Ionic Liquids in Analytical Chemistry: Fundamentals, Technological Advances, and Future Outlook

Introduction

The development of new analytical methods most often focus on novel materials used to impart selectivity or sensitivity to the protocol. Ionic liquids (ILs) are a class of solvents that have been extensively explored as promising materials for various applications and continue to be explored due to their tunable physicochemical properties. These materials possess melting temperatures below 100 °C and can interact with analytes through a multitude of interactions afforded by their readily tunable chemical structure.¹ These interactions include electrostatic, dispersive, hydrogen bonding, π–π, and dipolar interactions and can be modulated or strengthened based on the functional groups present within the chemical structure.² ILs consist predominately of organic cations and either inorganic or organic anions, both of which can be functionalized with desired moieties. Common cation and anions found in IL chemical structures are presented in Figure 1. The unique polarity afforded by the ionic structure has also led to their increasing use in areas including sample preparation, chemical separations, electrochemistry, mass spectrometry, and spectroscopy.³⁻⁷

Figure 1.

Chemical structures of commonly used IL cations and anions with R groups consisting of mainly alkyl chains or the benzyl moiety. Only the most commonly used cations and anions are depicted here and some IL chemical structures discussed in the text are not depicted in the figure.

ILs are often referred to as “designer solvents” because most of their physicochemical properties can be tuned by interchanging different cations and anions. Specific analytical applications may require solvents to exhibit a certain melting temperature, viscosity, volatility, conductivity, and/or solubility to meet the constraints of the method, and often these requirements cannot be achieved with traditional solvents. To overcome the limitation of organic solvents, many studies have sought to understand the influence that the IL chemical structure plays in dictating their behavior.⁸⁻¹¹ In this review, specific properties of ILs are discussed when related to the success of the application, but readers are encouraged to explore the chapter by Zhou et al. in Ionic Liquids Further UnCOILed: Critical Expert Overviews for further information regarding relationships between IL chemical structure and physicochemical properties.¹²

By incorporating certain functional groups into the IL chemical structure, subclasses of ILs have emerged including polymeric ionic liquids (PILs),^13,14 magnetic ionic liquids (MILs),¹⁵ zwitterionic ionic liquids (ZILs),¹⁶ dicationic liquids (DILs),¹⁷ chiral ionic liquids (CILs),¹⁸ and fluorescent ionic liquids (FILs).^19,20 Polymerizable IL monomers can be designed by incorporating reactive functional groups into the IL chemical structure, which subsequently undergo polymerization to form PILs. PILs have been applied in various geometries including thin films,²¹ cylindrical columns,²² and spheres.²³ They offer improved thermal and chemical stability over traditional ILs and have been employed in a variety of analytical applications ranging from sorbents in sample preparation methods to signal enhancers in mass spectrometry (MS) and spectroscopy methods.^24,25 ZILs consist of chemically bonded cations and anions and exhibit IL-like properties, such as low volatility, lower melting points, and moderate to high thermal stability.²⁶ Most notably, the higher viscosity and increased polarity of ZILs have led to their use as extraction solvents as well as gas and liquid chromatographic stationary phases.²⁷⁻²⁹ DILs, consisting of two tethered cations, have been studied in a variety of applications from extractions to separations, and have become a popular choice as charge inverters to improve sensitivity in the MS detection of negatively charged analytes in positive ionization mode.^17,30,31 CILs are a distinct class of ILs consisting of cations or anions with a chiral center. The specific stereochemistry of the CIL allows for stronger interactions with a targeted enantiomer, resulting in enantiomeric separations.¹⁸ CILs are often employed in chiral separations with cyclodextrins to enhance enantioseparations.³² Lastly, FILs have been uniquely designed with fluorescent anions in their chemical structure to improve the detection of analytes.²⁰ FIL-based detection methods have also been used with smartphone detectors or colorimetric assays for methods involving point-of-care and on-site detection.^33,34

The current review provides an update on the application of ILs in analytical chemistry since our last review published in 2019.³⁵ While this review is meant to be comprehensive, specific emphasis is given to studies demonstrating innovative applications of ILs in each of the subdisciplines within the field of analytical chemistry. The review is organized into three main topics, (1) chemical separations including sample preparation, chromatographic separations, membrane separations, and electrokinetic separations; (2) electrochemical sensing; and (3) other methods using mass spectrometry and spectroscopy. Table 1 defines common terminology used throughout this review and within the analytical chemistry disciplines; abbreviations and terminologies related to ILs will be defined within the text. To keep IL abbreviations consistent through the review, a uniform abbreviation style is used and may differ slightly from that reported in the original published articles.

Table 1. Abbreviations Used Throughout This Review Article to Refer to Materials, Techniques, and Methods Described within the Text.

Sample Preparation
LLE	Liquid–Liquid Extraction
ME	Microextraction
LPME	Liquid Phase Microextraction
DLLME	Dispersive Liquid–Liquid Microextraction
SDME	Single Drop Microextraction
HS	Headspace
DI	Direct Immersion
SPME	Solid Phase Microextraction
TFME	Thin Film Microextraction
MEPS	Microextraction by Packed Sorbent
SPE	Solid Phase Microextraction
CPME	Capsule Phase Microextraction
ABS	Aqueous Biphasic System

Chromatography
GC	Gas Chromatography
μGC	Micro Gas Chromatography
LC	Liquid Chromatography
HPLC	High-Performance Liquid Chromatography
UHPLC	Ultrahigh Performance Liquid Chromatography
IEC	Ion Exchange Chromatography
RP-LC	Reverse Phase Liquid Chromatography
HILIC	Hydrophilic Interaction Chromatography

Membrane Separations
LM	Liquid Membranes
ELM	Emulsion Liquid Membranes
BLM	Bulk Liquid Membranes
SLIM	Supported IL Membranes
ILPMS	IL Composite Polymer Membrane
MMM	Mixed Matrix Membrane
ILMMM	IL Mixed Matrix Membrane
PILM	PIL Membranes
ILGM	IL Gel Membranes
ILMC	IL Membrane Contactors

Electroseparations
CE	Capillary Electrophoresis
EKC	Capillary Electrokinetic Chromatography
MEKC	Micellar Electrokinetic Chromatography
HI-EKC	Hydrophilic Interaction Capillary Electrokinetic Chromatography
EI-FFF	Electric Field-Flow Fractionation

Detection Methods
MS	Mass Spectrometry
ESI	Electrospray Ionization
MALDI	Matrix Assisted Laser Desorption/Ionization
MSI	Mass Spectrometry Imaging
SERS	Surface-Enhanced Raman Spectroscopy

IL Characterization
DFT	Density Field Theory
NMR	Nuclear Magnetic Resonance (Spectroscopy)
TGA	Thermogravimetric Analysis
IR	Infrared Spectroscopy

Analytical Terminology
LOQ	Limit of Quantification
LOD	Limit of Detection

Commonly Mentioned Materials and Compounds
PDMS	Polydimethylsiloxane
PVA	Poly(vinyl alcohol)
PEG	Polyethylene Glycol
PET	Polyethylene Terephthalate
NP	Nanoparticles
SDS	Sodium Dodecyl Sulfate
PAH	Polyaromatic Hydrocarbon
DNA	Deoxyribonucleic Acid

Subclasses of ILs
CIL	Chiral Ionic Liquid
DIL	Dicationic Liquid
FIL	Fluorescent Ionic Liquid
MIL	Magnetic Ionic Liquid
PIL	Polymeric Ionic Liquid
ZIL	Zwitterionic Liquid

Sample Preparation

Sample preparation is often a crucial step in chemical analysis as it separates target analytes from interfering substances and concentrates them for improved detection. It is especially critical for biological and environmental samples as direct introduction of these complex matrices into analytical instrumentation is often undesirable due to inherent disadvantages, and analyte concentrations are often too low for detection by common analytical instrumentation.^36,37 ILs have been long used as solvents for the extraction of analytes from complex matrices. ILs have been employed in liquid–liquid extraction (LLE) as a selective and environmentally friendly alternative to conventional organic solvents.^38,39 However, the high viscosity and costs of ILs compared to traditional organic solvents are often seen as limitations for their use in extractions that require larger volumes. These challenges have been mitigated by employing ILs in microextraction (ME) procedures, allowing for the full exploitation of their solvation power.⁴⁰ Microextractions employ very small volumes of extraction solvent relative to the sample volume and are commonly applied as a preconcentration method prior to analysis.⁴¹ ILs have gained widespread popularity in both solvent-based and sorption-based ME techniques, owing to their distinctive characteristics and overall versatility.

Liquid Phase Microextractions (LPMEs)

Dispersive liquid–liquid microextraction (DLLME) and single drop microextraction (SDME) have emerged as innovative techniques for applying ILs in ME studies.^42,43 DLLME was first introduced by Rezaee et al. in 2006 where they developed a simple and rapid method for extracting organic compounds from aqueous samples.⁴⁴ DLLME uses a ternary solvent system, where a water-immiscible extraction solvent (commonly denser than water) is mixed with a water-miscible disperser solvent. The mixture is quickly injected into the aqueous sample, causing the extraction solvent to disperse into fine droplets and a cloudy solution to be formed. The contact area between the solvent and sample is increased resulting in accelerated equilibrium between the two phases. Advantages of DLLME methods include simplicity of operation, speed, low cost, and high analyte recovery and enrichment factors. However, DLLME is frequently criticized for the use of harmful chlorinated solvents for extraction.

ILs have emerged as alternative solvents due to their lower toxicity (compared to chlorinated solvents), high structural tunability, and higher density compared to water. Liu et al. was the first to report the use of ILs in DLLME for detecting four heterocyclic insecticides in water samples.⁴⁵ Numerous studies have explored and modified IL-based DLLME methods, leading to exciting improvements and applications. Various approaches have been developed to eliminate the use of organic disperser solvents, including ultrasound-assisted, vortex-assisted, microwave-assisted, and air-assisted DLLME techniques; however, these often require external energy for dispersion of extraction solvents.⁴⁶ Piao et al. reported for the first time an acidic task-specific IL-based effervescence-assisted ME method to determine triazine herbicides in tea beverages⁴⁷ in which the herbicides are known to have potential adverse effects, including hormone disruption, birth defects, and reproductive cancers.⁴⁸ In effervescence-assisted ME methods, the extraction solvents or adsorbents are dispersed using carbon dioxide bubbles produced from a straightforward reaction between carbonate and acid in an aqueous solution. This method utilized the 1-butyl-3-methylimidazolium hydrogen sulfate ([C₄MIm⁺][HSO₄^–]) IL, where the cationic group acted as the extractant while the anionic group served as a substitute for traditional acids. This reaction enhanced mass transfer between the extraction solvent and analytes without requiring an external energy source. Following dispersion, the ion-exchange reagent ammonium hexafluorophosphate was introduced, resulting in replacement of the hydrophilic IL with the hydrophobic [C₄MIm⁺][PF₆^–] IL, allowing for easy recovery from the aqueous solution.

Effervescence-assisted DLLME requires centrifugation to collect the extraction solvent, which is often regarded as the most time-consuming step in IL-DLLME. Thus, recent studies have focused on utilizing MIL-based DLLME, where MILs can be easily separated using a simple permanent magnet. MILs are a subclass of ILs that incorporate paramagnetic atoms (i.e., transition metals or lanthanide metals) within their chemical structure. These tunable materials retain the defining properties of ILs while possessing unique physicochemical characteristics that make them responsive to external magnetic fields.⁴⁹ Fiorentini et al. developed a MIL-based DLLME method where the trihexyl(tetradecyl)phosphonium tetrachloroferrate (III) ([P_6,6,6,14⁺][FeCl₄^–]) MIL was used as an extraction solvent for the capture and determination of trace levels of arsenic (As(III)) in honey.⁵⁰ As(III) was preconcentrated by chelating with ammonium diethyldithiophosphate under acidic conditions and then extracted by the MIL with acetonitrile as a dispersive solvent. Magnetic separation of the analyte-containing MIL phase eliminated the need for centrifugation. Trujillo-Rodríguez et al. developed an approach where a new class of MILs was used for in situ DLLME to achieve higher enrichment factors than conventional DLLME, while also removing the centrifugation step.⁵¹ Commonly employed MILs in analytical methodologies contain paramagnetic anions such as tetrachloroferrate(III) ([FeCl₄^–]), bromotrichloroferrate(III) ([FeBrCl₃^–]), tetrachloromanganate(II) ([MnCl₄^2–]), tris(hexafluoroacetylaceto)nickelate(II) ([Ni(hfacac)^3–]), or tris(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac)^4–]). These anions often render MILs unsuitable for in situ applications for many reasons, but most apparent is that the paramagnetic component can be exchanged during the metathesis reaction, hindering subsequent magnetic separation. The newly designed MILs consist of cations featuring nickel(II) centers coordinated with four N-alkylimidazole ligands and chloride anions, which can undergo a metathesis reaction with the bis[(trifluoromethyl)sulfonyl]imide ([NTf₂^–]) anion. This research led to a study by Bowers et al. utilizing the concept of MIL-based in situ DLLME for the extraction of long and short double-stranded DNA.⁵² A very recent study by Qiao et al. reported the use of a multimagnetic center MIL (MMIL) featuring paramagnetic metals in both the cation and anion as extractants in DLLME for the determination of parabens in beverages. To further enhance paraben enrichment, the method incorporated a back-extraction step through in situ decomposition of the MMIL.⁵³

Another LPME technique that has also gained traction for its unique advantages is single microdroplet microextraction (SDME). Although SDME was the first LPME technique introduced, research on this topic has been less prevalent in recent years compared to DLLME. In this method, a drop (typically few microliters) of a water-immiscible solvent serves as the extraction phase and is suspended from a syringe needle. The microdroplet is either immersed in a stirred aqueous solution or exposed to the headspace for a specific duration, after which it is retracted and analyzed.⁵⁴ SDME has gained popularity due to its low cost, significant reduction in sample size, and minimal use of extraction solvents, all while offering high analyte enrichment. ILs have been investigated as alternative extraction solvents given their low vapor pressure and high viscosity that minimize solvent evaporation and enhance droplet stability.⁵⁵ Li et al. reported the use of the 1-butyl-3-methylimidazolium ([C₄MIm⁺][NTf₂^–] IL as extraction solvent for the determination of trace methanesulfonates using headspace SDME. Methanesulfonates are potential genotoxic agents formed through the reaction between residual solvents and methanesulfonic acid during synthesis and the manufacturing of drug substances.⁵⁶ The selective and sensitive determination of methanesulfonates, such as methylmethanesulfonate, ethylmethanesulfonate and isopropyl methanesulfonate, in drug substances possess significant challenges due to the complexities of the drug matrix. Previously proposed solutions were ineffective in mitigating the matrix effects; however, utilizing the [C₄MIm⁺][NTf₂^–] IL in headspace SDME mode after derivatizing the analytes not only eliminated the matrix effect but also resulted in good recoveries.

Sorbent Microextractions

PILs are formed through the polymerization of IL monomers, with or without an IL cross-linker. PILs have gained prominence as sorbents in various ME techniques, including solid-phsae microextraction (SPME),^27,57 thin film microextraction (TFME),^58,59 and microextraction by packed sorbent (MEPS).^60,61 Among these techniques, SPME has been widely used owing to its ability to detect a broad range of analytes in food, environmental, biological, and pharmaceutical samples.⁶²⁻⁶⁴ In SPME, small volumes of sorbents are either coated or immobilized on a solid support, which enhances their stability during the extraction process. It is a preconcentration technique based on the partitioning of analytes between an extraction phase and sample matrix, either in headspace or direct-immersion mode. SPME offers significant advantages over traditional extraction methods since it is rapid and integrates sample collection, extraction, and analyte enrichment from the sample matrix into a single step.⁶⁵ It is also simple and easy to automate and is fully compatible with chromatographic systems. Despite these benefits, SPME is limited by the number of commercially available sorbent coatings, which has driven increased research into designing new sorbents.²⁷ PILs have emerged as promising alternatives not only due to the tunable chemical structures inherent to IL monomers and cross-linkers but also due to their superior thermal and chemical stability compared to neat ILs.⁶⁶ These properties have led to widespread and innovative application of PILs as sorbents in SPME, enhancing the selectivity and efficiency of analyte detection across a broad range of fields. In a study by Yavir et al., PIL sorbent coatings were synthesized with nickel metal centers to extract volatile and semivolatile amines from water samples using HS-SPME.⁶⁷ The study highlights the use of the nickel-based PIL, composed of (tetra(3-vinylimidazolium)nickel bis[(trifluoromethyl)sulfonyl]imide ([Ni(VIM)₄][NTf₂]₂) IL monomer, as having unique selectivity toward amines as observed with nickel-containing ILs. Amines are toxic and hazardous to humans and animals and can react with nitrosylating agents to form carcinogenic N-nitroamines, further emphasizing the significance of the study.⁶⁸

Although SPME most commonly uses a fiber-type geometry, several innovative formats, such as in-tube SPME, have been developed. In-tube SPME was designed to facilitate direct, online coupling of SPME with high performance liquid chromatography (HPLC) systems. This system employs a capillary column segment as the extraction device, where analytes from the diluted sample are concentrated into the stationary phase through repeated draw/eject cycles or by flowing through the capillary using a microflow pump or autosampler. The extracted analytes are then eluted off by the mobile phase and transferred to the HPLC column, thereby reducing analysis times and often improving accuracy and precision.⁶⁹ In-tube SPME has seen numerous innovative applications since its introduction in the 1990s. A recent, notable advancement by Souza et al. involved developing PIL coated open tubular capillary columns for online in-tube SPME combined with ultrahigh performance liquid chromatography (UHPLC) coupled to MS/MS. This method was used to analyze endocannabinoids (eCBs) in plasma samples from patients with Parkinson’s disease.⁷⁰ The endocannabinoid system plays an important role in controlling signals in the brain, helping to regulate key functions in the nervous system. Research has shown a link between the endocannabinoid system and neurological disorders, such as Parkinson’s disease, where patients have been identified to have higher levels of endocannabinoids in their blood compared to healthy individuals.^71,72 PILs were synthesized using the 1-vinyl-3-hexylimidazolium chloride [C₆VIm⁺][Cl^–], 1-vinyl-3-hexadecylimidazolium bromide [C₁₆VIm⁺][Br^–] IL monomers and the 1,10-di(3-vinylimidazolium)decane dibromide [(VIm)₂C₁₀⁺][Br^–]₂ IL cross-linker via in situ thermal-initiated polymerization in a fused silica capillary column to effectively enrich eCBs due to nonspecific dispersive interactions and the IL hydrogen-bond basicity. More recently, Souza et al. reported a study on a new cross-linked zwitterionic PIL coating for fiber-in-tube SPME, which merges the features of fiber and in-tube SPME.⁷³ A schematic of this setup is shown in Figure 2. A zwitterionic PIL sorbent synthesized from the 1-vinyl-3-(propanesulfonate)imidazolium ([VIm⁺C₄SO₃^–]) IL and the 1,12-di(3-vinylimidazolium)dodecane dibromide ([(VIm)₂C₁₂⁺][Br^–]₂) IL cross-linker was coated on nitinol wires and packed into a polyether ether ketone capillary to obtain fiber-in-tube SPME. This setup was used to preconcentrate amyloid β-peptides (Aβs), biomarkers of Alzheimer’s disease in artificial samples, followed by quantification using UHPLC-MS/MS for protein binding studies. The zwitterionic PIL coating enabled preconcentration through ion-exchange and dispersive interactions. The authors also demonstrated the direct coupling of the developed fiber-in-tube SPME with MS/MS, allowing for sensitive detection of Aβ peptides at trace levels in cerebrospinal fluid samples without the need for chromatographic separation.⁷³

Figure 2.

Depiction of the online fiber-in-tube SPME configuration coupled to MS/MS detection for the preconcentration of amyloid β-peptides. Reprinted from Talanta, Vol. 254, Souza, I. D.; Anderson, J. L.; Tumas, V.; Queiroz, M. E. C. Direct coupling of fiber-in-tube solid phase microextraction with tandem mass spectrometry to determine amyloid beta peptides as biomarkers for Alzheimer’s disease in cerebrospinal fluid samples, pp. 124186 (ref (73), copyright 2023, with permission from Elsevier).

Thin film microextraction (TFME), a methodology similar to SPME, has also been explored in different ways using PILs for MEs. This technique improves the surface area-to-volume ratio of the sorbent coating, leading to enhanced mass transfer kinetics and faster extraction equilibration compared to SPME.⁵⁸ Shahriman et al. reported a study where they developed a paper-based TFME approach by grafting the PIL (poly(methyl methacrylateIL)) on the surface of commercial filter paper by using a dipping method to extract sulfonamides in environmental water samples.⁷⁴ The IL monomer used was 1-vinyl-3-hexylimidazolium bromide and the new method proved to be faster, more cost-effective, and simpler than previous techniques for determining sulfonamides in environmental samples, achieving low limits of detection (LOD) and small relative standard deviation values. However, the device’s reusability is limited due to potential leaching of the extractive phase, an issue that will likely be addressed in future design improvements. TFME has also been applied for rapid DNA isolation and recovery in downstream amplification assays. Eitzmann et al. developed reusable TFME devices coated with PILs and demonstrated efficient DNA extraction and compatibility with quantitative polymerase chain reaction (qPCR) analysis.⁵⁸ These devices enable quicker DNA recovery with less harmful solutions for qPCR and loop-mediated isothermal amplification (LAMP). Using a customized LAMP assay, TFME achieved 100% positive detection of a SARS-CoV-2 DNA sequence in artificial oral fluid samples, compared to 66.7% with SPME at a clinically relevant DNA concentration levels.

Microextraction by packed sorbent (MEPS), a miniaturized format of SPE, is another form of sorbent ME that has also been used in several innovative ways with ILs and other subclasses of ILs. Its configuration employs a sorbent-packed syringe instead of a typical cartridge that typically reduces solvent usage, simplifies the overall workflow, minimizes errors, and generates much less waste.^60,61 Jordan-Sinisterra et al. demonstrated the potential of MEPS when combined with ILs and their subclasses using the 1-vinyl-3-(butanesulfonate)imidazolium ([Vim⁺C₄SO₃^–]) ZIL supported on silica functionalized with graphene oxide via covalent bonding, as a sorbent for the extraction of pesticides from Brazilian coffee samples.⁶¹ The same ZIL supported on silica was employed to extract polycyclic aromatic hydrocarbons (PAHs) from coffee samples from both Colombia and Brazil. This approach not only facilitated the detection and quantification of harmful PAHs but also demonstrated the durability of the packed MEPS device, which could be reused over 100 times without compromising its extraction efficiency. These studies highlight the versatility and effectiveness of MEPS in combination with ILs for analyzing complex food matrices.⁶⁰

3D printed devices

PIL-based SPME coatings have shown wide applicability across various fields. However, the current reliance on manual coating methods presents obstacles to large-scale production. The key challenge lies in achieving high-throughput fabrication of PIL-coated fibers with consistent extraction efficiency, as ensuring uniform and customizable coating thickness across numerous fibers remains difficult. To address this, Hsieh et al. modified a commercial resin 3D printer to reduce prepolymer material usage in the production of sorbent-based extraction devices. Using this innovative platform, two imidazolium-based IL monomers were successfully printed in blade-type geometries for TFME and fiber-type geometries for SPME. Images of the 3D printed TFME blades are shown in Figure 3. The SPME PIL sorbents were applied to extract a variety of organic contaminants, including plasticizers, antimicrobial agents, UV filters, and pesticides from water, followed by HPLC analysis.⁷⁵ The results showed consistent extraction efficiencies across all sorbents with no significant differences in performance, demonstrating the potential of this technique to enhance the functionality and scalability of IL applications in environmental monitoring and analytical chemistry.

Figure 3.

Photographs of the miniaturized printing platform, modified for a liquid-crystal display 3D printer and used to create PIL thin film blades. (a) A thin film blade, measuring 15 mm × 2.5 mm × 0.5 mm, prepared from a 1 mL mixture of 50% (w/w) [C₈Vim⁺][Br^–], 47% (w/w) diurethane dimethacrylate (DUDMA) cross-linker, and 3% (w/w) diphenyl(2,4,6-trimethylbenzyl)phosphine (TPO) photoinitiator. (b) Simultaneous printing using two miniaturized platforms and two mL volume resin tanks. (c) Depiction of batch printing achieved during a single print period for blade and fiber geometries. Ten blades and 12 fibers consisting of PIL sorbents were simultaneously printed on their respective platforms. Reproduced from Hsieh, S.; Shamsaei, D.; Ocaña-Rios, I.; Anderson, J. L. Batch Scale Production of 3D Printed Extraction Sorbents using a Low-Cost Modification to a Desktop Printer. Anal. Chem. 2023, 95, 13417–13422 (ref (75)). Copyright 2023 American Chemical Society.

Capsule Phase Microextraction (CPME)

CPME is a ME technique that offers an alternative to traditional sample preparation approaches designed to address the need for a device capable of withstanding complex matrices without damage to the sorbent coating while allowing for easy retrieval and seamless integration with analytical instrumentation.⁷⁶ The extraction process relies on ME capsules composed of the following three key components: a sol–gel hybrid sorbent embedded in a porous polypropylene capillary membrane, porous polypropylene membranes, and a cylindrical magnet. The capsules are formed by welding two porous polypropylene capillary tubes, with one housing the magnet and the other containing the sorbent. The magnet allows the capsule to spin on a magnetic stirrer while the porous membrane functions as a built-in filtration system, eliminating the need for sample pretreatment like filtration or protein precipitation.⁷⁷ Manousi et al. developed a device incorporating an IL/Carbowax 20M-functionalized sol–gel sorbent into the lumen of porous polypropylene tube⁷⁸ and was employed for the extraction of three phosphodiesterase-5 inhibitors from human serum and urine followed by LC-MS analysis. Given their widespread presence in both legitimate and illicit products, there is a critical need to monitor these compounds in complex matrices such as biological and food samples. To enhance the sorbent’s selectivity, the N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate ZIL was incorporated to facilitate ion-dipole interactions with the target analytes. The device demonstrated remarkable reusability as it could be reused 25 times for both urine and serum samples. The proposed method was also evaluated for its green characteristics, where it exhibited reduced solvent use, minimal waste generation, cost-efficiency, and operational simplicity. Ntorkou et al. employed a similar strategy in developing a CPME device coupled with LC-post column derivatization for the determination of lanreotide (LAN), a somatostatin analogue, in human urine.⁷⁸ LAN is widely used to treat acromegaly and alleviate symptoms of neuroendocrine tumors.⁷⁹ Since LAN can be detected in the body for at least 11 days following administration, monitoring its distribution during therapy is crucial for assessing treatment effectiveness. The device incorporated a sol–gel Carbowax 20M-ZIL composite sorbent and the 3-[benzyl (dimethyl) ammonio]propane-1-sulfonate ZIL to enhance the extraction recovery of LAN by facilitating π–π interactions and ion-dipole interactions.

Aqueous Biphasic Systems (ABS) Using ILs

ABSs are widely utilized for the separation, purification, extraction, and enrichment of various biomolecules including proteins, animal cells, nucleic acids, and pharmaceuticals.⁸⁰ ABSs are liquid–liquid systems formed by mixing at least two water-soluble components. When these components exceed certain concentrations under specific temperature and pH conditions, they separate into two immiscible phases.⁸¹ Depending on the system, ABSs can be created using polymer–polymer, polymer-salt, or salt–salt mixtures dissolved in an aqueous medium.⁸² Extractions are largely driven by either the salting-out process or interactions between hydrophobic and hydrophilic regions within the polymer’s micellar framework.⁸³ Traditional polymer-based ABSs offer a narrow polarity range between their coexisting phases, which limits their ability to achieve high extraction efficiency and selectivity in a single step. This limitation can be addressed by incorporating ILs as phase-forming components in ABSs, thereby enabling improved performance. González-Martin et al. reported a study where an IL-based ABS system was employed as a one-step platform for cleanup, ME, and preconcentration of bisphenols as representative salivary biomarkers for their improved determination.⁸¹ In this study, a miniaturized ABS system was developed using the low cytotoxic butylguanidinium chloride ([C₄Gu⁺][Cl^-]) IL and dipotassium phosphate as the salting-out agent. Bisphenols were selected as salivary biomarkers due to their growing endocrine-disrupting effects. Sample cleanup was achieved by precipitating most of the interfering salivary proteins into a protein-enriched solid interphase of the ABS, while extraction and preconcentration of bisphenols were achieved in the IL-rich phase. Bisphenols were then determined using HPLC and fluorescence detection and the method obtained higher green scores when evaluated for different greenness metrics. Similarly, Ferreira et al. reported ABS systems composed of cholinium-based ILs and polypropylene glycol 400 to pretreat human serum for isolating E. coli genomic DNA from human serum albumin (HSA), thereby minimizing interference in DNA quantification by real-time PCR.⁸² The pretreatment was carried out at different pH values, revealing that HSA precipitated under low pH conditions, while no precipitation occurred at neutral pH. The most effective system used cholinium glycolate at pH 5, which resulted in complete HSA precipitation at the ABS interface while the IL-rich phase was enriched with high purity stable DNA. This IL-based ABS offered an effective way to prepare human serum samples, enabling the isolation of bacterial DNA and improving bacterial infection monitoring.

Flora et al. utilized an IL-based ABS coupled to a bead-based microfluidic immunofluorescent assay to extract and detect prostate-specific antigens from human serum.⁸⁴ The tetrabutylammonium chloride [N₄₄₄₄⁺][Cl^-] and tetrabutylphosphonium bromide [P₄₄₄₄⁺][Br^-]-based ABSs with phosphate buffer demonstrated reduced background fluorescence from interfering matrix components, and higher enrichment of the antigens into the IL-rich phase allowed for an LOD of 4.9–5.1 ng mL^–1 compared to the PEG (molecular weight 1000 g mol^–1)-ABS system with citrate buffer reaching 12.3 ng mL^–1. A very recent study by Phakoukaki et al. reported an innovative approach by combining IL-based ABS systems with small channels for continuous plug-flow extraction of l-tryptophan, showcasing the potential of ABS technology in diverse applications.⁸⁵ This method extracted l-tryptophan using channels with internal diameters of 0.5 mm and 0.8 mm, employing different IL-ABS formulations composed of varying wt % of the [C₄MIm⁺][Cl^-] IL and potassium phosphate. The extractions were performed under plug flow conditions, where fluid moves in separate plugs without much mixing. Higher concentrations of salt and IL improved the partitioning of the amino acid into the IL-rich phase. Overall, the mass transfer rates in this system were significantly faster than those in traditional solvent extraction methods making it highly efficient.

Chromatographic Separations

Gas Chromatography (GC)

GC is an analytical technique that separates volatile and semivolatile compounds based on their affinities for a stationary phase. Current commercial GC phases often have difficulty in separating highly basic or acidic compounds with acceptable peak symmetry and provide low chromatographic resolution for samples with a wide range of functional groups. Recently, there has been an increase in the utilization of ILs as chromatographic stationary phases due to their distinct properties. ILs possess unique and tunable selectivity, low vapor pressure, and high thermal stability and are advantageous for use in GC, as they separate a wide range of analytes with different functionality, can be utilized at high oven temperatures, and provide high separation efficiencies.^86,87 ILs are capable of separating both polar and nonpolar molecules through simultaneous dipolar interactions, electrostatic interactions, hydrogen bonding, and dispersion interactions that can be tuned by modifying their chemical structure. These advantages have led to an increased interest in ILs as stationary phases in GC.⁸⁷

Microfabricated Columns

One application in which ILs are utilized in GC is as stationary phases for microfabricated columns. Microfabricated columns are typically used in μGC, which is a miniaturized form of GC that utilizes short columns, allowing for portable, in-field separations and short analysis times. However, the separation power becomes limited as the length of μGC columns are decreased, requiring new and highly selective stationary phases. Gholizadeh et al. developed a new column configuration, shown in Figure 4, utilizing three, 30 cm long semipacked GC columns with IL stationary phases in tandem to maximize separation performance.⁸⁸ In this work, the following three ILs were synthesized: 1-butylpyridinum [NTf₂^–] (IL1), 1-(2-hydroxyethyl)-3-methylimidazolium [NTf₂^–] (IL2), and methyltrioctylammonium [NTf₂^–] (IL3). The IL stationary phases were designed for increased analyte selectivity in a mixture consisting of (1) saturated alkanes (C₇–C₃₀), (2) analytes with boiling points ranging from 80 to 238 °C, and (3) analytes with varying degrees of polarity. While none of the columns separated all components of the 46-compound mixture, complete separation of all analytes was achieved when the three IL columns were placed in a parallel configuration (i.e., IL1 v. IL2, IL2 v. IL3, and IL1 v. IL3), wherein the columns behaved as a pseudo-2D GC method. By adding the IL2 column as a second dimension to the IL1 column in a parallel configuration, previously coeluting compounds (pentanone and ethylbenzene) were separated. Using the method, a 46-compound mixture was fully separated within 4 min, ultimately preserving the short analysis times of μGC. Similarly, Meziani et al. introduced the use of a GC column with radially elongated pillars as a second column in GC × μGC.⁸⁹ This column was statically coated with a medium polarity IL based on a monocationic phosphonium derivative and a Supelco Equity-1 (OV-1701) column was used for the first dimension. Lower theoretical plate heights were achieved for the μGC column containing the IL stationary phase compared to other prepared columns, except for the polydimethylsiloxane (PDMS) column of similar film thickness. However, the 2D system was able to effectively separate alkanes and aromatics, though better separation was achieved using a PDMS stationary phase. Interestingly, a reversal of elution order was observed for the polar polyethylene glycol (PEG) and midpolar IL stationary phases tested compared to PDMS, in which alkanes were less retained. Issues concerning wrap-around were also noted, but further reduction in the column length was hypothesized to solve this problem.

Figure 4.

SEM images show (A) the top view of semipacked μGC column (200 μm scale), (B) a zoomed-in view of the silicon micropillars and channel (150 μm scale), (C) a cross view of pillar coated with [C₄Pyr⁺][NTf₂^–] IL (16 μm scale), and (D) a magnified view of the uniform IL coating on a micropillar (3 μm scale). Golay plots (E) for [C₄Pyr⁺][NTf₂^–], [C₂OHMIm⁺][NTf₂^–], and [N₁₈₈₈⁺][NTf₂^–] IL are shown under the following conditions: injection volume, 0.1 μL; split ratio, 200:1; inlet temperature, 280 °C; detector temperature, 300 °C; and oven temperature, 100 °C. Reproduced from Gholizadeh, A.; Chowdhury, M.; Agah, M. Parallel Ionic Liquid Semi-Packed Microfabricated Columns for Complex Gas Analysis. Anal. Chem. 2020, 92, 10635–10642 (ref (88)). Copyright 2020 American Chemical Society.

Traditional GC stationary phases, such as PDMS or PEG, are susceptible to hydrolysis and oxidation when less expensive, nonultra-high purity carrier gases are used, which can lead to higher column bleed, increased peak broadening and tailing, and stationary phase deterioration.⁸⁷ To combat these issues, Li et al. used phosphonium ILs as μGC stationary phases to decrease stationary phase moisture and oxygen uptake, thereby increasing the separation performance.⁹⁰ By coating a chip-based IL column, they were able to completely separate polar and nonpolar compounds, such as mixtures of alcohols, fatty acid methyl esters (FAMEs), chloroalkanes, alkanes, and aromatics. To verify the ILs’ resilience to hydrolysis and oxidation, the prepared columns were then exposed to moisture and oxygen, where they showed high stability when comparing retention times and full-width half maximums of peaks between the dry injections and moisture injections, indicating low stationary phase degradation. While only phosphonium-based ILs were studied, the authors noted that other ILs could be introduced for more specific and selective separations.

A new μGC stationary phase was introduced by Bae et al. consisting of the [C₄MIm⁺] tetrafluoroborate [BF₄^–] and dimethylpolysiloxane incorporated into a 3 m long hybrid gel microcolumn for the identification of narcotic substances.⁹¹ The IL-based hybrid gel microcolumn was able to separate eight volatile organic compounds and seven narcotic substance mixtures with high column efficiency and temperatures up to 240 °C. Volatile organic compounds were separated due to increased π–π interactions and hydrogen bonding compared to a pure dimethylpolysiloxane reference column. For the narcotic substances, the prepared column was shown to increase the retention times for ketamine, cocaine, and 3,4-methylenedioxymethamphetamine compared to the reference dimethylpolysiloxane column due to increased π–π interactions, ion-dipole interactions, and ion-induced interactions in the prepared hybrid gel column. Additionally, the prepared hybrid gel column showed increased column efficiency and decreased peak tailing compared to the dimethylpolysiloxane column, overall proving to be more effective in the separation of drug molecules.

Patrushev et al. reported for the first time ILs as stationary phases for multicapillary columns in traditional GC, providing short analysis times and operation over a wide range of flow rates.⁹² These multicapillary columns consisted as packs of 1,375 capillary columns that were 40 μm in diameter and a length of 23 cm. The high number of capillaries allows for multicapillary columns to be able to withstand high sample loading, while also being able to utilize high flow rates and high separation speeds. The prepared columns were coated with the 1,2-dimethyl-3-propylimidazolium ([C₃M(M)Im⁺]) [NTf₂^–], 4-methyl-N-propylpryidinium ([C₃MPyr⁺]) [NTf₂^–], and 6-methyl-N-hexylquinolinium ([C₆MQu⁺]) [NTf₂^–] ILs. van Deemter plots were generated for the three stationary phases in which the [C₃MPyr⁺][NTf₂^–] IL showed the lowest theoretical plate heights at higher flow rates and the [C₆MQu⁺][NTf₂^–] showed the lowest efficiencies at higher flow rates. The [C₃MPyr⁺][NTf₂^–] IL separated seven dimethylphenols within 45 s, and C₁₆–C₂₄ FAMEs within 75 s.

Framework-Embedded Ionic Liquid Stationary Phases

In recent years, there has been increased research where ILs have been added to structurally rigid macrocyclic molecules for chromatographic analysis of structurally similar isomers, including alkyl benzenes, halogenated benzenes, anilines, and phenols.⁹³ The three-dimensional structure introduces cavities in which analytes are able to enter and interact with the IL-functionalized sites of the molecule, allowing for stronger interactions and higher selectivity. Yu et al. presented an IL GC stationary phase consisting of two imidazolium cations attached to a triptycene (TP) framework.⁹⁴ TP has a three-dimensional structure consisting of three benzene rings and three open π-electron rich cavities, allowing for high thermal stability and multiple sites for functionalization.^93,94 This stationary phase was shown to baseline resolve anilines and phenols as well as positional isomers with good peak symmetry–a task otherwise hard to accomplish.⁹⁴ The new structure allows for a synergistic effect of multiple molecular interactions involving hydrogen bonding, dipole–dipole, cation-π/anion-π, and π–π stacking interactions originating from the unique amphiphilic structure. He et al. also introduced a TP-based stationary phase by combing the rigid backbone of the TP with three flexible octyl-imidazolium side chains to increase selectivity of analytes with high similarity.⁹³ The stationary phase achieved higher resolution of critical m-/p-halobenzene compounds, acidic and basic isomers, and showed selectivity for polar molecules.

Yuan and Qi created a new form of a TP-based IL stationary phase by bonding it to a dicationic ionic liquid (DIL).⁹⁵ The addition of a guanidinium (G) DIL provided higher selectivity toward a range of positional and structural isomers. The TPG stationary phase separated mixtures of phenols, anilines, and weakly polar aromatic isomers with high resolution, showing that the amphiphilic selectivity of the TPG column results from the triptycene framework and the guanidinium cations. In addition, the steric hindrance introduced by the triptycene moiety produced intermolecular space that provided easier access to isomers with relatively less molecular volume, leading to the different retention of the alkane isomers and pentanol. In a separation of mint essential oils, the TPG column was able to resolve 30 components, 12 more than the reference DB-35MS column with (35% phenyl)methyl polysiloxane as the stationary phase, indicating higher selectivity and separation capability. Similar to the aforementioned studies, Ba et al. introduced a novel GC stationary phase framework in which they attached an octyl-imidazolium [NTf₂^–] IL with a C₄ linker to a pillar[6]arene, a complex with 12 available sites for functionalization and π-electron-rich cavities.⁹⁶ In this study, the prepared column was found to differentiate between phenol, aniline, aldehyde, alkane, and aromatic isomers, as well as cis-/trans- isomers. The excellent resolving performance is attributed to the comprehensive interactions of the rigid three-dimensional structure of the pillar[6]arene, providing interactions with the aromatic cavities and the polar imidazolium ILs. The above studies clearly indicate that introducing ILs into rigid, three-dimensional structures enhance the selectivity and resolution of otherwise hard to separate isomers when applied as stationary phases in GC.

Increasing Stationary Phase Thermal Stability

GC utilizes high temperatures for separation and elution of analytes; therefore, thermally stable stationary phases are required. Phosphonium IL-based stationary phases exhibit lower maximum allowable operating temperatures (MAOTs) in the range of 180–200 °C, which limits their use as routine GC stationary phases.⁸⁶ Cagliero et al. studied the influence of immobilization of phosphonium-based IL stationary phases on the MAOT of the column.⁸⁶ By immobilizing the stationary phase to the inner wall of the capillary using a proprietary method, an overall increase in the MAOT was observed. In addition, when compared to the same stationary phase that was not bonded, the immobilized phosphonium IL-based stationary phase presented a negligible difference in selectivity, indicating that the interaction mechanism is maintained after immobilization. Another method for increasing the thermal stability of IL-based GC stationary phases was introduced by Odugbesi et al. and described perarylated ILs consisting of sulfonium and phosphonium cations with the [NTf₂^–] anion.⁹⁷ Perarylated ILs are much less susceptible toward undergoing Hofmann elimination (particularly with weakly nucleophilic anions), making them much more thermally stable. Unlike columns reported by Cagliero et al., these stationary phases were not immobilized to the capillary wall and provided MAOTs of 290–350 °C with improved selectivity for heavier polycyclic aromatic hydrocarbons.

Separation of Fragrance and Oil Mixtures

While the above studies provide a small introduction into IL applications in GC, a prominent area of interest is the use of ILs in the separation of fragrance and oil mixtures. Fragrances and oils consist of complex mixtures of structurally similar compounds, making their separation difficult and many contain environmental pollutants that are challenging to quantify due to mixture complexity. New stationary phases with unique selectivity are of great interest in the fragrance and oil field due to the large number of analytes consisting of a wide range of functional groups, making ILs promising stationary phases for their analysis due to their tunable chemical structures.⁹⁸ Mazzucotelli et al. investigated the effect of the anion on separation selectivity for phosphonium-based ILs.⁹⁸ For the separation of both sage and vetiver essential oils, the IL stationary phase with [Cl^–] anions separated monoterpenoid and sesquiterpenoids based on functional groups, separating ketones, esters, and alcohols sequentially. The IL stationary phase with [NTf₂^–] anions did not separate the components based on functional groups but instead separated monoterpenoid hydrocarbons from sesquiterpenoids and oxygenated compounds as a function of analyte polarity and volatility. Crucello et al. proposed a new method for the separation and quantification of polychlorinated biphenyls in insulating oils using two-dimensional GC (GC × GC).⁹⁹ GC × GC is a chromatographic method that combines two capillary columns featuring complementary selectivity for highly effective separations. In this method, the commercially available IL column SLB-IL59, consisting of the ditripropylphosphonium)dodecane ([(P_3,3,3)₂C₁₂⁺]) [NTf₂^–] IL allowed for rapid elution of aliphatic hydrocarbons due to low dispersive interactions as well as dipole–dipole interactions, thereby increasing retention of aromatic hydrocarbons. Using the SLB-IL59 column, a sample preparation step that is typically required for the analysis of biphenyls could be eliminated and lower pressures could be utilized to promote overall greener analysis. Additionally, Nan et al. provided a first look into the use of imidazolium-based ZILs possessing sulfonate anions as stationary phases for the separation of volatile carboxylic acids (VCAs) in GC.²⁸ VCAs are important molecules in the pharmaceutical and flavor and fragrance industries,¹⁰⁰ but are difficult to quantify as they tend to hydrogen bond with exposed silanol groups on the surface of fused silica capillary, leading to high peak asymmetry. ZIL stationary phases provided excellent peak shapes compared to the commercial column SLB-IL111, consisting of the 1,5-di(2,3-dimethylimidazolium)pentane ([(M(M)Im)₂C₅⁺]) [NTf₂^–] IL, as well as higher retention and unique selectivity.

High-Performance Liquid Chromatography

The use of ILs in chromatography is a continuously growing field due to their previously mentioned advantageous properties. Their use as HPLC stationary phases and mobile phase additives has given rise to new separation capabilities, providing an overall increase in resolution and selectivity for otherwise hard to separate analytes. IL-modified stationary phases interact with analytes through various mechanisms that can be tailored to favor different modes of separation.¹⁰¹ These different modes are dependent on the target analyte, which overall dictates the stationary phase and the employed mobile phase. The most common separation modes include reverse phase liquid chromatography (RP-LC), hydrophilic interaction chromatography (HILIC), and mixed-mode chromatography, all of which will be discussed in this section.

Reverse Phase HPLC Stationary Phases and Mobile Phase Additives

Due to the complex structure with the potential of multiple functionalities that ILs possess, their use in HPLC focuses on mixed-mode chromatography. However, there are studies in which ILs have been applied for use in RP-LC. ILs were introduced as mobile phase additives in HPLC for the improvement of column efficiency and peak shape due to their ability to suppress interactions with silanol groups present on the stationary phase.¹⁰² Treder et al. exhaustively investigated the effect of IL functionality as RP-LC mobile phase additives.¹⁰² In this work, 17 ILs were studied for the separation of anthracycline antibiotics. It was found that the identity of the cation did not matter as much as its size with larger cations resulting in lower retention, due to increased hydrophobicity. In addition, anions that exhibited low adsorption to the C₁₈ stationary phase were the best in separating anthracyclines, and the retention mechanism of anthracyclines could be controlled to improve the separation. In a separate study, Treder et al. investigated the use of ILs as mobile phase additives when aromatic stationary phases were employed.¹⁰³ They studied the effect of anion identity with imidazolium and pyridinium cations for 13 different ILs in the separation performance of anthracyclines. With the aromatic stationary phases, π–π interactions are possible with the aromatic anthracyclines compared to the previously studied C₁₈ stationary phase, thereby promoting increased retention. Interestingly, they found that the addition of an IL into the mobile phase reduced retention of anthracycline antibiotics on the aromatic stationary phases compared to the C₁₈ stationary phase. Specifically, anthracycline retention decreased in the order of [PF₆^–] > [CF₃SO₄^–] > [BF₄^–] > [CH₃SO₄^–] > [Cl^–] for each stationary phase. This effect was ascribed to the ability of the IL additives to undergo π–π interactions with the anthracycline analytes, thus decreasing the π–π interactions with the stationary phase.

Tereba-Mamani et al. introduced a new RP-LC mobile phase consisting entirely of sodium dodecyl sulfate (SDS) and 1-ethyl-3-methylimidazolium [C₂C₁IM⁺] [Cl^–], 1-butyl-3-methylimidazolium [C₄C₁Im⁺] [Cl^–], and 1-hexyl-3-methylimidazolium [C₆C₁IM⁺] [Cl^–], utilizing no organic solvent for the separation of basic analytes.¹⁰⁴ The mobile phase allowed for basic compounds to be attracted to the anionic portion of SDS while being repelled by the IL cation, which allows for modulation of retention with varying IL/SDS concentration. This mobile phase was applied in the separation of β-adrenoceptor antagonists; high retention was obtained with SDS and the IL individually and modulated when both were present.

While ILs were originally introduced as mobile phase additives in RP-LC, IL research has expanded into their use as stationary phases.^102,105 Jiang et al. functionalized carbon dots with 1-vinyl-3-octadecylimidazolium [C₁₈VIm⁺] [Br^–] and grafted them onto silica, allowing for increased separation of polar analytes.¹⁰⁶ Carbon dots exhibit high stability, low toxicity, biocompatibility, and modifiability.¹⁰⁶ The prepared Sil-ImC₁₈/carbon dot stationary phase separated alkyl benzenes, PAHs, aromatic amines, and phenols.

Stationary Phases for Hydrophilic Interaction Chromatography (HILIC)

HILIC is a HPLC mode that typically utilizes a high proportion of acetonitrile (ACN) to water as the mobile phase for the separation of polar and hydrophilic compounds.¹⁰⁷ It is hypothesized that HILIC operates by creating a stagnant water layer around the polar stationary phase, into which the polar and hydrophilic analytes partition before interacting with the stationary phase. Similar to the work completed by Jiang et al., Song et al. introduced imidazolium IL-derived carbon dots grafted to silica for use as a stationary phase in HILIC mode.¹⁰⁸ The prepared column, referred to as Sil-ImCDs, exhibited higher hydrophilic interactions compared to an imidazolium IL-functionalized stationary phase and an aminopropyl silica stationary phase. In addition, the prepared stationary phase exhibited decreased peak tailing and increased column efficiency, all of which were ascribed to the imidazolium, carboxyl acid, and hydroxyl functional groups present on the carbon dots.

Guo et al. investigated the use of imidazolium-based ILs with a chloride anion as stationary phases for HILIC mode.¹⁰⁹ In this study, typical compounds such as saccharides, nucleosides, and nucleobases were used as target analytes. Monosaccharides exhibited short retention, disaccharides moderate retention, and trisaccharides high retention, indicating that retention is influenced by the number of hydroxyl groups present and the polarity of the analytes. Since mobile phase composition is an important factor in HILIC, the effect of decreasing the proportion of ACN was investigated. At a 75% ACN composition, the saccharides could not be resolved, but at 95% ACN composition, the saccharides were fully resolved at shorter analysis times compared to a commercial reference column. This study also investigated the difference of the anion present in the IL, in which [NTf₂^–], [BF₄^–], and [Cl^–] were utilized. The [Cl^–] and [BF₄^–] anions exhibited better resolution and increased retention compared to [NTf₂^–] under the same chromatographic conditions. This effect is ascribed to the stronger dipoles and induced field strength of ion-pair in [Cl^–] and [BF₄^–], leading to overall stronger interactions with the anions compared to [NTf₂^–].

Mixed-Mode Stationary Phases

Mixed-mode chromatography utilizes a single stationary phase capable of providing multiple interaction modes for the separation of highly complex analyte mixtures.^105,110 These stationary phases are typically amphiphilic to induce hydrophilic and hydrophobic interactions and may possess aromatic moieties to induce π–π interactions. Due to the various interactions possible, mixed-mode chromatography has a high separation power and selectivity for analytes with differing functionality.¹⁰⁵ These clear advantages make development of mixed-mode stationary phases an important area of research. Luo et al. introduced hydrophilic carbonyldiimidazolium [CDI⁺] [Cl^–] and hydrophobic dodecyl (DD) together onto the surface of silica for a new mixed-mode stationary phase to improve the separation selectivity of environmental endocrine disruptors, including alkylphenols, bisphenols, phthalates, and steroidal hormones in RP mode.¹¹¹ When separations were performed in RP mode, retention increased as hydrophobicity of the analytes increased, except in the case of bisphenols, whose elution order was independent of hydrophobicity. When separations were performed in HILIC mode, nucleosides and bases were baseline resolved, without the addition of buffer salts to control pH. While this stationary phase was able to separate the hydrophilic nucleosides and nucleobases, it exhibited poor selectivity for other hydrophilic analytes. To overcome this limitation, the same group then introduced a novel imidazolium IL embedded multifunctional stationary phase.¹⁰⁵ In this IL stationary phase, 1-allyl-3-vinyl-imidazolium was reacted onto thiolated silica gel, which was then reacted further with stearyl thioglycolate to embed the imidazolium moiety. The prepared stationary phase, Sil-AVI-ST, was able to separate hydrophilic sulfonamides, vitamins, and nucleosides and nucleobases as well as hydrophobic phthalates, bisphenols, alkylphenols, and steroid hormones. The observed selectivity was attributed to the imidazolium cations being capable of multiple interactions with the target analytes, as well as the polar vinyl groups imparting increased hydrophilic interactions.

Wang et al. developed two IL stationary phases consisting of a dicationic imidazolium cation paired with [NTf₂^–] anions with two (DPE-DIL) and four (BND-DIL) aryl groups for increased separation selectivity for food additives.¹¹² The two prepared stationary phases were able to resolve nucleobases and nucleosides with low tailing factors when used in HILIC mode, but provided different elution orders for cytidine and inosine, indicating different selectivity. Under RPLC mode, the two stationary phases were able to separate three terphenyl isomers and triphenylene. Again, differences in selectivity between the two stationary phases were observed, as m-terphenyl and p-terphenyl coeluted on the DPE-DIL stationary phase, but were fully resolved on the BND-DIL stationary phase. Thus, the BND-DIL stationary phase was utilized for the separation of food additives. Nitrites and nitrates were able to be detected from different food samples with recoveries from 96.4 to 103.8%.

Many studies have investigated the effect of IL cation placement for mixed-mode stationary phases. One investigation conducted by Fan et al. studied the effect of spacer alkyl chain length on the retention of alkylbenzenes, nucleoside bases, and inorganic ions among three IL stationary phases, Sil-C₄Im, Sil-C₇Im, and Sil-C₁₀Im with [NTf₂^–] anions, under various HPLC modes to mimic mixed-mode conditions.¹⁰¹ In RPLC, the selectivity of aromatic molecules increased as the spacer alkyl chain length between the silica and the imidazolium cation was increased. Interestingly, the retention factors increased when the spacer chain length was lengthened from C₄ to C₇, but did not increase linearly from C₇ to C₁₀. They hypothesized this to be due to the C₄ and C₇ molecules lying flat on the stationary phase due to shorter spacer alkyl chains resulting in higher electrostatic attraction between the imidazolium cation and the dissociated silanol on the silica surface, while the C₁₀ lays in a vertical state on the stationary phase due to the longer chain shielding electrostatic attraction and increasing retention. In HILIC mode, the five nucleoside bases were able to be separated completely in order of increasing polarity. When employed in anion exchange chromatography (AEX), the [BrO₃^–], [NO₃^–], [I^–], and [SCN^–] compounds were fully separated, with the C₁₀ exhibiting the highest selectivity due to the longer spacer alkyl chain.

Luo et al. studied three regioisomeric IL silane stationary phases, in which the position of the imidazolium cation was translated along the stationary phase moiety; the three stationary phases were referred to as Sil-C₂Im-C₈, Sil-C₆Im-C₄, and Sil-C₉Im-C₁.¹¹⁰ All three stationary phases exhibited retention of hydrophilic nucleobases under HILIC conditions as well as increased retention of hydrophobic analytes under RPLC conditions. They found that due to the long alkyl chain of Sil-C₂Im-C₈, hydrophilic interactions between the analytes and the imidazolium cation were hindered, producing the lowest retention and thus hydrophilicity under HILIC conditions. Interestingly, the strongest hydrophilicity was exhibited by Sil-C₆Im-C₄ as nucleotides were most strongly retained on this stationary phase. The increased separation performance of alkyl benzenes and PAHs in RPLC mode was observed when the imidazolium moiety was embedded on the outermost part of the alkyl chain, due to an increase in aromatic system accessibility.

A similar study was conducted by Wang et al. investigating the relative length of two ILs consisting of imidazolium cations and [NTf₂^–] anions and a secondary carboxylic acid functionalized silane stationary phases on the separation performance of mixed-mode separations in HPLC.¹¹³ The stationary phases, referred to as Sil-C₄Im-C₉Co and Sil-C₉Im-C₄Co, were both thought to retain solutes through hydrophobic, hydrophilic, ion-exchange, and π–π stacking interactions. The study found that the anion strength was greater for Sil-C₉Im-C₄Co, as evidenced by the higher retention times of acids, whereas the Sil-C₄Im-C₉Co stationary phase offered higher retention of amines. In addition, under RPLC conditions, Sil-C₉Im-C₄Co completely resolved five alkylbenzenes and four PAHs, which was not observed for the Sil-C₄Im-C₉Co stationary phase and indicates a difference in hydrophobicity based on cation location.

In addition to an investigation of cation placement and alkyl chain length, the bonding of ILs to nontraditional silica particles or nonsilica particles has also been investigated. These materials are typically resistant to acid and base and allow for even functionalization, as well as decreasing silanol interactions for nontraditional silica particles.¹⁰⁷ Chen et al. introduced gold nanoparticles (NPs) that were covalently bonded to an IL bridged periodic mesoporous organosilica (PMO) stationary phase for per aqueous liquid chromatography (PALC).¹⁰⁷ PALC separates polar compounds using a high proportion of water, typically consisting of 90% or more as the mobile phase, making it an overall greener separation technique compared to HILIC. The prepared PMO-ILs-Au NPs column was studied under various modes to investigate the separation capability. In HILIC, the PMO-ILs-Au NP stationary phase increased retention of polar molecules as the mobile phase polarity was decreased, indicating a typical HILIC retention mechanism. When using 80/20 water/ACN as the mobile phase in PALC, no significant differences in analyte retention factor or peak shape were observed compared to HILIC with a 10/90 water/ACN mobile phase, indicating that this stationary phase in PALC could be used as a new chromatographic separation mode for the separation of polar molecules. From this finding, the authors then utilized PALC mode for detection and recovery of eight biogenic amines, in which recoveries of the method ranged from 63.52 to 93.08%. PALC mode was determined to be a simple, accurate, and rapid method complementary to RPLC and an alternative to HILIC that is capable of effectively separating polar analytes.

PILs have also increased in popularity due to their increased column stability under a wide range of chromatographic conditions. Liu et al. introduced a new classification of stationary phase for the mixed-mode separation of phospholipids by polymerizing phosphonium ILs onto silica microspheres.¹¹⁴ Structurally, phosphonium-based ILs and phospholipids are similar as they both contain a charged, hydrophilic phosphorus headgroup and hydrophobic alkyl chains, allowing for excellent separation selectivity of phospholipids. In preliminary studies, the stationary phase exhibited both hydrophilic and hydrophobic separation capabilities. Due to the strong hydrophobicity of the stationary phase and similar structure to phospholipids, excellent separation selectivity for phospholipid classes compared to a commercial amino column was achieved. Peng et al. prepared polymeric IL microspheres as HPLC stationary phases for separations in mixed-mode chromatography.¹¹⁵ PIL microspheres were determined to provide high surface area, allowing for increased interaction sites, as well as stationary phase stability. The monodisperse microspheres separated mixtures of alkylbenzenes, nucleosides, and alkaloids in RPLC and HILIC mode. In HILIC specifically, uracil and cytosine were able to be separated effectively in pure water, allowing for greener separations with the use of the prepared PIL microspheres.

HPLC is often employed in the separation of biological compounds, which may be chiral. Chiral chromatography is often used to separate enantiomers and utilizes silica particles functionalized with a chiral selector, such as cyclodextrin (CD).¹¹⁶ The separation of enantiomers is important as many biological and drug molecules are made up of enantiomers that have different chemical properties. Zhou et al. functionalized a C₁₈ stationary phase with the 3-n-octadecyl-1-vinylimidazolium bromide IL and 6-(1-allylimidazolium)-cyclodextrin tosylate monomers as a mixed-mode stationary phase for achiral and chiral separations.¹¹⁶ The stationary phase separated PAHs, alkylbenzenes, and terphenyls completely in RPLC as well as nucleosides and nucleotides in HILIC mode with better peak shapes and shorter separation times compared to a commercial ZORBAX NH₂ column. In ion-exchange chromatography (IEC), the stationary phase was also able to separate benzoic acids and phenols. For use in chiral separations, the β-CD functionalized IL stationary phase was able to separate enantiomers of 1-phenylpropanol, warfarin, and styrene oxide through anionic exchange interactions. Another study attached an imidazolium IL functionalized poly(quinine) to silica as a mixed-mode stationary phase.¹¹⁷ By combining quinine with imidazolium ILs in a chromatographic separation, aromatic compounds were completely separated in IEC, sulfanilamides, nucleosides, and nucleobases were separated in HILIC, and alkylbenzenes, benzene, and PAHs were separated in RPLC. In addition, quinine is an excellent chiral selector that resulted in the separation of three pairs of enantiomers with high selectivity. This stationary phase overall exhibited dipole–dipole, hydrophilic, anion-exchange, and chiral separation capabilities.

ILs have also been applied as part of monolithic stationary phases in capillary columns.¹¹⁸ Unlike traditional HPLC column consisting of packed silica particles, monolith columns consist of connected silica or polymer skeletons that provide good permeability and lower backpressures compared to traditional packed HPLC columns.¹¹⁹ As a result, they provide fast mass transfer and high permeability, making their application promising for the development of HPLC stationary phases.¹²⁰ Moravcová et al. introduced a monolith rod modified with a phosphonium-based IL for use in capillary liquid chromatography.¹¹⁸ The prepared stationary phase, trioctyl(3/4-vinylbenzyl)phosphonium chloride, was found to exhibit mixed-mode interactions, including hydrophobic, hydrophilic, and electrostatic interactions, allowing high separation and selectivity for compounds with differing substitution patterns on alkylbenzenes, functionalities, and different groups of analytes.

Polyhedral oligomeric silsesquioxane (POSS) is an easy to modify, three-dimensional structure and is robust to a wide pH and temperature range, making POSS ideal for chromatography. By introducing an IL to the POSS structure, multiple interaction mechanisms, including π–π, ion exchange, hydrophobicity, hydrophilicity, and hydrogen bonding can be achieved. Chen et al. introduced an allyl vinyl imidazolium (AVI) and D-2-allylglycine (AG) functionalized POSS monolithic column for the separation of phenols, alkylbenzenes, aromatic amines, nucleobases, amides, and thioureas.¹²⁰ Alkylbenzenes, amines, and phenols were separated through the RPLC retention mechanism and an increase in retention as the hydrophobicity of the alkylbenzene increased was observed. Nucleobases, amides, and thioureas were baseline resolved through HILIC retention mechanisms, and provided high separation efficiencies. The prepared column was then utilized to successfully separate cytochrome c tryptic digests and egg white protein extraction, indicating a promising future in the separation of macromolecules. Similarly, Zhou et al. functionalized a POSS methacryl substituted (POSS-MA) with 1-vinyl-3-dodecylimidazolium bromide (VDI) to form a POSS-VDI monolithic column.¹²¹ Alkylbenzenes were able to be separated through the RPLC retention mechanism with analysis times under 8 min, high efficiencies, and tailing factors not exceeding 1.29. Hydrophilic analytes, such as thioureas, were baseline separated within 5.5 min and achieved high separation efficiency, showing that POSS-VDI separated both hydrophobic and hydrophilic analytes. Furthermore, the separation of Fangji and the Roots of Kudzu Vine components was achieved for active ingredient identification.

Membrane Separations

Separation technologies often involve methods like crystallization, fractional distillation, solvent extraction, and chromatography, which can be complex, energy consuming, and lead to high solvent waste.¹²² To address these issues, more advanced and efficient techniques have been developed utilizing membrane-based approaches. A membrane is a thin layer that separates substances according to their physical and chemical characteristics when subjected to a driving force, such as a gradient in chemical potential (either concentration or pressure) or electrical potential.¹²³ A membrane can be homogeneous or heterogeneous, symmetric or asymmetric, and can take the form of a solid or liquid made from organic or inorganic materials. It may be neutral or charged and can contain functional groups for specific binding or complexing.¹²⁴ In recent years, separation via liquid membrane (LM) has become popular and valuable in fields like biotechnology, organic chemistry, chemical engineering, and wastewater treatment. A LM consists of a thin, uniform, nonporous layer of organic liquid positioned between two aqueous or gas phases of different compositions.¹²⁵ Generally, LMs are categorized into two types: supported and nonsupported. Nonsupported LMs include emulsion liquid membranes (ELMs) and bulk liquid membranes (BLMs), while supported LMs include hollow fiber and flat sheet configurations and are stabilized by capillary forces within the pores of polymeric or inorganic films. The field of membrane separation is vast, and extensive research has been conducted in this area. Although much of the focus has been on CO₂ capture, membrane separation has also been applied in wastewater treatment,¹²⁶ removal of toxic pollutants,¹²⁷ heavy metal separation,¹²⁸ and biofuel recovery,¹²⁹ among other applications.

CO₂ Capture

As concerns about global warming and rising carbon dioxide (CO₂) emissions from fossil fuels continue to grow, research into CO₂ capture has gained significant attention.^130,131 CO₂ is emitted in the form of various mixtures, including CO₂/N₂, CO₂/CO/N₂, CO₂/CO/H₂, and CO₂/CH₄. CO₂ capture is generally categorized into three main types: precombustion capture (CO₂/H₂), postcombustion capture (CO₂/N₂), and oxygen-enriched combustion (O₂/N₂). Compared to precombustion and oxy-combustion methods, postcombustion CO₂ capture offers significant advantages due to its ease of operation and compatibility with existing production processes.¹³² Furthermore, it addresses over 40% of global carbon emissions, making it a crucial segment in carbon capture efforts.¹³³ Amine solvent absorption is the most common postcombustion CO₂ capture method but is limited by high costs and energy use.^134,135 Membrane technologies are generally viewed to be more energy-efficient and scalable. To selectively separate CO₂, a range of polymer-based membranes, such as polysulfone, poly(vinyl alcohol), polyacrylamide, and polyvinylamine, have been widely utilized. However, many of these membranes face a trade-off between selectivity and permeability, leading to an ongoing search for alternative materials that can provide more efficient membrane performance.¹³⁶ ILs have drawn considerable interest for CO₂ capture due to their distinct properties, such as high thermal stability, low volatility, and the ability to tailor their structure through different cation and anion choices, resulting in excellent CO₂ solubility and selectivity. Pure ILs offer limited surface area and low CO₂ capture capacity, and their high viscosity, costly production, uncertain toxicity, and potential environmental impacts further limit their industrial applications.^137,138 Consequently, recent research has increasingly shifted toward incorporating ILs into membrane processes to enhance their performance and overcome these limitations. These include supported IL membranes (SILMs), IL composite polymer membranes (ILPMs), IL composite mixed matrix membranes (ILMMMs), poly(IL) membranes (PILMs), IL gel membranes (ILGMs), and IL membrane contactors (ILMCs). Such approaches enhance CO₂ capture efficiency by minimizing the required amount of active phase for specific processes while also facilitating the recovery and reusability of ILs.

SILMs represent one of the most popular membrane-based approaches for CO₂ capture and consist of a porous support material that traps an IL within its pores, allowing for greater interaction with CO₂. During operation, the membrane is compressed between two cells: one containing the CO₂ feed phase and the other filled with a receiving or stripping agent. A recent study by Mulk et al. addressed the challenges of high energy requirements for solvent regeneration and its toxicity in industrial CO₂ capture by utilizing the tributyl-tetradecyl-phosphonium chloride ([P₄₄₄₁₄⁺][Cl^–]) IL, as a solvent.¹³⁹ This IL was chosen for its long alkyl chain substituent, which enhances CO₂ solubility in postcombustion capture. Of the three hydrophobic supports tested, the polytetrafluoroethylene-supported membrane performed the best, showing high CO₂ permeability and selectivity over N₂ without IL leaching. Additionally, its durability suggests it could be effective for long-term CO₂ capture from large volume flue (exhaust) gases, making it a promising alternative to traditional amine-based solvents. Zhang et al. designed and prepared SILMs containing ILs with a series of imidazolium cations paired with phenolated anions that have dual-site interaction centers to isolate CO₂ from N₂.¹⁴⁰ 1,3-Dialkylimidazolium cations paired with basic anions have shown great potential as absorbents for CO₂ because of their ability to react with CO₂ to form carbene–CO₂ adducts. However, the strong interaction between carbene and CO₂ makes it challenging to release the absorbed CO₂.^141,142 By pairing imidazolium cations with phenolated anions, the CO₂ could be more easily desorbed due to the transfer of CO₂ from carbene to phenolated anion. Remarkably high CO₂ permeability and excellent selectivity for CO₂ over N₂ was also achieved. Although SILMs have been employed in various innovative applications for CO₂ capture, they do have some limitations. For instance, high pressure can lead to the leakage of the IL from the membrane’s supporting pores, which can compromise their long-term stability.

Ionic liquid mixed matrix membranes (ILMMM) represent an innovative approach to overcome limitations of SILMs. Mixed matrix membranes (MMMs) are produced by incorporating porous fillers like zeolites, graphene oxide, carbon nanotubes, metal oxides, and metal–organic frameworks (MOFs) into a polymer matrix. By combining the high selectivity of these fillers with the processability of polymers, MMMs offer enhanced performance in gas separations.¹⁴³ Habib et al. reported a study where a specific IL, 1-methyl-1-propyl pyrrolidinium dicyanamide ([C₃MPyr⁺][DCA^–]), was introduced as a filler in MMMs for the first time because of its high CO₂ solubility.¹⁴³ The membranes consisted of this IL with a MOF (MIL-101(Cr)), known for its large surface area and strong interactions with CO₂, that were blended with varying amounts of a polymer called Pebax. The prepared MMMs were very effective at separating CO₂ from nitrogen and methane. In a study by Chang et al., a MMM was created by first modifying covalent organic frameworks (COFs) with an imidazolium-based IL, 1-ethyl-3-methylimidazolium ([C₂MIm⁺]) [NTf₂^–], and then incorporating this modified COF into a polymer, known as PIM-1.¹⁴⁴ This modification increased the COF’s affinity for CO₂, reduced its pore size, and improved its compatibility with PIM-1. As a result, the developed membranes achieved significantly enhanced CO₂/N₂ separation, exceeding previous performance benchmarks.

Other membrane separation processes such as ILPMs, PILMs and ILGMs have also been used for CO₂ capture. Klepić et al. reported the preparation and testing of a stable ILPM made from PVA and the [C₂MIm⁺][DCA^–] IL to effectively separate CO₂ from H₂, taking advantage of PVA’s polar nature.¹⁴⁵ The membranes were prepared by mixing different amounts of IL into PVA. As the amount of IL increased, the membranes improved significantly in their ability to permeate CO₂ and H₂. Specifically, when the IL content exceeded 20 wt %, the membranes exhibited a phenomenon known as “reverse selectivity”, where CO₂ permeates faster than H₂. Conversely, at lower concentrations of IL, the membranes allowed H₂ to permeate more easily. This versatility in gas separation under different conditions opens new possibilities for practical applications. Zhang et al. reported the design and synthesis of novel PILs containing amino functional groups, which were then used to create composite membranes by casting them on polysulfone membranes.¹⁴⁶ This approach resulted in highly CO₂ selective membranes with excellent CO₂/N₂ separation performance. Yu et al. presented a simple and eco-friendly method to prepare double-network (DN) ion gel membranes with excellent mechanical properties and CO₂/N₂ separation performance.¹⁴⁷ Using a “one-pot” method, the researchers combined two networks with an IL and prepared the DN membrane with heat and UV light. The design boosted both the strength and CO₂ separation by adjusting the network density and IL content, surpassing previous performance standards for CO₂/N₂ separations. Numerous studies have focused on advancing CO₂ capture and separation technologies. Within this review, several unique IL-based approaches have been highlighted, but for further in-depth information on the topic, reviews by Solangi et al.¹⁴⁸ and Yan et al.¹³⁸ are highly recommended.

Water Contaminants and Environmental Pollutants

Membrane separation processes extend well beyond CO₂ capture and offer solutions in diverse fields. In a recent study, Imdad et al. developed a novel approach by transforming polyethylene terephthalate (PET) bottles into polymeric membranes, which served as supports for the Aliquat 336 IL in fabricating a SILM.¹²⁸ These PET-derived SILMs were then applied to efficiently remove hexavalent chromium (Cr (VI)), a hazardous pollutant, from contaminated water. This method addresses both plastic waste management and water pollution simultaneously. A study by Khalid et al. presented an innovative approach where an imidazolium-based IL, functionalized with biocompatible hydroxyapatite derived from fish scales, was incorporated into cellulose acetate to create three distinct IL membranes.¹²⁷ These membranes achieved efficient removal of toxic dyes from wastewater, eliminating 98% of the cationic dye crystal violet and 96% of the anionic dye congo red within 24 h, and also demonstrated strong antibacterial activity. This research offers practical lab protocols and industrial applications for sustainable environmental pollution reduction. Merlet et al. developed a separation process to selectively remove butanol from an acetone-butanol-ethanol solution using a tubular membrane and an IL for in situ extraction.¹⁴⁹ The objective was to ensure selectivity to prevent the extraction of acetone, ethanol, and water, thus eliminating the need for additional purification steps. The experiments demonstrated the successful separation of butanol from aqueous solutions, providing valuable insights for biofuel extraction by integrating membrane technologies with green solvents. Furthermore, the system has the potential for direct coupling to a fermenter to enhance efficiency.

Electroseparations

Capillary Electrophoresis (CE) and Electrokinetic Chromatography (EKC)

CE is a commonly used separation technique for biomolecules that relies on the electrophoretic mobility of the analytes and the electroosmotic flow (EOF) induced by an applied voltage and the surface charge of the capillary.¹⁵⁰ Electrophoretic mobility is dependent on the hydrodynamic radius of the analyte as well as its charge and is generally unique for each analyte. In CE, the EOF is the bulk flow of the background electrolyte (BGE) solution when a voltage is applied, and is dependent upon the charge of the inner capillary wall. When the capillary surface is negatively charged (as in the case of unmodified capillary) positive ions from the BGE form a double layer on the surface and the freely mobile, hydrated cations migrate toward the cathode when the voltage is applied, producing a bulk flow that carries analytes toward the detector. However, the direction of the EOF may be reversed by altering the capillary surface charge either through the use of permanent or dynamic capillary coatings.¹⁵¹ Suppressed or reversed EOF can have advantages of improved selectivity and sharper peaks depending on the analytes and experimental conditions.¹⁵² CE is known for its high separation efficiency, faster analysis times, and low sample volumes compared to HPLC, but has suffered from challenges due to internal temperature gradients and inconsistent surface charges.¹⁵³ However, improvements to CE methodologies have been achieved using ILs and have mainly centered around separations involving EKC.

Chiral Ionic Liquids

ILs in CE first gained attention when used as coatings to modify the capillary surface and control the EOF.^154,155 Research in this area dwindled over the last five years with greater focus on ILs as pseudostationary phases for EKC. In EKC, the BGE contains an additive to which analytes partition to or interact with, resulting in a more complex separation mechanism involving the electrophoretic mobility of the free analyte, the mobility of the analyte-additive complex, and binding constants/partitioning coefficients of the analytes. Common applications of EKC include enantioseparations achieved by adding chiral selectors, such as CDs to the BGE.¹⁵⁶ In this case, racemic mixtures can be separated based on the chirality of the enantiomer and its interactions with the CD chiral selector. EKC offers a significant advantage over traditional chromatographic methods as enantiomers with equal binding constants can still be separated based on the varying electrophoretic mobilities of the formed complex,¹⁵⁷ which is especially important for pharmaceutical compounds that are chiral and whose enantiomers have different biological or pharmacological activity.¹⁵⁸ Recently, a synergistic effect between CILs and CD has been observed¹⁵⁹ and are thought to result from specific ion-pairing interactions. Often no enantioselectivity is achieved by using the CIL alone, but significant improvements in selectivity and peak shape can be achieved by a combination of CIL and CD compared to the CD alone.³²

Zhang et al. explored the effect of CILs consisting of the tetramethylammonium ([N₁₁₁₁⁺]) cation and amino acid anions, l-glutamate [l-Glu^–], l-arginine [l-Arg^–], and l-proline [l-Pro^–], on the enantioseparation of model drug compounds (i.e., amlodipine, citalopram, nefopam, tryptophan, and sulconazole) compared to α-CD chiral selector under aqueous conditions.¹⁶⁰ Incorporation of the CIL was observed to decrease the EOF, affording more opportunities for analyte-CD interactions, but was determined to not be the main cause of the synergistic effect observed with CIL/α-CD systems. Additionally, higher resolution was achieved for all five model drugs when the CIL was included in the BGE compared to when [N₁₁₁₁–OH⁺] and [l-Arg^–] were included, suggesting that this effect is not a result of the individual CIL components alone. The inclusion complex formed between the enantiomers and the α-CD was instead thought to be stabilized by interactions of the [N₁₁₁₁⁺] cation with α-CD, thereby preventing interactions between the enantiomers and the surface of the α-CD macrocycle. In the work by Salido-Fortuna et al., the synergistic effect of additional amino acid CILs, [N₁₁₁₁⁺][l-Lys^–], [N₁₁₁₁⁺][l-Glu^–], tetrabutylammonium ([N₄₄₄₄⁺]) [l-Lys^–], and [N₄₄₄₄⁺][l-Glu^–], were explored along with ten different CD chiral selectors to improve the enantioseparation of seven drugs molecules (i.e., nadolol, metoprolol, terbutaline, duloxetine, verapamil, econazole, and sulconazole).¹⁶¹ The best chiral discrimination was realized with the 2-hydroxypropyl-β-CD (HP-β-CD) under acidic conditions resulting in discrimination of five of the seven racemic mixtures. When 5 mM of HP-β-CD was combined with 30 mM of the CIL, a 2-fold improvement in enantioresolution was achieved although better resolution was observed with the [N₄₄₄₄⁺] CILs than the [N₁₁₁₁⁺] CILs for the studied drug mixture. Migration times were also significantly increased with some even greater than 60 min. Ultimately, separations featuring higher selectivity allow for more efficient separations to be achieved since higher voltages can be employed without risking loss of resolution, though faster separations of ibrutinib enantiomers were separated in another study by employing 2-sulfated-γ-CD (S-γ-CD) in negative polarity mode.¹⁶²

Nonaqueous CE conditions, in which the BGE consists of an organic solvent instead of water, can sometimes be employed to improve the solubility of analytes, increase compatibility with MS detectors, reduce analysis times, or alter the selectivity of the separation.¹⁶³ In a study by Ren et al., tetraalkylammonium amino acid CILs were employed with β-CD to separate dansyl-amino acid enantiomers.¹⁶⁴ In this work, N-methylformamide was used as the organic solvent in the nonaqueous buffer solution to improve the solubility of β-CD and allowed for higher β-CD concentrations to be used. By employing a 100 mM β-CD concentration under nonaqueous conditions, six dansyl-amino acids could be separated with resolution between 1.29 and 1.84 compared to a β-CD concentration of 10 mM in aqueous conditions, in which the enantiomers could not be separated. For a synergistic system with the [N₁₁₁₁⁺][l-Arg^–] IL, significant improvements in resolution, peak shape, and separation efficiency were also noted. Through molecular docking simulations, association between [N₁₁₁₁⁺][l-Arg^–] and β-CD was shown to favor analyte binding, but the presence of the CIL appeared to strengthen inclusion complexation of the dansyl-amino acids. Slight enantioseparation of dansyl-amino acid enantiomers was also achieved for the first time using the [N₁₁₁₁⁺][l-Arg^–] IL as the sole chiral selector, suggesting that CILs can participate in enantiorecognition. Zhang et al. expanded the list of possible CILs to include tartaric acid with various cation combinations and demonstrated that the cation can affect enantiorecognition of ten amino alcohols.¹⁶⁵ The CIL was used in this case as the chiral selector in the aqueous–organic BGE consisting of 80–90% methanol. When [N₁₁₁₁⁺], [N₂₂₂₂⁺], and [N₄₄₄₄⁺] cations were used with a tartaric acid anion, resolution of amino alcohol enantiomers was improved compared to the tartaric acid chiral selector alone and dicationic CILs afforded even greater enantioseparations.¹⁶⁶ Compared to other cations (including imidazolium, pyridinium, phosphonium, pyrrolidinium, piperidinium, and cholinium), significant differences in selectivity, peak shape, and migration times were observed, attributed to better separations of larger cations with enantioresolution decreasing in the order of [C₄MPyr⁺]₂[l-TT^–], [C₄MPip⁺]₂[l-TT^–], and [C₄MIm⁺]₂[l-TT^–].¹⁶⁵ The mechanism influencing enantioselectivity was thought to be due to a steric hindrance effect, but additional studies are needed to confirm this and assess other possible contributing factors.

To further enhance enantioseparations, micellar electrokinetic chromatography (MEKC) has been employed and has a similar separation mechanism to EKC, but also relies on the partitioning of the analyte-chiral selector complex to the micellar phase. In MEKC, surfactants are present in the BGE above the critical micelle concentration and form micelles in the buffer solution that act as a pseudostationary phase. Feng et al. used the 1-butyl-3-methylimidazolium dodecyl sulfate ([C₄MIm⁺][C₁₂SO₄^–]) IL with a clindamycin phosphate chiral selector to separate six racemic drug molecules.¹⁶⁷ Compared to the MEKC separation using SDS, the enantioselectivity of the IL-based method was increased and resulted in significantly better peak shapes.

Monolithic IL Columns

Monolithic columns containing ILs have shown promise in EKC methods.¹⁶⁸ In general, monolithic columns consist of a highly porous, interconnected polymer and have become of interest in chromatographic separations due to their high permeability, greater accessible surface area, and rapid analyte mass transfer.¹⁶⁹ Research studies have explored modifications to the macropore structure of monolithic columns that control these properties.¹⁷⁰ Huang et al. developed a novel polyhedral oligomeric siloxane-based zwitterionic monolithic capillary columns featuring retention-independent plate heights and found that the [C₆MIm⁺][BF₄^–] IL porogen played an important role in controlling the mesopore structure¹⁷¹ responsible for analyte retention and column selectivity.^172,173 While the mesoporous structure (2–50 nm) can be detrimental to small-molecule separations in HPLC due to poor stationary phase mass transfer, this is not the case in electrokinetic separations due to the presence of an EOF facilitating highly efficient separations.¹⁷⁰ Huang et al. applied this column for hydrophilic interaction capillary electrokinetic chromatography (HI-EKC) and explored the separation mechanism using benzoic acids, nucleosides, nucleobases, and glycopeptide antibiotics.¹⁷¹ Mixed-mode retention mechanisms were confirmed in both HILIC and RP mode due to additional ion-exchange and electrostatic interactions. A strong anodic EOF was also achieved, especially under lower pH conditions, allowing for faster separations of negatively charged analytes with narrower peaks and suitable run-to-run and column-to-column repeatability under HI-EKC conditions.

Monolithic porous layer open tubular (PLOT) columns for EKC have also become of interest as higher EOF and lower back pressure are achieved and allow for higher loading capacities and high separation efficiencies. In a study by Zhou et al., the 1-allyl-methylimidazolium chloride IL ([AMIm⁺][Cl^–]) monomer was copolymerized with styrene and ethylene dimethacrylate cross-linker to form a monolithic PLOT column for EKC-MS.¹⁷⁴ A strong anodic EOF was achieved between pH 2–8 and could be modulated by controlling the amount of IL in the polymer. By modifying the EOF through the IL concentration, separation of amino acids was accomplished in which the enhanced EOF superseded the electromigration rate of the amino acids. When applied for the analysis of neutral analytes, separation of various model analytes was achieved through hydrophobic and π–π interactions, and when applied for the separation of parabens, baseline resolution of methyl, ethyl, and propyl paraben was achieved within 12 min that could not be achieved using CE alone.

Other Electromigration Applications

While CE and CE-related methods are the most employed electromigration techniques, the term at its core refers to the movement of an ion or charged particle under the influence of an electric field. Due to their conductive properties, ILs have been employed in various techniques in which electric fields are applied, including ABS, membranes, and microfluidic devices.^175−177 In the case of ABS, the applied voltage aided to induce phase separation between the kosmotropic salt phase and the IL-containing phase,¹⁷⁵ allowing for more rapid extractions. In free-flowing IL membranes, the permeability and selectivity for CO₂ capture were improved and controlled by applying an electric field, resulting in denser packing of the migrating IL molecules near one side of the membrane.

Microfluidic Devices

Microfluidic devices are designed for handling small liquid sample volumes using micrometer sized channels and chambers, often for the analysis of cells and biomolecules.¹⁷⁸ Microfluidic devices can be integrated with electrodes for the capture of charged particles/molecules within the chambers for subsequent analysis.¹⁷⁹ One such application designed a device featuring microchip capillary electrophoresis for the separation and online detection of flavins.¹⁷⁷ In this method, the 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([C₄MIm⁺][NTf₂^–]) IL was used as a hydrophobic, low viscosity liquid to compartmentalize the sample and minimize changes in sample volume. Compartmentalization occurred electrokinetically, and the IL plugs were determined to be optimal at 1–10x the capillary diameter for repeatable sample introduction. Once the IL migrated into the microchip, surface adsorption prevented the IL from traveling further, resulting in the sample breaking through the IL plug due to the maintained EOF. Results showed a 6.3-fold improvement in peak intensity compared to conventional electrokinetic injections. However, higher relative standard deviation values were obtained for this method and may be due to residual IL effects on the EOF.

ILs have also been used in microfluidic devices to seal chambers after capturing analyte(s) of interest.¹⁸⁰ Banovetz et al. utilized dielectrophoresis to capture tumor cells (MDA-MB-231) into microfluidic chambers for single cell analysis through the use of the 1-decyl-3-methylimidazolium ([C₁₀MIm⁺]) [NTf₂^–] IL.¹⁸¹ Due to the conductive nature of the IL, cell lysis was possible for a subsequent enzymatic assay to assess β-galactosidase activity, a prominent biomarker used for early detection of breast cancer. The method was able to demonstrate a high degree of heterogeneity in the expression of β-galactosidase among 258 individual cancer cells, suggesting that different subpopulations of tumor cells may require different types of treatments.

Electric Field-Flow Fractionation (EI-FFF)

Liu et al. using an IL/mesoporous silica coated electrodes for the separation of microparticles by EI-FFF.¹⁸² In traditional FFF, a liquid solution is passed through a narrow channel, and separation occurs based on the particles’ mobilities under an applied field (electric, magnetic, gravitational, thermal, hydraulic, or centrifugal).¹⁸³ These fields are often applied perpendicular to the laminar flow of the sample to cause the particles to accumulate near the wall of the channel, resulting in a concentration gradient based on translational diffusion coefficients. Since the flow velocity near the wall is slower than near the center of the channel, separation based on analyte diffusion can be achieved as smaller, more diffuse particles are more likely to migrate away from the wall into the higher velocity flow region. In EI-FFF, an applied voltage results in the migration of the charges particles to the accumulation wall and separation depends on the diffusivity of the particle as well as its electrophoretic mobility. Recently, cyclical EI-FFF (CyEI-FFF) was developed and utilizes stepwise switching of the electric field’s polarization to produce higher separation efficiencies.¹⁸⁴ Liu et al. employed a stainless steel channel coated with the IL/mesoporous silica material acting as an electrode and containing a second platinum electrode coated with the same material.¹⁸² The methyltri-N-octylammonium bis(trifluoromethanesulfonyl)imide ([N₁₁₁₈⁺][NTf₂^–]) IL was chosen for its wide electrochemical window and resulted in the formation of annular channels in which higher laminar flow velocities existed in the center of the two electrodes. The particles oscillated within the annular channel between the two electrodes as the alternating current was applied and separation occurred based on the particles size, charge, and morphology due to influences of electrophoretic force, electrostatic force, and viscous force. Two operation modes were examined and were applied for the separation of polystyrene particles of various sizes. The separation mechanism for this method is shown in Figure 5 with separation based on particle size being achieved with resolution values greater than 1.22.

Figure 5.

(A) OTR-CyEIFFF instrument used for online separation of polystyrene particles. (B) Schematic depicting the microchannel column with inner and outer IL/mesoporous silica modified platinum and stainless-steel electrodes and (C) the cross-sectional view. (D) Diagram showing the influence of the electric field, electrophoretic forces (F_EP), electrostatic forces (f′), and viscous drag forces (f) on positively and negatively charged particles. (E) Separation mechanism for the OTR-CyEIFFF method is shown, including the formation of an annular array, in which smaller sized particles travel faster and closer to the platinum wire electrode under laminar flow. Reproduced from Liu, L.; Yang, C.; Liu, C.; Piao, J.; Kaw, H.Y.; Cui, J.; Shang, H.; Ri, H.C..; Kim, J.; Jin, M.; Li, D. Open-tubular Radially Cyclical Electric Field-flow Fractionation (OTR-CyElFFF): An Online Concentric Distribution Strategy for Simultaneous Separation of Microparticles. Lab Chip 2020, 20, 3535–3543 (ref (182)). Copyright 2020 Royal Society of Chemistry.

Electrochemical Sensing

Among the various physical properties of ILs discussed so far, their high conductivity, excellent electrochemical stability, and wide electrochemical window make them well-suited for numerous electrochemical applications.^185−187 As such, ILs have been utilized in the development of ion-selective sensors, reference electrodes, voltametric sensors, gas sensors, and biosensors.^188−190 This section focuses on the application of ILs in electrochemical sensors with an emphasis on their incorporation into reference electrodes and ion-selective electrodes.

A reference electrode provides a stable and known reference potential that enables measuring potential changes of the working electrode.¹⁹¹ The Ag/AgCl electrode is one of the most widely used reference electrodes in electrochemistry due to its stability and simplicity. The reference electrode contacts the sample solution through a salt bridge filled with an internal electrolyte solution consisting of nearly equal transference numbers for cations and anions. Typically, concentrated KCl is used to minimize the liquid junction potential at the interface with the sample and provides a stable, sample-independent reference potential. However, this type of electrode and required set up has certain drawbacks, such as variations in KCl concentration, the need for regular maintenance, dependence of the liquid junction potential on the junction material type, and challenges with miniaturization.¹⁹² An approach to address these limitations is the development of an IL-doped reference electrode membrane, which eliminates the need for a conventional aqueous salt bridge.¹⁹³ The interface between the IL and the aqueous sample solution creates a consistent interfacial potential that remains unaffected by the concentration and ionic strength of the aqueous phase. Herein, we describe a few recent applications employing ILs in the manufacturing of reference electrodes.

Kuczak et al. examined basic physicochemical properties of various ILs on the performance of polymeric membrane reference electrodes.¹⁹⁴ Seven ILs containing cations featuring different hydrophobicity, including trihexyltetradecylphosphonium, 1-hexyl-1-methylpiperidinium, 1-pentyl-1-methylpiperidinium, 1-benzyl-3-methylimidazolium ([BzMIm]), 1-(2-methoxyethyl)-3-methylimidazolium, 1-(2-hydroxyethyl)-3-methylimidazolium ([C₂OHMIm]), and triethylsulfonium cations with [NTf₂^–] anions were incorporated into polyurethane/o-nitrophenyl octyl ether (PU/o-NPOE) (1:2) membranes. The stability of the measured potential for the membrane-based reference electrodes was then tested under different conditions and indicated that hydrophobicity differences between IL cations and anions contributed to a strong potentiometric response for lipophilic ions. The study also evaluated the potential stability of IL-based polymeric membrane reference electrodes in aqueous solutions containing different concentrations of KCl and NaCl as well as their long-term stability in a solution with 125 mM NaCl and 5 mM KCl. Although all the IL-based electrodes provided better stability in comparison with a blank membrane, electrodes containing membranes of [BzMIm⁺][NTf₂^–] and [C₂OHMIm⁺][NTf₂^–] provided the best stability. By testing membranes containing 1%, 2%, and 3% IL, the study showed that varying IL concentrations within the polymer matrix did not significantly affect the electrode capacity for maintaining stable potential in different solutions. Since leaching of plasticizer from polymeric membrane electrodes can be problematic membranes containing only PU and IL were tested and it was found that the membranes without plasticizer can be applicable in the development of reference electrodes.

Chen and co-workers employed a biocompatible reference electrode membrane doped with an IL.¹⁹⁵ Among the seven silicone materials tested, poly(3,3,3-trifluoropropylmethylsiloxane) (referred to as fluorosilicone 1) doped with the [C₈MIm⁺][NTf₂^–] IL was the only membrane matrix that performed effectively as a reference electrode and provided a stable potential, as shown in Figure 6a. The use of silicone as a membrane matrix not only eliminated the need for plasticizers but is also biocompatible. To investigate the effects of IL chemical structure and hydrophobicity on the stability and potential drift of the reference electrode, fluorosilicone 1-based membranes doped with four different ILs containing cations of varying hydrophobicity and paired with the [NTf₂^–] anion were tested. The results showed that membranes doped with [C₈MIm⁺][NTf₂^–], [C₁₀MIm⁺][NTf₂^–], and [C₁₂MIm⁺][NTf₂^–] ILs exhibited minimal potential changes across concentrations of KCl, as shown in Figure 6b. Finally, long-term stability tests indicated that fluorosilicone 1 reference electrodes doped with the [C₈MIm⁺][NTf₂^–] IL exhibited minimal potential drift, measuring 20 μV h^–1 in artificial blood and 112 μV h^–1 in serum over 8 and 5.8 days, respectively. Figure 6c represents the potential stability over time of the optimized electrode.¹⁹⁵

Figure 6.

(a) Represents the effect 1.0–16 mM KCl on the potential of reference electrodes incorporating [C₈MIM⁺][NTf₂^–]-doped silicone membranes compared to a free-flow double junction reference electrode. (b) Effect of 1.0–16 mM KCl on the potential of Fluorosilicone 1 reference electrodes doped with various ILs including [C₈MIM⁺][NTf₂^–], [C₁₀MIM⁺][NTf₂^–], [C₁₂MIM⁺][NTf₂^–], and [N₁₄₄₄⁺][NTf₂^–] ILs. (c) Shows long-term EMF measurement results conducted using a solid-contact reference electrode prepared with the [C₈MIM⁺][NTf₂^–] IL immersed in artificial blood electrolyte solutions. Reproduced from Chen, X.V.; Stein, A.; Bühlmann, P. Reference Electrodes Based on Ionic Liquid-Doped Reference Membranes with Biocompatible Silicone Matrixes. ACS Sens. 2020, 5, 1717–1725 (ref (195)). Copyright 2020 American Chemical Society.

The development of reference electrodes incorporating silicone membranes doped with ILs has extended beyond the aforementioned study due to the promising results in miniaturization and biocompatibility of these electrodes. Dong et al. introduced an innovative fabrication method that uses PDMS membranes doped with ILs, combined with colloid-imprinted mesoporous carbon (CIM) as a solid contact layer.¹⁹⁶ The study identified that the catalyzed polymerization of the silicone causes the hydroxyl-terminated polydimethylsiloxane oligomers to cross-link, forming large structures that cannot penetrate the pores of the CIM carbon. Consequently, after the solvent evaporates, the pores of the CIM carbon are filled exclusively with the IL. The depletion of ILs in the reference membrane increases the membrane’s resistance and compromises overall electrode function. To address this, two strategies were investigated including presaturation of mesoporous carbon with IL before adding the silicone-based solution and increasing the IL concentration to counter the sequestration effect. These approaches allowed for the creation of stable reference electrodes with enhanced long-term performance and potential stability, especially for applications in biocompatible and miniaturized sensors.

Potentiometry with Ion Selective Electrodes

ILs have also been investigated in potentiometry applications with ion-selective electrode (ISEs). ISEs enable detection of ionic chemical species with good selectivity and low detection limits. Solid-state ISEs are often designed with an intermediate layer to facilitate the connection between electronic conductors and the ion-selective membrane. Due to their ionic nature and ability to polymerize ILs have proven to be interesting materials for preparing ISE membranes with enhanced stability and ion transport. The most recent applications of ILs in solid-state ISEs are presented in this section

Wardak et al. reported a lead-sensing method using solid-state ISEs in which the polymeric membrane was modified with a nanocomposite of carbon nanofibers and an IL.¹⁹⁷ While the use of the [C₆MIm⁺][PF₆^–] IL as a solvent for lead preconcentration and as a lipophilic component in lead-selective membranes has been previously demonstrated,¹⁹⁸ this study leveraged π-electrons in the imidazolium ring to interact with the π-surface of carbon nanofibers. This interaction was found to facilitate the formation of an electrochemical stabilization and steric nanocomposite, which was then used to modify the polymer membrane in a solid-contact lead ion-selective electrode. For membrane preparation, the nanocomposite was combined with low-molecular-weight poly(vinyl chloride) (PVC), bis(1-butylpentyl) adipate (BBPA), 2-nitrophenyl octyl ether (NPOE), and a lead ionophore, with the proportion of nanocomposite tested ranging from 0–9% by weight. The modification enhanced sensitivity, broadened the measurement range, increased the membrane’s hydrophobicity, and improved selectivity coefficients. Additionally, the modified membrane reduced leaching of active ingredients, thereby enhancing the electrode’s long-term stability.

In another study, Wardak et al. employed a nanocomposite of multiwalled carbon nanotube (MWCNTs) and the [C₄MIm⁺][PF₆^–] IL for copper ISEs.¹⁹⁹ MWCNTs were found to facilitate charge transfer between the internal electrode and the membrane, while the IL reduced resistance and improved ion transport. The copper ISE demonstrated enhanced selectivity, lower detection limits, and a broader measurement range compared to its unmodified counterpart. Similarly, Pietrzak et al. developed a solid-state nitrate ISE using a nanocomposite of MWCNTs and the trihexyltetradecylphosphonium chloride ([P₆₆₆₁₄⁺][Cl^–]) IL to examine the effects of MWCNT dimensions (length and diameter) on electrode performance.²⁰⁰ The nanocomposite-based electrodes showed a broader linear range and better calibration slope compared to nonmodified electrodes and the results indicated that the porosity, surface area, and homogeneity of MWCNTs influenced the electrode’s response, with higher porosity and structural homogeneity positively impacting electrode performance.

Inorganic materials can also be used for modifying SC-ISEs, based on redox capacitance or double-layer capacitance transduction mechanisms. Zeng et al. proposed using Ag@AgCl/1-tetradecyl-3-methylimidazolium chloride (Ag@AgCl/[C₁₄MIm⁺][Cl^–]) as an inorganic redox buffer in solid-contact Ca²⁺-selective electrodes via a redox capacitance mechanism.²⁰¹ The [C₁₄MIm⁺][Cl^–] IL provided a stable source of chloride ions and enhanced the conductivity properties of the buffer. The electrode’s performance was evaluated in terms of stability, response time, impedance, and resistance to interference from light, oxygen, and carbon dioxide. The Ag@AgCl/[C₁₄MIm⁺][Cl^–] buffer exhibited high redox capacitance, offering stable potentials and rapid response times. Finally, the electrode’s performance was successfully demonstrated in measuring calcium ions in seawater samples.

Mass Spectrometry

In MS, ILs are generally utilized to increase signal intensity. The most common way ILs are used to achieve signal enhancement are as matrices for matrix-assisted laser desorption ionization (MALDI)-MS and as complexing agents for electrospray ionization (ESI)-MS. This section discusses recent literature on ILs for MS covering new applications, sample types, and interface designs.

Dicationic Liquids for Electrospray Ionization

In ESI, ILs can be introduced during the ionization process to act as a complexing agent; this is specifically done with DILs. When an anionic analyte undergoes complexation with a DIL, the resulting species carries a positive charge, allowing the analysis to be carried out in positive ion mode (i.e., measuring positive analytes).³¹ This process is called polarity switching or a charge inversion reaction and provides signal enhancement due to the minimized effect of the corona discharge in positive versus negative ion mode.

A common application of polarity switching is for the measurement of per and polyfluorinated compounds (PFCs). Li et al. described the use of the 1,1-bis(3-methylimidazolium-1-ly)butylene difluoride ([(MIm)₂C₄²⁺] 2[F^–]) IL complexed with PFCs for ionization and signal enhancement.²⁰² They measured a mixture of ten PFCs first using supramolecular solvent based extraction and ultrahigh-performance supercritical fluid chromatography. A makeup liquid comprised of [(MIm)₂C₄²⁺] 2[F^-] and acetonitrile was then mixed with the effluent post column. The mixture was ionized via ESI prior to MS analysis using a triple quadrupole mass analyzer. Results showed that using the DIL complexing agent in positive mode provided more sensitive detection of PFCs with one to 2 orders of magnitude signal enhancement compared to performing measurements in negative ion mode with no complexing agent. Li et al. used DIL-based charge inversion and a miniaturized MS to measure PFCs in biological matrices.²⁰³ This work also used an in capillary dispersive magnetic SPME method based on aptamer-functionalized polymer-modified magnetic nanoparticles that allowed for analyte enrichment prior to MS analysis. The same capillary system used for extraction also functioned as a nano ESI emitter. In this system, DILs were introduced inside of the capillary extraction system in a plug near the capillary tip. Ten different DILs with varying side groups and linkage chains were tested to investigate their binding affinity for PFCs; the dicationic IL structure was found to be crucial for the charge inversion reaction. Their results showed that the [(MIm)₂C₄²⁺] 2[F^–] IL gave the highest signal intensity with 6.3–28.9 times the signal intensity in positive ion mode using DIL-based charge inversion. Furthermore, the method examined detection of PFCs in human blood. DIL based charge inversion has also been used for the measurement of haloacetic acids with ESI-MS.²⁰⁴

Charge inversion reactions based on DILs can also be used with ionization techniques outside of ESI. Guo et al. used the technique for matrix-assisted ionization in the analysis of PFCs within environmental water samples.²⁰⁵ In this technique, the sample followed by the DIL matrix ([(MIm)₂C₄²⁺] 2[F^–] and acetonitrile) were spotted onto paper and placed near the MS inlet. The intrinsic vacuum from the inlet led to sublimation of the matrix and analyte while at the same time forming positively charged complexes. From sampling to results, the approach took only 1 min and provided signal enhancements up to 2 orders of magnitude in positive ion mode. PFCs were also measured using a DIL for ionization enhancement with easy ambient sonic-spray ionization MS.²⁰⁶

Xi and Muddiman utilized DIL charge inversion reactions post MALDI to expand metabolomic coverage for mass spectrometry imaging (MSI).²⁰⁷ In this work, the 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride ([(BPyr)₂C₅²⁺] 2[F^–]) IL was introduced after MALDI using an ESI interface. This technique permitted measurement of 73 negative analytes complexed with the DIL along with 164 positively charged analytes, providing a 44% increase in molecular coverage. Figure 7 shows mass spectra from this study comparing the results of positive ion mode with and without the use of DIL for charge inversion, highlighting the increased molecular coverage. This study also demonstrated the applicability of their technique for MSI of a hen ovary, showing a rapid and effective way to detect positive and negative analytes of interest in one measurement.

Figure 7.

Mass spectra showing positive ion mode with [(BPyr)₂C₅²⁺] 2[F^–] complexation (purple, top) and positive ion mode (orange, bottom). (A) Not reproduced. (B,C) Zoomed in sections from (A). Labeled m/z values correspond to DIL complexes with analytes from rat liver sections. Reproduced from Xi, Y.; Muddiman, DC. Enhancing Metabolomic Coverage in Positive Ionization Mode Using Dicationic Reagents by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization. Metabolites 2021, 11, 810 (ref (207)). Copyright 2021 by authors licensed to MDPI.

Polymeric Ionic Liquids for Electrospray Ionization

Hu at al. demonstrated the use of an IL-based organic polymer for online coupling of in-tube SPME with MS using the 1-allyl-methylimidazolium chloride ([AlMIm⁺][Cl^–]) IL.²⁴ A custom MS/extraction interface using a fused silica capillary for the in-tube SPME and as the ESI emitter tip was created followed by mounting of the coated capillary onto a translational stage. When moved away from the MS inlet, the high voltage connection was broken, and the sample was loaded into the capillary for extraction. The interface was then moved toward the MS inlet to enable the connection of the high potential for ESI. Elution solvents were introduced, and analytes were then sprayed out from the capillary tip for ionization. The IL polymer provided a highly porous structure that not only provided high extraction efficiency but also enhanced the ionization efficiency of the ESI process. The polymer’s monolithic structure created microchannels at the tip of the ESI capillary, which provides multiple spray orifices for ESI creating smaller liquid droplets that were easier to evaporate and convert to gas phase ions. This study found that ionization efficiency was enhanced at optimized flow rates for the SPME capillary compared to more traditional ESI capillary geometries. Validation studies were conducted on nonsteroidal anti-inflammatory drug compounds producing high linearity from 0.1 to 200 ng mL^–1 of ketoprofen and flurbiprofen, LODs from 0.02 to 0.03 ng mL^–1, and limits of quantitation (LOQs) from 0.08 to 0.10 ng mL^–1. The adaptability of this technique shows promise for applications toward numerous analytes by simply changing the IL polymer.

Ionic Liquid Matrices for Matrix-Assisted Laser Desorption/Ionization

MALDI-MS is an MSI technique that uses a UV laser to desorb small amounts of sample and matrix. MALDI-MS allows for the detection of intact large molecules making it ideal for metabolomics, proteomics, and lipidomics. The ionization mechanism in MALDI is complicated and not widely understood.²⁰⁸ Primary ionization occurs via proton transfer from an acidic matrix to the analyte molecules, and secondary ions form due to thermal ionization and continuing reactions in the desorption plume. The uncertainty and complexity in the MALDI process can lead to an unrepresentative sample to be measured. In combination with low mass range ions derived from the matrix and inconsistent shot-to-shot reproducibility due to heterogeneity in the film, MALDI-MS cannot be used for quantitative measurements. Ionic liquid matrices (ILMs) are promising alternatives to overcome both obstacles when used for MALDI-MS. The studies discussed in this section focus on using ILMs to better understand the ionization process, to overcome heterogeneity in the matrix coating, and enable the detection of low m/z molecules.

Kobylis et al. performed an extensive study examining the effect of ionic character/iconicity of protic ILs on the ionization process of MALDI.²⁰⁹ The iconicity of each protic IL was characterized using Walden plots, density functional theory studies, nuclear magnetic resonance spectroscopy, and thermal gravimetric analysis coupled to infrared spectroscopy prior to MALDI-MS analysis in negative and positive ion modes. The study found that the ionization process of MALDI is independent of the ionicity of the ILM and further studies are required to understand the ionization mechanism of ILMs in MALDI-MS.

Lin et al. explored the use of ILMs for the quantitative analysis of adenosine nucleotide ratios, which can provide valuable information on energy transfer in metabolism.²¹⁰ In this study, the fragmentation yield, UV absorption, shot-to-shot reproducibility, signal intensity, and adenosine nucleotide ratios were examined with a traditional MALDI matrix and four ILMs. The 2,5-dihydroxybenzoic acid pyridine (DHBP) ILM was found to be the most effective due to lower UV absorption of the 355 nm laser and provided a milder ionization process which reduced fragmentation of the phosphate group, thereby providing higher signal intensities of the analyte ions. Additionally, the DHBP ILM also provided better shot-to-shot reproducibility which allowed for quantitative analysis of the adenosine nucleotides.

Mernie et al. explored the use of ILs for the measurement of oligosaccharides.²¹¹ To overcome significant challenges of MALDI-MS in measuring complex mixtures of carbohydrates related to their low ionization efficiency and inability of the mass analyzer to separate oligosaccharides with high structural similarity, ILs were used to stabilize the DHB metallic-nanoparticle (MNP) conjugated matrix that was coated on thin layer chromatography plates. The addition of ILs led to a more homogeneous DHB-MNP coating, resulting in more efficient ionization and fragmentation capabilities with up to a 28-fold increase in the intensity of precursor and fragment ions. The analysis scheme was applied to human milk oligosaccharide profiling and 25 oligosaccharides were detected using this one-step approach.

ILMs have also been utilized to measure high molecular weight molecules. A study by Yamazaki et al. used 3-aminoquinoline-based ILMs to measure cyclodextrin-based polyrotaxane (CD-PR) molecules up to 700 kDa using a MALDI time-of-flight MS with a high mass detector.²¹² The ILM differentiated double-stranded and aggregated forms of the CD-PR. Additionally, the ILM preferentially created singly charged ions that are desired at higher mass ranges.

Spectroscopy and Optical Sensors

Recent studies have highlighted the remarkable potential of ILs in optical sensing applications. The design and development of sensors necessitates the careful consideration of factors such as selectivity, sensitivity, response time, the ability to differentiate between various analytes, and consistency in response.²¹³ In addition to meeting the aforementioned sensor characterization requirements, IL-based sensors offer unique advantages including vast structural tunability and synthetic versatility, enabling the design of probes with specific anions and cations tailored to sensing needs.^214−216 Furthermore, the high vapor pressure of ILs minimizes the effects of concentration changes due to evaporation, thereby enhancing detection stability. As a result, IL-based sensors have attracted significant attention, leading to numerous research efforts in this area. In this section, we will focus on the design of fluorescence and colorimetric sensors as well as their application in surface-enhanced Raman spectroscopy (SERS) in recent years.

Ionic Liquids in Fluorescence and Colorimetric Detection

Fluorescence detection is considered one of the most sensitive and selective optical sensing methods. ILs have contributed to the design of such sensors through various mechanisms, including the incorporation of fluorophores into the IL chemical structure, facilitating interactions between ILs and analytes, modifying the fluorescence properties of ILs upon analyte interaction, and enabling charge transfer processes.²¹⁷ These interactions have driven the development of several IL-based sensors capable of detecting a wide range of targets, including gases, ions, organic compounds, inorganic compounds, and biological molecules.

Fluorescent Ionic Liquid (FIL) Probes

FILs have attracted significant attention for their unique optical properties, with various types being synthesized for applications in chemical sensing. For example, benzobis(imidazolium) ILs, salicylate-bearing FILs, quinolizinium-based FILs, metal-ion-coordinated FILs, and polyamidoamine dendrimer-derived FILs are among some of the FILs introduced in recent years.²¹⁸ Building on this progress, Gan et al. designed and developed the [P₆₆₆₁₄⁺][HQS^–] FIL for the sensitive and selective detection of Al³⁺.²¹⁸ This innovative probe integrated 8-hydroxyquinoline-5-sulfonic acid [HQS^–] as the anion, thereby leveraging its fluorophore and binding moiety properties. Compared to the traditional HQS probe, the [P₆₆₆₁₄⁺][HQS^–] FIL exhibited enhanced sensitivity, selectivity, and binding strength and achieved a LOD of 5.5 × 10^–8 M, which is significantly lower than HQS (2.4 × 10^–7 M), and a binding constant of 7 × 10⁴, also much higher than HQS (6 × 10³). These improvements were attributed to modified charge distribution in the FIL, which creates a stronger interaction with Al³⁺ and stabilizes the probe-metal complex. The detection mechanism relied on enhancing the fluorescence characteristics, wherein the fluorescence intensity of the probe increaed upon interaction with the analyte, enabling quantitative measurement. Additionally, the [P₆₆₆₁₄⁺][HQS^–] FIL demonstrated faster response times and superior fluorescence enhancement at low Al³⁺ concentrations, making it highly effective for practical applications in aqueous environments.

FIL probes have also demonstrated exceptional potential in dual-channel sensing, incorporating both colorimetric and fluorescence detection, leading to significant advancements in the rapid and on-site determination of environmental pollutants. Che et al. introduced the FIL probe containing the [P₆₆₆₁₄⁺] cation with a fluorescein-based anion [Fluo^–] for the sensitive and selective detection of paraquat, a widely used herbicide with significant toxicity.²¹⁹ The anion-functionalized probe enhanced analyte enrichment through strong electrostatic attractions to cationic the paraquat, improving both sensitivity and selectivity by excluding uncharged pesticides. Additionally, this design reduced the distance between the probe and analyte by extracting the analytes from the bulk, enabling faster response times. In fluorescence detection, the probe-paraquat interaction resulted in fluorescence quenching, yielding a LOQ of 64.0 nM. In colorimetric detection, the solution’s color transitioned from green to orange and finally to pink with increasing paraquat concentration, achieving an LOQ of 100 nM. This dual-channel capability enabled real-time visual monitoring of paraquat.

Another example highlighting the development of real-time visual monitoring using FIL probes was reported by Che et al. focusing on monitoring trace amounts of mercury(II) in environmental and biological samples.²²⁰ A different FIL probe was constructed using a physical mixture of [P₆₆₆₁₄⁺] [RDB^–] (rhodamine B) and 7-hydroxycoumarin, while incorporation of rhodamine B into the IL chemical structure enhanced the electronegativity of the oxygen atom on its phenoloxy group, thereby increasing its affinity for mercury(II) and subsequently improving the selectivity of the probe. The addition of 7-hydroxycoumarin expanded the colorimetric range of the probe in the presence of Hg²⁺. While the [P₆₆₆₁₄⁺][RDB^–] IL probe alone exhibited a slight color change from yellow to orange, the combination of [P₆₆₆₁₄⁺][RDB^–] and 7-hydroxycoumarin provided for a distinct color transition from light white to opera pink upon interaction with mercury(II), significantly enhancing the performance of the colorimetric channel. Furthermore, the probe’s potential for practical applications was demonstrated through its integration into a paper-based sensor, enabling on-site visual monitoring of Hg²⁺.

Biocompatible FIL probes have further expanded their applications. A curcumin (Cur)-based FIL probe, [N₃₃₃₃⁺][Cur^–], exhibited good sensitivity for benzoyl peroxide (BPO) detection.²²¹ The probe was constructed using the [N₃₃₃₃⁺][Cur^–] IL and upon adding BPO, a distinct color change from blood red to nearly colorless was observed and attributed to a redox reaction, enabling its use in colorimetric detection. Furthermore, the presence of BPO induced fluorescence quenching from bright yellow to colorless when illuminated with 365 nm UV light. Fluorescence-based detection with a reported LOD as low as 10 nM was achieved.

Ionic Liquid-Modified Carbon Dots and Photonic Spheres

Carbon dots are a relatively new class of fluorescent dyes with diameters of less than 10 nm.^222,223 They have garnered significant attention due to their advantages, including tunable photoluminescence, biocompatibility, and resistance to photobleaching compared to organic dyes and metallic quantum dots. However, their full potential has been hindered by their tendency to aggregate and relatively low quantum yields.²²⁴ ILs have been shown to offer promising solutions to these limitations when used in the modification of carbon dots by improving their detection limits and quantum yields. Wang et al. reported an IL-CD probe for the determination of vitamin B6 in milk.²²⁵ The probe was prepared using a solvothermal method by mixing the [C₈MIm⁺][BF₄^–] IL and o-phenylenediamine, followed by heating the mixture at a specific temperature for a defined period of time. In the presence of vitamin B6, a static quenching mechanism led to a significant reduction in fluorescence intensity, enabling sensitive detection with a LOD of 5 × 10^–5 mg mL^–1. Additionally, the probe demonstrated good selectivity for vitamin B6 over other vitamins and ions commonly found in milk, due to the specific analyte-probe complexation that occurred.

Liu et al. introduced PIL-based photonic spheres as sensing elements for the colorimetric determination of five explosive compounds, including 4-nitrophenol, picric acid, 2,4,6-trinitroresorcinol, 2,4-dinitrophenol, and 1,3,5-trinitrotoluene.²²⁶ For PIL synthesis, an imidazolium derivative functionalized with a urea group as the monomer and hexamethylene diisocyanate as the cross-linker was used. The incorporation of urea-functionalized PILs resulted in strong hydrogen bonding between the urea motifs and the nitro groups of the analytes, offering high affinity to the PIL spheres. The direct binding mechanism enabled straightforward detection of the explosives, thereby avoiding complicated analysis procedures. Additionally, the ILs exhibited diverse intermolecular interactions allowing for single photonic sphere to exhibit versatile responses to a broad range of analytes.

Surface-Enhanced Raman Spectroscopy (SERS)

ILs and deep eutectic solvents (DESs) have demonstrated promising results in SERS by serving as agents to enhance the production and performance of nanostructured substrates. The following section explains some of the roles of ILs in SERS. Using the ability of ILs to stabilize nanoparticles, Li et al. synthesized a SERS substrate based on the 1-methyl-3-hexyl imidazole ferric tetrachloride [C₆MIm⁺][FeCl₄^–]) MIL gold nanoparticles (Au NPs) using a microwave-assisted method.²²⁷ These substrates were successfully employed for the sensitive detection of clopidol residues in egg samples. The study reported that the MIL played a role in providing uniform particle distribution and sizes that resulted in an amplified SERS signal and further guided the synthesis of Au NPs with high performance.

Carreón et al. demonstrated the use of a deep eutectic solvent (DES)-like IL in the development of self-assembled bimetallic Ag–Au NP films.²²⁸ The DES was prepared by combining choline chloride and urea in a 1:2 molar ratio, which functioned as both a soft template and substrate and facilitated the controlled self-assembly of Ag–Au NPs during the thermal evaporation process, ensuring precise nanostructure formation. The resulting Ag–Au NP film-based SERS substrate demonstrated high sensitivity for detecting crystal violet and nanoplastics, providing a LOD of 10^–14 M for crystal violet and 1 μg mL^–1 for PET. Zhang et al. highlighted the potential of IL-based isotropic platforms for highly sensitive SERS applications and employed ILs as carriers in their study to create an isotropic SERS platform, shown in Scheme 1.²²⁹ The N,N′-bis(10-undecenyl)-2-methylimidazolium bromide ([MImV₁₁,V₁₁⁺][Br^–]) IL was used in which the coordination effect and anion exchange capability of the IL was exploited. This approach enabled the successful growth and stabilization of Au NPs on the polymerized surface of the IL. A LOD as low as 10^–12 M was achieved with an enhancement factor of 3.04 × 10⁷ for methyl orange, demonstrating the platform’s high sensitivity and potential for trace detection.

Scheme 1. Depiction of the Coassembly Strategy of [MImV₁₁,V₁₁⁺][Br^–] IL Segments and Further Synthesis Conducted to Prepare the Ternary Isotropic SERS Platform.

Reprinted from J. Mol. Liq., Vol. 391, Zhang, D.; Zhang, H. Highly sensitive SERS platform on isotropic ionic liquid-based liposome, pp. 123311 (ref (229)). Copyright 2023, with permission from Elsevier.

Conclusions and Future Outlook

ILs remain as promising solvents and materials for a wide range of analytical applications. Over the last five years, the design of more selective ILs has been evident for many applications. Optical sensors have been designed using FILs to detect analytes within complex samples and enantioseparations using CILs have shown enhanced selectivity due to synergistic effects. IL membranes have also shown improved selectivity for CO₂ capture and present innovative solutions for environmental as well as industrial challenges with implications in sustainability. PILs have also been an important subclass of ILs with broad applicability from sample preparation to separations to detection.

Rapid extraction methods using greener solvents remain of interest to achieve high throughput and reduce the use of toxic organic solvents. Miniaturized analytical devices and instruments, ranging from μGC columns and microfluidic devices to mini mass spectrometers and smartphone detectors, have become more prevalent. Additionally, point-of-care and on-site detection methods continue to be a growing area of interest for clinical diagnostics and environmental monitoring. Most prominently, many studies have also demonstrated the cross-disciplinary advantages of their devices, using them for both extraction/separation and detection. This has been observed by coupling in-tube SPME with MS detection, yielding methods that demonstrate high analyte enrichment and lower LODs and LOQs. The coupling of LLE and ABS approaches with microfluidic devices may also offer significant advantages for rapid extraction and detection. Simplification of analytical methods and workflows has advantages in reducing loss of analyte during transfer and sampling errors and in reducing the overall cost of the method.

This review has highlighted many advancements that have been made in the creation of IL-based polymer systems for targeted analytical applications. The versatility of reaction strategies used to prepare PILs have grown significantly in the past decade and will continue to be employed in chemical analysis platforms to prepare polymeric materials at different length scales. It is anticipated that 3D printing approaches employing stereolithography and fused deposition modeling will facilitate more widespread studies using PILs, particularly since there have been tremendous gains in the resolution of commercial printers within the last 5–8 years. Investigations into the design and use of monolithic columns featuring PIL stationary phases for complex chemical separations will continue to expand, along with the development of new approaches to produce PIL microspheres for sample preparation. More sensitive colorimetric and fluorescent assays will be produced using FILs as synthetic chemistry methods will permit a greater mixing and matching of cations/anions to produce compounds with desired optical characteristics.

Within the past 5 years, a greater number of studies have explored deep eutectic solvents (DESs) as sustainable solvents within the field of analytical chemistry compared to the preceding time period. DESs are generally more straightforward to prepare compared to ILs and require little to no purification and cleanup steps. Additionally, their solvation characteristics can often be modulated by choosing the appropriate hydrogen bond donor and hydrogen bond acceptor, which provides them some important advantages over ILs. However, DESs have limited utility at elevated temperatures and their long-term stability need to be carefully monitored. Future attention should be given into understanding the role and tunability of IL chemical structure in the developed methodologies as it pertains to designing better extraction/separation/detection methods and contributing to a deeper knowledge of IL behavior. Advanced analytical methods that can measure important physicochemical properties of ILs (e.g., viscosity, glass transition temperature, conductivity, thermal stability, UV absorption profile, water content) while requiring very little sample will be impactful and can be coupled with advanced computational methods to aid in predicting their properties based on chemical structures and anion/cation combination.

Biographies

Victoria R. Zeger obtained her B.S. degree in chemistry from West Virginia University in 2019. She was a Ph.D. student in Prof. Jared Anderson’s group at Iowa State University from 2019-2024 and defended her Ph.D. in analytical chemistry in December 2024. Her research interests include the development of new microextraction procedures using ionic liquids and developing new gas chromatography stationary phases using ionic liquids and ionic liquid derived materials.

Bhawana Thapa obtained her bachelors degree from Rajiv Gandi University of Health Sciences in India and her M.S. degree from the University of Tartu, Estonia/Uppsala University, Sweden. In January 2021, she joined the Ph.D. program at Iowa State University and has been involved in several research areas in Prof. Jared Anderson’s group related to solid phase microextraction, 3D printing, and using ionic liquids as selective stationary phases for gas chromatography and high performance liquid chromatography.

Danial Shamsaei obtained his bachelors and masters degrees from Isfahan University of Technology in Iran. In fall 2021, he joined the chemistry Ph.D. program at Iowa State University. His research in Prof. Jared Anderson’s group has focused on developing smartphone-based luminescence detectors for chromatographic separations and employing ILs and deep eutectic solvents as materials for 3D printing applications in HPLC and GC.

Jessica F. DeLair obtained her B.A. degree in biochemistry in May 2023 from Augustana University in Sioux Falls, South Dakota. Upon joining the chemistry Ph.D. program at Iowa State University, she has worked in Prof. Jared Anderson’s group focusing on the synthesis and characterization of zwitterionic liquids as a new class of ultrapolar stationary phases for gas chromatography.

Tristen L. Taylor received her bachelor’s degree in forensic chemistry from the University of Providence in 2020. She went on to complete her Ph.D. in 2024 under Alexander Gundlach-Graham at Iowa State University. Her area of specialty is inductively coupled plasma mass spectrometry with an emphasis on instrumentation and methods for liquid sample introduction and single particle analysis.

Jared L. Anderson is the Alice Hudson Professor of Chemistry at Iowa State University and is also a Faculty Scientist at Ames National Laboratory, U.S. Department of Energy. His research focuses on the development of stationary phases for multidimensional chromatography, alternative approaches for sample preparation, particularly in nucleic acid isolation and purification, and analytical tools for trace-level analysis within active pharmaceutical ingredients.

The authors declare no competing financial interest.

Special Issue

Published as part of Analytical Chemistryspecial issue “Fundamental and Applied Reviews in Analytical Chemistry 2025”.