# Unravelling dispersion forces in liquid-phase enantioseparation. Part III: Interplay between dispersion, repulsion, and hydrophobic forces in the enantioseparation of planar chiral 3-substituted-1-iodoethynylferrocenes

Barbara Sechi, Alessandro Dessì, Roberto Dallocchio, Saba Jorbenadze, Natia Tchanturia, Bezhan Chankvetadze, Sergio Cossu, Iza Matarashvili, Victor Mamane, Paola Peluso

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## ► To cite this version:

Barbara Sechi, Alessandro Dessì, Roberto Dallocchio, Saba Jorbenadze, Natia Tchanturia, et al. Unravelling dispersion forces in liquid-phase enantioseparation. Part III: Interplay between dispersion, repulsion, and hydrophobic forces in the enantioseparation of planar chiral 3-substituted-1-iodoethynylferrocenes. Analytica Chimica Acta, 2025, 1382, pp.344853. 10.1016/j.aca.2025.344853. hal-05354500

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Submitted on 12 Nov 2025

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# Unravelling dispersion forces in liquid-phase enantioseparation. Part III: Interplay between dispersion, repulsion, and hydrophobic forces in the enantioseparation of planar chiral 3-substituted-1-iodoethynylferrocenes

Barbara Sechi<sup>a, ID</sup>, Alessandro Dessì<sup>a</sup>, Roberto Dallocchio<sup>a ID</sup>, Saba Jorbenadze<sup>b</sup>, Natia Tchanturia<sup>b</sup>, Bezhan Chankvetadze<sup>b ID</sup>, Sergio Cossu<sup>c</sup>, Iza Matarashvili<sup>b</sup>, Victor Mamane<sup>d, *, ID</sup>, Paola Peluso<sup>a, ** ID</sup>

## HIGHLIGHTS

* Enantioseparation of planar chiral ferrocenes on polysaccharide selectors.

* Dependence of enantiomer elution order on mobile phase and selectand structures.

* Impact of adding water to the mobile phase on selector-selectand affinity.

* Deconvolution of hydrophobic and dispersion forces in liquid chromatography.

## GRAPHICAL ABSTRACT

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## ARTICLE INFO

**Keywords:**

Enantioseparation
High-performance liquid chromatography
Planar chiral ferrocenes
Noncovalent interactions
Polysaccharide-based chiral stationary phases

## ABSTRACT

**Background:** The detection of dispersion forces in enantioselective liquid chromatography (LC) is rather challenging because selectand, selector and mobile phase molecules may participate in multiple types of intra- and intermolecular noncovalent interactions of different strength. In this frame, the LC system can be used as a tool to evaluate the impact of changing the structures of selectand, chiral selector and separation medium, through fast analytical screenings. In the last few years, we studied the enantioseparations of chiral compounds containing the ethynylferrocene moiety as a test probe to identify dispersion forces in LC by using amylose carbamate-based chiral stationary phases (CSPs).

<footnote><sup>a</sup> Istituto di Chimica Biomolecolare ICB-CNR, Sede secondaria di Sassari, Traversa La Crucca 3, Regione Baldinca, Li Punti, 07100, Sassari, Italy</footnote>

<footnote><sup>b</sup> Institute of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Tbilisi State University, Chavchavadze Ave 3, 0179, Tbilisi, Georgia</footnote>

<footnote><sup>c</sup> Dipartimento di Scienze Molecolari e Nanosistemi DSMN, Università Ca' Foscari Venezia, Via Torino 155, Mestre, I-30172, Venezia, Italy</footnote>

<footnote><sup>d</sup> Institut de Chimie de Strasbourg, UMR CNRS 7177, Equipe CLIC, 1 rue Blaise Pascal, Cedex, Strasbourg, 67008, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.
E-mail addresses: vmamane@unistra.fr (V. Mamane), paola.peluso@cnr.it (P. Peluso).</footnote>
**Results:** The results of the study have been reported in a series of three papers. In Part II, we confirmed that the high affinity observed for the second eluted ($R_p$)-enantiomer of the 1-(iodoethynyl)-3-phenylferrocene toward amylose phenylcarbamate-based selectors could be reasonably based on dispersion forces. In the present Part III of the series, we focus on the impact of changing the 3-aryl substituent of 1-(iodoethynyl)-3-arylferrocenes on the enantioseparation, considering the 1-(iodoethynyl)-3-phenylferrocene as reference for comparison. On this basis, the enantioseparations of seven planar chiral ferrocenes were performed and compared by using amylose-based CSPs. n-Hexane-based mixtures, polar organic solvents and aqueous organic mixtures were used with the aim of evaluating the impact of mobile phases of different polarity on the enantioseparations. The results of the chromatographic analyses confirmed the high and unique affinity of the second eluted ($R_p$)-enantiomer of the 1-(iodoethynyl)-3-(4-t-butyl)phenylferrocene toward the amylose-based CSPs.

**Significance:** By using 1-(iodoethynyl)-3-arylferrocenes as test probes, this study confirmed that dispersion forces may turn steric repulsion into attraction with a strength depending on the structure of the 3-aryl group and on mobile phase polarizability. Furthermore, the possibility to deconvolute hydrophobic and dispersion forces in aqueous mobile phases was also demonstrated.

# 1. Introduction

Dispersion or London forces are "attractive forces between apolar molecules, due to their mutual polarizability" [1] and are formed between induced dipoles of two or more interacting atoms or molecules. The induced dipoles result from the oscillation of electrons between different states, a phenomenon that may take place when two interacting apolar chemical species are in proximity [2]. Due to its character, a single interaction based on dispersion forces is weak. However, being additive, dispersion forces can stabilize medium- and large-sized polarizable molecular systems, making their interaction energy more negative [3]. On this basis, the practical effect of dispersion forces in molecular binding and recognition is to turn steric hindrance into attraction [4].

The first studies considering the possibility that dispersion forces may also act in liquid chromatography (LC) date back to the late 1970s [5,6]. Due to the complexity of the noncovalent interaction patterns underlying binding and selectivity in a LC system, deconvoluting dispersion forces between an analyte and a stationary phase (selector + supporting phase) is rather challenging. Indeed, selectand, selector, and mobile phase molecules may participate in multiple types of intra- and intermolecular noncovalent interactions of different strength [7]. On the other hand, the use of the LC system allows to evaluate the impact of changing the structures of the compounds used as selectands and of the stationary phase used as interaction counterpart through fast analytical screenings. Furthermore, mobile phase composition can be tuned with great flexibility in LC. Thus, the impact of mobile phase polarity on the strength of noncovalent interactions underlying binding and enantior-ecognition can be effectively screened.

Over time, experimental studies focused on the contribution of dispersion forces to the binding of apolar compounds and apolar stationary phases in aqueous mobile phases were reported in the literature [8–10]. Aqueous media were preferentially used due to the lower ability of water as dispersion (energy) donor compared to other solvents used in LC like methanol, acetonitrile, and n-hexane. Indeed, water has lower polarizability because of the lower oscillation capability of its electrons [11]. This results in a lower ability to form induced-dipoles and to participate in dispersion-based interactions involving the surrounding molecules. On the other hand, hydrophobic effects also contribute to selectand-stationary phase binding in water-containing media. Thus, the contributions of dispersion and hydrophobic forces is not easy to deconvolute [12]. In HPLC, dispersion forces were shown to contribute to the isotope effect and the separation of H/D isotopologues [13–16]. Recent investigations demonstrated the usefulness of deuterium substituents as probes to deconvolute the contributions of hydrophobic and dispersion forces occurring in chromatography in aqueous organic mixtures used as mobile phases [17–19].

While the contribution of dispersion forces to selectand-stationary phase binding in LC is still an open issue, whether dispersion forces can contribute to enantioselectivity is even more challenging. Very few studies focused on this issue [11,16–23].

Ferrocenes have remarkable capability to exert dispersion forces due to the presence of a large, electron-rich, and polarizable electron system [24]. This feature is enhanced if substituents containing additional π-electron clouds are introduced in the cyclopentadienyl ring(s) [25,26]. Recently, HPLC enantioseparations of planar chiral ferrocenes containing extended π-electron clouds were performed by using amylose-based chiral stationary phases (CSPs). Peculiar enantioselectivity results were obtained in these cases depending on mobile phase polarity and on structure of the ferrocene derivative [11,22,27,28].

In the last few years, our groups studied the enantioseparations of chiral compounds containing the ethynylferrocene moiety as a test probe to identify dispersion forces in HPLC by using amylose-based CSPs. The results of this study have been reported in a series of three papers. In the first paper of the series [22], the enantioseparation of the axially chiral 3,3'-dibromo-5,5'-bis-ferrocenylethynyl-4,4'-bipyridine and the 3,3'-dibromo-5,5'-bis-phenylethynyl-4,4'-bipyridine were examined and compared. This study demonstrated the superiority of the ferrocenyl group compared to the phenyl group, as a structural element favouring enantiodifferentiation. In the second paper [11], we considered the enantioseparations of planar chiral 1-(iodoethynyl)-3-arylferrocenes (3-aryl = phenyl, 2-naphthyl) to understand the molecular bases of the high affinity observed for the second eluted ($R_p$)-enantiomer of these analytes toward amylose phenylcarbamate-based selectors. This phenomenon was enhanced by using methanol-containing mixtures as mobile phases. In the present third paper of the series, we focus on the impact of changing the 3-aryl substituent of 1-(iodoethynyl)-3-arylferrocenes on the enantioseparation, considering the 1-(iodoethynyl)-3-phenylferrocene 1 as reference compound for comparison (Fig. 1). For this purpose, the enantioseparations of the planar chiral ferrocenes 1-7 were performed and compared by using amylose-based CSPs (Fig. 2) and mobile phases with different polarity (n-hexane-based mixtures, polar organic solvents and aqueous organic

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Structures of planar chiral ferrocenes 1-7. ($R_p$)- and ($S_p$)-enantiomers are depicted for the general structure of 1-(iodoethynyl)-3-arylferrocenes.</figcaption></figure>
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Lux Amylose-1, Lux i-Amylose-1: R', R" = Me
Lux i-Amylose-3: R' = Me; R" = Cl

<caption>**Fig. 2.** Amylose-based chiral columns and selectors used for the enantioseparations of chiral planar ferrocenes **1-7**.</caption>

mixtures). Furthermore, the molecular properties of ferrocenes **1-7** were computed by quantum mechanics at density functional theory (DFT) level to evaluate their possible impact on the enantioseparation outcomes. In this regard, it is worth mentioning that computational-aided studies on dispersion forces were reported to a small degree in the field of chromatography [29,30].

The first aim of the present investigation was to evaluate which structural modification of compound **1** could enhance or weaken the dispersion-based affinity exhibited by this compound toward the amylose-based stationary phases as interaction counterparts. The second aim of this study was to develop analysis methodologies for the enantioseparation of the new planar chiral ferrocenes by using different elution modes. Indeed, very few systematic analytical studies on the enantioseparation of planar chiral ferrocenes were reported so far [11, 22, 27, 28, 31].

## 2. Materials and methods

### 2.1. Reagents and chemicals

Compounds *rac-1*, *rac-4*, *rac-5* and their (*S*<sub>p</sub>)-enantiomers were prepared and characterized as previously reported [11,32]. Compounds *rac-2*, *rac-3*, *rac-6*, *rac-7* and their (*S*<sub>p</sub>)-enantiomers were prepared in the frame of this study, and all details on their syntheses and characterization are reported in the Supplementary data. HPLC grade *n*-hexane, methanol (MeOH), propan-2-ol (2-PrOH), acetonitrile (ACN), and water were purchased from Sigma-Aldrich (Taufkirchen, Germany). Experimental details about X-ray diffraction (XRD) analyses of (*R*<sub>p</sub>)-5 are reported in the Supplementary data.

### 2.2. Chromatography

An Agilent Technologies (Waldbronn, Germany) 1100 Series HPLC system (high-pressure binary gradient system equipped with a diode-array detector operating at multiple wavelengths [220, 254, 280, and 360 nm], a programmable autosampler with a 20-µl loop, and a thermostated column compartment) was employed. Data acquisition and analyses were carried out with Agilent Technologies ChemStation Version B.04.03 chromatographic data software. The UV absorbance is reported as milliabsorbance units (mAU). Lux Amylose-1 (coated) and Lux i-Amylose-1 (immobilized) [amylose tris(3,5-dimethylphenylcarbamate)], and Lux i-Amylose-3 [amylose tris(3-chloro-5-methylphenylcarbamate)] (5 µm) (Phenomenex Inc., Torrance, CA, USA) were used as chiral columns (250 × 4.6 mm) (Fig. 2). The samples were prepared at 1 mg·ml<sup>-1</sup> in the mobile phase. Analyses were performed in isocratic mode at 25 °C. The flow rate was set at 0.8 ml/min, if not indicated otherwise. Dead time (*t*<sub>0</sub>) was measured by injection of tri-

tert-butylbenzene (Sigma-Aldrich) as a non-retained compound [33]. The van't Hoff experiments were conducted at 5, 10, 15, 20, 25, 30, 35, 40, and 45 °C in normal phase elution and at 10, 15, 20, 25, 30, 35, 40, 45, and 50 °C with MeOH/water 95:5 v/v by using a thermostat jacket equipped with a RE104 Lauda circulating water bath (Lauda, Königshofen, Germany) (resolution 0.1 °C; accuracy ±0.4 °C; temperature control ±0.02 °C). In all cases, when the temperature was changed, the column was allowed to equilibrate for 1 h before injecting the samples. Thermodynamic parameters were derived from the slopes and the intercepts of the van't Hoff plots by linear regression analysis (see Supplementary data for theory details). For all analytes, the enantiomer elution order (EEO) was determined by injecting (*S*<sub>p</sub>)-enantiomers prepared by asymmetric syntheses [11,32] (see Supplementary data for **2**, **3**, **6** and **7**). Original chromatographic traces are reported in the Supplementary data (section S5.4 Chromatographic traces).

### 2.3. Computations

Geometry optimization and vibrational frequencies of ferrocenes **1-7**, methyl 3,5-dimethylphenylcarbamate, and methyl 3-chloro-5-methylphenylcarbamate were computed in vacuum at density functional theory (DFT) level with the B3LYP functional and the def2TZVPP basis set by using Gaussian 16W (Wallingford, CT 06492 USA) [34]. Electrostatic potential extrema on the molecular electron density isosurfaces (maxima and minima) (*V*<sub>S,max</sub> and *V*<sub>S,min</sub>) (au, electrons/bohr) were calculated by using Gaussian 16W, at the DFT (B3LYP/def2TZVPP). Search for the exact location of *V*<sub>S,max</sub> and *V*<sub>S,min</sub> was made through the Multiwfn code [35] and through its module enabling quantitative analyses of molecular surfaces (isovalence: ferrocenes, 0.001 au; carbamates, 0.002 au) [36]. The electrostatic potential in a point r [*V*(*r*)] is a real physical property which may provide information on specific regions of the molecules. Indeed, regions with high (with nucleophile character) and low (with electrophile character) electron density can be identified by negative *V*<sub>S,min</sub> and positive *V*<sub>S,max</sub> values, respectively [37].

## 3. Results and discussion

### 3.1. Structural features of selectors, selectands, and mobile phases

The first requirement of a study aiming at investigating noncovalent interactions through liquid phase separation techniques is a careful selection of the three pivotal components of the separation system: selectand(s), selector(s) and mobile phase(s). The structures of these three components must be selected based on the noncovalent interaction target under investigation in an on-off perspective; this means that the identification of certain noncovalent interactions is made by exploiting chromatographic systems designed on rational bases to enhance, weaken, or suppress these interactions.

Given dispersion forces as the target of our investigation, amylose carbamate-based selectors (Fig. 2) were chosen. These chiral selectors feature an extended surface and multiple interaction sites [38,39]. Furthermore, the amylose-based polymers present smaller chiral cavities compared to cellulose-based selectors favouring close contacts [40, 41]. In previous studies, the higher ability of ferrocene to interact with an amylose tris(3,5-dimethylphenylcarbamate) nonamer compared to benzene was demonstrated by molecular dynamics [22]. Moreover, the tight contact between the amylose tris(3,5-dimethylphenylcarbamate) nonamer and compound **1** was also demonstrated [42]. In the present study, we considered methylated (Lux Amylose-1 and i-Amylose-1) and methylchlorinated (Lux i-Amylose-3) columns to evaluate if different stereoelectronic properties of the chiral selectors could impact the phenomenon under investigation. Indeed, amylose tris(3,5-dimethylphenylcarbamate) as the Amylose-1 selector is a stronger hydrogen bond (HB) acceptor (*V*<sub>S,min</sub> (HNC=O) = -0.0625 au) and a weaker HB donor (*V*<sub>S,max</sub> (H-NCO) = 0.0843 au) compared to the amylose tris(3-chloro-5-methylphenylcarbamate) as the Amylose-3 selector (*V*<sub>S,min</sub>
(HNC=O) = -0.0595 au, $V_{S,max}$ (H-NCO) = 0.0914 au) (Table S1, Supplementary data). Furthermore, the impact of the structural modification due to immobilization (Amylose-1 vs i-Amylose-1) on enantioselectivity could be also examined.

To inspect the electronic properties of the main interaction sites featuring compounds 1-7, $V_{S,max}$ and $V_{S,min}$ values were also computed and compared (Table S2, Supplementary data). As shown in Fig. 3, these ferrocene derivatives share four regions having high electron charge density due to the presence of (conjugated) π-electron clouds (associated with the triple bond, the two cyclopentadienyl rings and the 3-aryl substituent), a region of high electron charge density on the nucleophilic belt of iodine, and an electrophilic region corresponding to the iodine σ-hole. Given the $V_{S,max}$ value computed for iodine in compound 1 as reference for comparison ($V_{S,max}$ (I) = 0.0707 au), the electrophilicity of the halogen substituent increased for compounds bearing 3-aryl groups with an electron-withdrawing character (3-aryl: p-FC₆H₄ (2); anthracene-9-yl (6); phenanthrene-9-yl (7), 0.0708 ≤ $V_{S,max}$ (I) ≤ 0.0723 au). Conversely the electrophilicity of the halogen substituent decreased for compounds containing electron-donor substituents on the 3-aryl group (3-aryl: p-MeOC₆H₄ (3); p-MeC₆H₄ (4); p-tBuC₆H₄ (5), 0.0693 ≤ $V_{S,max}$ (I) ≤ 0.0697 au). Consistently, the electron charge density on the triple bond, on the cyclopentadienyl rings, and on the negative belt of the iodine increased for compounds 3-5 and decreases for 2, 6, and 7 compared to ferrocene derivative 1. The electron charge density on the 3-aryl substituent was lower only for compound 2 compared to 1, whereas it was higher for all the other compounds. Thus, the electronic properties of the group at the 3-position of the substituted cyclopentadienyl ring impacted the overall distribution of the electron charge density in the corresponding molecule.

It is worth mentioning that, among the considered planar chiral ferrocenes, both enantiomers of 5 (3-aryl = *p*-t-BuC₆H₄) showed a crystallographic packing featuring a highly ordered structure containing close contacts based on dispersion between the cyclopentadienyl rings and between the *tert*-butyl groups (Fig. S1, Supplementary data) [11]. Coherently, the highest value of zero-point vibrational energy (ZPVE), as a descriptor of the oscillation frequencies of a molecular system, was computed for this compound (226.16 kcal·mol⁻¹) compared to the other ferrocene derivatives of the series (150.92 ≤ ZPVE ≤ 214.67 kcal·mol⁻¹) (Table S3, Supplementary data). Furthermore, in preliminary experiments performed in LC by using amylose-based CSPs and methanol-containing mobile phases, higher retention factors of the second eluted ($R_p$)-enantiomer for compound 5 compared to compound 4 (3-aryl = *p*-MeC₆H₄) were observed, despite the higher steric hindrance exerted by the *tert*-butyl group. Based on the recognized ability of

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Main interaction sites of 1-(iodoethynyl)-3-phenylferrocenes 1-7. Compound 1 is shown as a representative compound.</figcaption></figure>

this group to behave as dispersion interaction donor [43], we aimed in the present study at verifying if a particular behaviour in LC could be observed exclusively for compound 5 compared to the other members of the series.

Given the pivotal function of the mobile phase in LC, we performed the analytical screening by using mobile phase with different polarity and polarizability: *n*-hexane-based mixtures, pure MeOH, pure ACN, aqueous ACN- and MeOH-based mixtures. These elution modes were selected based on the observation that polarizability, and thus the ability as dispersion interaction donor, decreases following the order *n*-hexane > ACN > MeOH > water [11]. On this basis, a different ability to favour or to weaken/suppress selectand-selector dispersion forces could be expected for these solvents.

## 3.2. Comparative enantioseparation of ferrocenes 1-7 in normal-phase mode

In a first screening, the enantioseparations of compounds 1-7 with Lux Amylose-1, Lux i-Amylose-1, and Lux i-Amylose-3 were studied and compared by using *n*-hexane/2-PrOH 95:5 v/v as mobile phase (Fig. 4 and Table S4, Supplementary data). The enantioseparation outcomes, in terms of retention factors ($k_1$ and $k_2$) and separation factor ($\alpha$), were dependent on the substituents at the 3-position of the ferrocene cyclopentadienyl ring and on selector structure.

In almost all cases, $k_1$ and $k_2$ values increased following the order i-Amylose-3 < i-Amylose-1 < Amylose-1 confirming the polar nature of the interactions contributing to selector-selectand binding in normal phase. Only in the case of compound 1, the second eluted enantiomer showed slightly higher $k_2$ values with the i-Amylose-1 ($k_2$ = 2.46) compared to the coated column ($k_2$ = 2.28).

On each column, the retention factors of the seven compounds appeared to be dependent on the polarity and on the steric hindrance of the substituents on the 3-aryl group. On one side, compound 5, bearing the bulky *tert*-butyl group on the 3-phenyl ring and presenting the iodine with the lowest electrophilic properties ($V_{S,max}$ (I) = 0.0697 au), was the less retained on all columns. On the other side, compound 7 featuring the largest surface area (385.82 Å²) and the iodine with higher electrophilic properties ($V_{S,max}$ (I) = 0.0708 au) was the most retained in almost all cases. Furthermore, compound 4 (3-aryl = *p*-MeC₆H₄) showed higher retention and selectivity compared to the bulkier 5 (3-aryl = *p*-tBuC₆H₄), whereas compound 3 (3-aryl = *p*-OMeC₆H₄) showed higher retention than compounds 4 and 5 bearing apolar alkyl groups as distinctive substituents.

For 1, 3, 4, and 6 selectivity increased followed the order Amylose-1 < i-Amylose-1 < i-Amylose-3 showing an opposite trend compared to retention factors. Thus, the chloromethylated i-Amylose-3 column provided better $\alpha$ values for compounds 1, 3, 4, and 6 compared to the dimethylated i-Amylose-1, whereas better selectivity was obtained for 2, 5, and 7 with this latter column. The immobilized i-Amylose-1 provided better $\alpha$ values for all compounds compared to the coated column except for 2 and 7. Compound 1 showed the highest enantioselectivity with all columns. This meant that a) under normal phase elution conditions the substituents introduced on the 3-aryl moiety had a detrimental effect on enantioselectivity, and b) the amylose-based columns, irrespective of their distinctive stereoelectronic structure, exhibited the highest enantioseparation performance toward compound 1.

The peculiar behaviour observed for compounds 6 and 7 deserves to be highlighted. Indeed, whereas for compounds 1-5 baseline enantioseparations were obtained within 15 min in all cases (1.27 ≤ $\alpha$ ≤ 2.85), for the anthracenyl derivative 6 baseline enantioseparation could be obtained with i-Amylose-3 exclusively ($\alpha$ = 1.36). The phenanthrenyl derivative 7 was baseline separated with Amylose-1 ($\alpha$ = 1.28), partially separated with i-Amylose-1 and not enantioseparated with i-Amylose-3. Furthermore, the EEO was ($R_p$)-($R_p$) for all compounds with the three chiral columns except for compound 6 showing ($R_p$)-($S_p$) as EEO with all columns. In this regard, it is worth mentioning that compounds 6 (area
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Comparison of retention factors of first ($k_1$) and of second ($k_2$) eluted enantiomers, and separation factor ($\alpha$) of compounds 1-7 with Lux Amylose-1, i-Amylose-1, and i-Amylose-3 as chiral columns by using n-hexane/2-PrOH 95:5 v/v and n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v as mobile phases (flow rate = 0.8 ml/min, T = 25 °C). For chromatographic parameters see Tables S4 and S5, Supplementary data.</figcaption></figure>

= 379.19 Å²; volume = 407.11 Å³) and 7 (area = 385.82 Å²; volume = 408.09 Å³) are the largest sized of the series, and the compact structure of the amylose-based selectors is, in general, less prone to accommodate large molecules [39,44]. It is interesting to stress that compounds 5, 6, and 7, having the largest surface areas and volumes, presented either the lowest retention (compound 5) or the lowest average selectivity (compounds 6 and 7).

### 3.2.1. Impact of adding methanol to the binary hexane/propan-2-ol mixture as mobile phase

The enantioseparation trends described in the previous paragraph changed completely when the MeOH-containing mixture n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v was used as mobile phase (Fig. 4 and Table S5, Supplementary data). In most cases, retention and selectivity factors increased by using the MeOH-containing ternary mobile phase. Remarkably, a huge increase of the retention of the second eluted

enantiomers was observed for 1 (+79.04 min) and 5 (+23.94 min) by introducing small amounts of MeOH in the mobile phase. For compounds 1-5 baseline enantioseparations were obtained in all cases (1.56 ≤ $\alpha$ ≤ 9.13), whereas for the anthracenyl derivative 6 baseline enantioseparation could be obtained again with i-Amylose-3 exclusively ($\alpha$ = 1.41). The introduction of MeOH in the mobile phase increased the enantioselectivity for the phenanthrenyl derivative 7 with i-Amylose-1 ($\alpha$ = 1.22) and i-Amylose-3 ($\alpha$ = 1.34), obtaining baseline enantioseparation in both cases. In this regard, it is worth mentioning that the phenanthrenyl derivative 7 was only partially separated on the i-Amylose-1 ($\alpha$ = 1.06) and unresolved on the i-Amylose-3 by using n-hexane/2-PrOH 95:5 v/v as mobile phase. Interestingly, a huge increase of the enantioselectivities was observed for compounds 1 (3.92 ≤ $\alpha$ ≤ 8.79) and 5 (6.17 ≤ $\alpha$ ≤ 9.13) with the MeOH-containing mobile phase compared to the binary n-hexane/2-PrOH 95:5 mixture (1: 2.25 ≤ $\alpha$ ≤ 2.88; 5: 1.28 ≤ $\alpha$ ≤ 1.61). In particular, adding MeOH to the mobile
phase made the α values higher for compound 5 compared to the ferrocene derivative 1 with the immobilized columns. This result appeared very interesting if we consider that compound 5 exhibited the lowest selectivity factors when n-hexane/2-PrOH 95:5 v/v was used as mobile phase. Apparently, this showed that the addition of MeOH switched on a new mechanism based on nonpolar interactions which enhanced the enantioseparation of compound 5. At least two factors could contribute to this result: a) the lower polarizability of MeOH compared to n-hexane and 2-propanol, and b) the lower polarity of the immobilized columns compared to the coated Amylose-1. Indeed, immobilization procedures may induce subtle alterations in the selector generating a chiral platform with different recognition capability given that some covalent immobilization technologies require the use of under-derivatized polysaccharides in order to attach the selector to the silica surface [45]. Our previous studies shown that immobilization of the amylose tris(3,5-dimethylphenylcarbamate) selector affects the ability of the carbamate moiety to exert HBs [46].

For compounds 1-5, the addition of MeOH to the n-hexane-based mobile phase did not change the EEO on any of the used columns [(Sₚ)-(Rₚ)]. A MeOH-dependent reversal of EEO was observed for compound 6 with i-Amylose-1 [(Rₚ)-(Sₚ)]n-hexane/2-propanol → (Sₚ)-(Rₚ)]n-hexane/2-propanol/MeOH, whereas a pendant group-dependent reversal of EEO occurred for compound 7 by changing i-Amylose-1 [(Sₚ)-(Rₚ)] to i-Amylose-3 [[(Rₚ)-(Sₚ)].

The thermodynamic parameters (Table 1) associated with the enantioseparations collected with the i-Amylose-1 column by using n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v as mobile phase were determined through van't Hoff analysis at different temperatures from 5 to 45 °C in 5 °C increments (Tables S6 and S7, Fig. S2 and S3, Supplementary data).

Interestingly, positive ΔΔS and the highest ΔΔH values were determined for compounds 4, 6, and 7 which also showed the lowest selectivity values with the i-Amylose-1/MeOH-containing mobile phase chromatographic system. In particular, whereas the enantioseparations were enthalpy-driven for compounds 1-3 and 5-7 (1.87 ≤ Q ≤ 4.65) [Q = ΔΔH/(ΔΔS-298.15)], an entropy-driven thermodynamic was derived for the methylated derivative 4 (Q = 0.06). These results evidenced that repulsive forces weakened enantioselective interaction of 4, 6, and 7 with the CSP compared to the other members of the series. For these three compounds, especially for 4, this was reflected in a higher level of disorder for the adsorption of the second eluted enantiomer compared to the first eluted one. As mentioned above, this may be due to the steric effect weakening the interaction of the large-sized 6 and 7 with the stationary phase. Even for the smaller compound 4, no reason other than the steric effect due to the presence of the methyl group on the 3-aryl moiety could justify the observed distinctive behaviour.

### 3.2.2. Enantiomer elution order of ferrocenes 6 and 7

As mentioned above, (Sₚ)-(Rₚ) as EEO was observed for compounds 1-5 with all six chromatographic systems obtained by combining the three amylose-based columns and the two n-hexane-based mobile

phases. In contrast, several types of EEO reversals could be observed for compounds 6 and 7 (Fig. 5): a) a dependence of the EEO on the structure of analytes could be observed by using Amylose-1 with both mobile phases and i-Amylose-1 with the binary mobile phase; b) an immobilization-dependent reversal of EEO (Amylose-1 vs i-Amylose-1) for 6 with the MeOH-containing mobile phase; c) two pendant group-dependent EEO reversals (i-Amylose-1 vs i-Amylose-3) for both 6 and 7 with the MeOH-containing mobile phase; d) a mobile phase-dependent EEO reversal for 6 on i-Amylose-1.

To gain further insights into the thermodynamic bases of these results (Table 2), the thermodynamic parameters associated with the enantioseparations collected with the three amylose-based columns by using the two n-hexane-based mobile phases were determined by studying temperature dependence of the chromatographic retention factor (k) and separation selectivity (α) from 5 to 45 °C in 5 °C increments (Tables S8 and S9, and Fig. S4, Supplementary data).

With the binary MeOH-free mobile phase, the EEO for 6 was (Rₚ)-(Sₚ) with all the three columns and the enantioseparations resulted entropy-driven (0.65 ≤ Q ≤ 0.93) in all cases. In the case of 7, the opposite EEO observed with the Amylose-1 and i-Amylose-1 columns [(Sₚ)-(Rₚ)] compared to compound 6 [(Rₚ)-(Sₚ)] corresponded to a change of the thermodynamics resulting in enthalpy-driven enantioseparations. Interestingly, compound 7 could be partially separated on the i-Amylose-3 by changing the temperature within both enthalpy- (T = 5 °C) and entropy-driven (T ≥ 30 °C) domains. In this case, the two domains were observed due to fact that the isoenantioselective temperatures fell within the operative temperature range (21.7–25.0 °C) (Table S9). The observed EEO was (Sₚ)-(Rₚ) in the enthalpic domain, and (Rₚ)-(Sₚ) in the entropic domain, and the ln α vs 1/T plot showed the characteristic V shape (Fig. S4). Even for 6 on i-Amylose-1, the EEO reversal by changing the binary mobile phase [(Rₚ)-(Sₚ)] to the tertiary mobile phase [(Sₚ)-(Rₚ)] corresponded to a change of the thermodynamic domain from entropy- to enthalpy-driven.

With the MeOH-containing mobile phase, the enantioseparations were enthalpy-driven with all columns for both compounds. On this basis, the analyte structure-dependent EEO reversal observed for compounds 6 and 7 on the Amylose-1 with the MeOH-containing mixture occurred in the same enthalpy-driven domain (Q (6) = 2.07; Q (7) = 2.27). The immobilization-dependent EEO reversal (Amylose-1 vs i-Amylose-1) observed for 6 with the MeOH-containing mixture also occurred in the same enthalpy-driven domain. However, in this case the contribution of entropy to the enantioseparation free energy was negative with the coated column (ΔΔS = -0.22 cal K⁻¹·mol⁻¹) and positive with the immobilized column (ΔΔS = 0.01 cal K⁻¹·mol⁻¹). Finally, for both compounds 6 and 7, the pendant group-dependent EEO reversal (i-Amylose-1 vs i-Amylose-3) occurred again within the enthalpy-driven domain, with a positive entropy contribution to the enantioseparation free energy for the i-Amylose-1 column (ΔΔS (6) = 0.01 cal K⁻¹·mol⁻¹; ΔΔS (7) = 0.11 cal K⁻¹·mol⁻¹) and negative for the i-Amylose-3 column (ΔΔS (6) = -4.08 cal K⁻¹·mol⁻¹; ΔΔS (7) = -7.10 cal K⁻¹·mol⁻¹). Coherently, a more negative enthalpy contribution could be determined

<caption>**Table 1**</caption>

Thermodynamic parameters associated with the differences of adsorption of the two enantiomers onto the CSP derived from the van't Hoff plots (temperature range 278.15–318.15 K) for analytes 1-7 [EEO = (S)-(R)] on Lux i-Amylose-1 (flow rate = 0.8 ml/min). Mobile phase: n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v.

<table><thead><tr><th>Fc<sup>a</sup></th><th>3-aryl</th><th>ΔΔH (cal·mol<sup>-1</sup>)</th><th>ΔΔS (cal·K<sup>-1</sup>·mol<sup>-1</sup>)</th><th>T<sub>ISO</sub>, K</th><th>Q<sup>b</sup></th><th>α<sup>c</sup></th></tr></thead><tbody><tr><td>1</td><td>Ph</td><td>-1432.43</td><td>-1.03</td><td>1386.3</td><td>4.65</td><td>7.00</td></tr><tr><td>2</td><td>p-FC<sub>6</sub>H<sub>4</sub></td><td>-1067.02</td><td>-1.38</td><td>772.1</td><td>2.59</td><td>3.09</td></tr><tr><td>3</td><td>p-MeOC<sub>6</sub>H<sub>4</sub></td><td>-1529.59</td><td>-2.74</td><td>559.0</td><td>1.87</td><td>3.29</td></tr><tr><td>4</td><td>p-MeC<sub>6</sub>H<sub>4</sub></td><td>22.65</td><td>1.34</td><td>16.9</td><td>0.06</td><td>1.88</td></tr><tr><td>5</td><td>p-tBuC<sub>6</sub>H<sub>4</sub></td><td>-2242.53</td><td>-3.41</td><td>657.2</td><td>2.20</td><td>7.63</td></tr><tr><td>6</td><td>Anthracen-9-yl</td><td>-27.92</td><td>0.01</td><td>-</td><td>-</td><td>1.05</td></tr><tr><td>7</td><td>Phenanthren-9-yl</td><td>-81.27</td><td>0.11</td><td>-</td><td>-</td><td>1.22</td></tr></tbody></table>

<footnote>a Fc, Ferrocene.</footnote>

<footnote>b Q = ΔΔH°/(298.15 × ΔΔS°), thermodynamic ratio.</footnote>

<footnote>c α, separation factor.</footnote>
<figure><img src="image_9.png" /><figcaption>Fig. 5. Reversals of EEO observed in the enantioseparations of ferrocene derivatives 6 and 7 with Lux Amylose-1, i-Amylose-1, and i-Amylose-3 by using n-hexane/2-PrOH 95:5 v/v and n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v as mobile phases (flow rate = 0.8 ml/min, T = 25 °C).</figcaption></figure>

for the enantioseparation on i-Amylose-3 (ΔΔΗ (6) = -1427.68 cal mol⁻¹; ΔΔΗ (7) = -2294.39 cal mol⁻¹) compared to i-Amylose-1 (ΔΔΗ (6) = -28.26 cal K⁻¹·mol⁻¹; ΔΔΗ (7) = -81.27 cal K⁻¹·mol⁻¹).

Based on the reported results, two types of relationships between EEO reversal and thermodynamics could be identified: a) the EEO reversal was due to a change of the thermodynamic domain of the enantioseparation. This meant that the EEO reversal was due to a change of the noncovalent interaction pattern underlying enantioseparation; b) the EEO reversal occurred in the same thermodynamic domain. This meant that the EEO reversal was due to a change of the strength (not type) of the noncovalent interactions underlying enantioseparation.

These results confirmed the ability of MeOH to switch to a new mechanism compared to that operating with the MeOH-free binary mobile phase.

### 3.3. Comparative enantioseparation of ferrocenes 1-7 with polar organic and aqueous organic mixtures as mobile phases

With the aim to evaluate the dependence of retention and selectivity on polarity and polarizability of the mobile phases, we also performed the enantioseparations of compounds 1-7 by using pure MeOH and the mixture MeOH/water 95:5 v/v as mobile phases (Table S10, Supplementary data). The Lux Amylose-1 was used as chiral column. With both mobile phases, the EEO was always the same [(S<sub>p</sub>)-(R<sub>p</sub>)] and no EEO reversal was observed under these elution conditions. In Fig. 6, the dependence of the separation factors on mobile phase polarizability (MeOH/water > MeOH > n-hexane/2-PrOH/MeOH > n-hexane/2-PrOH) is shown. A huge increase of the separation factors could be observed for compounds 1 and 5 by using pure MeOH as mobile phase (α (1) = 11.75; α (5) = 13.42), and even more with MeOH/water 95:5 v/v (α (1) = 11.90; α (5) = 16.17), compared to the other compounds of the series (2-4, 6, 7) (1.60 ≤ α ≤ 5.60). The large separation factors observed for 1 and 5 were due to the huge increase of the affinity of the second eluted (R<sub>p</sub>-)enantiomers with pure MeOH and even more in aqueous MeOH (k<sub>2</sub> (1) = 55.97; k<sub>2</sub> (5) = 56.64). In this regard, it is

worth noting that quite large retention factors for the second eluted enantiomers were also obtained for compounds 2-4, 6, and 7 in the aqueous MeOH as mobile phase, but to a smaller degree (4.87 ≤ k<sub>2</sub> ≤ 19.75).

To gain further information on the distinctive chromatographic results observed for compounds 1 and 5, the thermodynamic parameters associated with the enantioseparations collected with the Amylose-1 column by using MeOH/water 95:5 v/v as mobile phase were determined by studying temperature dependence of the chromatographic k and α from 10 to 50 °C in 5 °C increments (Tables S11 and S12, Supplementary data). The enantioseparations resulted enthalpy-driven in all cases except for that of compound 4 which provided an entropy-driven separation (Q = 0.72). The most negative enthalpy and entropy contributions to the enantioseparation free energy were determined for compound 5 (ΔΔΗ = -2344.86 cal·mol⁻¹; ΔΔS = -2.32 cal·K⁻¹·mol⁻¹), whereas the most positive values were obtained for compound 4 (ΔΔΗ = 1113.69 cal·mol⁻¹; ΔΔS = 5.22 cal·K⁻¹·mol⁻¹). A positive value of ΔΔS (1.74 cal·K⁻¹·mol⁻¹) was also obtained for compound 1. Thus, the presence of distinctive substituents at the 3-position of the cyclopentadienyl ring had a great impact on the thermodynamics of the enantioseparation in the aqueous-organic mixture.

This trend was confirmed by comparing the entropy-enthalpy compensation (EEC) graphs collecting the thermodynamic quantities (ΔΔΗ and ΔΔS) contributing to the enantioseparation free energy differences and determined for compounds 1-7 by using MeOH/water 95:5 v/v (Fig. 7A) and n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v (Fig. 7B). This comparison may present some limitations because different CSPs (Amylose-1 and i-Amylose-1, respectively) and different temperature ranges (5–45 °C vs 10–50 °C) were used to determine the thermodynamic quantities in the two cases. Despite these operative differences, some aspects deserve to be mentioned. In both graphs, it is evident that the different thermodynamics observed for compounds 5 and 4 and reflected in the chromatographic outcomes was already present, to a small degree, when n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v was used as mobile phase. This difference was even more evident when MeOH/
<caption>**Table 2**</caption>

van't Hoff thermodynamics (temperature range 278.15–318.15 K) and enantiomer elution order determined for the enantioseparation of analytes **6** and **7** on Lux Amylose-1, i-Amylose-1, and i-Amylose-3 (flow rate = 0.8 ml/min). Mobile phases: n-hexane/2-PrOH 95:5 v/v and n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v.

<table><thead><tr><th>Fc<sup>a</sup></th><th>column</th><th>Mobile phase</th><th>EEO<sup>b</sup></th><th>Thermodynamics</th></tr></thead><tbody><tr><td rowspan="3">6</td><td>Amylose-1</td><td rowspan="3">n-hexane/2-PrOH 95:5</td><td>(R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Entropy-driven</td></tr><tr><td>i-Amylose-1</td><td>(R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Entropy-driven</td></tr><tr><td>i-Amylose-3</td><td>(R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Entropy-driven</td></tr><tr><td rowspan="3">7</td><td>Amylose-1</td><td></td><td>(S<sub>p</sub>)-(R<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td>i-Amylose-1</td><td></td><td>(S<sub>p</sub>)-(R<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td>i-Amylose-3</td><td></td><td>(S<sub>p</sub>)-(R<sub>p</sub>) &amp; (R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Enthalpy/Entropy-driven</td></tr><tr><td rowspan="3">6</td><td>Amylose-1</td><td rowspan="3">n-hexane/2-PrOH/MeOH 95:2.5:2.5</td><td>(R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td>i-Amylose-1</td><td>(S<sub>p</sub>)-(R<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td>i-Amylose-3</td><td>(R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td rowspan="3">7</td><td>Amylose-1</td><td></td><td>(S<sub>p</sub>)-(R<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td>i-Amylose-1</td><td></td><td>(S<sub>p</sub>)-(R<sub>p</sub>)</td><td>Enthalpy-driven</td></tr><tr><td>i-Amylose-3</td><td></td><td>(R<sub>p</sub>)-(S<sub>p</sub>)</td><td>Enthalpy-driven</td></tr></tbody></table>

water 95:5 was used as mobile phase. In the aqueous-organic mixture, compound **1** presented a much less negative ΔΔH value (−883.02 cal·mol⁻¹) compared to **5** (−2344.86 cal·mol⁻¹). However, the positive ΔΔS value (1.74 cal·K⁻¹·mol⁻¹) determined for **1** made the free energy difference of enantioseparation (ΔΔG = −1404.80 cal·mol⁻¹) of this compound slightly lower than that of compound **5** (ΔΔG = −1653.15 cal·mol⁻¹). Compared to **1**, compound **3** presented a more negative ΔΔH value (ΔΔH = −1329.82 cal·mol⁻¹), but a higher entropy penalty (ΔΔS = −1.26 cal·K⁻¹·mol⁻¹) that made the enantioseparation process more thermodynamically disfavoured (ΔΔG = −954.15 cal·mol⁻¹). The relative positions of the EEC points corresponding to compounds **6** and **7** were also interesting. In normal phase (Fig. 7B), these points are rather close to that referring to compound **4**. As already mentioned, this reveals that steric hindrance opposes to binding and recognition more in these cases compared to the other compounds. In the EEC graph referred to the aqueous mobile phase enantioseparations (Fig. 7A), the two points move toward more negative enthalpy and entropy regions showing that stronger interactions enhanced binding and recognition in hydrophobic environment, but in a less effective way compared to compound **5**. As expected, polar compounds like **2** and **3** showed less negative enthalpy in aqueous environment compared to normal phase. Interestingly for compound **5**, more negative enthalpy (ΔΔH = −2344.86 cal·mol⁻¹) and less negative entropy (ΔΔS = −2.32 cal·K⁻¹·mol⁻¹) were observed with

the chromatographic system MeOH/water 95:5/Amylose-1 compared to the system n-hexane/2-PrOH/MeOH/i-Amylose-1 (ΔΔH = −2242.53 cal·mol⁻¹; ΔΔS = −3.41 cal·K⁻¹·mol⁻¹).

### 3.4. Comparative enantioseparation of ferrocenes 1-7 with aqueous acetonitrile mixtures as mobile phases

The higher retention times observed for compounds **1** and **5** by introducing 5 % water in MeOH did not allow to study the effect of adding water to the mobile phase. However, given that water has very low polarizability, we were interested in evaluating the dependence of retention and selectivity on increasing water percentages in the mobile phase. On this basis, we considered ACN as organic component of the aqueous mobile phase in place of MeOH with the aim to study the effect of adding water in a feasible way in terms of elution times (Table S13, Supplementary data). Although higher retention and selectivity were also obtained by adding 5 % water to ACN, lower retention times were obtained by changing MeOH to ACN as organic solvent. As shown in Fig. 8, with ACN-based mobile phases all compounds showed lower separation factors, except for **4** in ACN and ACN/water 95:5, and **3** in ACN/water 95:5. Also with ACN-based mobile phases compounds **1** and **5** showed the highest separation factors.

The enantioseparation of compounds **1-7** was also screened by

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Dependence of separation factor α determined for the enantioseparation of compounds **1-7** with Lux Amylose-1 on mobile phase composition (flow rate = 0.8 ml/min, T = 25 °C). For chromatographic parameters see Table S10, Supplementary data.</figcaption></figure>

<footnote><sup>a</sup> Fc, Ferrocene.</footnote>

<footnote><sup>b</sup> EEO, enantiomer elution order.</footnote>
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Enthalpy-entropy compensation for compounds 1-7: (A) Lux Amylose-1, MeOH/water 95:5 v/v, 0.8 ml/min, temperature range 283.15–323.15 K; (B) Lux i-Amylose-1, n-hexane/2-PrOH/MeOH 95:2.5:2.5 v/v/v, 0.8 ml/min, temperature range 278.15–318.15 K.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Comparison of separation factors α determined for the enantioseparation of compounds 1-7 with Lux Amylose-1 by using MeOH vs ACN (A) and MeOH/water 95:5 v/v vs ACN/water 95:5 v/v (B) (flow rate = 0.8 ml/min, T = 25 °C). For chromatographic parameters see Tables S10 and S13, Supplementary data.</figcaption></figure>

adding 10 %, and 20 % of water to ACN (Table S13, Supplementary data). For all compounds, retention factors increased with increasing the content of water in the mobile phase due to a clear hydrophobic effect likely related to the hydrophobic structure of this class of ferrocenes. We also considered the difference between the retention factors of the second eluted enantiomers of each substituted derivative and that of the phenyl derivative, $\Delta k_2 = k_2(n) - k_2(1) (n = 2-7)$, as descriptor to

<figure><img src="image_13.png" /><figcaption>**Fig. 9.** Dependence of the variations of retention factors of the second eluted enantiomers determined for compounds 2-7, given $k_2$ of compounds 1 as reference for comparison ($\Delta k_2 = k_2(n) - k_2(1)$, n = 2-7), on the percentage content of water in ACN/water mixtures used as mobile phases (flow rate = 0.8 ml/min, T = 25 °C). For chromatographic parameters see Table S13, Supplementary data.</figcaption></figure>

quantify the impact of the distinctive substituent on the affinity of the second eluted enantiomers (Fig. 9). As a result, an opposite behaviour was observed for the tert-butyl derivative 5 compared to the other compounds: a positive difference when dispersion-based forces were dominant (5: $\Delta k_2 = 138.0$) and negative differences when repulsive forces and/or compound polarity opposed to hydrophobic and dispersion forces (1-4, 6, 7: -29.7 $\le \Delta k_2 \le -7.8$).

In Fig. 10, the overlapped chromatographic traces obtained for the enantioseparation of 1 and 3-5, as representative compounds, with Amylose-1 in ACN/water 80:20, are reported. Some aspects deserve to
<figure><img src="image_14.png" /><figcaption>Fig. 10. Chromatographic traces for the enantioseparation of compounds 1, 3, 4, and 5 with Lux Amylose-1 and ACN/water 80:20 v/v as mobile phase (flow rate = 0.8 ml/min, T = 25 °C). For chromatographic parameters see Table S13, Supplementary data.</figcaption></figure>

be highlighted: a) the huge elution time observed for the (R<sub>p</sub>)-enantiomer of compound 5 (t = 971.37 min), almost five time higher compared to the reference compound 1 (t = 198.97 min); b) the first eluted (S<sub>p</sub>)-enantiomers presented retention times increasing following the order 3 < 1 < 4 < 5 based on an evident hydrophobic effect. Indeed, being the most polar compound, the methoxy derivative 3 was eluted as first, whereas 5 showed the highest retention time as the less polar compound; c) the elution sequence observed for the second eluted (R<sub>p</sub>)-enantiomers could be rationalized on a different base. Indeed, it appeared not strictly related to the hydrophobic effect but rather derived from the balance between hydrophobic, repulsive and dispersion forces, these latter being dominant in the case of compound 5. As a result, polar (for 3) and repulsive (for 4) forces, opposing to hydrophobic and dispersion-based effects, contributed to shift the second eluted (R<sub>p</sub>)-enantiomers of compounds 3 and 4 at lower retention times compared to 1 and even much more compared to 5. The higher affinity of (R<sub>p</sub>)-1 and (R<sub>p</sub>)-5 toward the amylose-based selector could be explained as derived by a suitable balance between steric hindrance, in terms of isosurface volume, and dispersion ability, in terms of ZPVE. Indeed, 1 showed the lowest ZPVE value (156.12 kcal·mol<sup>-1</sup>) and the smallest volume (307.90 Å<sup>3</sup>), thus a low steric hindrance compensated the lower dispersion donor ability favouring the penetration of the selectand in the selector cavities. For 5, the highest ZPVE value (226.16 kcal·mol<sup>-1</sup>) and the largest volume (385.29 Å<sup>3</sup>) were determined by computation compared to the other three derivatives used as reference for comparison, thus the high dispersion donor ability turned steric hindrance into attraction in this case.

The behaviour observed in the liquid phase for compound 5 appeared consistent with its ability to form highly ordered crystal packing at the solid state based on the cooperation of multiple dispersion forces (Fig. S1, Supplementary data) [11].

## 4. Conclusions

The enantioseparation of the 1-(iodoethynyl)-3-arylferrocene derivatives 1-7 was investigated by using three amylose-based chiral columns and six mobile phases featuring different polarizability. Baseline enantioseparations were obtained in all cases for derivatives 1-5, whereas the elution conditions required a more careful selection for the anthracenyl derivative 6 and the phenanthrenyl derivative 7. On this basis, compounds 1-5 could be conveniently enantioseparated by using n-hexane/2-PrOH as mobile phase, whereas in polar organic solvents like ACN or MeOH, and in the related aqueous mixtures, these derivatives presented very high affinity toward the amylose-based

seyectors. This high affinity resulted in too long elution times. The addition of a small quantity of MeOH to the n-hexane/2-PrOH mobile phase had a beneficial effect for the enantioseparation of compounds 6 and 7 with the amylose-based columns.

Remarkably, this study conclusively demonstrates that 1-(iodoethynyl)-3-arylferrocenes may be involved in dispersion-based interactions with amylose-based chiral selectors irrespective of the steroelectronic properties of the distinctive pendant groups of the selector. Indeed, a huge increase of the affinity of the second eluted (R<sub>p</sub>)-enantiomers was observed with Lux Amylose-1, i-Amylose-1, and i-Amylose-3. Among the studied ferrocene derivatives, the 1-(iodoethynyl)-3-(4-t-butyl)phenylferrocene 5 exhibited a huge affinity toward the Amylose-1 chiral selector, especially under hydrophobic conditions. Under these elution conditions, it was possible to deconvolute the hydrophobic effect underlying the affinity of the first eluted (S<sub>p</sub>)-enantiomers of 5 and its analogues from the effect related to dispersion forces underlying the affinity of the second eluted (R<sub>p</sub>)-enantiomers. In water-containing chromatographic systems, the balance between repulsive, hydrophobic and dispersion forces was shown to determine the thermodynamics of binding and recognition and, in turn, the chromatographic outcomes. The polarizability of the mobile phase and the distinctive structure of the ferrocenyl selectands contributed to determine the chromatographic outcomes. In the case of derivative 5, dispersion forces were shown to be dominant compared to repulsive forces, whereas the opposite occurred for the other compounds of the series, including compound 4. This observation was distinctive for compound 5, and this was not surprising because dispersion forces take place under strict structural requirements able to produce a subtle balance between surface extension, electron oscillation ability due to the presence of extended π-electron clouds, and steric hindrance resulting in successful attraction. On the other hand, the highest value of ZPVE was computed for 5 (226.16 kcal·mol<sup>-1</sup>).

Finally, the findings of this study contribute to confirm the following general principles and observations: a) although dispersion forces are classically considered weak, these noncovalent interactions are additive and can contribute to enantioselective recognition with remarkable strength. For this purpose, a careful and fine design is necessary to enhance the dispersion-based interaction ability of a molecular system by balancing repulsion and attraction effects [3,4]; b) aromatic moieties containing bulky alkyl groups can be used as design elements to improve the ability of selector and selectand to participate in dispersion forces. This strategy was successfully used in catalysis [3,47]; c) as already demonstrated in other fields of chemical sciences [26], the ferrocenyl moiety and compounds containing extended π-electron clouds can
effectively participate in dispersion-based interactions in liquid-phase separation. For instance, brush-type stationary phases based on pendant groups featuring extended π-electron clouds were found to function through dispersion forces [29,30]; d) it should be carefully considered that dispersion forces were demonstrated to impact on the electronic description of π-π stacking and strongly contribute to this type of noncovalent interactions [2,24].

These considerations pave the way to the design and utilization of new molecular systems tailored for dispersion-driven interactions even in enantioseparation science.

### CRediT authorship contribution statement

**Barbara Sechi:** Formal analysis, Data curation. **Alessandro Dessi:** Formal analysis. **Roberto Dallocchio:** Formal analysis. **Saba Jorbenadze:** Writing - review & editing, Formal analysis. **Natia Tchanturia:** Formal analysis, Data curation. **Bezhan Chankvetadze:** Writing - review & editing, Resources. **Sergio Cossu:** Writing - review & editing, Data curation. **Iza Matarashvili:** Writing - review & editing. **Victor Mamane:** Writing - review & editing, Project administration, Funding acquisition. **Paola Peluso:** Writing - original draft, Project administration, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

We thank Consiglio Nazionale delle Ricerche (Grant no.: SAC_CNR-SRNSF_GEORGIA_URI) and SRNSFG (Shota Rustaveli National Science Foundation of Georgia) (Grant no.: CNR-24-134) for the Italy-Georgia Joint Bilateral Agreement, Shota Rustaveli National Science Foundation of Georgia (grant N. FR-24-1766), and the University of Strasbourg, the Centre National de la Recherche Scientifique (CNRS), and the French National Research Agency (Grant no.: ANR-21-CE07-0014) for financial support.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2025.344853.

### Data availability

Data will be made available on request.

### References

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