, the development of an EC-CARS approach could be expected to hold interesting opportunities for electrochemists: By adding an electrical contact to a membrane or catalyst as a working electrode, one could monitor potentialcontrolled chemical processes inside nanoporous systems. 3D EC-CARS imaging could reveal spatial heterogeneities in the potential-triggered reactivity of functional membrane or catalyst materials. From a fundamental knowledge point of view, such experiments could greatly contribute to our understanding of the concepts of the electrochemical interface, double layer formation and local potential distribution inside electroactive nanopores.


extfieldsofstudy: Physics Chemistry

text:
Application of silicene, germanene and stanene for Na or Li ion storage: A theoretical investigation

Silicene, germanene and stanene likely to graphene are atomic thick material with interesting properties. We employed first-principles density functional theory (DFT) calculations to investigate and compare the interaction of Na or Li ions on these films. We first identified the most stable binding sites and their corresponding binding energies for a single Na or Li adatom on the considered membranes. Then we gradually increased the ions concentration until the full saturation of the surfaces is achieved. Our Bader charge analysis confirmed complete charge transfer between Li or Na ions with the studied 2D sheets. We then utilized nudged elastic band method to analyze and compare the energy barriers for Li or Na ions diffusions along the surface and through the films thicknesses. Our investigation findings can be useful for the potential application of silicene, germanene and stanene for Na or Li ion batteries.

Introduction
The interest toward two-dimensional (2D) materials was raised by the great success of graphene [1][2][3]. Graphene is a zero-gap semiconductor that present outstanding mechanical [4] and heat conduction [5] properties, surpassing all known materials.
Silicon based anodes due to the high specific capacity of 4200 mAh/g [21,22] are considered as an alternative to the conventional graphite based anode materials. The degradation of silicon and its drastic volume changes during the battery operation nevertheless question the commercialization of this battery technology [23][24][25].
Having similar atomic and electronic structures as that of the silicon, germanium and tin are also expected to present high capacity for Li-ion batteries. Silicene, germanene and stanene, the 2D form of these element, owing to their large surface area, high mechanical flexibility and higher electron mobility can be also therefore considered as interesting candidates to use in secondary batteries [20]. Nonetheless, because of experimental complexities, theoretical studies can be considered as an alternative to evaluate the application prospect [25][26][27][28][29]. This study consequently aims to probe the applicability of silicene, germanene and stanene for Li or Na ions storage using DFT calculations.

Modelling
All DFT calculations in the present study were carried out using Vienna ab initio simulation package (VASP) [30,31] using the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation exchange-correlation functional [32]. The projector augmented wave method [33] was employed with an energy cutoff of 450 eV. In this work, van der Waals interactions have been included using the semiempirical correction of Grimme [34], as it is implemented in VASP. This method have been shown to be a valid choice for calculating the binding energies [35,36]. We applied periodic boundary conditions in all directions with a 17 Å vacuum layer to avoid image-image interaction along the sheet thickness. In order to simulate adatoms adsorption, we included 60 atoms for the pristine silicene, germanene and stanene sheets. The planar size of these constructed periodic cells were about 20 Å×19.24 Å, 20.85 Å×20.07 Å and 23.92 Å×23 Å, for silicene, germanene and stanene membranes, respectively. We first identified the strongest binding sites for Na or Li ions and then we gradually increased the ions concentrations by randomly but uniformly locating them on the pre-identified sites. After automatically positioning the ions, conjugate gradient method energy minimization was performed with a 10 -4 eV criteria for energy convergence using a 5×5×1 k-point mesh size for Brillouin zone sampling. A single point calculation was finally achieved to report the energy and electronic density of states (DOS) of the system at which the Brillouin zone was sampled using a 13×13×1 k-point mesh size. Tetrahedron method was employed for the calculation of DOS. For the evaluation of charge transfer between the ions and substrate, we performed Bader charge analysis [37]. Climbing-image nudged elastic band (NEB) [38] method was utilized to obtain diffusion pathways and the corresponding energy barriers. For the NEB calculations, we included 12 atoms for the films with a Monkhorst Pack mesh [39] of 15×15×1 special points. We note that we also compared the energy of the several systems with different number of k-points along the sheets thickness and we found that a single k-point yields absolutely accurate results.

Results and discussions
We first evaluate the most stable adsorption sites for a single Na or Li atoms on silicene, germanene and stanene sheets. A relatively large energy for the ion adsorption on an anode material is a highly demanding factor. To find the most stable adsorption site, we considered three different initial positions: above the top of 5 an atom, above the middle-point of a bond and finally above the hollow site in the hexagonal lattice. In order to accurately find the adsorption site, we used a relatively strict energy criteria of 10 -5 eV for the conjugate gradient method energy minimization termination. Interestingly, for the both Na and Li atoms on silicene, germanene and stanene, we found that the hollow sites present the maximum binding energy. This observation is in agreement with previous studies for Li atom adsorption on silicene [20,43]. For these hollow sites, in Fig. 2   increasing the atoms content, the anode films undergo deformation and lattice distortion. For Na or Li atoms adsorption on silicene, however an almost fully saturated structure (Fig. 4d) presents a more uniform structure in comparison with a film with lower concentration of adatoms (Fig. 4c). This can be explained due to the fact that for a fully covered film, the structure is more symmetrical. On another hand, for the stanene sheet covered with Li atoms, we found that in general by increasing the ions content the distortion increases ( Fig. 4f and Fig. 4h). In this case as it is shown in Fig. 4e, due to a much larger hollow site, the Li atoms are adsorbed almost inside the film. By increasing the ions concentration, consequently the new Li ions push more the surrounding Sn atoms inducing higher distortions in the lattice.
Therefore, for a particular adatom, if the strongest adsorption site is placing upper than the film, the lattice distortion upon the ion insertion reduces. We should remember that the amount of distortion also correlate to the stiffness of a material.
For silicene, germanene and stanene, in general, the stiffness has been reported to reduce considerably with increasing atomic weights (from Si to Sn) [45,46]. Lower stiffness rigidity can also well explain the higher distortion of the lattices.
Nevertheless, by increasing the adatoms concentration on the considered films, the buckling length increases gradually. We mention that Na atom because of a larger radius adsorbs on the hollow site, upper than the Li atom. For Na atoms consequently the change in the buckling length is lower. Because of the free surfaces and the flexibility of the considered films, the maximum stresses induced in the structures are always below 0.72 GPa.nm. This stress is clearly low, implying that the structures cannot be degraded mechanically upon the ions intercalation. Fig-4, Samples of lithiated silicene and stanene films.
Diffusion of ions inside an anode plays an important role in the batteries performance. We next shift our attention to the diffusion pathway of a single Na or Li ions on silicene, germanene and stanene films. This was achieved by calculating the variations in energy as an adatom moves between equivalent adsorption sites.
Diffusion process involves simultaneous motion of several atoms, including the Na or Li adatom and their Si, Ge or Sn neighbours [20]. Two representative diffusion pathways were considered, planar path which happens along the surface of the films and the other pathways is occurring along the sheet thickness by passing through the hexagonal hollow. In the all studied cases for the diffusion along the surface, first Li or Na atom at the hollow site jump over a Si, Ge or Sn atom, then they move to the next hollow site (Fig. 5a and Fig. 5c). Therefore, diffusion along the surface follows a zigzag pathway. The diffusion energy barrier for Na ion along the surface is predicted to be slightly smaller than that for Li ion. Na atom because of the larger radius is placed higher out of the hollow site and it can diffuse and facing less repulsive forces. Electronic conductivity is among the important factors for the application prospect of an electrode material. The internal electronic resistance of a battery correlates strongly to the electronic conductivity of the electrode materials. Moreover, the ohmic heating generated during the charging or discharging process is also proportional to the electronic conductivity of the active material particles. We therefore briefly explore the electronic responses of studied systems. The calculated electronic DOSs are plotted in Fig. 6. For the all studied pristine films without Na or Li adatoms, at the zero state energy (Fermi level) the DOS is zero, this reveals zerogap semiconducting electronic nature. Nevertheless, only in the case of silicene we found that upon lithiation, the DOS undergoes a transformation from a zero-gap to a semiconductor with a finite band gap. This finding is in agreement with the previous theoretical studies for lithiated silicene [20,43]. For the rest of studied systems, we found that upon the adatom adsorptions, total DOS for valence/conduction band around the Fermi level are increased. In these cases, at the Fermi level the band gap 9 is disappeared, which implies that the structures are presenting metallic electronic response. It can be therefore concluded that only the silicene present semiconducting properties upon the Li atoms adsorption, which is not a desirable behaviour for its practical application as an anode material for Li ion batteries. In summary, we performed DFT calculations to investigate the application of silicene, germanene and stanene for Na or Li ion batteries. We found that in all cases the hexagonal hollow sites offer the maximum binding energies for ions. We predicted that the binding energy almost remains constant by increasing the ions concentration up to the full coverage of the films. Based on the Bader charge analysis results we predicted high charge capacities of around 954 mAh/g, 369 mAh/g and 226 mAh/g for Li or Na ions storage using single-layer silicene, germanene and stanene, respectively. We found that adatoms adsorption induce lattice distortion and in addition by increasing the ions coverage the buckling length of the sheets increases gradually. We estimated that the studied films structures cannot degrade mechanically upon the ions intercalation. We then probed the diffusion pathways and the corresponding energy barriers of Li and Na ions. We studied the diffusion pathways over the surface and also through the thickness of the films. Finally, we explored the electronic DOS of the studied films upon Li or Na ions adsorption. Our findings can be useful for the possible application of silicene, germanene and stanene for Na or Li-ion batteries.