Organic Modifier Concentration Research Articles

Hydrophilic Interaction Liquid Chromatography for the Analysis of Pharmaceutical Formulations

Abstract: For a long time, Reversed-Phase Liquid Chromatography (RPLC) was the most dominant technique for the analysis of pharmaceutical compounds, but with poor efficiency in the separation of small polar molecules. From the efforts to solve the problem of insufficient retention of these molecules, during the last decades, a mode of liquid chromatography named Hydrophilic Interaction Liquid Chromatography (HILIC) has experienced vast expansion. It is based on the use of a highly hydrophilic stationary phase along with an aqueous mobile phase with high organic modifier content. In this review, the characteristics of stationary and mobile phases used in HILIC are described, and corresponding separation mechanisms are discussed. An overview of recently published papers dealing with the application of HILIC in analyzing pharmaceuticals in biological and non-biological samples is provided. Besides, the application of HILIC systems in the determination of the physicochemical properties of compounds is described.

Assessing the potentialities of an easy-to-use sample treatment strategy: Multivariate investigation on “Moka extraction” of typical ingredients from dietary supplements

Mr. Bialetti invented Moka in 1933 and it still represents the most common way to prepare coffee at home. The process through which Mokas extract components from the ground coffee is a solid-liquid extraction which occurs at high pressure and temperature. These features are desirable in simple sample treatment strategies, since they allow good extraction efficiencies in a short time. Herein, for the first time, Moka-pot extraction was considered as an alternative processing protocol to extract polar compounds from dietary supplements. The effect of four experimental variables on extraction efficiency was evaluated through a fractional factorial design of experiments applied to a pooled matrix. In particular, solvent pH and its content of organic modifier, heating temperature and sample mass (reflecting the ratio to the amount of solvent which has to be kept fixed due to practical needs) were considered. The performances of the best conditions were then validated by determining recoveries (between 52 and 134 %, except for acetylsalicylic acid) and matrix effects (resulting always negligible or moderate at 100-fold dilution) of a spiked matrix which did not present any of the target analytes. They were finally applied to real samples, allowing to quantify some compounds, including artificial sweeteners, methylxanthines and taurine. Results were then compared with the quantities declared on the labels and those obtained with a Salt-Assisted Liquid-Liquid Extraction (SALLE), previously developed. Interestingly, the two methods were comparable for most compounds, but Moka extraction allowed to quantify taurine, which was not recovered with the SALLE. This promising result encourages further work to extend the use of the simple Moka device to other analytes and further matrices.

Combination of large-volume sample stacking with polarity switching and micelle electrokinetic chromatography for the analysis of anion compounds in Yangxinshi tablets.

Yangxinshi tablet (YXST) is a traditional Chinese medicine preparation characterized by its high efficacy and safety for the treatment of cardiovascular diseases. Anionic compounds have been revealed as potential active components. However, there is currently limited research regarding its quality control. We aimed to establish a strategy for the simultaneous separation and determination of five key anionic compounds in YXST. A sensitive and efficient analytical method was developed and applied for the simultaneous separation and determination of five key compounds in YXST using large-volume sample stacking with polarity switching and micelle electrokinetic chromatography (LVSS-PS-MEKC) coupled with diode array detection. Crucial parameters, including sample volume, applied voltage, composition and pH of the running buffer, concentration of organic modifier, and switching time of the polarity, were systematically evaluated and optimized using a single variable method to enhance separation performance. Furthermore, the impact of cyclodextrin and sodium dodecyl sulfate as electrolyte modifiers was also investigated. Under the optimal conditions, baseline separation of the five compounds (daidzein, puerarin, glycyrrhiztinic acid, chlorogenic acid, and salvianolic acid B) was achieved within 20 min. In comparison to the conventional MEKC mode, the constructed LVSS-PS-MEKC method exhibited a more than sixfold increase in the enrichment factor. The method was validated in terms of linearity, precision, accuracy, 24 h stability, and recovery and successfully applied to analyze YXST samples. A sensitive strategy was developed for the simultaneous separation and determination of five key anionic components in YXST, offering a robust and efficient strategy for pharmaceutical analysis.

Features and Advantages of the Recurrent Approximation of Retention Times in Reversed-Phase High-Performance Liquid Chromatography

Background: Recurrent approximation of retention parameters in reversed-phase HPLC is effective for revealing anomalies that are otherwise difficult to detect, namely, the reversible hydration of analytes. This was demonstrated previously for restricted sets of analytes with acetonitrile–water eluents. Expanding the number of analytes and eluents seems to be a topical problem. Two kinds of derivatives of aromatic carbonyl compounds were characterized: unsubstituted hydrazones and oximes. Methods: If analyte demonstrates no anomalies in dependences of retention times vs. concentration of organic modifier, the recurrent approximations of these dependences are linear. To explain the features of recurrent approximations, the numerical experiments were proposed and considered. The artificial shifting of one, two, or more points allows for the modeling the different kinds of deviations of approximations from linearity. Results: It was shown that hydrazones are the class of analytes having no anomalies of retention parameters. On the contrary, several anomalies were detected for oximes. Downward deviations of points in the plots of recurrent approximations of retention times are the signs of reversible hydration. This effect for methanol–water eluents was detected for the first time. Some of oximes underwent hydrolysis. Conclusions: Recurrent approximation of retention times allows detecting chemical transformations of analytes during RP HPLC analysis.

Modification of gradient HPLC method for determination of small molecules' affinity to human serum albumin under column safety conditions: Robustness and chemometrics study

In the early stages of drug discovery, beyond the biological activity screening, determining the physicochemical properties that affect the distribution of molecules in the human body is an essential step. Plasma protein binding (PPB) is one of the most important investigated endpoints. Nevertheless, the methodology for measuring %PPB is significantly less popular and standardized than other physicochemical properties, like lipophilicity. Here, we proposed how to modify protocols presented by Valko into column safety conditions and evaluated their robustness using fractional factorial design. For robustness testing, four factors were selected: column temperature, mobile phase flow rate, maximum isopropanol concentration in the mobile phase, and buffer pH. Elaborate methods have been applied for the analysis of HSA affinity for three groups of antibiotic-oriented substances that vary in chemical structure: fluoroquinolones, sulfonamides, and tetrazole derivatives. Furthermore, based on the reversed-phase chromatography the workflow of pilot studies was proposed to select molecules that have high affinity to HSA and can not be eluted from the HSA column using the concentration of organic modifier recommended by the column manufacturer.

Comparison of ZIC-HILIC Columns for the Simultaneous Analysis of Antiviral Drugs in Dosage Forms by Hydrophilic Interaction Liquid Chromatography

Background:: Antiviral drugs are vital since many viruses can produce fatal infections, as we have recently seen with the COVID-19 pandemic. Antiviral drugs can battle viruses at multiple stages of their life cycles, including neuraminidase, nucleic acid synthesis, protease, and virion fusion or entry [1]. Objective:: Antiviral drugs have poor retention in reversed-phase liquid chromatography, which makes it difficult to analyze a mixture of antiviral medications using high-performance liquid chromatography. Using zwitterionic hydrophilic interaction liquid chromatography (ZICHILIC), the paper highlights the simultaneous quantification of three antiviral drugs as active constituents in pharmaceutical formulations. Moreover, the influence of the length of the spacer between the two charges in two stationary phases on the retention behavior of antiviral drugs has been discussed. Methods:: Two homemade stationary phases (ZIC1-HILIC and ZIC5-HILIC) were utilized for the qualitative and quantitative analysis of three antiviral drugs, and UV was used as the detector. Several chromatographic conditions were examined, such as the organic modifier concentration, buffer concentration, and pH value. Results:: After optimizing the parameters, the devised method was applied to analyse three antiviral medications quantitatively. The initial results demonstrated the current procedure for separating and determining these three antiviral drugs to be sensitive, robust, and effective. Consequently, the present method has shown excellent repeatability, a broad linear range (0.1- 16.5 μgml-1), and excellent sensitivity (LOD 0.04-0.072 μgml-1). The RSD value of the method was less than 1. Conclusion:: A mixed mode of hydrophilic and ion exchange interactions was the predominant mode of antiviral medications with two ZIC-HILIC stationary phases. The ZIC5-HILIC stationary phase had a lower detection and limit of quantitation for three antiviral drugs and a prolonged retention time compared to the ZIC1-HILIC stationary phase with a shorter chain length.

Effect of Organic Additives on the Intensity of Metal Lines in the Emission Spectrum of a Drop-Spark Discharge upon Sample Introduction into an Electrolyte Anode

The effect of the nature and concentration of organic medium modifiers on the intensity of lines of some heavy metals (Ag, Cd, Hg, Pb, Tl, Zn) in the spectrum of a drop-spark discharge upon sample introduction into an electrolyte anode is studied. It is shown that additives of 0.1–6 wt % of polar organic compounds greatly increase the intensity of Pb, Tl, and Zn lines and reduce the intensity of Ag lines. The greatest effect, a twenty-fold enhancement of zinc lines, is observed upon the introduction of methanol into dilute acids. The cadmium signal is insensitive to organic impurities in dilute acids, but is enhanced in concentrated salt solutions.

Mobile phase and column chemistry selection for high sensitivity non-targeted LC/ESI/HRMS screening of water

Systematic selection of mobile phase and column chemistry type can be critical for achieving optimal chromatographic separation, high sensitivity, and low detection limits in liquid chromatography electrospray high resolution mass spectrometry (LC/MS). However, the selection process is challenging for non-targeted screening where the compounds of interest are not preselected nor available for method optimization. To provide general guidance, twenty different mobile phase compositions and four columns were compared for the analysis of 78 compounds with a wide range of physicochemical properties (logP range from −1.46 to 5.48), and analyte sensitivity was compared between methods. The pH, additive type, column, and organic modifier had significant effects on the analyte response factors, and acidic mobile phases (e.g. 0.1% formic acid) yielded highest sensitivity. In some cases, the effect was attributable to the difference in organic modifier content at the time of elution, depending on the mobile phase and column chemistry. Based on these findings, 0.1% formic acid, 0.1% ammonia and 5.0 mM ammonium fluoride were further evaluated for their performance in non-targeted LC/ESI/HRMS analysis of wastewater treatment plan influent and effluent, using a data dependent MS2 acquisition and two different data processing workflows (MS-DIAL, patRoon 2.1) to compare number of detected features and sensitivity. Both data-processing workflows indicated that 0.1% formic acid yielded the highest number of features in full scan spectrum (MS1), as well as the highest number of features that triggered fragmentation spectra (MS2) when dynamic exclusion was used.

Semi-empirical description of a retention of small organic probes on AtlantisTM Premier BEH C18 AX mixed-mode column under varying pH and organic modifier conditions

Mixed-mode chromatography is a widely used analytical method that utilizes multiple separation mechanisms in one chromatographic column; frequently a reversed-phase and ion-exchange interactions. Adequate understanding of how the individual interactions influence the overall retention of the analyte is important for fast and effective design of mixed-mode-based methods. Previous attempts to derive semi-empirical mathematical models of mixed-mode chromatography were limited to a narrow range of analytes and did not consider the effect of organic modifier in the mobile phase. In this paper, we attempt to improve upon the previous models by extending the range of classes of analytes to weak acids and bases and including the effect of organic modifier. An improved semi-empirical model was derived that captures all important retention trends of the combined effect of reversed-phase and ion-exchange retention mechanisms for all tested classes of analytes and up to 30% (v/v) organic modifier content.

A chiral metal-organic framework synthesized by the mixture of chiral and non-chiral organic ligands for enantioseparation of drugs by open-tubular capillary electrochromatography

A chiral metal-organic framework L-Histidine-Zeolitic imidazolate framework-67 (L-His-ZIF-67) was synthesized by the mixture of chiral organic ligand L-histidine and non-chiral organic ligand 2-methylimidazole directly, and to the author's knowledge, the chiral L-His-ZIF-67 coated capillary column we prepared has still not been reported to date in the field of capillary electrophoresis. This chiral metal-organic frameworks material was used as the chiral stationary phase for enantioseparation of drugs by open-tubular capillary electrochromatography. The separation conditions such as pH value, buffer concentration and proportion of organic modifier were optimized. Under optimal conditions, the established enantioseparation system achieved good separation effect, and the resolution of five chiral drugs: esmolol (7.93), nefopam (3.03), salbutamol (2.42), scopolamine (1.08) and sotalol (0.81). In addition, the chiral recognition mechanism of L-His-ZIF-67 was elucidated by a series of mechanism experiments, and the specific interaction force was preliminarily speculated.

Evidence for the Hydration of Some Organic Compounds during Reverse-Phase HPLC Analysis.

Some polar analytes (X) can reversibly form hydrates in water-containing eluents under the conditions of reversed-phase HPLC analysis, X + H2O ⇄ X × H2O. One of the methods to detect their formation is the recurrent approximation of the net retention times of such analytes, tR(C + ΔC) = atR(C) + b, where ΔC = const is the constant step in the variation of the organic modifier content of an eluent. These dependencies are linear if hydrates are not formed, but in the case of hydrate formation, they deviate from linearity under high water content. It has been shown that UV spectroscopic parameters, namely, relative optical densities: Arel = A(λ1)/A(λ2), depend on eluent composition for some organic compounds, but their variations cannot be used as indicators for hydrate formation. The coefficients that characterize the dependence of the analyte retention indices on the organic component concentration of an eluent, dRI/dC, appeared to be the most informative additional criterion for hydration. The values of these coefficients for most polar analytes are largely negative (dRI/dC < 0), whereas, for nonpolar compounds, they are largely positive (dRI/dC > 0).

SIMULTANEOUS DETERMINATION OF MONOCAFFEOYLQUINIC ACIDS AND CAFFEINE BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELUENTS BASED ON PROPANOL-2 AND ETHYL ACETATE (REJECTION OF ACETONITRILE)

The purpose of the study is development of conditions for the simultaneous determination of caffeine and three isomers of monocaffeoylquinic acids (esters of caffeic and quinic acids: 3CQA, 4CQA and 5CQA) in coffee using "green" reverse-phase HPLC, in which an expensive and environmentally unfavorable acetonitrile is replaced with ethyl acetate as an organic modifier of mobile phases. An approach which provides comparing the efficiency and selectivity of the substance separation in a wide range of concentrations of organic modifiers of selected eluent systems is proposed. It is shown that the replacement of acetonitrile with propanol-2 or ethyl acetate slightly changes the selectivity of the separation of three isomeric chlorogenic acids, but significantly reduces the relative retention of caffeine due to better solvation of caffeine with an organic modifier. This makes it possible to change the position of caffeine elution relative to chlorogenic acids in a targeted way to avoid coelution of caffeine with other extractive substances by changing the concentration and type of the organic modifier of the mobile phase. Ethyl acetate-based eluents are shown to be convenient for simultaneous determination of caffeine and monocaffeoylquinic acids in conditions of reverse-phase HPLC. The replacement of acetonitrile with ethyl acetate makes it possible to re-extract mainly caffeine and monocaffeoylquimc acids from concentrating cartridges (DIAPAK C18) during sample preparation. More lipophilic extractive substances still remain on the concentrating cartridge, which provides the possibility of using a simple isocratic mode thus reducing the duration of analysis and consumption of the organic modifier of the mobile phase.

Studies on the Effects of Process Conditions on Separation of B1, B2 and B3 Vitamin Mixture Using HILIC and RPLC Chromatography

In this work, a series of experiments on the retention of B1 (riboflavin), B2 (thiamine) and B3 (nicotinic acid) vitamins in the HILIC and RPLC conditions have been performed involving the effects of organic modifier type and content, pH of the eluent and buffering salt (ammonium acetate) concentration in the mobile phase as well as temperature of the system. For the needs of this study, three columns of different features have been chosen: Acclaim™ Mixed–Mode HILIC–1 (Dionex, Sunnyvale, CA, USA), Eurospher II 100–5 HILIC (Knauer, Berlin, Germany) and the RPLC column Nucleodur® C18 Gravity-SB (Macherey-Nagel, Duren, Germany). The influence of acetonitrile and methanol content in the eluent and process temperature have been tested and, based on that, the most promising systems have been selected regarding the possible separation of the vitamin mixture. Both the pH and buffering salt concentrations in the eluent have been adjusted in order to indicate the most effective system, which turned out to be the one involving the Nucleodur column and the eluent with 90% methanol, at pH 6 and Cbuff = 20 mmol/dm3, which enables separation of the mixture within a time as short as 2.5 min at a 1.0 mL/s flowrate in isocratic conditions.

Standard-Free Quantification of Dicarboxylic Acids: Case Studies with Salt-Rich Effluents and Serum.

The present study evaluates the ionization efficiency (IE) of linear and branched C2-C14 dicarboxylic acids (DCAs) by electrospray ionization (ESI) under different conditions. The influence of the concentration of organic modifier (MeOH); mobile phase additive; and its concentration, pH, and DCA structure on IE values is studied using flow injection analysis. The IE values of DCAs increase with the increase of MeOH concentration but also decrease with an increase of pH. The former is due to the increase in solvent evaporation rates; the latter is caused by an ion-pairing between the diacid and the cation (ammonium), which is confirmed by the study with different amines. The investigation of DCA ionization in the presence of different acidic mobile phase additives showed that a significant improvement in the (-)ESI responses of analytes was achieved in the presence of weak hydrophobic carboxylic acids, such as butyric or propanoic acid. Conversely, the use of strong carboxylic acids, such as trichloroacetic acid, was found to cause signal suppression. The results of the IE studies were used to develop the liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method that provided instrumental limits of detection in the range from 6 to 180 pg. Furthermore, upon applying the nonparametric Gaussian process, a model for the prediction of IE values was developed, which contains the number of carbons in the molecule and MeOH concentration as model parameters. As a case study, dicarboxylic acids are quantified in salt-rich effluent and blood serum samples using the developed LC-HRMS method.

Polydopamine Coating Doped with Graphene Oxide Enhances Enantioseparation of Capillary Column.

How to improve the enantiomer separation efficiency of drugs is a hot topic. In this paper, polydopamine (PDA) coating doped with graphene oxide (GO) by physical adsorption was used to modify the capillary column to enhance the enantioseparation efficiency of the drugs. In the capillary electrochromatography (CEC) system, the novel capillary column with carboxymethyl-β-cyclodextrin (CM-β-CD) as a chiral selector has completed the enantioseparation of four basic drugs (propranolol, metoprolol, amlodipine and chlorpheniramine). The optimum separation conditions were obtained by optimizing the pH of the buffer, the concentration of organic modifier, the concentration of the chiral selector and the voltage, and the resolution and peak shape were significantly improved compared with uncoated bare-fused column. The stability and reproducibility of the new capillary column were satisfactory and the relative standard deviation of intra-day and inter-day was <3.2%, and of column-to-column was <4.8%. The rich functional groups of GO are key factors to improve the enantioseparation efficiency, which also indicates that nanomaterials with easy modification of functional groups and large specific surface area are excellent resources for capillary modification applications.

Pre-column Derivatization and Separation of Diastereomeric-Derivatives of Racemic Mexiletine and Confirmation of Elution Order and Molecular Configuration

Present study describes the synthesis of cyanuric chloride based four active chiral reagents (ACRs) and their application in the enantiomeric separation of (RS)-mexiletine. Herein, four cyanuric chloride-based ACRs were prepared by introducing L-proline derivatives under nucleophilic substitution reaction. The synthesized ACRs were characterized by different spectroscopic techniques. Racemic mexiletine hydrochloride was used for the enantio-recognition study. All the four ACRs were used to convert (RS)-mexiletine into related diastereomeric derivatives and then separated on the C18-column of RP-HPLC. The different parameters such as sample amount, the concentration of mobile phase, organic modifier and pump pressure were varied to optimize separation conditions. The energy-minimized structures of synthesized diasteromeric derivatives (DDs) were developed using DFT calculations. The validation study was conducted for the developed method and correlation-coefficient, calibration range, LOD and LOQ calculated. The stability and recovery were calculated by inter and intraday assay.

Separation of pharmaceutically active compounds by multimodal chromatography with ultraviolet detection

AbstractThis research focused on pharmaceutically active ingredients analyzed by mixed‐mode liquid chromatography. For this purpose, 16 analytes were chosen with acidic, basic, or neutral properties that were analyzed under different conditions. These analytes play an important role in the pharmaceutical industry and improved separation will make drug control more efficient. Mixed‐mode chromatography is becoming an increasingly popular method for the analysis of diverse compounds due to the process being based on the combination of two or more interaction modes. The use of the mixed‐mode stationary phase can be especially beneficial for the development of new drugs which may have specific physical and chemical properties. Data obtained from measurements on three different mixed‐mode columns (WAX‐1, WCX‐1, and HILIC‐1) were used to construct the plots illustrating the relationship between the retention factors of the analytes and organic modifier content, buffer concentration, and buffer pH. As a result of the known chemical structures of the analyzed molecules and analysis of these plots, possible interaction mechanisms in various chromatographic systems were proposed. Conclusions enabled suggestions for the optimization of chromatographic conditions to be made as well as the improved selection of the stationary phase for different types of analytes.

Analytical quality by design in the development of a solvent-modified micellar electrokinetic chromatography method for the determination of sitagliptin and its related compounds

A solvent-modified micellar electrokinetic chromatography method was developed following the Quality by Design approach for the simultaneous determination of sitagliptin (SIT), an oral antihyperglycemic drug, and its main impurities derived from the synthesis process. The separation system was identified in the scouting phase and was made by sodium dodecyl sulphate (SDS) micelles with the addition of n-butanol and methanol. The knowledge space was investigated through an asymmetric screening matrix, taking into consideration eight critical method parameters (CMPs) involving the composition of the background electrolyte in terms of buffer concentration and pH, the concentration of surfactants and organic modifiers, and voltage. The critical method attributes (CMAs) were identified as analysis time and the distance between the tail of the electroosmotic flow system peak and the front edge of impurity I1 (sitagliptin triazole hydrochloride). A Box-Behnken Design was used in response surface methodology for calculating the quadratic models relating the CMPs to the CMAs. From the models it was possible to compute the method operable design region (MODR) through Monte-Carlo simulations. The MODR was identified in the probability maps as the multidimensional zone where the risk of failure to achieve the desired values for the CMAs was lower than 10 %. The experimental conditions corresponding to the working point, with the MODR interval, were the following: background electrolyte, 14 (10−18) mM borate buffer pH 9.20, 100 mM SDS, 13.6 (11.1–16.0) %v/v n-butanol, 6.7 (4.5–8.8) %v/v methanol; voltage and temperature were set to 28 kV and 22 °C, respectively. The developed CE method was validated in accordance with International Council for Harmonisation guidelines and was applied to the analysis of SIT tablets. The routine analysis for the quality control of the pharmaceutical product could be conducted in about 11 min.

Features of the Recurrent Approximation of Retention Parameters of Polyfunctional Compounds in Reversed-Phase High-Performance Liquid Chromatography

Five antitumor drugs are selected as examples of polyfunctional organic compounds to test the possibilities of approximating their retention parameters in reversed-phase high-performance liquid chromatography using recurrent relationships of the form of tR(C + ΔС) = atR(C) + b, where C is the concentration of organic modifier in the composition of the eluent (acetonitrile), ΔC = const is a constant step of its change in concentration, and a and b are coefficients calculated using the least squares method. Examples of both ideally linear dependences in all ranges of acetonitrile concentrations and deviations from linearity in different ranges of its concentrations are revealed. Discussion of the possible reasons for such deviations (both structural factors and physicochemical properties) suggests that acid–base equilibria in the eluent are not the main ones, the positions of which depend on the concentration of the organic component. The reversible formation of hydrated forms typical of compounds whose molecules contain such polar fragments as amide ‒CO–NH– and sulfonamide –SO2–NH groups, seems more likely. Recurrent approximation of the retention parameters can be considered the simplest and most obvious way to reveal the specific interactions of the test compounds with components of the eluent.

Tetraalkylammonium-l-tartrate ionic liquids as sole chiral selectors in capillary electrophoresis

In the field of separation science, the use of tartaric acid-based ionic liquids (TTILs) for chiral separation has not yet been reported. In this work, six novel mono- and di-tetraalkylammonium l-tartrate ILs (TAA-l-TTs) with different alkane chain length were evaluated for their potential as sole chiral selectors in capillary electrophoresis (CE). In comparison with conventional l-tartaric acid (l-TT) chiral selector, these IL-type chiral selectors exhibit remarkably improved enantioseparation performance, which is even better than most previously reported TT ester-type chiral selectors. Major factors influencing CE separation were investigated, including the background electrolyte (BGE) composition, ILs concentration, type and proportion of organic modifier, buffer pH, etc. The results show that the l-TT-based IL-type chiral selectors prefer a weak acid buffer (150 mM boric acid at pH 4.5) with a high proportion of methanol (80%-90%). The best resolutions (Rs) were obtained when the IL concentration was set at 60 mM. We further carried out a series of comparative experiments and a molecular simulation study to explore the enantiorecognition mechanism of TTILs. The result indicates that the TAA-l-TTs cannot be considered as a simple combination of TAA salt and l-TT. The dissociation degree of TTILs may be an important factor influencing the enantiorecognition process.