Article pubs.acs.org/ac
Molecular Shape Recognition through Self-Assembled Molecular Ordering: Evaluation with Determining Architecture and Dynamics Abul K. Mallik,† Hongdeng Qiu,† Tsuyoshi Sawada,†,‡ Makoto Takafuji,†,‡ and Hirotaka Ihara*,†,‡ †
Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan ‡ Kumamoto Institute for Photo-Electro Organics (Phoenics), Kumamoto 862-0901, Japan S Supporting Information *
ABSTRACT: The relationship between molecular gel-forming compound-based double-alkylated L-glutamide-derived functional group-integrated organic phase (Sil-FIP) structure and chromatographic performance is investigated and compared with widely used alkyl phases (C30, polymeric and monomeric C18) as references. The functional group-integrated molecular gel on silica is chemically designed newly in a way that the weak interaction sites are integrated with high orientation and high selectivity can be realized by multiple interactions with the solutes. Its functions can be emphasized by being immobilizable with a terminal carboxyl group and the fact that five amide bonds including β-alanine subunit are integrated per molecule. Furthermore, its self-assembling function can be detected by monitoring of the chiroptical property. Temperature-dependent circular dichroism (CD) intensity was determined as an indicator of chirality for the gel forming compounds. 13C cross-polarization magic angle spinning (CP/MAS) NMR spectra of the Sil-FIP phase indicate that predominance of gauche conformations exists at higher temperature (above 30 °C). 29Si CP/MAS NMR were carried out to investigate the degree of cross-linking of the silane and silane functionality of the modified silica. Temperature-dependent 13C CP/MAS NMR and suspended-state 1H NMR measurements of the Sil-FIP phase exhibit the dynamic behavior of the alkyl chains. To correlate the NMR and CD results with temperature-dependent chromatographic studies, standard reference materials (SRM 869b and SRM 1647e), column selectivity test mixture for liquid chromatography was employed. Additional shape selectivity text mixtures were also used to clarify the mechanism of shape selectivity performance of Sil-FIP compared with commercially available columns. The evaluation with the spectroscopic and chromatographic analyses presents very important information on the surface morphology of the new organic phase and the molecular recognition process. Integrated and ordered functional groups were investigated to be the main driving force for very high molecular shape selectivity of the Sil-FIP phase.
L
better molecular shape selectivity for the separations of polycyclic aromatic hydrocarbons (PAHs) isomers can usually be achieved with polymeric stationary phases compared with monomeric one.5−7 There are some other factors, which influences molecular shape recognition in LC, including stationary phase bonding density,1,8−10 alkyl-phase chain length,11,12 column temperature,3,13−17 or architecture and dynamics of the bonded interphases.18,19 Longer alkyl chain phases (C30 and C34) were also developed for the separation of shape-constrained larger biomolecules, such as isomers of carotenoid and tocopherol.17,20,21 On the other hand, in the last two decades, molecular gel systems with chiral low-molecular-weight compounds have
iquid chromatography has become an indispensable tool for both routine analysis and research in the pharmaceutical, biomedical, and biotechnology industries. On an analytical level reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widespread technique, probably owing to the broad applicability of that mode of separation to a wide range of compounds and sample matrices. Therefore, the development of new chemically bonded stationary phases for reversed-phase liquid chromatography (RPLC), engineered for solving specific separation problems, has led to improved analyses of a broad range of compounds. The majority of bonded phases employed in RP-HPLC are still of the reversedphase n-alkyl type, mostly octadecylsilylated silica (C18) and C8. Two types of chromatographic sorbents can be distinguished on the basis of bonding chemistry (monomeric and polymeric C18). The separation of a certain class of isomers are important but challenging due to similar molecular shape.1−4 Generally, © 2012 American Chemical Society
Received: March 29, 2012 Accepted: June 24, 2012 Published: June 24, 2012 6577
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Figure 1. Synthesis of N′,N′-dioctadecyl-Nα-[(4-carboxybutanoyl)-β-alanyl]-L-glutamide (6)-grafted silica or Sil-FIP phase.
To study the surface morphology of alkyl stationary phase, solid-state NMR spectroscopy is one of the most powerful tools. NMR experiments can be designed to probe conformational structure and dynamic aspects as well as bonding chemistry of immobilized alkyl ligands through observation of 1 H, 13C, and 29Si nuclei present in the interphases. A number of investigations of organic phases have been reported that the combination of cross-polarization (CP) with magic angle spinning (MAS) allows acquisition of high-resolution NMR spectra of low-abundance heteronuclei (e.g., 13C and 29Si) in reasonable measuring times.42−46 In this study, a stationary phase is newly created with immobilization of molecular gel-forming compounds onto silica. The functional groups were integrated with ordered structure on silica in a way that (with the addition of β-alanine subunit on double-alkylated L-glutamide-derivative) highly ordered and concentrated interaction sites were responsible for very high molecular shape selectivity of double-alkylated Lglutamide-derived low-molecular organogelator-based organic phase even at high temperature (50 °C). The relationship between the new organic phase structure or morphology and chromatographic performance was clarified by comparing with alkyl phases (C30, polymeric C18 and monomeric C18). The influence of alkyl chain conformational order of RP materials on their separation behavior in liquid chromatography has already been investigated for C18, C22, and C30 bonded phases.19,47,48 Here we present the application and correlation of physical properties by analytical and spectroscopic methods (solid-state NMR spectroscopy, suspended-state 1H NMR, diffuse reflectance infrared Fourier transform (DRIFT), and liquid chromatography) for the study of shape selectivity with functional group-integrated organic phase on silica (Sil-
been widely investigated. These gels have attracted interest because of the fact that gelation is induced by the formation of a three-dimensional network with nanofibrillar aggregates that are based on highly ordered structures with a unique chirality.22−25 One of the successful results is seen in Lglutamide-derived amphiphiles. For example, dialkyl L-glutamide-derived amphiphilic lipids form nanotubes,26 nanohelices,27 and nanofibers23 based on bilayer structures in water and on the fact that intermolecular hydrogen bonding among the amide moieties not only contributes self-assembly27 but also shows a very unique secondary chirality with extremely strong circular dichroism (CD) signals.27 Another unique selforganization has been realized by lipophilic derivatives of Lglutamide even in organic solvents.28 Self-assembling integrated materials have also been used for molecular recognition.29−31 A number of efforts have been done toward developing an understanding of shape selective interactions between the stationary phase and the analyte and have already been reported for alkyl phases.5,6,12,32−34 However, a systematic theory describing the molecular interaction between analytes and bonded phase ligands has not been firmly established because of difficulties encountered in the surface characterization of these C18 stationary phases. However, in the past 20 years, various newly synthesized bonded phases have been introduced as stationary phases, which include synthetic and bonding chemistries.35−41 Because these new stationary phases could be synthesized according to the design of the bonded phase ligands, the systematic characterization of stationary phases has become increasingly important for the development of more powerful stationary phases with desirable separation performance for specific uses. 6578
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FIP).29Si CP/MAS NMR, 13C CP/MAS NMR, suspended-state 1 H NMR, and differential scanning calorimentry (DSC) experiments were used to characterize chain conformational structure and dynamic aspects. Circular dichroism (CD), transmission electron microscope (TEM), DSC, and FT-IR measurements were carried out to study the gelation property of the gel-forming compound before immobilization onto silica. Lastly, separations of Standard Reference Materials (SRM869b and SRM1647e) at variable temperature as shape-selective probe compounds were performed on Sil-FIP, C30, C18 (poly), and C18 (mono) columns for correlation of chromatographic retention behavior with the measured physical properties and evaluated retention mechanisms.
UV−vis photodiode array detector. As the sensitivity of UV detector is high, 5 μL sample was injected through a Reodyne Model 7125 injector. The column temperature was maintained by using a column jacket with a circulator having heating and cooling system. A personal computer connected to the detector with JASCO-Borwin (Ver 1.5) software was used for system control and data analysis. Methanol was used as a mobile phase for the separation of SRM 869b, β-carotene isomers and other PAH isomers at 10−50, 40, and 15 °C, respectively. Separations of SRM 1647e and estradiol isomers were carried out using a 80:20 and 60:40 (volume fraction) methanol/water mobile phase at 50 and 35 °C, respectively. All separations were at a flow rate of 1 mL/min. UV detection for the separation of SRM 869b, 16 PAHs (SRM 1647e), and other PAHs was at 254 nm and for β-carotene and estradiol isomers was at 450 and 226 nm, respectively. The retention factor (k) measurement was done under isocratic elution conditions. The separation factor (α) is the ratio of the retention factor of 2 solutes that are being analyzed. The chromatography was done under isocratic elution conditions. The retention time of D2O was used as the void volume (t0) marker (The absorption for D2O was measured at 400 nm). All data points were derived from at least triplicate measurements; with retention time (tR) value varying ±1%.
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EXPERIMENTAL SECTION Materials and Reagents. Standard reference material SRM 869b, Column Selectivity Test Mixture for Liquid Chromatography, and SRM 1647e, Priority Pollutant Polycyclic Aromatic Hydrocarbons, were obtained from the Standard Reference Materials Program (NIST, Gaithersburg, MD). The tocopherol isomers were obtained from CalBiochem, U.S.A. β-Carotene was purchased from Sigma, USA. 17α-Estradiol and 17βestradiol were purchased from Dr. Ehrennstorfer GmbH (Augsburg, Germany) and Sigma (St. Louis, MO, U.S.A.), respectively. L-Glutamic acid, stearylamine, diethylphosphorocyanidate (DPEC, peptide synthesis reagent), triethylamine (TEA), and β-alanine were purchased from Wako (Japan) and used without further purification. The functional groupintegrated organic phase on silica (Sil-FIP) stationary phase was synthesized, characterized, and packed into stainless steel column (150 × 4.6 mm i.d.). YMC silica (YMC SIL-120-S5 having a 5 μm diameter, a 12 nm pore size, and surface coverage 300 m2 g−1) was used. In contrast, we used commercial monomeric and polymeric C18 columns (Inertsil, ODS 3, column size 150 × 4.6 mm i.d. with a 5.5 μm particle size, a 10 nm pore size, and surface coverage 450 m2 g−1 and a highly loaded (%C 29.0) Inertsil, ODS-P, column size 150 × 4.6 mm i.d. with a 5.5 μm particle size, a 10 nm pore size, and surface coverage 450 m2 g−1 from GL Sciences, respectively), and C30 (column size 150 × 4.6 mm i.d., a 5 μm particle size, a 10 nm pore size, and surface coverage 297 m2 g−1 from Nomura Chemical Co., Ltd.) column for the comparison of chromatographic results. For the sample preparation, trans-βcarotene was photoisomerized based on a literature procedure.49 HPLC-grade solvents were used in chromatographic separations. Synthesis of the Molecular Gel-Forming Compound (N′,N′-Dioctadecyl-Nα-[(4-carboxybutanoyl)-β-alanyl]-Lglutamide (6))-Grafted Silica (Sil-FIP). The compound 6 was synthesized from N-benzyloxycarbonyl-L-glutamic acid through alkylation, debenzyloxycarbonylation and again alkylation and debenzyloxycarbonylation of β-alanine, and finally ring-opening reaction with glutaric anhydride to obtain 6 as shown in Figure 1. The compound 3 (N′,N′-dioctadecyl-Lglutamide) was synthesized according to previously reported methods.50 The detailed synthetic procedure of 6, immobilization onto silica, and other experimental details are given in the Supporting Information. Liquid Chromatography. The chromatographic system consists of a Gulliver PU-1580 intelligent HPLC pump a Rheodyne sample injector having 20 μL loop. The chromatograph included a JASCO 1580 pump and a JASCO MD-1510
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RESULTS AND DISCUSSION Self-assembled gel-forming compound (6) was designed, synthesized, and immobilized onto the silica surface (Figure 1) and used as packed column for liquid chromatography. Functional groups were integrated in such a way that five amide bonds including β-alanine subunit per molecule were grafted onto silica surface as weak interacting sites. Before immobilization, the self-assembling functions of the molecule (6) were detected by monitoring of the temperature-dependent chiroptical property. The circular dichroism (CD) intensity of 6 showed clear temperature-dependency, which is an indicator of chirality (Supporting Information, Figure S1). NK-77 as a cationic dye has no CD signal on its absorption band because NK-77 is an achiral molecule (Supporting Information, Figure S2). However, when NK-77 was mixed with anionic assemblies from 6, extremely large and positive CD signals around the absorption bands of NK-77 were detected at temperatures below 55 °C. The molecular elipticity [θ]552 reached 1.8 × 106 deg cm2 dmol−1. This large value cannot be explained by simple electrostatic interaction of NK-77 with chiral 6.27,51,52 This result indicates that NK-77 interacts with the chiral molecular gel to produce R-chirally stacked dimers or polymers. Furthermore, from this measurement we observed a phase transition between ordered and disordered structures because the CD intensity showed remarkable temperature-dependency with a bending point around 55 °C and a CD intensity reduced to 1/100 at temperatures above the bending point. To clarify the phase transition temperature, differential scanning calorimentry (DSC) was measured and it showed that the thermogram bending point was closely related to the peaktop temperature in the phase transition (Supporting Information, Figure S1). These were the evidence of selfassembling ability with chirally ordered structures of compound 6. Additionally, 6 formed a gel containing nanometer-scale organized assemblies in organic solvent. TEM image of 6 shows a fibrous aggregate prepared from benzene (Supporting Information, Figure S3). Intermolecular hydrogen bondings among the amide bonds are mainly responsible for the 6579
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Figure 2. FT-IR and DRIFT spectra of 6 and Sil-FIP in THF (solution) (a, c) and in benzene (gel state) (b, d), respectively. The solutions and suspensions were prepared at a concentration of 2 mmol and 50 wt % respectively.
presence of grafted amide bonded lipids on the silica surface. Equally important is the appearance of N−H stretching (3288 cm−1) in the spectrum for Sil-FIP, providing further evidence that FIP was successfully grafted onto the silica surface. 29 Si CP/MAS NMR was carried out to investigate the degree of cross-linking of the silane and silane functionality of the modified silica. NMR spectra of the Sil-FIP phase, Sil-APS, and the native silica are shown in Figure S6 (Supporting Information). It was reported that the signals of trifunctional species (Tn) appear in the range of −49 to −66 ppm and signals from the native silica (Qn) from −91 to −110 ppm.19 Obviously Sil-FIP phase possess high cross-linking according to the signals at −56 ppm (T2) and at −65 ppm (T3), while there is no signal for T1 species visible in the spectrum. The intensity of signal at −56 ppm for T2 is very low compared to T3 meaning these stationary phase contain trifunctional species with very high degree of cross-linking even higher than the best C22 bonded phase reported by Pursch et al..19 These resuts agree with the C/N value of elemental analysis results as discussed before. The absence of T1 groups on the grafted material proves the successful grafting and furthermore implies the high stability of the phase. In 29Si spectrum of native bare silica, the Q4 (tetrasiloxane), Q3 (hydroxysiloxane), and Q2 (dihydroxysiloxane) were detected at intense signal of −110, −101, and −92 ppm, respectively.19 However, in the spectrum of Sil-FIP dihydroxysiloxane (Q2) or geminal silanol groups is almost undetectable indicating low degree of silanol activity for the grafted materials. The reduced signal for Q3 species compared to native silica gave an insight about the lower amount of free OH-groups on the surface, which lead to less silanophilic interactions in HPLC.19,55 The solid-state 13C CP/MAS NMR56 and suspended-state 1 H NMR45 measurements were carried out at different temperatures from 20 to 50 °C to investigate the conformations and mobility of the long alkyl chains, respectively of the grafted organic phase. Under the condition of magic angle spinning and dipolar coupling of protons, the chemical shift of methylene groups in 13C CP/MAS NMR spectroscopy depends largely on the conformation of alkyl chains (CH2)n. In general, the 13C signals for alkyl chains is observed at two resonances, one is at
aggregation. We have observed that 6 can form gel before and even after immobilization onto silica as shown in Figure 2. The FT-IR and diffuse reflactance infrared Fourier transform (DRIFT) spectra have a characteristic peaks for 6 and Sil-FIP carbonyl groups at 1647 and 1642 cm−1 in THF (sol state). The peaks shift to 1634 and 1637 cm−1 in benzene (gel state), respectively, which are attributed to the hydrogen bond formation of carbonyl groups (Figure 2). We have reported that L-glutamide derivative without headgroup form globular structure. However, modification of head-groups with hydrogen bonding sites gives self-assembling aggregates.53,54 The molecular gel-forming 6 was immobilized onto 3aminopropyltrimethoxysilane (APS)-grafted silica by covalent linkages to obtain a functional group-integrated organic phase on silica (Sil-FIP) (Figure 1). The elemental analysis results for Sil-APS and Sil-FIP are shown in Table 1. From the elemental Table 1. Elemental Analysis and TGA Data of Sil-APS and Sil-FIP Stationary Phase
SilAPS SilFIP
%C
%H
%N
C/N
surface coverage (μmol m−2)
grafting (%) TGA
7.85
1.96
2.55
3.08
7.32
8.49
20.71
3.92
3.32
6.24
2.34
23.79
analysis results, the load of APS and lipid attached to the silica surface were calculated by the previous method45 as 7.32 and 2.34 μmol m−2 for Sil-APS and Sil-FIP, respectively. The C/N value of Sil-APS is 3.08, which indicates that almost all of the methoxy groups of APS were consumed for silanation to silica, for cross-linking, or for both. 29Si CP/MAS NMR can give more information about silanation and cross-linking and will be discussed in the later part. Similar amount of grafting was also confirmed by the TGA measurements (Supporting Information, Figure S4). Grafting of organic molecules on a silica surface was also confirmed by DRIFT measurement (Supporting Information, Figure S5). A group of peaks at 2923 and 2853 cm−1, respectively, were attributed to the C−H bond stretching of the long alkyl chain for Sil-FIP. The spectrum of Sil-FIP showed intense bands at 1634 and 1540 cm−1, indicating the 6580
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Figure 3. Phase selectivity (αTBN/BaP = kTBN/kBaP) plotted as a function of temperature. Column, Sil-FIP, C30, C18 (poly), and C18 (mono); Chromatograms for the separation of SRM 869b at 35 °C.
integrated and ordered functional groups activity, and molecular shape selectivity in RPLC. The stationary phases (Sil-FIP, C30, C18 (poly), and C18 (mono)) were characterized with the separation of SRM 869b as a column (shape) selectivity test mixture for liquid chromatography.60 This material consists of 1,2:3,4:5,6:7,8-tetrabenzonaphthalene (TBN), phenanthro[3,4-c]phenanthrene (PhPh), and benzo[a]pyrene (BaP) and was developed after evaluation of over 100 polycyclic aromatic hydrocarbons (PAHs) solutes; the three solutes (PAHs) selected provided the most sensitive indication of changes in selectivity due to solute shape. It was demonstrated that the elution order of these solutes is correlated with the type of surface modification chemistry used to prepare the stationary phases and the overall shape recognition ability for C18 phases.61,62 The ratio (α) of retention factors (k) for TBN and BaP (i.e., αTBN/BaP = kTBN/ kBaP) provides a measure of shape selectivity that is useful for column intercomparisons. Generally, polymeric C18 columns and longer alkyl chain columns exhibit a high degree of shape selectivity, and αTBN/BaP values typically fall within the range of 0.3−1.0. Most of the monomeric columns have lower shape recognition capabilities and routinely have αTBN/BaP values of greater than 1.0. Columns that are considered intermediate in their shape recognition abilities have αTBN/BaP values of that range from 1.0 to 1.7. Typically, at higher temperatures the shape selectivity decreases for all alkyl stationary phases, as indicated by an increase in the value of αTBN/BaP.13,19 We have observed selectivity coefficients with Sil-FIP phase ranging from 0.16 (at 10 °C) to 0.49 (at 50 °C) (Figure 3), which exhibited very high shape selectivity of Sil-FIP even at higher temperature (50 °C), although the alkyl chains were completely disordered as determined by solid-state 13C CP/MAS NMR, suspended-
32.6 ppm attributed to trans conformation, indicating crystalline and rigid state, and the other at 30.0 ppm corresponding to gauche conformation, indicating disordered and mobile state.57 Solid-state 13C CP/MAS NMR spectroscopy reveals that in Sil-FIP the conformation of alkyl chains (CH2)n can be attributed to about 50% trans and 50% gauche conformation at 20 °C (Supporting Information, Figure S7). While temperature increases crystalline state changes to amorphous structure or disordered alkyl chains. At 50 °C completely disordered alkyl chains were observed in NMR measurements. Similar results were also obtained by suspended-state 1H NMR45 in methanol (Supporting Information, Figure S8), indicating that the mobility (intensity) of alkyl chains are low at lower temperature and increased at higher temperature and the results agrees with the results of solid-state 13 C CP/MAS NMR spectroscopy. Additionally, DSC of Sil-FIP was conducted in methanol and observed that it can stay ordered crystalline state below 50 °C and disordered mobile state above 50 °C. DSC theromgram of Sil-FIP showed and endothermic peak at 49.6 °C (Supporting Information, Figure S9) that aggress with the results of solid-state and suspendedstate NMR spectroscopy. Previously, we have reported that the alkyl chains in polymeric C18 is almost all trans (ordered or very low mobility of alkyl chains) and in monomeric C18 almost all gauche (disordered or mobile) conformations in solid-state 13C CP/MAS NMR and suspended-state 1H NMR spectroscopy.45 Other groups also have investigated that NMR spectra of bonded phases with extended alkyl chains (C18 to C34), alkyl chain order increases with increasing chain length and molecular shape selectivity increases with increasing chain order.58,59 In the following sections we will discuss about our findings regarding the correlation among alkyl chain order, 6581
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state 1H NMR, and DSC measurement. If we compare the shape selectivity values with the commercial alkyl phase, we have found 0.51 (at 10 °C) to 1.8 (at 50 °C), 0.28 (at 10 °C) to 0.76 (at 50 °C), and 1.93 (at 10 °C) to 1.95 (at 50 °C) for C30, C18 (poly), and C18 (mono), respectively. These results indicate much lower shape selectivities of alkyl phases and almost no shape selectivity (except C18 (poly)) at higher temperature compared to Sil-FIP phase. Figure 3 also shows the comparative chromatograms for the studied phase with the separation of SRM 869b, only on Sil-FIP and C18 (poly) BaP elutes last (increased shape recognition) and on other phases TBN elutes after BaP (low shape recognition) at 35 °C. Among the alkyl phases C18 (poly) showed highest shape selectivity, which derived from the higher bonding density, as well as ordering of the alkyl chains as described by many groups.1,8,13,18 The reasons for the best shape selectivity of the Sil-FIP phase among the four columns will be discussed in the later part. To better assess selectivity differences of the four columns, additional extended PAH mixtures were composed and the selectivity for two-dimensional shape was studied with a molecular shape descriptor, such as molecular length and length-to-breadth (L/B) ratio. The first examination was carried out with two nonlinear and linear PAHs. Sil-FIP showed very high selectivity for benzo[a]anthracene (1) and naphthacene (2), which have the same number of carbon atoms and π-electrons but differ only in their molecular shape such as the length and aspect ratio (L = 11.1 Å, L/B = 1.60 and 12.1 Å, L/B = 1.90, respectively). The separation factor (α2/1) reaches 5.50 with Sil-FIP. On the other hand, C30 and C18 phases, which are the most widely used adsorbents for HPLC showed only α2/1 = 1.75, 1.55, and 1.13 in their C30, C18 (poly), and C18 (mono), respectively (Supporting Information, Figure S10). Other chromatographic tests have been established to assess shape recognition. For example, the selectivity for o-terphenyl (L/B = 1.11, L = 10.5 Å) and triphenylene (L/B = 1.12, L = 11.5 Å) probes with similar L and L/B values, number of carbon atoms, and π-electrons but different molecular planarity, was introduced for the evaluation of the planarity recognition capability of C18 phases by Tanaka et al.63 and Jinno et al..40 Here, we have used another mixture of o- (nonplanar), m(nonplanar), and p-terphenyl (almost planar) isomers and triphenylene (planar) to investigate both planarity and linearity selectivity with our studied columns (Supporting Information, Figure S11). Dramatic differences in selectivity were observed for the Sil-FIP, C30, C18 (poly), and C18 (mono) phases. In C18 (mono), m- and p-terphenyl was eluted together. In C30, the mixture was separated successfully in the order of o-, m-, and pterphenyls and triphenylene; however, in Sil-FIP and C18 (poly), the retention order was changed so that p-terphenyl was eluted after triphenylene, which again suggesting very high linearity/slenderness selectivity of Sil-FIP and C18 (poly) phases. Both p- (L/B = 2.34, L = 15.6 Å) and o-terphenyls (L/B = 1.11, L = 10.5 Å) possess the same number of carbon atoms and π-electrons but differ in molecular linearity or slenderness. Sil-FIP (αp‑terphenyl/o‑terphenyl = 24.9) showed a remarkably enhanced selectivity to o- and p-terphenyls than C30 (αp‑terphenyl/o‑terphenyl = 2.70), C18 (poly) (αp‑terphenyl/o‑terphenyl = 7.09), and C18 (mono) (αp‑terphenyl/o‑terphenyl = 1.45) (Supporting I nf o r m a t i o n , F i g u r e S 1 1 ) . F u r t h e r m o r e , Si l - F I P (αtriphenylene/o‑terphenyl = 11.90) showed much enhanced ability to recognize molecular planarity compared with C 30 (αtriphenylene/o‑terphenyl = 3.85), C18 (poly) (αtriphenylene/o‑terphenyl = 5.87), and C18 (mono) (αtriphenylene/o‑terphenyl = 1.95). Therefore,
Sil-FIP phase exhibits excellent linearity and planarity selectivity toward PAHs. It was demonstrated that the longer chain C30 bonded phases permit separations of carotenoids20 and tocopherols 64 compared to C18 sorbents and here we have utilized β-carotene isomers as another class of rigid, extended solutes to evaluate shape selectivity of Sil-FIP phase. Excellent separation of major isomers (13-cis, trans, 9-cis) was observed on Sil-FIP (Supporting Information, Figure S12). The difficulty in the separation of these isomers is that they differ only in the characteristics of their molecular shape, such as linearity or some bending. They clearly show that before irradiation one peak of trans was recorded; however, after irradiation several isomers were separated completely with intense peaks of major isomers (Supporting Information, Figure S12). We have also reported about the baseline separation of β- and γ-tocopherol isomers with molecular-gel forming compound-grafted silica phase.65 The separation of β- and γ-tocopherol isomers thought to be one of the most challenging separation due to their structural similarity.64 The relationship between molecular gel-forming compoundbased organic phase structure and excellent shape-selective chromatographic performances compared with alkyl phases (C30, polymeric and monomeric C18) must need to discuss here. In general, the molecular shape selectivity in the C18 and C30 phases increases with increasing carbon loading, alkyl chain length, decreasing column temperature, or depending on the architecture and dynamics of the phases as we mentioned earlier.8−19 All of these have been attributed by slight increase of alkyl chain ordering, but not for direct interaction with guest molecules. For example, increasing carbon loading and chain length as well as decreasing column temperature increases chain ordering and consequently increases shape selectivity. However, Sil-FIP showed better selectivity, regardless of the fact that it had similar/lower carbon loading (%C 12.9) than C30 (%C 17.8), C18 (poly) (%C 29.0), and C18 (mono) (%C 13.8), and the alkyl chains of Sil-FIP are not ordered completely and are rather flexible at high temperature as indicated by the NMR spectroscopy. However, the gel-forming ability or chirally ordered functional groups of glutamide derive lipid is available even at higher temperature (
