Electrochemically deposited conductive composite sorbent

for highly efficient online solid-phase microextraction of

jasmonates in plant samples

Xu Linga,b, Zilin Chen *a,b

aKey Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan

University), Ministry of Education, and Wuhan University School of Pharmaceutical

Science, Wuhan 430071, ChinabState Key Laboratory of Transducer Technology, Chinese Academy of Sciences,

Beijing 10080, China

Corresponding authorDr. Zilin ChenLuojia Chair Professor Vice Dean and Institute Director School of Pharmaceutical Sciences, Wuhan UniversityWuhan, 430071CHINAPhone: 86-27-68759893Fax:   86-27-68759850Email: chenzl@whu.edu.cn

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Supplementary data

The Supplementary data provides additional relevant details of this work, including:

Optimization of the polymerization conditions

Optimization of parameters for online solid-phase microextraction-HPLC

Figure. S1. Liquid chromatograms of jasmonic acid and methyl jasmonate

after SPME with PEDOT-SG and PEDOT respectively

Figure. S2. Cyclic voltammogram of electropolymerization

Figure. S3. Optimization for polymerization

Figure. S4. SEM image of pure PEDOT on carbon fiber bundle

Figure. S5. Optimization for extraction

Table. S1. Table S1. Comparison of PEDOT-Sulfonated graphene based

SPME-HPLC for jasmonates with other current Methods

Table S2. Recoveries of jasmonic acid and methyl jasmonate by online

SPME-HPLC

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Optimization of polymerization conditions

The polymerization process of the sulfonated graphene-PEDOT hybrid composite on

carbon fiber may be strongly influenced by many factors, such as monomer and

supporting analyte concentration, scan rates. To obtain higher extraction efficiency,

several main affecting factors were investigated as discussed as follows.

Effect of sulfonated graphene concentration. Sufficient sulfonated graphene in the

supporting electrolyte solution is a key factor for the extraction of analytes. Therefore,

it is necessary to investigate the effect of sulfonated graphene concentration on

extraction performance. In general, the solubility of sulfonated graphene in aqueous

solution is 2 mg⋅mL⁻¹. In our experiment we investigated five different

concentrations gradient and triple respectively for each concentration. From the result

shown in Figure. S2A, we can see that when the concentration is blew 2 mg⋅mL⁻¹,

the extraction efficiency kept increasing gradually. However, concentration of higher

than 2 mmol⋅L⁻¹ cannot dissolve and uniformly distribute in the solution, which may

cause waste of synthesized sulfonated graphene. As a result, we chose 2 mg⋅mL⁻¹ of

sulfonated graphene for our experiment.

Effect of scan rates. Scanning rates is a vital kinetic parameter, which helps to

increase the homogeneity of the coating obviously, thus improving extraction

efficiency and reducing extraction time. The effect of the scanning rate (0.025, 0.05,

0.075, 0.1, 0.2 V⋅s⁻¹) on the extraction efficiency of jasmonates was investigated.

Results in Figure. S2B shows that the peak areas for both jasmonic acid and methyl

jasmonate reached its maximum amounts at a scan rate of 0.025 V⋅s⁻¹. The results

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revealed that slow scan rate leads to better the extraction efficiency. However, it

would take longer time to coat the fiber bundle. Therefore, slower scan rates less than

0.025 V⋅s⁻¹ were not tested and it was chosen as an optimal option.

Effect of scan segments. The thickness of the electro-polymerized hybrid

membrane is influenced by scan segments in cyclic voltammetry. When it was varied

between 70 and 130, the extraction efficiency varied slice as Figure. S2C showed.

When scan segments increased from 130 to 150, the extraction efficiency increased

sharply simultaneously. We deduce that an increase of cyclic voltammetry segments

up to 150 gave rise to a rougher surface of the hybrid film on the fiber bundle, as

more sulfonated graphene are immobilized during polymerization. Concerning about

the time for modification, we choose 150 scan segments for next step.

Optimization of parameters for online solid-phase microextraction-HPLC

The sorption of jasmonates molecules onto the surface of modified fibers is mainly

due to the hydrophobic interaction, hydrogen bonds and π-πelectrostatic force, which

may be affected by pH of the solution to a great extent. Therefore, pH value of sample

solution is an important parameter for SPME. The pH values among 3.0-8.0 were

investigated, and the pH value was adjusted by 0.01 mol⋅L⁻¹ NaOH and HCl solution.

Sample solutions of 10 mL were loaded onto the polymer sorbent at a constant flow

rate (1 mL⋅min⁻¹). Total peak areas were calculated at each pH value and the results

are shown in Figure. S4A. The extraction efficiency of both jasmonic acid and methyl

jasmonate increased along with the increase of pH in the range of 3.0–6.0. A

maximum for jasmonic acid was observed in pH 6 and for methyl jasmonate it was

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pH 7. Higher pH value over 7 would result in lower extraction efficiency. This can be

explained by the dissociation state of carboxyl group on jasmonic acid molecules and

sulfonic acid group of sulfonated graphene. Acidic condition will restrain the

dissociation of acid group, making the analytes and the sulfonated graphene to be in

the electro-neutral state. As a result, the hydrophobic heterocyclic rings of the

jasmonates molecule were absorbed onto the conjugate carbon backbone. Concerning

insolubility, partial acidic condition may be advantageous for the hydrophobic

interaction. Dissociation of the jasmonates molecules would increase its solubility and

electrostatic force, but the hydrogen bonds may be destroyed in strong solution. In

general, mild aqueous condition is advantageous for the formation of hydrogen bonds

between jasmonates and sulfonated graphene. In our experiment we noticed that

jasmonates were better extracted in aqueous solution than in acetonitrile. However,

anions such as OH− are easy to react with jasmonic acid and help for dissociation of

sulfonic group. The extraction efficiency is the result of equilibrium of these three

kinds of interactions. With all factors taken into consideration, pH 6.0 is selected in

the following studies.

The pre-extraction solution is pushed through the packed PEEK tube loop, thus the

sample flow rate has potential effect on the contact between jasmonates and the

adsorbent. In our experiment, the effect of sample flow rate was studied; the values

were ranged from 0.5 to 0.9 mL⋅min⁻¹ controlled by a syringe pump. As shown in

Figure. S4B, peak areas fell off gradually in the examined sample flow rate, indicating

that sample flow rate has big influence on extraction efficiency. Considering analysis

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time and pressure, which might increase along with the flow rate, 0.5 mL⋅min⁻¹ of

flow rate was applied for further studies.

In-tube SPME based on packed sorbents is a nonequilibrium absorption process

and the extraction efficiency is closely related to sample volume. Volumes of pre-

extraction solution in the range of 2.5–25 mL were loaded onto the PEEK tube

separately, and the extraction efficiencies were investigated. As shown in Figure. S4C,

the peak areas increase rapidly along with increase of the sample volume from 2.5 to

20 mL and increased slowly from 20 to 25 mL. Sample volume of 20 mL was selected

for ultimate extraction.

Figure. S1. Liquid chromatograms of jasmonic acid and methyl jasmonate after SPME with PEDOT-SG and PEDOT respectively. Sample: 500 ng⋅mL⁻¹ pH 7 standard aqueous solution, 20 mL loaded at 0.8 mL⋅min⁻¹. Peak identification: 1 jasmonic acid, 2 methyl jasmonate.

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Figure. S2. Cyclic voltammogram of modification. (monomer

concentration：5 mmol⋅L⁻¹, scan rate: 0.025 V⋅s⁻¹, 150 scan

segments)

Figure. S3. Optimization for polymerization: (A) sulfonated graphene concentration, (B) scan rates, (C) scan segments. 20 ml sample solution

(10 ng⋅mL⁻¹, pH 7) was loaded at 1 mL⋅min⁻¹ by syringe pump.

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Figure. S4. SEM image of pure PEDOT on carbon fiber bundle

Figure. S5. Optimization for extraction: (A) Sample pH, (B) Sample flow rates, (C) Sample volume.

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Table S1. Comparison of PEDOT-Sulfonated graphene based SPME-HPLC

for jasmonates with other current Methods

Method Sorbents LOD (ng⋅mL⁻¹)

This method PEDOT-sulfonated graphene 0.01-0.1

Film Extraction-GC [4] Polydimethylsiloxane 0.2

PMME-HPLC [7] MAA-EGDMA 0.5-2.0

SPE-HPLC [10] β-CD modified silica, 4-VP-EGDMA 2.0-3.0

HSPME-GC-MS [11] Polydimethylsiloxane 1.3

Table S2. Recoveries of jasmonic acid and methyl jasmonate by online

SPME-HPLC

*Mean value. N=6.

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AnalytesConcentration

(ng⋅mL⁻¹)Added

(ng⋅mL⁻¹)Found*

(ng⋅mL⁻¹) Recovery %RSD%

Jasmonic acid 60.7 10.0 69.9 92.4 1.84

Methyl jasmonate 26.3 10.0 36.1 98.9 2.96

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