Biodegradable herbicidal ionic liquids based on synthetic auxins and analogues of betaine

Article abstract of DOI:10.1039/c7nj01474k

In this manuscript we present the synthesis and characterization of herbicidal ionic liquids (HILs) based on betaine derivatives (N-dodecylbetaine or N-(3-cocoamidopropyl)betaine) playing the role of the cation and four firmly established synthetic auxin

Full text of DOI:10.1039/c7nj01474k

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The role of atropisomers on the photo-reactivity and fatigue of
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Biodegradable herbicidal ionic liquids based on
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DOI: 10.1039/C7NJ01474K
synthetic auxins and analogues of betaine
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Michał Niemczak,* ,† Łukasz Chrzanowski, † Tadeusz Praczyk ‡ and Juliusz Pernak†
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† Department of Chemical Technology, Poznan University of Technology, Poznan 60-965,
Poland
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‡ Institute of Plant Protection – National Research Institute, Poznan 60-318, Poland
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ABSTRACT. In this manuscript we present the synthesis and characterization of herbicidal
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ionic liquids (HILs) based on betaine derivatives (N-dodecylbetaine DOoI:r10.10N39-/(C37N-J01474K
cocoamidopropyl)betaine) playing role of cation and four firmly established synthetic auxin
herbicides (2,4-D, MCPA, MCPP or dicamba) applied as anions. The syntheses were conducted
in two steps: protonation of alkylbetaines with hydrochloric acid followed by the ion exchange
reaction between formed intermediates and potassium salts of herbicides. All the synthesized
salts were classified as ionic liquids (ILs) owing to their melting points below 100 °C. The
products exhibited high thermal stability with decomposition temperatures (Tonset) varied from
240 to 273 °C. The herbicidal efficacy experiments performed under greenhouse and field
conditions confirmed their biological activity. The enhancement of their herbicidal activity
results from the potent surface-active properties generated by alkylbetaine cations. The spray
solutions of investigated HILs display low contact angle (approx. 50-62 °) and surface tension
(approx. 30-33 mN·m-1) contributing to the increase of contact surface between leaves and
active ingredient and thereby the increase of absorption of herbicide into the plant. The highest
susceptibility to biodegradation (approx. 60-75% efficiency) was proven for salts containing
phenoxyacetates (2,4-D and MCPA) anions.
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KEYWORDS. Herbicidal ionic liquids, 2,4-D, MCPA, MCPP, dicamba, weed control,
dodecylbetaine, cocoamidopropylbetaine.
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INTRODUCTION
One of the newest, promising possibilities of using ionic liquids (ILs) is to incorporate a herbicidal
anion into their structure. This action leads to the formation of a new group of compounds defined
as herbicidal ionic liquids (HILs).1,2 Due to their activity against unwanted vegetation, they may
be applied as anti-weed agents in crop protection causing the improvement of both quality and
quantity of crops.3 HILs belong to the third generation of ILs; they simultaneously exhibit targeted
biological activity and selected physical and chemical properties.4 The anions used for synthesis
of HILs are popular and commercially available herbicides such as phenoxyacids derivatives:
2,4-D,2,3 MCPA,1,4-6 MCPP1,4,6 and MCPB7. Other utilized herbicides include derivatives of
benzoic acid, e.g. dicamba,8 nicotinic acid, e.g. clopyralid,9 sulfonylureas, e.g. metsulfuron-methyl
(MSM),10 bentazone,11 fomesafen12 or one of the most popular non-selective herbicides –
glyphosate13. The greatest advantage of HILs in comparison to commercial herbicidal formulations
is their low volatility.8 Hence, the negative influence of toxic vapors is reduced due to the ionic
form of the pesticide which does not spread to the neighboring fields minimizing the risk of
environmental pollution. HILs also possess unique physicochemical properties, including high
thermal stability and reduced water solubility which minimizes the risk of unintended drift.6-8,13,14
The first reports on HILs focused on economically reasoned cations such as cationic surfactants,
quaternary ammonium or phosphonium halides. Their high surface activity resulted in enhanced
herbicidal efficiency and allowed to reduce significantly the required dose of active ingredient per
hectare. This strategy eliminates the necessity of using additional adjuvants often recommended
for commercial crop protection agents, which are often characterized by an unspecified toxicity
and negative environmental impact.1-3 Recently, the second generation of herbicidal ionic liquids
has been described and characterized. The firmly established herbicidal anions was successfully
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combined with a 2-chloroethyltrimethylammonium cation likewise exhibiting biological activity
as a plant growth regulator15,16 or fungicide, such as tebuconazole or propiconazole17. Moreover,
the chemical structure of the cation may be modified to regulate the toxicity and biodegradability
of HILs.1,14,18-20 Therefore, the reports describing HILs derived from renewable sources such as
betaine and carnitine21 or D-glucose22 started to appear. Recently, ionic liquids have been proved
to be effective plant resistance inducers; they are able to stimulate the resistance system of the
plant to act against pathogenic microorganisms, especially viruses - due to the fact there are no
plant protection agents acting directly on them.23,24
N-Dodecylbetaine and N-(3-cocoamidopropyl)betaine are two well-known representatives of
amphoteric surfactants, which structures are similar to amino acid in proteins.25,26 As zwitterionics,
they exist in aqueous solution in the form of inner salt possessing both the carboxylate anion as
well as a quaternary ammonium cation.27 (Fig. 1).
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Figure 1. Structures of N-dodecylbetaine (A) and N-(3-cocoamidopropyl)betaine (B), where coco
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is a mixture of alkyl substituents C8-C18.
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Alkylbetaines are commonly used as foam boosters in shampoos, shower gels, liquid soaps, hand
cleaners or as antistatic agents mostly due to their very low irritation levels and excellent capacity
to promote mildness in cosmetic formulations.27-30 Comparing to cationic surfactants,
alkylbetaines also have the advantages of good water-solubility, high biocompatibility and
biodegradability as well as fine stability in a wide pH range.25,26 The unique, multifunctional
properties allowed to successfully apply them as solubilizers of phosphatidylcholine liposomes,30
components enhancing the resonance light scattering (RLS) signals in the quantitative analysis of
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nucleic acids,25 additives to anionic surfactants promoting the surface elasticity31,32 or adjuvants
facilitating the mixing, application or effectiveness of agrochemicals (e.g., phenoxyacids,
glyphosate)32-35
.
The idea of incorporating alkylbetaines into HILs structure appeared as the consequence of
substantial benefits obtained from utilizing the cationic surface active compounds as the source of
the cation.1-3,8,10-13,36 Considering several reports describing excellent biodegradability of
amphoteric surfactants,37,38 we expected that alkylbetaine-based HILs will preserve this feature.
Here we present a convenient synthesis methodology of biologically active ILs consisting of cation
derived from cost-effective and easily available commercial amphoteric surfactants. This study
involves also an evaluation of the influence of the structure of both cation and anion on the HILs
physicochemical properties (i.e., solubility, thermal stability, surface activity), their herbicidal
efficiency (determined in greenhouse and field experiments) as well as susceptibility to
biodegradation.
MATERIALS AND METHODS
Materials
N-Dodecylbetaine ([C12Bet], CAS Registry No. 66455-29-6, 35% aqueous solution,
EMPIGEN® BB detergent) and N-(3-cocoamidopropyl)betaine ([CAPBet], CAS Registry No.
61789-40-0, 30% aqueous solution, Dehyton® PK 45) were purchased from Sigma Aldrich and
BASF, respectively. After dehydration of both solutions, betaines were dissolved in methanol.
Then, inorganic contaminants were separated via vacuum filtration and the filtrate was thoroughly
evaporated. Purified betaines were dried in vacuum for 24 h. Hydrochloric acid, potassium
hydroxide and solvents (methanol, DMSO, acetonitrile, acetone, isopropanol, ethyl acetate,
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chloroform, toluene, hexane), were purchased from Sigma Aldrich and used without further
purification. All herbicidal acids (2,4-D, MCPA, MCPP and dicamba) were supplied by Organika-
Sarzyna S.A. Poland and PESTINOVA Poland.
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Preparation of HILs
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Protonation of N-dodecylbetainium chloride [C12Bet][Cl]
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N-dodecylbetaine (0.20 mol) and 200 cm3 of acetone were placed in a 500 cm3 Erlenmeyer flask
equipped with a mechanical stirrer and the obtained mixture was vigorously stirred. Then,
stoichiometric amount (0.20 mol) of hydrochloric acid (36%) was added and the reaction was
conducted at 60 °C for 1 h. As a result of betaine protonation , a white solid of N-dodecylbetainium
chloride precipitated, which was filtered off and washed with small amounts of acetone.
Afterwards, the product was dried under reduced pressure at 50 °C for 24 h. The melting point of
N-dodecylbetainium chloride was in the range 158 – 160 °C.
Protonation of N-(3-cocoamidopropyl)betainium chloride [CAPBet][Cl]
N-(3-cocoamidopropyl)betaine (0.20 mol) and 200 cm3 of acetone were placed in a 500 cm3
Erlenmeyer flask equipped with a mechanical stirrer and the obtained mixture was vigorously
stirred. Then, stoichiometric amount (0.20 mol) hydrochloric acid (36%) was added and the
reaction was conducted at 60 °C for 3 h. Then, acetone and water were removed using a rotary
evaporator. In order to evaporate traces of water and hydrochloric acid the product was dried under
reduced pressure at 60 °C for 120 h. The obtained N-(3-cocoamidopropyl)betainium chloride was
a yellowish grease at room temperature.
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Anion exchange reactions
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All reactions were conducted in methanol using an EasyMax™ reactor. The appropriate amount
(0.05 mol) of N-dodecylbetainium chloride or N-(3-cocoamidopropyl)betainium chloride was
dissolved in 25 cm3 of methanol. Afterwards, 25 cm3 of the methanolic suspension containing the
potassium salt of 2,4-D (0.05 mol) or the methanolic solution of MCPA, MCPP or dicamba
potassium salts (0.05 mol) were slowly added. The reaction was conducted under constant stirring
at 60 °C for 3 h. As a result of the anion exchange reaction, the by-product (potassium chloride)
precipitated from the reaction solution. After cooling the post-reaction mixture to 0 °C, the
inorganic salt was separated by filtration and methanol was evaporated from the filtrate. Then, the
raw product was dried and dissolved in 50 cm3 of anhydrous acetone. The obtained precipitate was
separated via vacuum filtration and the solvent was evaporated. Finally, the product was dried
under reduced pressure at 50 °C for 24 h.
Methods
General
1H NMR spectra were recorded on Mercury Gemini 300 and Varian VNMR-S 400 MHz
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spectrometers operating at 300 MHz and 400 MHz with TMS as the internal standard. C NMR
spectra were obtained with the same instruments at 75 and 100 MHz, respectively. Elemental
analyses (CHN) were performed at the Adam Mickiewicz University, Poznan (Poland). NMR
spectra descriptions as well as elemental analysis results are provided in the Supporting
Information (Table S1 and Fig. S1-S20). The water content was determined using an Aquastar
volumetric Karl Fischer titration with Composite 5 solution as the titrant and anhydrous methanol
as a solvent. The water content in synthesized products was determined by Karl-Fischer
measurements and was found to be between 1000 – 1500 ppm.
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Density measurements
Density was determined using an automatic Density Meter DDM2911 with a mechanical oscillator
method. The density of the samples (approx. 2.0 cm3) was measured with respect to temperature
controlled conditions via Peltier, from 20 to 80 °C. The used apparatus was calibrated using
deionized water as the reference substance.
Viscosity measurements
Viscosity was determined using a rheometer (Rheotec RC30-CPS) with cone-shaped geometry
(C50-2). The viscosity of the samples (approx. 1.5 cm3) was measured with respect to temperature,
from 20 to 80 °C. The uncertainty of the viscosity measurement was estimated to be less than 10-
4 Pa·s.
Refractive index measurements
Refractive index was determined using Automatic Refractometer J357 with electronic temperature
control in range from 20 to 80 °C. The accuracy of the temperature stabilization was 0.02 °C and
uncertainty of the refractive index measurement was less than 0.00005.
Thermal analysis
Differential scanning calorimetry (DSC) and thermogravimetric (TG) analyses were performed on
Mettler Toledo Stare TGA/DSC1 unit (Leicester, UK) under nitrogen at a heating/cooling rate of
10 °C∙min-1. During DSC analyses samples between 5 and 15 mg were placed in aluminum pans
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and were heated from 25 to 160 °C and cooled to -100 °C. During TG experiments samples
between 2 and 10 mg were placed in aluminum pans and were heated from 30 to 500 °C.
Solubilities
The solubilities of the obtained products in ten representative solvents were determined according
to the protocols in Vogel’s Textbook of Practical Organic Chemistry.39 The solvents chosen for
study were selected in order of decreasing polarity expressed as the Snyder polarity index: water,
9.0; methanol, 6.6; DMSO, 6.5; acetonitrile, 6.2; acetone, 5.1; isopropanol, 4.3; ethyl acetate, 4.3;
chloroform, 4.1; toluene, 2.3; hexane, 0.0. A 0.1 g sample of each salt was added to a certain
volume of solvent and the samples were thermostated in water bath MEMMERT model WNB 7
at 25 °C. On the basis of the volume of solvent used, three types of behaviors were recorded:
‘soluble’ applies to compounds which dissolved in 1.0 cm3 of solvent, ‘limited solubility’ – applies
to compounds that dissolved in 3.0 cm3 of solvent, and ‘not soluble’ – applies to the compounds
which did not dissolve in 3.0 cm3 of solvent.
Greenhouse Experiments
Common lambsquarters (Chenopodium album L.), cornflower (Centaurea cyanus L.) and rapeseed
(Brassica napus L.) were used as test plants. The plants were grown in 0.5 dm3 plastic pots
containing commercial peat-based potting material under controlled environmental conditions:
temperature of 20(±2) °C, humidity of 60%, and a photoperiod of 16/8 h day/night. After
emergence, the plants were thinned to 5 plants in each pot. Treatments were applied at the 4–6
leaves growth stage. Commercial products: Aminopielik Standard 600 SC (600 g dimethylamine
salt of 2,4-D in 1 dm3), Chwastox Extra 300 SL (300 g of sodium and potassium salts of MCPA
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in 1 dm3) and Dikamba 700 SC (700 g sodium salt of dicamba in 1 kg) were used as reference
compounds. The synthesized salts were applied at a dose corresponding to 400 g of active
ingredient per 1 hectare for compounds with 2,4-D, MCPA, and MCPP or 200 g of active
ingredient per 1 hectare for compounds with dicamba. Reference herbicides were used at the same
doses. All tested salts were dissolved in a mixture of water and ethanol (1:1 v/v), whereas reference
compounds were dissolved in water. The applications were conducted using a moving sprayer
(APORO, Poznan, Poland) with a TeeJet® VP 110/02 (TeeJet Technologies, Wheaton, IL, USA)
flat-fan nozzle capable of delivering 200 dm3·ha-1 of spray solution at 0.2 MPa operating pressure.
The sprayer was moved above the plants (40 cm distance) at a constant speed of 3.1 m·s-1. After
treatment, the plants were again placed in a greenhouse under the environmental conditions
mentioned above. A weed control was evaluated visually two weeks after treatment (2 WAT) using
a scale of 0 (no effect) to 100% (complete weed destruction). The study was carried out in four
independent experiments in a randomized setup. Tukey’s test was used for the comparison of
averages and lowest significant difference (LSD) was determined at the level of 5%. All
calculations were performed using Agriculture Research Manager (ARM) software. To compare
the studied treatments, a multiple Tukey’s post hoc test (α=0.05) was applied.
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Field Experiments
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The field trials were carried out in spring barley in 2014 and 2015 at the Experimental Station of
Institute of Plant Protection in Winna Gora (E: 17°26', N: 52°12'). Cereals were cultivated
according to the local agricultural practice. The plot size was equal to 16.5 m2. The experimental
design was based on a randomized block with four replications. The plots were infested by
cornflower (Centaurea cyanus L.) in 2014 and oilseed rape volunteers (Brasica napus L.) as well
as common lambsquarters (Chenopodium album L.) in 2015. The treatments were applied at the
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stage of the end of crop tillering (BBCH 29) using a knapsack sprayer (APORO, Poznan, Poland)
equipped with TeeJet® AXR 110/03 flat-fan nozzles (TeeJet Technologies, Wheaton, IL, USA)
delivering 200 dm3·ha-1 of spray solution at 0.3 MPa of operating pressure. A weed control was
evaluated visually five weeks after a herbicide application (5 WAT) using a scale of 0 (no effect)
to 100% (complete weed destruction). Commercial products: Aminopielik Standard 600 SC (600
g dimethylamine salt of 2,4-D in 1 dm3), Chwastox Extra 300 SL (300 g of sodium and potassium
salts of MCPA in 1 dm3) and Dikamba 700 SC (700 g sodium salt of dicamba in 1 kg) were used
as reference compounds. Each error margin range represents standard errors of the mean (SEM).
The SEM values were calculated according to equation 1:
Equation 1. Calculation of SEM values.
푠
푆퐸푀 =
푛0.5
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where SEM – standard error of the mean, s – sample standard deviation, n – number of samples.
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Surface Activity
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Surface tension and contact angle measurements were carried out using a DSA 100 analyzer
(Krüss, Germany, accuracy ±0.01 mN·m-1), at 25 °C. The surface tension was determined using
the shape drop method. Basically, the principle of this method is to form an axisymmetric drop at
the tip of a needle of a syringe. The image of the drop (3 cm3) is taken with a CCD camera and
digitized. The surface tension (γ in mN·m-1) was calculated by analyzing the profile of the drop
according to the Laplace equation. Temperature was controlled using a Fisherbrand FBH604
thermostatic bath (Fisher, Germany, accuracy +0.1 °C). The determination of the contact angle
was based on the sessile drop method, i.e. drops of liquid are deposited on a solid surface (paraffin).
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Acute Oral Toxicity Test Towards Rats
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Wistar rats (symbol lmp: WIST, outbred) used in the studies originated from a culture in the
Medical Institute of Work in Lodz/Poland and were kept in cages of the conventional type. Before
the study, the animals were quarantined for a minimum 5 days and observed daily during this
period. The animals were marked individually. During quarantine and the experiments, the animals
were kept in a conditioned room of the following parameters: temperature 21-22 °C, relative air
humidity 40-75%, and artificial illumination 12 h light/12 h darkness. Rats were kept in cages with
plastic bottom and wired superstructure, with the dimensions of 58 x 37 x 21 cm (length x width
x height). The animals were kept in cages individually (in the observation study the rats received
a dosage of 2000 or 300 mg·kg-1 b.w.) or 4 rats per cage (in the main study the dose of 300 mg·kg-
1 b.w.). UV-sterilized wooden shavings were used as litter. Each cage was equipped with a label
containing the following information: name of test material, study code, used dose, start date and
planned ending date of the experiment, sex, and animal numbers. The rats were given standard
granulated GLM fodder and tap water ad libitum.
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Ready Biodegradability According To The OECD 301 F Test – Manometric Respirometry
The test was performed according to OECD guideline for 301 F test. The biological oxygen
demand (BOD) was determined every 24 h for 28 days using the OxiTop system (WTWGmbH
Weilheim Germany) in a thermostated incubator (IKA Germany) covered with aluminium foil.
The activated sludge was collected from a local municipal wastewater treatment plant
(Kozieglowy, Poznan, Poland). Prior to use, the activated sludge was aerated for 7 days in mineral
medium which was also used for subsequent tests. The mineral medium consisted of KH2PO4 - 85
mg·dm-3, K2HPO4 – 220 mg·dm-3, Na2HPO4·2H2O – 220 mg·dm-3, NH4Cl – 17 mg·dm-3,
CaCl2·2H2O – 37 mg·dm-3, MgSO4·2H2O – 23 mg·dm-3, FeCl3– 0.25 mg·dm-3. The measured pH
was at 7.2. The test was performed in brown glass bottles containing mineral medium, inoculum
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(cell density at approx. 106 cells·cm-3 determined with plastic Paddle Tester for aerobic bacteria,
Hach, USA) and tested ILs at a concentration of approx. 10-30 mg·dm-3, which was equal to 100
mg·dm-3 of Theoretical Oxygen Demand (ThOD, calculated based on equation 1).
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Equation 1.Theoretical Oxygen Demand for a chemical formula given as CcHhNnOoPp:
1
2
5
2
mg
mg
(
)
16[2c + h − 3n + p − o]
ThOD =
mmol
mg
molecular mass of the test substance
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Allylthiourea (1.16 mg·dm-3) was added to inhibit nitrification. All samples were analyzed in
triplicates together with controls (sodium benzoate without inoculum, tested substances without
inoculum) and blanks (medium and inoculum without tested substances). Gas tight flasks were
equipped with a CO2 trap (solid NaOH) and incubated in the dark at 20 °C for 28 days. The
biodegradation efficiency was calculated based on the oxygen uptake in each bottle (measured
automatically by the electronic OxiTop head) and corrected for the oxygen demand of the blank,
with the respect to the Theoretical Oxygen Demand and the amount of ILs tested.
RESULTS AND DISCUSSION
Synthesized ILs
The substrates used for the syntheses were commercially available products containing common
amphoteric
surfactants:
N-dodecylbetaine
[C12Bet]
(CAS:
66455-29-6)
and
N-(3-cocoamidopropyl)betaine [CAPBet] (CAS: 61789-40-0), where coco refers to the mixture of
saturated linear alkyl substituents C8−5%, C10−6%, C12−50%, C14−19%, C16−14%, C18−10%. In
the first stage, both dehydrated substances have been protonated with hydrochloric acid. As a result
of the protonation reaction N-dodecylbetainium chloride – [C12Bet][Cl] and N-(3-
cocoamidopropyl)betainium chloride - [CAPBet][Cl] were obtained and then separated from the
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reaction mixture, in accordance with scheme 1. Reaction yields for both precursors were 90 and
92%, respectively.
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Scheme 1. Synthesis of alkylbetainium HILs via protonation followed by an anion exchange
reaction, where R is dodecyl or 3-cocoamidopropyl and A is 2,4-D, MCPA, MCPP or dicamba.
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In the next step, the chloride anion was exchanged for herbicidally active anions (2,4-
dichlorophenoxyacetate - 2,4-D, 4-chloro-2-methylphenoxyacetate – MCPA, 2-(4-chloro-2-
methylphenoxy)propionate – MCPP and 3,6-dichloro-2-methoxybenzoate - dicamba). Structures
of used herbicides are presented in Fig. 2. All salts were obtained in high yields exceeding 90%
(Table 1).
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Figure 2. Structures of used herbicidal acids.
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Table 1. Synthesized alkylbetainium salts 1-8.
Yield State Melting pointb
Salt
R
Anion
Abbreviation
(%) at 25°C
(°C)
C12H25 2,4-D
C12H25 MCPA
C12H25 MCPP
[C12Bet][2,4-D]
[C12Bet][MCPA]
[C12Bet][MCPP]
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Solid
64-66
1
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3
4
5
6
7
8
Solid
37-40
---
Liquid
Solid
C12H25 dicamba [C12Bet][Dicamba]
28-30
---
CAPa 2,4-D
CAP MCPA
CAP MCPP
[CAPBet][2,4-D]
[CAPBet][MCPA]
[CAPBet][MCPP]
Wax
Liquid
Liquid
Liquid
---
---
CAP dicamba [CAPBet][Dicamba]
---
291
a 3-cocoamidopropyl; b METTLER TOLEDO MP 90 melting point system
292
293
294
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296
297
298
299
300
301
302
303
304
305
One of the obtained compounds (5) was a wax and four (3, 6-8) were liquids at room temperature.
Any of the synthesized salts did not undergo phase transfer to the solid state at 100 °C, therefore
they could be classified as ionic liquids (ILs). Salts with N-dodecylbetainium cation and anions
2,4-D (1), MCPA (2) and dicamba (4) were solids at room temperature with melting points at
approx. 65, 39 and 29 °C, respectively. Moreover, owing to their low melting points 3 and 5-8, are
classified as room temperature ionic liquids (RTILs). Therefore, the 3-cocoamidopropyl
substituent was more suitable for synthesis of RTILs compared to dodecyl chain. This
phenomenon is mainly associated with the presence of an amide group affecting the geometric
arrangement of the whole molecule and resulting in a decrease of its lattice energy. Furthermore,
we discovered that alkyl chain elongation in betaine type cation may result in a decrease of HILs
melting point, which is also consistent with data collected for betaine (N,N,N-trimethylglycine)-
based HILs recently described by our research group.21 This characteristic behaviour of ILs has
been described repeatedly in the literature40,41 and explains the differences between betainium and
N-dodecylbetainium cations. Further reduction of the melting point between 1-4 and 5-8 is due to
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the enhanced asymmetry caused by the presence of the amide group in the N-(3-
cocoamidopropyl)betainium cation.
On the basis of 1H NMR spectra it was established that the structure of the anion generally had a
marginal influence on the chemical shift values originating from protons in the cation (Table S2,
Supporting Information). However, the main differences between precursors and synthesized ILs
have been noted for protons from the methylene group (N-CH2-COOH), as both chlorides were
characterized by approx. 0.4-0.7 ppm higher values compared to 1-8. The observed shifts on NMR
spectra may become an evidence of efficient anion exchange as well as the absence of trace
substrates in the obtained ILs. A similar analysis was conducted for 13C NMR spectra (Table S3).
We found that the structure of the anion has a noticeable influence on the values of chemical shifts
only between precursors and the appropriate ILs, which were obtained from them. The signals
from the methylene group (N-CH2-COOH) for 1-8 occurred at approx. 63 ppm, whereas the same
signal from precursors appeared at lower values of approx. 61 ppm.
320
321
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325
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327
328
Physicochemical properties
Physicochemical properties, such as viscosity, density and the refractive index, were determined
for the ILs in the range from 20 to 80 °C (Figs. 3-5; collected physicochemical data and calculated
linear regression coefficients were presented in the Supporting Information in Tables S4-S8). The
greasy state of N-(3-cocoamidopropyl)betainium 2,4-dichlorophenoxyacetate (5) did not allow to
determine its viscosity and density. Moreover, measurements for ILs 1, 2 and 4 were performed at
temperatures above their melting points.
According to the obtained viscosity results, it was found that all analysed ILs are Newtonian fluids
and their sheer stress values increases linearly with the increase of shear rate – the correlation
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coefficients (R2) exceeded 0.99 (see Fig. S21, ESI). The influence of temperature on the viscosity
values for [C12Bet][2,4-D] (1), [C12Bet][MCPA] (2), [C12Bet][MCPP] (3), [C12Bet][Dicamba] (4),
[CAPBet][MCPA] (6), [CAPBet][MCPP] (7) and [CAPBet][Dicamba] (8) is presented on Fig. 3.
332
333
Figure 3. Influence of temperature on the viscosity values for ILs (1-4, 6-8).
334
335
336
337
338
339
340
341
342
343
344
345
346
At 20 °C values of viscosity for the synthesized RTILs differed from 64.7 Pa∙s for 3 to 3461.8 Pa∙s
for 8 (Table S4, ESI). However, the measured values for both RTILs comprising MCPP anion (3
and 7) were significantly lower compared to RTILs with MCPA (5) as well as dicamba anion (8).
We also noted that the viscosity of the obtained RTILs was notably higher compared to the
previously described HILs,5,7,14 yet, they were similar to results obtained for betaine and carnitine-
based HILs.21 We explained this phenomenon as the effect of the considerable amount of hydrogen
bonds between the ions caused by the presence of carboxyl group in the ILs cation. Moreover, the
collected values for betaine 2-(4-chloro-2-methylphenoxy)propionate amounted to approx. 60 and
10 Pa∙s at temperature values of 20 and 30 °C,21 accordingly, and were lower in comparison to 3
comprising a long dodecyl substituent instead of a methyl group. Hence, we may assume that the
straight alkyl chain elongation in ILs with betaine type cation resulted in an increase of viscosity
parameter, which complies with other reports about ILs.7,42 It is worth noting that ILs with the
dicamba anion (4 and 8) possessed relatively higher viscosities than ILs comprising phenoxyacids.
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As for other viscous ILs, the viscosity of the obtained ILs decreased nonlinearly with increasing
temperature. With regard to literature, further extension of the alkyl chain as well as the presence
of the amide group (capable of forming subsequent intermolecular hydrogen bonds) should cause
a substantial rise of viscosity. Except for ILs with dicamba (4 and 8), the obtained results are
compatible with the foregoing assumption. Finally, on the basis of the obtained results, it is
possible to classify all tested ILs as high viscosity ionic liquids.
The density of prepared RTILs 3 and 5-8 at 20 °C was higher compared to the density of pure
water (Fig. 4). The obtained values varied from 1.068 g∙cm-3 for 3 to 1.156 g∙cm-3 for 8 (Table S5,
ESI) and were similar to other described HILs.5,7,14
356
357
Figure 4. Influence of temperature on the density values for ILs (1-4, 6-8).
358
359
360
361
362
363
364
This shows that the density of alkylbetaine ILs is decreasing with elongation of the alkyl chain
from methyl to dodecyl or 3-cocoamidopropyl, which is consistent with the findings on ILs
containing ammonium cation.43 The density values at room temperature lead to the conclusion that
the presence of MCPP anion in the IL results in lower density values in comparison to ILs
containing MCPA or dicamba. A similar tendency was observed for ILs based on esterquats.14 As
expected, the increase of temperature led to a linear decrease of density and the line describing this
relationship was expressed as the equation y = a·x + b (Table S6, ESI), where a was equal to
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approx. -0.0007 while b ranged from 1.08 g∙cm-3 for HIL with MCPP anion (3) to 1.17 g∙cm-3 for
both ILs comprising dicamba (4 and 8). As an effect of increasing the temperature from 20 to 80
°C, the analysed parameter was reduced by approx. 0.04 g∙cm-3 for all RTILs, similarly to our
previous reports.7,21 The presented results confirm the previous observation that dicamba-based
ammonium ILs possess higher density values in comparison to phenoxyacids.14
The refractive index, as a value characterizing the deviation of a light beam during propagation
from one medium to another, is an important property not only as the characteristic value used for
verification the identity or purity of each IL, but also as the measure of ILs electric
polarizability.44,45 The measured indices of ILs often vary in the range from 1.40 to 2.20 depending
on the structure of both the cation as well as the anion.7,46 Refractive indices of the synthesized
RTILs (3, 5-8) at 20 °C ranged from 1.499 for 6 to 1.515 for 8 (Table S7, ESI). Collected data
presented in Fig. 5 coincide with the previous reports,5,7,14,21 in which the determined values for
different ammonium HILs varied from approx. 1.50 to 1.54.
378
379
Figure 5. Influence of temperature on the refractive index values for ILs (1-8).
380
381
382
Heating all samples caused, similarly as in case of density measurements, a linear and slight
decrease of analysed parameter. The linear regression allowed to describe this relationship using
the equation y = a·x + b (Table S8, ESI), where a varied from -0.00035 for 8 and 7 to -0.00030
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392
393
394
395
396
397
for 5 and 7. The b parameter ranged from 1.5048 for 3 to 1.5219 for 4 and 8. Eventually, at 80 °C,
the refractive indices were lower by approx. 0.020 and reached values below 1.500 for all ILs.
In general, the refractive index of ILs increases with the increase of their density. The refractive
index of a substance is higher when its molecules are more tightly packed, i.e., when the compound
possesses higher density.44 Taking into account this hypothesis, we observed that the refractive
indices presented in this work are partly correlated with the values of ILs density described above.
The highest densities for dicamba-based ILs (4, 8) correspond with their values of refractive index.
Therefore, IL 3 was characterized by the lowest values of refractive index as well as density. The
relationship ‘density-refractive index’ additionally explains why the alkyl substituent elongation
in ILs comprising ammonium as well as betaine type cations caused a decrease of their refractive
indices.
Thermal Properties of synthesized ILs
Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) data for the
synthesized ILs are presented in Table 2.
Table 2. Thermal properties of the obtained ILs.
a
b
c
d
e
Tg Tcryst Tm Tonset5% Tonset
IL
(°C) (°C) (°C) (°C)
(°C)
-14
-20
-17
-6
70
---
---
33
---
---
---
---
64
37
---
27
---
---
---
---
228
230
219
200
171
159
185
184
250
1
2
3
4
5
6
7
8
272
260
240
273
265
263
246
-6
-13
-11
-9
b
c
398
399
aTg – glass transition temperature; Tcryst – temperature of crystallization; Tm – melting point;
dTonset5% – decomposition temperature of 5% sample; eTonset – decomposition temperature
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403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
Among all the prepared ILs, five (3, 5-8) were liquids below 20 °C. For each of them neither
melting nor crystallization were observed in the analysed temperature range. The lowest melting
point was detected for 4 (Tm = 27 °C), which additionally possessed a crystallization event on
cooling at 33 °C. Other ILs with N-dodecylbetainium cation (1 and 2) melted at higher
temperatures amounting to 37 °C for 2 and 64 °C for 1, respectively. None of the obtained
compounds exceeded the threshold Tm value of 100 °C, therefore all may be classified as ILs.
Moreover, the crystallization temperature (Tcryst) for 1 was equal to 70 °C, however, this parameter
could not be determined for the obtained IL with the MCPA anion (2). The supercooling behaviour
of 2, for which a melting transition was observed on first heating but could not be reproduced
within several heating/cooling cycles, was also noted for other HILs with betainium and
carnitinium cations21 as well as HILs comprising cationic surfactants as cations1,2,8,10,13
.
The glass transition temperatures (Tg) determined for each of the obtained ILs ranged from -20 to
-6 °C. Among ILs derived from phenoxyacids (2,4-D, MCPA and MCPP), ILs with the N-(3-
cocoamidopropyl)betainium cation (5-8) exhibited higher Tg values, ranging from -13 to -6 °C,
whereas in case of ILs with the N-dodecylbetainium cation (1-3) the values were slightly lower
(from -20 to -14 °C). Likewise, the structure of the anion influenced the glass transition
temperature values for both cations, which can be displayed by the following order: 2,4-D >
MCPP> MCPA. However, due to the lack of the long alkyl substituent in HILs derived from
betaine, their Tg values were notably higher in comparison to HILs presented in this manuscript
and exceeded 0 °C. Thus, when analysing the influence of the cation structure on glass transition,
we may arrange them in the order of decreasing Tg values: betainium > N-(3-
cocoamidopropyl)betainium > N-dodecylbetainium.
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423
424
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427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
Analysis of structure vs glass transition confirmed that alkyl chain elongation in the cation of ILs
results in lowering their glass transition temperatures. In literature this is mainly caused by
disruption of the crystal packing and reduction of the lattice energy due to the extension of
substituent attached to the nitrogen atom.47 However, it should be mentioned that the further
increase of alkyl chain length may enhance the attractive van der Waals interactions and then led
to increased Tg values.48 These findings explain why ILs with N-dodecylbetainium cation are
characterized
by
lower
glass
transition
temperatures
compared
to
N-(3-
cocoamidopropyl)betainium-based ILs.
The thermogravimetric analysis results confirmed the high stability of the obtained ILs (Table 2).
The measured Tonset5% values were in a range from 159 for 6 to 230 °C for 2. The ILs comprising
the N-dodecylbetainium cation were characterized by a higher thermal stability, since their Tonset5%
values were higher by 14 to even 60 °C compared to ILs based on the N-(3-
cocoamidopropyl)betainium cation. The reason for such behaviour is the presence of an amide
group in the cation which is more susceptible to thermal degradation in comparison to straight
alkyl chain. Among ILs with the N-dodecylbetainium cation (1-4), the phanoxyacetates (1 and 2)
exhibited slightly higher Tonset5% values compared to IL with MCPP (3).
Generally, the temperature values of 50% decomposition of the synthesized ILs ranged from 240
to 273 °C. It was observed that the presence of phenoxyacids caused an increase of thermal stability
of 1-3 and 5-7 compared to ILs comprising the dicamba anion (4 and 8). This observation is
consistent with the previous papers regarding the lower thermal stability of HILs with the dicamba
anion compared to ILs comprising phenoxycarboxylates as anions.6,14,21 On the other hand, the
highest values of Tonset which exceeded 270 °C were observed for 2 and 5 - based on
phenoxyacetates. The results obtained for 5-8 were only slightly higher compared to 1-4 which
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447
448
449
450
451
suggest that in this case the influence of the cation structure on the Tonset parameter is
inconsiderable.
Solubilities
The solubilities of the prepared ILs were tested in ten representative solvents, varying from high
polarity to low polarity. The obtained results are presented in Table 3.
Table 3. Solubility of prepared salts at 25 °C.
IL
9.0a 6.6 6.5 6.2 5.1 4.3 4.3 4.1 2.3 0.0
–
–
±
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
+
+
±
+
+
+
+
+
+
+
–
–
–
±
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
–
–
–
+
+
+
+
±
+
+
+
+
±
+
+
+
+
+
+
–
–
+
±
–
–
±
–
1
2
3
4
5
6
7
8
452
a Snyder polarity index + complete solubility; ± limited solubility; – insoluble
453
454
455
456
457
458
It was observed that all tested ILs are well soluble in organic solvents with high polarity, such as
methanol and DMSO, which corresponds well with our previous reports describing ILs based on
phenoxyacids or dicamba anions and long alkyl ammonium ester cations,14 bisammonium cations6
and betaine cation21. However, alkylbetaine-based ILs exhibited low solubility in water. The high
hydrophobicity of the obtained ILs may be explained by the presence of a long alkyl chain
(reported for other ILs)7,10,14 as well as a carboxylic group (reported for ILs with betainium
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460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
cation)21. Interestingly, both betaines in zwitterionic form as commercial surfactants are
characterized by excellent water solubility. Low water solubility may be a particularly useful
property in terms of controlling soil and groundwater mobility of a new herbicide or good wetting
of plants leading to a better substance absorption. Slightly less polar acetone and isopropanol also
dissolved all ILs regardless of both the cation and anion structure. However, N-(3-
cocamidopropyl)betainium cation caused a deterioration of ILs (5-8) solubility in other less polar
solvents, such as acetonitrile or ethyl acetate. Furthermore, despite similar Snyder polarity index
values (4.3) of isopropanol and ethyl acetate, the obtained results were significantly different.
Owing to the ionic bond in the structure of ILs, isopropanol as the semipolar protic solvent was a
better diluent than the aprotic semipolar ester - ethyl acetate. Due to presence of a long alkyl
substituent, the synthesized ILs were also soluble in nonpolar solvents, such as chloroform and
toluene. In addition, both ILs with MCPP anion (3 and 7) as well as N-dodecylbetainium
3,6-dichloro-2-methoxybenzoate (4) also dissolved in the most non-polar solvent - hexane, which
was previously reported only for esterquats HILs comprising a highly hydrophobic
di(tallowoyloxyethyl)dimethylammonium cation.14
Herbicidal Activity
The herbicidal activity of the obtained ILs was determined both in greenhouse and field tests. Their
efficiency was compared to the reference herbicides comprising dimethylammonium salt of 2,4-D
(1, 5), sodium-potassium salt of MCPA (2, 3, 6, 7) and sodium salt of dicamba (4, 8). The
herbicidal efficacy of the synthesized alkylbetainium ILs in the greenhouse experiments was
examined using common lambsquarters (Chenopodium album L.), rapeseed (Brassica napus L.)
and cornflower (Centaurea cyanus L.) as test plants. The results are shown in Fig. 6. No significant
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486
differences were observed in the case of results obtained for cornflower, whereas the results of
experiments on common lambsquarters and rapeseed differed notably depending on the salt used
for treatment. The main symptoms of plant damage such as deformation of shoots, leaves and
disruption of plant growth caused by auxin-like herbicides (i.e. phenoxyacids or dicamba) were
observed for the examined ILs during the experiment (see Fig. S22, ESI).
487
488
Figure 6. Herbicidal efficacy of tested ILs against common lambsquarters, cornflower and
489
rapeseed under greenhouse conditions.
490
491
492
493
494
495
496
497
Generally, all of the tested ILs demonstrated high efficacy (exceeding 90%) which underlines the
beneficial role of both utilized alkylbetainium cations. For example, ILs 1, 4, 5 and 8 were
characterized by higher herbicidal activity towards rapeseed compared to the applied standards.
According to the results of greenhouse experiments, none of the studied ILs, excluding salts with
MCPP anion (3, 7) toward common lambsquarters, exhibited lower efficacy in comparison to
reference herbicides. Moreover, it was noted that ILs with dicamba anion (4, 8) were characterized
by multiple times higher herbicidal activity compared to the reference herbicide towards rapeseed
at the applied dose of the active ingredient. Among all synthesized ILs, 6 turned out to be the most
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499
500
501
502
503
504
505
506
507
508
active – its efficiency was higher than 95% against all three plants. The presented studies confirm
that biological activity of the synthesized ILs has been preserved – they may be classified as new
herbicidal ionic liquids (HILs).
Biological activity of [C12Bet][MCPA] (2) has been tested in a preliminary field experiment in
spring barley in 2014. It was noted that this IL is characterized by better or comparable
effectiveness compared to the reference herbicide containing MCPA. This HIL reached an average
effectiveness equal to 100% towards common lambsquarters and 90% towards cornflower, while
the reference herbicide efficiency was lower and amounted to 90 and 70% for both weeds,
respectively. The next field experiment performed in 2015 in spring barley confirmed the
substantial enhancement of biological activity of the tested HILs caused by the presence of
alkylbetaine cation (Fig. 7).
509
510
Figure 7. Weed control in spring barley in 2015 against two weed species evaluated 5 weeks after
511
treatment (5 WAT).
512
513
All HILs demonstrated similar or significantly higher efficiency towards common lambsquarters
and rapeseed as compared to the applied reference herbicide. In case of 2,4-D-based HILs (1, 5)
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515
516
517
518
519
520
521
522
523
524
the control of common lambsquarters was five-times higher compared to 2,4-D reference
herbicide. The studied HILs in the field tests exhibited very high efficacy (> 90%) which indicates
that they could be used for successful weed control in spring cereal crops. The enhancement of
herbicidal efficiency caused by the incorporation of amphoteric surfactant (alkylbetaine) into HIL
is also consistent with literature, as a similar phenomenon has been noted for many HILs based on
cationic surfactants and phenoxyacids1-3,14,36 as well as well as dicamba8,14,36
.
Surface properties
The main surface activity parameters, such as contact angle (CA) and surface tension () of spray
solutions utilized for evaluation of herbicidal activity of the synthesized HILs, were determined
(collected values are provided in ESI – Table S9) and presented in Fig. 8.
525
526
Figure 8. Surface tension () and contact angle (CA) of spray solutions containing HILs (1-8) and
527
reference herbicides.
528
529
As the derivatives of amphoteric surfactants – all the obtained HILs were characterized by good
surface-active and wetting properties. Their values of surface tension between 30.4 for 8 and 32.6
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531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
mN·m-1 for 4 are comparable to the results obtained by using the most effective adjuvants.49,50 In
general, surface tension of HILs comprising phenoxyacids (1-3, 5-7) was over 40 mN·m-1 lower
compared to reference herbicides, whereas the difference in  values between dicamba-based HILs
(4, 8) and reference herbicide was approx. 34 mN·m-1.
Low values of contact angle and surface tension should enhance the wetting of plants, spray
retention, absorption, cuticle penetration and translocation of the active ingredient. This
assumption has been confirmed in previous reports regarding HILs comprising cationic surfactants
and once again underline the role of cation selection for synthesis of biologically active ILs.2,3
According to Fig. 8, the synthesized HILs were characterized by low values of contact angle
ranging from approx. 50° to 62°. The structure of the herbicidal anion had a minor influence on
the measured values, which differed by a few degrees. Furthermore, CA of solutions containing
the synthesized salts were substantially lower than values observed for standards which, due to
their lack of surface active properties, exceeded 100°. The differences in CA by 40-55° between
HILs and standards manifest in different shape of droplets of spray solution on the leaf of the weed
– droplets with HILs possess a greater contact area (see Fig. S23, ESI), which facilitates the
penetration of the active substance into the interior of the plant and also prevents droplets from
falling from the leaf.
Toxicity of selected HILs
Two HILs with the MCPA anion (2, 6) were selected for determination of LD50 value towards rats.
The presence of the alkylbetainium cation did not alter the toxicity level of the new herbicides in
comparison to MCPA in acidic form (Table 4).
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555
Table 4. Acute oral LD50 for rats of the synthesized alkylbetaine derivatives.
Category LD50
Compound
GHSa (mg·kg-1)
[C12Bet][MCPA] (2)
[CAPBet][MCPA] (6)
MCPA free acid
4
4
4
3
5
300-2000
300-2000
96251
7137,52
≥ 490053
N-dodecylbetaine
N-(3-cocoamidopropyl)betaine
556
a Globally Harmonized System of Classification and Labelling of Chemicals
557
558
559
560
561
562
563
564
565
566
567
568
569
570
Both tested betaine derivatives could be included into category 4. In case of 2 we found that the
combination of N-dodecylbetainium cation with MCPA anion resulted in the reduction of toxicity
in comparison to data reported for pure N-dodecylbetaine.37,52 Moreover, regarding our previous
results for HILs with the MCPA anion and betaine or carnitine cation21, it was found that the
influence of the presence of long alkyl chain (dodecyl as well as 3-cocoamidopropyl) in cation of
betaine has no considerable impact on the acute toxicity towards rats. Hence, all tested betainium-
based HILs were classified into 4 category thus far.
Susceptibility to biodegradation
Generally, betaine derivatives are considered as readily biodegradable compounds, which
distinguishes this group of surfactants. The primary biodegradation of N-dodecylbetaine and N-
(3-cocoamidopropyl)betaine was almost complete – results for both exceed 90% in the same
OECD test.37 This fact additionally showed us the significance and potential of utilization of
alkylbetainium-based HILs in plant protection research. The comparison of HILs with the same
29