Preparation of chitosan‐graft‐(β‐cyclodextrin) based sol‐gel stationary phase for open‐tubular capillary electrochromatography

A novel open‐tubular CEC column coated with chitosan‐graft‐(β‐CD) (CDCS) was prepared using sol‐gel technique. In the sol‐gel approach, owing to the 3D network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating isomers were shown. The column efficiencies of 55 000～163 000 plates/m for the isomeric xanthopterin and phenoxy acid herbicides using the sol‐gel‐derived CDCS columns were achieved. Good stabilities were demonstrated that the RSD values for the retention time of thiourea and isoxanthopterin were 1.3 and 1.4% (run to run, n = 5), 1.6 and 2.0% (day to day, n = 3), 2.9 and 3.1% (column to column, n = 3), respectively. The sol‐gel‐coated CDCS columns have shown improved separations of isomeric xanthopterin in comparison with CDCS‐bonded capillary column.     

Graphene oxide and reduced graphene oxide as novel stationary phases via electrostatic assembly for open‐tubular capillary electrochromatography

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open‐tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallyldimethylammonium chloride) (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet‐layer‐like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers, and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused‐silica capillary via electrostatic assembly.     

Nano‐amylose‐2,3‐bis(3,5‐dimethylphenylcarbamate)‐silica hybrid sol immobilized on open tubular capillary column for capillary electrochromatography enantioseparation

The chiral organic‐inorganic hybrid materials can exhibit a high loading, and the chiral selector nanoparticles can create efficient stationary phases for open‐tubular capillary electrochromatography (OT‐CEC). Hence, a novel protocol for the preparation of an OT column coated with nano‐amylose‐2,3‐bis(3,5‐dimethylphenylcarbamate) (nano‐ABDMPC)‐silica hybrid sol through in situ layer‐by‐layer self‐assembly method was developed for CEC enantioseparation. By controlling the assembly cycle number of nano‐ABDMPC‐silica hybrid sol, a homogeneous, dense and stable coating was successfully prepared, which was confirmed by SEM and elemental analysis. As the main parameter influencing the chiral separating effect, the nano‐ABDMPC bearing 3‐(triethoxysilyl)propyl residues concentration was investigated. The experimental results showed that 10.0 mg/mL nano‐ABDMPC bearing 3‐(triethoxysilyl)propyl residues coated OT capillary column possessed chiral recognition ability toward the six enantiomers (phenylalanine, tyrosine, tryptophan, phenethyl alcohol, 1‐phenyl‐2‐propanol, and Tröger's base) at some of the different conditions tested. Additionally, the coated OT column revealed adequate repeatability concerning run‐to‐run, day‐to‐day and column‐to‐column. These results demonstrated the promising applicability of nano‐ABDMPC‐silica hybrid sol coated OT column in CEC enantioseparations.     

金纳米粒子修饰的手性毛细管电色谱固定相的制备及性能表征

制备了粒径为15 nm的金纳米粒子(GNPs)并将其修饰到氨基衍生化的硅胶整体柱内,通过化学键合法将牛血清白蛋白(BSA)固载到GNPs的表面作为手性固定相。通过透射电子显微镜、扫描电子显微镜等方法进行表征,结果表明,GNPs分散性良好,并被成功地修饰到毛细管柱内,含量高达17.18%。优化了BSA手性柱的制备条件,最终确定了体积分数为10%的3-氨丙基三乙氧基硅烷(APTES)和15 g/L BSA为最佳反应条件。在毛细管电色谱分离模式下,对缓冲液pH值、电压等分离条件进行了考察,最终选择了10 mmol/L pH 7.4的磷酸缓冲液和15 kV运行电压作为最佳分离条件。手性柱对3种手性化合物(色氨酸、阿替洛尔和麻黄碱)有拆分效果,对色氨酸能实现基线分离。与物理吸附法相比,化学键合法制备的手性柱拆分效果好,分析物无需柱前衍生化,且色谱柱稳定性良好。该文的制备方法也为其他类型手性选择剂的引入提供了良好的思路。     

Sol‐gel technique for the preparation of β‐cyclodextrin gold nanoparticles as chiral stationary phase in open‐tubular capillary electrochromatography

A novel open‐tubular capillary electrochromatography column coated with β‐cyclodextrin was prepared using the sol‐gel technique. In the sol‐gel approach, owing to the three‐dimensional network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating enantiomers were shown. The influences of capillary inner diameter, coating time, organic modifier, buffer pH, and buffer concentration on separation were investigated. The sol‐gel‐coated β‐cyclodextrin column has shown improved enantioseparation efficiency of chlorphenamine, brompheniramine, pheniramine, zopiclone in comparison with the sol‐gel matrix capillary column. The migration time relative standard deviation of the separation of the enantiomers was less than 0.89% over five runs and 2.9% from column to column. This work confirmed that gold nanoparticles are promising electrochromatographic support to enhance the phase ratio of open‐tubular capillary electrochromatography column in capillary electrochromatography.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Use of cyclodextrin‐modified gold nanoparticles for enantioseparations of drugs and amino acids based on pseudostationary phase‐capillary electrochromatography

The application of chemical‐modified gold nanoparticles (GNPs) as chiral selector for the enantioseparation based on pseudostationary phase‐CEC (PSP‐CEC) is presented. GNPs modified by thiolated β‐CD were characterized by NMR and FT‐IR. The nanoparticle size was determined to be of 9.5 nm (+2.5 nm) by Transmission Electron Microscopy (TEM) and UV spectra. Four pairs of dinitrophenyl‐labeled amino acid enantiomers (DL‐Val, Leu, Glu and Asp) and three pairs of drug enantiomers (RS‐chlorpheniramine, zopiclone and carvedilol) were analyzed by using modified GNPs as the chiral selector in PSP‐CEC. Good theoretical plate number (up to 2.4×10⁵ per meter) and separation resolution (up to 4.7) were obtained even with low concentration of modified GNPs (0.8–1.4 mg/mL). The corresponding concentration of β‐CD in the buffer was only 0.30?0.53 mM, which was much lower than the optimum concentration of 15 mM if pure β‐CD was used as chiral selector. Our results showed that thiolated β‐CD modified GNPs have more sufficient interaction with the analytes, resulting in significant enhancement of enantioseparation. The study shed light on potential usage of chemical modified GNPs as chiral selector for enantioseparation based on PSP‐CEC.     

Separation of tryptic peptides of native and glycated BSA using open‐tubular CEC with salophene–lanthanide–Zn2+ complex as stationary phase

Open‐tubular CEC (OT‐CEC) with a new stationary phase, salophene–lanthanide–Zn²⁺ complex, has been applied to the separation of tryptic peptides of native BSA and BSA glycated by glucose and ribose. Glycation of proteins (non‐enzymatic modification by sugars) significantly affects their properties and it is of great importance from a physiological point of view. Separation of tryptic peptides of glycated BSA by CZE was poor because of their strong adsorption to the bare fused silica capillary. An improved separation of tryptic peptides of both native and glycated BSA was achieved by OT‐CEC in the fused silica capillary non‐covalently coated with salophene–lanthanide–Zn²⁺ complex, which suppressed the adsorption of peptides to the capillary and via specific interactions with some (glyco)peptides enhanced selectivity of the separation. Significant differences have been found in OT‐CEC analyses of tryptic hydrolysates of native and glycated BSA. In OT‐CEC‐UV profile of tryptic peptides of native BSA, 44 peaks could be resolved, whereas a reduced number of 38 peaks were observed in the profile of tryptic peptides of glucose‐glycated BSA and only 30 peaks were found in the case of ribose‐glycated BSA. The developed OT‐CEC can be potentially used for monitoring of protein glycation.     

One‐pot preparation of mercaptotetrazole‐silica hybrid monoliths by the thiol‐ene click reaction for mixed‐mode capillary liquid chromatography

A novel mercaptotetrazole‐silica hybrid monolithic column was prepared for capillary liquid chromatography, in which the thiol‐end mercaptotetrazole was mixed with hydrolyzed γ‐methacryloxypropyltrimethoxysilane and tetramethyloxysilane for the co‐polycondensation and thiol‐ene click reaction in a one‐pot process. The effects of the molar ratio of silanes, the amount of mercaptotetrazole, and the volume of porogen on the morphology, permeability and pore properties of the as‐prepared mercaptotetrazole‐silica hybrid monoliths were investigated in detail. A series of test compounds including alkylbenzenes, amides and anilines were employed for evaluating the retention behaviors of the mercaptotetrazole‐silica hybrid monolithic columns. The results demonstrated that the mercaptotetrazole‐silica hybrid monoliths exhibited hydrophobic, hydrophilic as well as ion‐exchange interaction. The run‐to‐run, column‐to‐column and batch‐to‐batch reproducibilities of the mercaptotetrazole‐silica hybrid monoliths were satisfactory with the relative standard deviations less than 1.4 (n  = 5), 3.9 (n  = 3) and 4.0% (n  = 5), respectively. In addition, the mercaptotetrazole‐silica hybrid monolith was further applied to the separation of sulfonamides, nucleobases and protein tryptic digests. These successful applications confirmed the promising potential of the mercaptotetrazole‐silica hybrid monolith in the separation of complex samples.     

A novel in situ strategy for the preparation of a β‐cyclodextrin/polydopamine‐coated capillary column for capillary electrochromatography enantioseparations

Inspired by the chiral recognition ability of β‐cyclodextrin and the natural adhesive properties of polydopamine under alkaline conditions, in this study, a rapid and in situ modification strategy was developed to fabricate β‐cyclodextrin/polydopamine composite material coated‐capillary columns for open tubular capillary electrochromatography. The results of scanning electron microscopy, FTIR spectroscopy, streaming potential, and electro‐osmotic flow studies indicated that β‐cyclodextrin/polydopamine was successfully fixed on the inner wall of the capillary column. This coating can be achieved within 1 h affording a greatly reduced capillary preparation time. The performance of the β‐cyclodextrin/polydopamine‐coated capillary was validated by the analysis of seven pairs of chiral analytes, namely epinephrine, norepinephrine, isoprenaline, terbutaline, verapamil, tryptophane, carvedilol. Good enantioseparation efficiencies were achieved for all. For three consecutive runs, the relative standard deviations for the migration times of the analytes for intraday, interday, and column‐to‐column repeatability were in the range of 0.41–1.74, 1.03–4.18, and 1.66–8.24%, respectively. Moreover, the separation efficiency of the β‐cyclodextrin/polydopamine‐coated capillary column did not decrease obviously over 90 runs. The strategy should also be feasible to introduce and immobilize other chiral selectors on the inner walls surface of capillary columns.     

Preparation and evaluation of a hydrophilic poly(2‐hydroxyethyl methacrylate‐co‐N,N′‐methylene bisacrylamide) monolithic column for pressurized capillary electrochromatography

A polar polymethacrylate‐based monolithic column was introduced and evaluated as a hydrophilic interaction CEC stationary phase. The monolithic stationary phase was prepared by in situ copolymerization of a neutral monomer 2‐hydroxyethyl methacrylate and a polar cross‐linker N,N′‐methylene bisacrylamide in a binary porogenic solvent consisting of dodecyl alcohol and toluene. The hydroxyl and amino groups at the surface of the monolithic stationary phase provided polar sites which were responsible for hydrophilic interactions. The composition and proportion of the polymerization mixture was investigated in detail. The mechanical stability and reproducibility of the obtained monolithic column preformed was satisfied. The effects of pH and organic solvent content on the EOF and the separation of amines, nucleosides, and narcotics on the optimized monolithic column were investigated. A typical hydrophilic interaction CEC was observed on the neutral polar stationary phase. The optimized monolithic column can obtain high‐column efficiencies with 62 000–126 000 theoretical plates/m and the RSDs of column‐to‐column (n = 9), run‐to‐run (n = 5), and day‐to‐day (n = 3) reproducibility were less than 6.3%. The calibration curves of these five narcotics exhibited good linearity with R in the range of 0.9959–0.9970 and linear ranges of 1.0–200.0 μg/mL. The detection limits at S/N = 3 were between 0.2 and 1.2 μg/mL. The recoveries of the separation of narcotics on the column were in the range of 84.0–108.6%. The good mechanical stability, reproducibility, and quantitation capacity was suitable for pressure‐assisted CEC applications.     

Alkylthiol gold nanoparticles in sol-gel-based open tubular capillary electrochromatography

A novel open-tubular capillary electrochromatography (OTCEC) column was prepared by immobilizing dodecanethiol gold nanoparticles on prederivatised fused-silica capillary columns with sol-gel technology. 3-Mercaptopropyl-trimethoxysilane (MPTMS) was selected as sol-gel precursor to develop a sol-gel layer on the inner wall of the capillary, prior to assembly of dodecanethiol gold nanoparticles onto the generated sol-gel layer through specific interaction between the gold nanoparticles and surface terminating thiol groups. The electrochromatographic behaviour of the sol-gel gold nanoparticle capillary was compared with a gold nanoparticle capillary prepared via MPTMS surface functionalisation, through variation of the percentage of the organic modifier, pH, and separation voltage. Efficient separation for a "reversed-phase" test mixture of thiourea, naphthalene, and biphenyl and for selected polycyclic aromatic hydrocarbons (PAHs) was obtained on the sol-gel based gold nanoparticle capillaries. OTCEC separations of three selected drug substances (propiophenone, benzoin, and warfarin) were also demonstrated. Scanning electron microscopy was used for the characterization of the sol-gel gold nanoparticle capillary surface. The results confirm that dodecanethiol gold nanoparticles, bound on the sol-gel-based inner layer of fused-silica capillary, can provide sufficient solute-bonded phase interactions for OTCEC with reproducible retention as well as characteristic reversed-phase behaviour.     

Preparation and application of trimethylamine amination polychloromethyl styrene nanolatex coated capillary column for the determination of bromate by field‐amplified sample stacking open‐tubular capillary electrochromatography

A new trimethylamine amination polychloromethyl styrene nanolatex (TMAPL) and TMAPL coated capillary column (ccc‐TMAPL) were successfully prepared. The TMAPL coating was characterized with reversed steady EOF values of ca. ?16.8 × 10^?5 cm² V^?1 s^?1. It was applied to establish open‐tubular (OT) CEC and field‐amplified sample stacking (FASS) OT‐CEC methods for the determination of bromate in tap water. Compared to OT‐CEC, the LOD with FASS‐OT‐CEC was improved from 80 to 8 ng/mL. The developed FASS‐OT‐CEC method was practically used for the analysis of bromate in tap water samples with recoveries ranging from 93.6 to 103.5%.     

Open tubular layer of S‐ofloxacin imprinted polymer fabricated in silica capillary for chiral CEC separation

An open tubular molecule imprinted polymer (OT‐MIP) capillary column has been prepared for chiral separation of ofloxacin enantiomers in CEC. The S‐ofloxacin imprinted OT column was fabricated by thermally initiated non‐covalent polymerization procedure inside a pretreated and silanized fused silica capillary. The template molecule was incorporated with methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA) and 4‐styrenesulfonic acid (4‐SSA) and dissolved in a porogen mixture of ACN/2‐propanol (9:1). The separation efficiency of the 4‐SSA MIP column was found quite better than that of the MIP column without 4‐SSA. It has been demonstrated that our OT‐MIP column can separate ofloxacin enantiomers with excellent chiral separation efficiency after tuning the various chromatographic conditions. The optimized chromatographic eluent was 85:15, v/v%, ACN/60 mM sodium acetate at pH 7. The separation efficiency and selectivity of chiral separation of this study were far better than those obtained by previous methods for chiral separation of R‐ and S‐ofloxacin.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

The Development of Reversed‐Phase Capillary Electrochromatography for the Separation of Steroids

This paper describes the preparation and optimization of packed capillary columns for reversed‐phase separation of steroids with CEC. The fabrication of on‐column frits is considered to be the most important step for obtaining a reproducible packed column for CEC separation. Porous silicate frits were generated in a fused‐silica capillary by heating the silica gel/sodium hydroxide solutions electrically. The optimized conditions involve silica gel (10.8%), sodium hydroxide (5.8%), and heating time (5 sec) with heating voltage (5V) for obtaining a 100‐μ end‐frit that can withstand pressure over 6000 psi. A HPLC pump was utilized to pack the 5‐μm ODS particle slurry into the capillary column. The ODS packed capillaries were then utilized for the separation of four anabolic cholesterols with a capillary electrophoresis system without pressurization of the column. The reproducibility of the packed columns was evaluated by measuring the relative standard deviations of four steroids. The relative standard deviations of migration time for column‐to‐column, day‐to‐day, and run‐to‐run are less than 7%, 2%, and 1% for four steroids, respectively.     

Pepsin‐modified chiral monolithic column for affinity capillary electrochromatography

Pepsin‐modified affinity monolithic capillary electrochromatography, a novel microanalysis system, was developed by the covalent bonding of pepsin on silica monolith. The column was successfully applied in the chiral separation of (±)‐nefopam. Furthermore, the electrochromatographic performance of the pepsin‐functionalized monolith for enantiomeric analysis was evaluated in terms of protein content, pH of running buffer, sample volume, buffer concentration, applied voltage, and capillary temperature. The relative standard deviation (%RSD) values of retention time (intraday <0.53, n = 10; interday <0.53, n = 10; column‐to‐column <0.70, n = 20; and batch‐to‐batch <0.80, n = 20) indicated satisfactory stability of these columns. No appreciable change was observed in retention and resolution for chiral recognition of (±)‐nefopam in 50 days with 100 injections. The proteolytic activity of this stationary phase was further characterized with bovine serum albumin as substrate for online protein digestion. As for monolithic immobilized enzyme reactor, successive protein injections confirmed both the operational stability and ability to reuse the bioreactor for at least 20 digestions. It implied that the affinity monolith used in this research opens a new path of exploring particularly versatile class of enzymes to develop enzyme‐modified affinity capillary monolith for enantioseparation.     

Preparation of a thiols β‐cyclodextrin/gold nanoparticles‐coated open tubular column for capillary electrochromatography enantioseparations

Inspired by the distinct chemical and physical properties of nanoparticles, here a novel open‐tubular capillary electrochromatography column was prepared by electrostatic assembly of poly(diallydimethylammonium chloride) onto the inner surface of a fused‐silica capillary, followed by self‐adsorption of negatively charged SH‐β‐cyclodextrin/gold nanoparticles. The formation of the SH‐β‐cyclodextrin/gold nanoparticles coated capillary was confirmed and characterized by scanning electron microscopy and energy dispersive spectrometry. The results of scanning electron microscopy and energy dispersive spectrometry studies indicated that SH‐β‐cyclodextrin/gold nanoparticles were successfully coated on the inner wall of the capillary column. The performance of the SH‐β‐cyclodextrin/gold nanoparticles coated capillary was validated by the analysis of six pairs of chiral drugs, namely zopiclone, carvedilol, salbutamol, terbutaline sulfate, phenoxybenzamine hydrochloride, and ibuprofen. Satisfactory enantioseparation results were achieved, confirming the use of gold nanoparticles as the support could enhance the phase ratio of the open‐tubular capillary column. Additionally, the stability and reproducibility of the SH‐β‐cyclodextrin/gold nanoparticles coated capillary column were also investigated. Then, this proposed method was well validated with good linearity (≥0.999), recovery (90.0–93.5%) and repeatability, and was successfully used for enantioseparation of ibuprofen in spiked plasma samples, which indicated the new column's potential usage in biological analysis.     

Chiral separation of amides of amino acid on a (S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamide‐bonded silica using nonaqueous capillary electrochromatography

(S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamine‐bonded silica was used as a chiral stationary phase for separation of a set of racemic π‐acidic and π‐basic α‐amino acid amides in electrolyteless ACN‐water eluents by CEC in the RP and polar organic (PO) modes. The effect of the amount of water in the ACN‐water eluent on chiral separation was examined. As water is added to ACN, retention was shortened but resolution and selectivity deteriorated severely. Retention, enantioselectivity, and resolution factors obtained in 100% ACN were compared with those in an n‐hexane‐isopropanol eluent with a small amount of water by normal phase (NP) CEC. Much shorter retention times with comparable enantioselectivities were observed with 100% ACN, demonstrating the advantage of separation on (S)‐N‐(DNB)leucine‐N‐phenyl‐N‐propylamine‐bonded silica in PO‐CEC over NP‐CEC.