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Review

Recent advances in chiral separation of amino acids using capillary electromigration techniques Alessandro Giuffrida a,∗ , Giuseppe Maccarrone b , Vincenzo Cucinotta b , Serena Orlandini c , Annalinda Contino b,∗∗ a

Institute of Biostructure and Bioimaging, National Research Council (CNR), via P. Gaifami 18, 95126 Catania, Italy Department of Chemical Sciences, University of Catania, viale A. Doria, 6, 95125 Catania, Italy c Department of Chemistry “U. Schiff”, University of Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy b

a r t i c l e

i n f o

Article history: Received 16 April 2014 Received in revised form 8 August 2014 Accepted 11 August 2014 Available online xxx Keywords: Amino acids Chiral separations Capillary electrophoresis Capillary electrochromatography Microchip electrophoresis Capillary electrophoresis–mass spectrometry

a b s t r a c t This review highlights recent progresses in the chiral recognition and separation of amino acid enantiomers obtained by capillary electromigration techniques, using different chiral selectors and especially cyclodextrins, covering the literature published from January 2010 to March 2014. Sections are dedicated to the use of derivatization reagents and to the possibility to enantioseparate underivatized amino acids by using either ligand exchange capillary electrophoresis (LECE) and capillary electrophoresis (CE) coupled on line with mass spectrometry. A short insight on frontier nanomaterials is also given. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Derivatized amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ligand exchange capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CE–MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Chiral molecules in nature exist almost exclusively as single enantiomers, a property that is critical for molecular recognition and replication processes and would thus seem to be a prerequisite for the origin of life [1]. Amino acids (AAs) are also chiral and until 50 years ago, scientists believed that only their l forms are of relevance

∗ Corresponding author. Institute of Biostructure and Bioimaging, National Research Council. Tel.: +390957385073. ∗∗ Corresponding author. Department of Chemical Sciences, University of Catania. Tel.: +390957385150. E-mail addresses: [email protected] (A. Giuffrida), [email protected] (A. Contino).

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in living organisms [2]. However studies in the last decades [3,4] have shown that d-AAs are widely present in the tissues of higher organisms, including humans, either as products of the vital activities of endogenous flora or during spontaneous racemization of l-AAs in the structure of polypeptides during ageing [5,6], as well as endogenous active substances and biomarkers [7,8]. On the other hand, alterations in d-amino acid concentrations may be related to different pathological states, as in Alzheimer’s disease [9,10]. Foodstuffs are the most significant sources of non-natural damino acids. In fact, modern food industry technology applies a different range of procedures to modify the characteristics of proteins to improve the flavour, consistency, and nonperishability of food and thus d-AAs generated during technological processing are found in commercial foodstuffs and alcoholic beverages. The

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presence of free d-AAs in foodstuff is an indication of microbial contamination, making these compounds as indicators of food quality [11]. In fact, even a minor degree of racemization on the proteins’ AAs is the cause of a reduced digestion of such proteins impairing the nutritional quality of an edible product. Detection of d-AAs in foodstuff also allows to assess authenticity and adulteration of foods and beverages [12], either as regard distinction between wild and transgenic materials [13,14]. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chromatography (GC) have been extensively used for the enantiomeric separation of single l-amino acids and their corresponding d-enantiomers, giving rise to high resolution values and detectability of amino acids in picomoles and femtomoles [15]. However nowadays there is no doubt that Capillary Electrophoresis (CE) has evolved as an attractive and powerful separation technique providing high separation efficiency and short migration times and needing low sample volumes [16,17]. In this field the use of single isomer chiral selectors [18] and the deep knowledge of solution equilibria involved in the separation processes have made possible a fine tuning of the experimental conditions used, allowing significant improvements in the chiral analysis of amino acids, especially if chiral separations exploit the formation of metal complexes, as in ligand exchange capillary electrophoresis (LECE), where the solution equilibria involved are well known in numerous cases [19]. This review focuses on recent results in the chiral separation of amino acids by capillary electromigration techniques, covering the literature published from January 2010 to March 2014. To make easier the discussion of the literature data we have divided them in four different sections dedicated to derivatized amino acids, ligand exchange capillary electrophoresis, CE coupled with mass spectrometry (MS) and nanomaterials in capillary electromigration techniques, respectively. We have summarized the different experimental conditions and performances in Table 1, which describes the different developments of the chiral separation of amino acids that were published in the investigated time interval.

2. Derivatized amino acids Enantiorecognition of amino acids by capillary electromigration techniques is accomplished in most cases on their derivatives. In fact, amino acids lack chromophores and fluorophores, except for the aromatic amino acids tryptophan, tyrosine and phenylalanine and in less extent histidine. Several reagents, such as o-phthaldialdehyde (OPA), benzoylchloride, naphthalene-2,3dicarboxaldehyde (NDA), fluorescein isothiocyanate (FITC), dansylchloride (Dns), and 9-fluoroenylmethylchloroformate (FMOC) have been synthesized for the derivatization of amino acids. This introduces an additional analytical step, but offer interesting advantages during the analysis of these molecules. Besides the sensitivity enhancement achieved by using adequate probes together with fluorescence detection, derivatization frequently facilitates the chiral separation of amino acids. In fact, enantiorecognition based on macrocyclic receptors involves multimodal interactions via hydrogen bonding, ␲–␲ interactions, steric hindrance, hydrophobic interactions and so on. For derivatized amino acids the inclusion in and the interaction with the chiral selector becomes more discriminating not only due to the increase in size but also due to the new interactions involving the label. Several derivatization labels and techniques have been employed. Jiang et al. [20] exploited the inclusion properties of a class of macrocyclic antibiotics achieving the enantioseparations of a series of N-benzoylated derivatives of four amino acids using a CE method, which combined the partial filling technique with the dynamic coating technique and the co-EOF

Fig. 1. Evolution of analyte zone in the separation and stacking of neuro-chemicals. (A) Filling of capillary with 1.5 MTB (pH 10) containing 12.5% (v/v) IPA; (B) injection of a large volume of anlaytes solution; (C) stacking of 16 analytes by PEO solution (150 mM TB pH 8.5, 35 mM STDC, 35 mM ␤-CD and 12.5% (v/v) IPA); (D) separation of the stacking CBI-dl-amino acids PEO. The ␮EOF and ␮EP represent the EOF mobility and the electrophoretic mobilities of cationic and anionic neurochemicals, respectively. The detection wavelength was set at 260 nm [23].

electrophoresis technique, highlighting the important role of non covalent interactions in the enatiorecognition process. Enantioseparation of 28 N-benzoylated amino acids was also investigated using the partial filling technique (PFT) on both polycationic polymer hexadimethrine bromide (HDB) modified capillary and eCAP neutral capillary, respectively and bromobalhimycin as CE additive [21]. In the eCAP neutral capillaries 26 of 28 tested racemic amino acid derivatives were almost baseline resolved without observing any interference from the front of the bromobalhimycin plug. Due to low cost of chemicals, short reaction times, high stable derivatives, and high yield derivatives, the FMOC group is one of the most useful labelling groups for ␣-amino acids, providing also the advantage of high sensitivity in fluorescence detection. Wang et al. [22] developed a highly sensitive method for enantioseparation of trace fenoprofen and amino acid derivatives by capillary electrophoresis with vancomycin as the chiral selector. By combining different techniques, i.e. large-volume sample stacking using EOF pump with anion-selective exhaustive injection (LVSEP–ASEI), they obtained more than 1000-fold enhancement in detection sensitivity compared with the normal injection mode. FMOC derivatization was also successfully used for the simultaneous separation and concentration of 9 pairs of amino acid enantiomers by combining poly (ethylene oxide) (PEO)-based stacking, ␤-cyclodextrin (␤-CD)mediated micellar electrokinetic chromatography (MEKC) [23]. The simultaneous use of the on-line sample preconcentration (Fig. 1) and the FMOC derivatization enabled detection at the nanomolar level (40–60 nM), as well as the determination of FMOC-derivatized dl-Trp, dl-Phe and dl-Glu in three different types of beer, demonstrating the potential of this method for food analysis. Fluorescein isothiocyanate (FITC) is a commonly used fluorescent labelling reagent for high sensitive detection by the application of laser induced fluorescence (LIF). This derivatization has been successfully used to test the performances of microfluidic glass chips with durable modification of the channel surface with the neutral hydrophilic-coating material poly(ethylene glycol) PEG-1M-100 in microchip electrophoresis (MCE) [24]. The results showed that both

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Table 1 Experimental conditions of enantiomeric separations of Amino Acids.

Derivatized amino acids

Analytes

Technical

Separation conditions

Detection

References

N-benzoylated-(Ala, Leu Met, Threo)

Partial filling technique, dynamic coating technique

DAD, 214 nm

[21]

FMOC-(Val, Met, Phe, Leu, Ser, Ala) FMOC-(Asn, His, Val, Leu, Trp, Phe, Glu, Asp, Lys)

Partial filling technique

50 mM Tris–phosphate buffer solution (pH 6.0) containing 0.001%, w/v hexadimethrine bromide (HDB); 8 mM balhimycin dissolved in running buffer was partially filled with a pressure of 50 mbar for various time 100 mmol/L Tris–H3 PO4 (pH 6.0) and 2 mmol/L vancomycin 1.5 M TB pH 10, 35 mM STDC, 35 mM ␤-CD and 12.5% (v/v) IPA, whereas buffer vials contain 150 mM TB pH 8.5, 35 mM STDC, 35 mM ␤-CD, 12.5% (v/v) IPA and 0.5% (w/v) PEO 10 mM Tris buffer pH 8.7 containing 1 mM ␤-CD

UV, 214 nm

[22]

UV, 260 nm

[23]

LIF: exc : 420–480 nm, em : 4515 nm

[24]

Polynorepinephrine coated capillaries, 20 mM (pH 7.5) PBS

Electrogenerated chemiluminescence detection

[26]

30 mM ␤-CD, 30 mM OS-␥-CD, 25 mM SDS, 40 mM sodium tetraborate and 17% isopropanol. GCD3AM and BCD3AM from 0.25 to 2 mM, 20 mM acetic acid/ammonium acetate solution (pH 6.4)

UV, 214 nm

[30]

PDA, 214 nm

[31]

Fluorescence detection at 520 nm

[32]

LIF: the excitation was performed by a 488 nm Ar-ion laser

[33]

UV, 260 nm

[35,37]

UV, 260 nm

[36]

UV, 260 nm

[38]

PDA, 214 nm

[39]

LIF: lexc 488, lem 520 nm

[40]

FITC-(Phe, Trp)

His, Phe Val

FMOC-(Tyr, Ser, Ala, Met, Phe, Ileu, Leu, Trp, Glu, Asp)

␤-CD-mediated MEKC

Hydrophilic coating, microchip electrophoresis Capillary electrochromatography coupled with electrogenerated chemiluminescence detection MicellarElectrokineticchromatography

FITC-(4F-dl-Ph-Gly, 4Cl-dl-Ph-Gly, 3-CF3-dl-Ph-Gly, 2-CF3-dl-Ph-Gly, 2-F-dl-Ph-Gly, 2-Cl-dl-Ph-Gly, Leu, His, Trp, Ala, Tyr, Ph-Gly, 4-NO2-dl-Phe) FITC-(Leu, Ala Glu, Asp)

CD-EKC

FITC-(Arg, Pro, Ala, Asp)

MEKC

Dns-(Aba, Aca, Met, Nrl, Nrv, Phe, Ser, Thr)

CD-EKC

Dns-(Aca, Nleu, Phe)

CD-EKC

Dns-(Aba, Nleu, Aca, Val, Leu, Met)

CD-EKC

Dns-(Asp, Met, Glu, Ser, Aba, Val, Leu, Nrv, Nrl, Phe)

CD-EKC

4-Fluoro-7-nitro-2,1,3benzoxadiazole-(Asp, Glu)

CD-EKC

MEKC

CE: 80 mM sodium borate buffer (pH 9.3) containing 12 mM ␤-CD and 18 mM sodium taurodeoxycholate (STC), SDME–CE: 2.5 nM FITC-AAs enriched by 10 min SDME 50 mM borate buffer at pH 9.6 containing 5 mM sodium dodecylbenzene sulphonate (SDBS), 10 mM␤-CD Mono-6A-[3-(4ammoniobutyl)-imidazol-1ium]-6A-deoxy-␤-cyclodextrin chloride (AMBIMCD), 50 mM acetate BAMIMCD in 50 mM pH 6.0 acetate buffer Mono-6A-(2-hydroxyethyl-1ammonium)-6A-␤cyclodextrin chloride (HEtAMCD), 50 mM NaH2 PO4 3-Deoxy-3-amino-2(S),3(R)-␥cyclodextrin (GCD3AM) and 6A,6D-dideoxy-6A,6D-N-[6,6 di-(␤-alanylamido)]-6,6 dideoxy-␣,␣’-trehalose]-␤-CD (THALAH), 20 mM of ammonium acetate at pH = 6.8 100 mM borate buffer pH8.0 containing 8 mM heptakis (2,6di-O-methyl)-␤-cyclodextrin (DM-␤-CD) and 5 mM 6-monodeoxy-6-mono(3hydroxy)propylamino-␤cyclodextrin (HPA-␤-CD)

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Carboxytetramethylrhodamine succinimidyl ester (TAMRA SE)-Ala, Ser, Trp, Thr, His, Leu, Ile, nor-Leu, tert-Leu, Tyr, Cys, Glu)

Ligand exchange capillary electrophoresis

CE-MS

Analytes

Technical

Separation conditions

Detection

Capillary array electrophoresis (CAE)

100 mM Tris–borate buffer (pH 10.0) containing 2 mM ␤-CD and 10 mM hexamethylenediamine (HDA).

LIF: 532 nm

[41]

[2,3,4,6-Tetra-O-acetyl-lthio-␤-glucopyranose (TATG)/Ophthaldialdehyde (OPA)]-Ala

Sequential sample injection and MEKC

SDS in the 40 mM borate buffer, OPA/TATG, organic modifier, acetonitrile, acetone or methanol

UV, 340 nm

[42]

Dns-(Ala, Tyr, Val, Met)

LECE

UV, 220 nm

[48]

Dns-(Trp, Met, Asp, ABA, Leu)

LECE

DAD, 208, 254 nm

[49]

His

LECE

PDA, 290 nm

[50]

Phe, Tyr, Trp

CD-LECE

PDA, 214 nm

[51]

Dns-(Ser, Met, Ile, Phe, Tyr, Cys, Asn, Arg, Ala, His, Thr, Asp, Leu, Lys)

LECE

UV, 254 nm

[52]

Dns-(Ser, Met, Ile, Phe, Tyr, Asn, Thr)

LECE

UV, 254 nm

[53]

Dns-(Ser, Met, Ile, Phe, Tyr, Asn, Thr, Asp, Ala)

LECE

UV, 254 nm

[54]

Dns-(Ser, Met, Ile)

LECE

Low molecular weight organogel (LMOG)-filled capillary. 10 mM d-valinol and 5 mM cupric acetate monohydrate in methanol 10 mM l-lysine, 5 mM copper(II) sulfate, 25 mM ammonium acetate l-Lys or l-Orn) (2.5–5.0 × 10−4 mol dm−3 ) in 2.0 × 10−2 mol dm−3 CH3 COONH4 (pH 7.6) in the presence of CuSO4 3A-deoxy-3A-[2-(4imidazolyl)ethylamino]2A(S),3A(R)-␤-cyclodextrin (CDhm3) (0.6–1.8 mM) in 0.02 M of CH3 COONH4 (pH 6.8) in the presence of CuSO4 [Cu2+ ]/CDhm3 1:1.2 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM zinc sulfate and 15.0 mM 1-butyl-3-methylimidazolium l-Orn salts ([BMIm]Orn), adjusted to pH 8.4 with Tris 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM [C6mim][l-Lys] at pH 8.2 25.0 mM Cu(Ac)2 , 50.0 mM [l-Pro][CF3 COO] and 20% (v/v) methanol at pH 4.0 5.0 mM ammonium acetate, 100.0 mM boric acid, 3.0 mM ZnSO4 , 6.0 mM l-arginine, and 20.0 mM 1-butyl-3-methylimidazolium chloride

UV, 254 nm

[55]

3,4Dihydroxyphenylalanine (DOPA), Phe, Tyr Trp

CE–MS

200 mM formic acid, 5 mM sulfated ␤-CD

ESI-MS

[61]

CD–LECE–MS

ESI-MS

[62]

Dns-(Phe, Ser, Thr, Val)

LE–CEC–MS

ESI-MS

[63]

DOPA, Glu, Ser

Microchip electrophoresis–mass spectrometric (MCE-MS) CE–MS2

6-Mono-deoxy-6-[4-(2aminoethyl)imidazolyl]-␤-CD (CDmh) derivative (0.25 mM) in 2 mM acetic buffer (pH 5 4.8) in the presence of CuSO4 [Cu(II)]/selector 1:1.2 ratio A silica-based monolithic column chemically modified with l-pipecolic acid 15 mM ammonium acetate/acetic acid buffer (pH 5.5) and methanol (1:1), sulfated ␤-CD 15 mM ␥-CD 5 mM, 50 mM ammonium carbonate (pH 10.0) buffer

Nano-ESI-MS

[64]

ESI-MS

[65]

Fused-silica capillary coated with HDB, 0.5 mM vancomycin in 50 mM ammonium formate pH 7.0 with 10% methanol

ESI-MS

[66]

FITC-(Ala, Asn, Asp, Gln, Glu, Leu, Met, Phe, Ser, Tyr, and His) FMOC-(Ala Asn Asp Cit Gln Glu Ile Leu Met Phe Pip Pro Ser Thr Trp Tyr Val)

CE–MS2

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References

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Table 1 (Continued) Analytes

Technical

Separation conditions

Detection

References

Dinitrophenyl-(Glu, Asp, Leu, Val)

CEC

UV, 360 nm

[68]

Trp

EKC

UV, 254 nm

[69]

Phenylthio-carbamyl-(Trp, Tyr, Leu, Ser, Phe, Asp, Ala, Thr, Arg)

CEC

UV, 254 nm

[70]

Trp

Chip-based enantioselective open-tubular capillary electrochromatography (OT-CEC) Chip-based enantioselective open-tubular capillary electrochromatography (OT-CEC)

50 mM sodium tetraborate solution with specific concentration of thiolated ␤-CD-modified gold nanoparticles (GNPs) Phosphate buffer solution (pH 7) with 30% (v/v) acetonitrile, containing 0.50 mg mL−1 surface molecularly imprinted silica nanoparticles (MI-SiNPs) Silica monolith modified with bovine serum albumin–gold nanoparticles (BSA-GNPs); mobile phases containing 60% MeOH and phosphate buffer at pH 7.5 ␤-Cyclodextrin (␤-CD) conjugated graphene oxide-magnetic nanocomposites (GO/Fe3 O4 NCs), 20 mM PBS (pH 7.17)

Amperometric detection

[71]

Phosphate-buffered solution (PBS) (20 mM) at pH 7.17, bovine serum albumin (BSA)-conjugated graphene oxide–magnetic nanocomposites (GO/Fe3 O4 ) as stationary phase Teicoplanin on N-(2-aminoethyl)-3aminopropyltrimethoxysilanemodified mesoporous silica Fe3 O4 magnetic nanoparticles (AEAPTMS-MSMNPs), 0.1 M, NaH2 PO4 , HClO4 buffer at pH 2.5, 40 mM ␣-CD Cinchonidine nanocrystals immobilized capillary, 10 mM phosphate buffer (pH 5.0)

Amperometric detection

[72]

UV, 214 nm

[73]

UV, 230 nm

[74]

Nanomaterials

Trp, Thr

Trp, Phe

CE

Phe, N-(3,5-dinitrobenzoyl)-Leu

CEC

FITC-labelled d,l-tryptophan and d,l-phenylalanine enantiomers could be separated by MCE using a coated chip at reversed polarity. Li et al. [25] used a high-sensitivity LIF detector with silver mirror coating detection window and small-angle optical deflection from collinear system for CE, by using FITC-labelled amino acids as model analytes to evaluate the performance of the apparatus and optimize the design factors. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems, thus providing a new strategy for miniaturized instruments in biological or chemical analysis at low cost. Liang et al. [26] succeeded in the separation of amino acid racemates by using a polynorepinephrine adhesive coating with an electrochemiluminescence detector. In this device the Ru(bpy)3 3+ species reacting with the amino acids are generated in situ in the reaction/observation flow cell, thus avoiding unstable post-column reagent addition and the problems of both sample dilution and band broadening. Cyclodextrins are the most powerful chiral selectors exploited in electromigration techniques and thus they are extensively used up to the present as testified by several reviews recently appeared in the literature [27–29]. Fradi et al. [30] developed a new chiral MEKC analytical method for the analysis of mixtures of AAs derivatized inside the capillary with FMOC, using ␤-CD and octakis(2,3-dihydroxy-6-O-sulfo)-␥-CD as chiral selectors, SDS as surfactant and isopropanol as organic modifier, leading to the simultaneous separation of 10 pairs of FMOC–AAs. In our laboratory [31] 13 enantiomeric pairs of ␣-amino acids derivatized with fluorescein isothiocyanate were successfully separated in CE using as chiral selectors the single isomer derivatives

(SID) 3-monodeoxy-3-monoamino-␤- and ␥-cyclodextrins. The availability of SID cyclodextrins allowed the authors to go inside the mechanism of the molecular recognition and to make a fine tuning and a better design of the properties of the receptors, highlighting the key role of the cavity size and optimizing the separation performances. FITC derivatization was also used in a highly sensitive method for chiral analysis of amino acids by in-line single drop microextraction (SDME) and chiral capillary electrophoresis with LIF detection [32]. In addition to serving as a labelling reagent providing high fluorescence signal, hydrophobic FITC was primarily used as a modifier aiding the extraction of zwitterionic amino acids by blocking the amino groups and increasing the hydrophobicity, yielding 220 times increase in extraction efficiency. Several 100-fold enrichments were achieved, yielding LODs of 30–60 pM and enabling direct analysis of d-AAs in a 99% enantiomeric excess mixture. In Ref. [33] a MEKC-LIF method was developed using sodium dodecylbenzene sulphonate (SDBS) as surfactant for the determination of chiral amino acids in pomegranate juices. The use of SDBS as the micellar medium enhanced the fluorescence intensities of amino acids derivatised with FITC and allowed their separation with less ␤-CD concentration, compared to the use of SDS. Dansyl derivatization has originally been applied to the sequential analysis of peptides and proteins and finds another use in biochemistry for the fluorogenic labelling of proteins and enzymes. Dansyl chloride is the most widely used reagent for the derivatization of amino acids, due to a simple derivatization procedure, very good stability, good reproducibility and a good limit of detection for the method. In fact, besides absorbing light in the UV region,

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Fig. 2. Schematic representation of enantioseparation of mandelic acid racemate based on SID HEtAMCD having three recognition sites [38].

dansyl group is also fluorescent and thus dansyl amino acids can be detected by a fluorimetric detector [34]. The group of W. Tang succeeded in placing both imidazolium and ammonium moieties in the sidearm on the CD’s primary ring synthetizing monosubstituted dually cationic cyclodextrins [35] and dicationic cyclodextrins as a single regioisomer [36,37]. These families of compounds show high enentioselectivity towards either dansyl amino acids and acidic racemates in aqueous capillary electrophoresis. The obtainment of single isomers derivatives allowed the authors to get a deep insight into the molecular recognition processes, ascribing the high enantioselectivity of the receptors either to inclusion phenomena and to the additional electrostatic interactions between the protonated CD and the anionic analytes. The same authors obtained a novel cationic cyclodextrin with three recognition sites: the cavity of ␤-CD, ammonium cation and hydroxy group in the sidearm to contribute three corresponding driving forces, i.e. inclusion, electrostatic interaction and hydrogen bonding (Fig. 2). This novel receptor exhibits outstanding enantioselectivities toward dansyl amino acids and acidic racemates in CE [38]. Dns-AAs were also used to test the possibility of separation for a sample consisting of 10 different enantiomeric pairs of ␣-amino acids in electrokinetic chromatography by using the SID 3-deoxy-3-amino-2(S),3(R)-␥-cyclodextrin (GCD3AM) and 6A,6D-dideoxy-6A,6D-N-[6,6 -di-(␤-alanylamido)]-6,6 -dideoxy␣,␣’-trehalose]-␤-CD (THALAH) as chiral selectors [39]. Through an accurate strategy of choice of the experimental conditions and the developing of a procedure of identification of the single peaks in the electropherograms called LACI (lastly added component identification), the authors succeeded observing all the peaks, though only partly resolved, due to the 20 analytes (Fig. 3). A rapid and highly selective method has been developed for the analysis of d-Asp and d-Glu in brain tissue samples, by utilizing a dual cyclodextrin system containing 6-monodeoxy-6-mono(3hydroxy)-propylamino-␤-CD and heptakis(2,6-di-O-methyl)-␤CD and a pre-column derivatization with 4-fluoro-7-nitro-2,1,3benzoxadiazole, a fluorogenic reagent offering rapid and efficient labelling reaction while providing relatively stable fluorescent products and laser-induced fluorescence detection [40]. The method was validated for biological application, showing limits of detection for d-Asp and d-Glu of 17 and 9 nM, respectively, and a limit of quantification for both analytes of 50 nM. Carboxytetramethylrhodamine succinimidyl ester (TAMRA SE) is a good alternative to the existing fluorescent probes, exhibiting

a combination of excellent properties, such as good photostabiltiy, high extinction coefficient, and high pH insensitive fluorescence quantum yield. In Ref. [41] high-throughput enantiomeric separations of 12 TAMRA SE-AAs by a homemade 532 nm capillary array electrophoresis CAE-LIF scanner are presented. Baseline separations were achieved in 100 mM Tris–borate buffer (pH 10.0) containing 2 mM ␤-CD and 10 mM hexamethylenediamine (HDA). In Ref. [42] a novel method for automatic online monitoring and analysis of the Ala racemization reactions was described by combining sequential sample injection and MEKC enantioseparation. The pair of Ala enantiomers in the sample vial can be sequentially injected into the capillary with rapid online derivatization with: [2,3,4,6-tetra-O-acetyl-l-thio-␤glucopyranose (TATG)/ophthaldialdehyde (OPA), and then efficient chiral separated and detected by MEKC technique. The method exhibits high reproducibility with relative standard deviation values (n = 20) of 1.35%, 1.98%, and 1.09% for peak height, peak area and migration time, respectively.

3. Ligand exchange capillary electrophoresis In the field of chiral recognition LECE has been widely used, as testified by the several reviews recently appeared in the literature [43–47]. In fact, if a molecule coordinates a metal ion, this corresponds to an additional recognition site, and the resulting complex has very strict coordination requirements. Thus, the formation of ternary metal complexes with the analytes induces fine tuning of the separation, realizing selectivity hardly achievable otherwise. Furthermore, the knowledge of the solution equilibria involved in the separation processes allows the optimization of the experimental conditions. Rizkov et al. [48] used a new family of copper ligand-exchange selectors, l- or d-␤-amino alcohols, for the chiral separation of d,l-dansyl-amino acids, unmodified phenylalanine and tryptophan racemates, and ␤-blocker l,d-propranolol by SDS–micellar electrokinetic chromatography and by electrophoretic chromatography in a low molecular weight organogel (LMOG)-filled capillary. They optimized the experimental conditions, finding high resolution values in a pH range wider than that used for the copper–valine selectors that could be used only under acidic conditions (pH 3.5). The group of G. Schmid [49] succeeded in chiral separation of dansylated amino acids by ligand-exchange capillary electrophoresis using l-phenylalaninamide, l-lysine and l-threonine as chiral

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l-Lys or l-Orn as background electrolytes and utilizing accurate ex ante calculations. This has been obtained by the addition to the BGE of NaClO4 which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances, thus exploiting the slight but still detectable stereoselectivity of the ternary complexes formed between the two enantiomers and the l-Lys or l-Orn copper(II) binary complexes. The same approach was used to enantioseparate underivatized tyrosine, tryptophan and phenylalanine enantiomeric pairs, by using the copper(II) complexes of a histamine derivative of ␤cyclodextrin functionalized at the secondary rim [51]. The accurate investigation of the solution equilibria of both the binary and ternary complexes allowed the authors to obtain satisfactory resolution at appropriate analytical concentration of the ionizable CD and at a 1:1.2 copper(II) ion/ligand ratio. Although the LECE technique is well acknowledged and widely used, it still faces the pressing challenge that suitable chiral ligands are still limited. Thus it is highly advantageous to explore novel chiral ligands for constructing efficient chiral LECE systems and broadening their application range. Ionic liquids (ILs), which are a group of organic salts with melting points close to or below room temperature, possess unique chemical and physical properties, including air and moisture stability, high solubility power, low vapor pressure, and controllable chemical structure, are promising compounds in chiral ligand exchange capillary electrophoresis (CLECE). The group of L. Qi synthetized a series of amino acid ionic liquids (AAILs), either with l-Orn [52] and l-Lys [53] as anion and l-Pro [54] and l-Arg [55] as cation, obtaining baseline resolution of several Dns-d,l-amino acids. For l-Lys system [53] a comparative study between the performance of Zn(II)-l-Lys and Zn(II)-[C6 mim][l-Lys] complex was conducted (Fig. 4), highlighting the unique role of AAILs that show improved chiral resolution properties.

4. CE–MS

Fig. 3. Electropherograms showing the LACI procedure in the presence of GCD3AM at C = 1.8 mM (pH = 6.8, V = 20 kV) [39].

selectors, carrying out experiments with different central metal ions such as Cu(II), Co(II), Cd(II), Ni(II) and Zn(II). Optimal conditions were found out by studying the effect of the pH and the selector molarity on the chiral resolution and the best separation was obtained for the Cu(II)/l-lysine complex, showing a chiral resolution up to 17 for Dns-dl-Met. Some of us [50] reported on the chiral separation of underivatized d,l-histidine with the binary complexes of copper(II) with

The use of MS detection spreads the applicability of CE technique in chiral amino acids analysis by providing a consistent increase of the detection sensitivity offering simultaneously high separation selectivity and specificity. Furthermore, the use of MS detection allows the AAs analysis, without the need of any derivatization procedure, and introduces the possibility of analyte structural characterization as can be deduced from the reviews written in the last 4 years [56–60]. A chiral capillary electrophoresis–tandem mass spectrometry (CE–MS/MS) method was developed for enantiomeric quantification of 3,4-dihydroxyphenylalanine (DOPA) and its precursors, phenylalanine (Phe) and tyrosine (Tyr), by using a negatively charged chiral selector, sulfated ␤-cyclodextrin [61]. Besides the simultaneous quantification of six stereoisomers, the high enantioseparation efficiency allowed detection of trace dDOPA in l-/d-DOPA mixtures, demonstrating the enantiospecific metabolism of DOPA in PC-12 nerve cells used as neuronal model. In order to separate underivatized amino acids the copper(II) complex of a modified cyclodextrin, namely 6-mono-deoxy-6[4-(2-aminoethyl)imidazolyl]-␤-CD (CDmh), was used as chiral selector in a ligand exchange capillary electrophoresis–mass spectrometry method (LECE–MS) [62]. The use of single isomer derivatives CDs and the accurate knowledge of the solution equilibria allowed a fine tuning of the experimental conditions, using very low concentration values of the selector and achieving a large selectivity. The values of LOD obtained by LECE–MS were significantly lower than those previously obtained by LECE-UV and thus, the use of ESI-MS detection widens the possible application of LECE

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Fig. 4. Electropherograms measured from mixed Dns-d,l-AAs using a running buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 , and 6.0 mM [C6mim][l-Lys] at pH 8.2. Peak identity: (A) (1) Dns-d-Ile, (1 ) Dns-l-Ile; (2) Dns-d-Met, (2 ) Dns-l-Met; (3) Dns-d-Ser, (3 ) Dns-l-Ser; (B) (4) Dns-d-Thr, (4 ) Dns-l-Thr; (5) Dns-d-Phe, (5 ) Dns-l-Phe; (6) Dns-d-Tyr, (6 ) Dns-l-Tyr; (7) Dns-d-Asn, (7 ) Dns-l-Asn [53].

to separate UV non-absorbing analytes, as well as to use strong UV-absorbing selectors. Another approach was used by Zhang and co-workers that developed a LE-CEC–MS method for chiral separation of racemic dansyl amino acids by preparing a silica monolithic column chemically modified with l-pipecolic acid, as chiral stationary phase [63]. This monolithic column can form a stable chelating complex with Cu(II) ions, allowing the performance of CEC (capillary electrochromatography) separation by mobile phases without Cu(II) ions with MS detection. The column shows reasonable repeatability and good stability, allowing to successfully separate by CEC Dns-d,l-Phe, Dnsd,l-Ser, Dns-d,l-Thr, and Dns-d,l-Val detected by MS. Microchip electrophoresis (MCE), which can be regarded as a miniaturized version of classical CE performed on microchips, has been proven to be a speedy and highly efficient separation technique with many attractive microfluidic features. Li et al. [64] developed a microchip electrophoresis–mass spectrometric (MCEMS) method for fast chiral analysis. Enantiomeric MCE separation was achieved by means of the partial filling technique. Enantiomeric separation of 3,4-dihydroxyphenylalanine (DOPA), as well as, for the first time, of native (i.e. un-labelled) glutamic acid (Glu), and serine (Ser) was achieved within 130 s. The system, that integrates on-chip cell culture, injection of the culture solution, chiral MCE separation, and specific MS detection into a single platform, was used to study the DOPA metabolism in human SH-SY5Y neuronal cells, showing that the cells metabolized only l-DOPA and left con-existing d-DOPA intact. In order to determine the degree of racemization of the free amino acids contained in different hydrolyzed protein fertilizers used as plant biostimulants two capillary electrophoresis–tandem mass spectrometry (CE–MS2) methods were optimized by Hernandez and co-workers [65]. Using a BGE containing ␤-CD or ␥-CD, the chiral separation and identification of 13 protein amino acids and ornithine was achieved. The sensitivity obtained allowed for LODs in the 0.02–0.8 ␮M range, which is enough to detect enantiomeric impurities less than 1%. Recently the same authors developed a chiral CE–MS2 methodology for the separation of amino acids using vancomycin (VC) as chiral selector, FMOC as derivatizing agent [66]. The simultaneous use of a coated capillary and of a partial filling technique allowed to avoid the VC adsorption onto the capillary wall that results in poor separation efficiency, and the contamination of the ionization source that may cause an increase in the noise as well as a decrease in the detection sensitivity. By an accurate optimization of the experimental conditions, the CE–MS2 methodology developed enabled the chiral separation and identification of 17 amino acids with good linearity and precision (Fig. 5).

5. Nanomaterials In separation science, significant advances have been made in electrophoresis and microchip separations employing nanoparticles. Some of the latest advances using nanoparticles include their use as stationary phases for liquid chromatography, gas chromatography, and capillary electrochromatography (CEC), as well as run buffer additives to improve separation abilities in capillary electrophoresis, and for separation and selectivity control in microchip separations. In fact, nanomaterials have large surface-to-volume ratio and peculiar physical and chemical properties and thus they have been widely exploited in the field of chiral recognition by electromigration tecniques [67]. Gold nanoparticles (GNPs) modified by thiolated ␤-CD have been used as chiral selector for the enantioseparation of four pairs of dinitrophenyl-labelled amino acid enantiomers (dl-Val, Leu, Glu and Asp) and three pairs of drug enantiomers (RSchlorpheniramine, zopiclone and carvedilol) [68]. The separation, based on pseudo stationary phase-capillary electro chromatography (PSP-CEC), in which the nanoparticles play the role of the chiral stationary phase without packing the capillary, show high efficiency and resolution at a much lower concentration than the system using only the ␤-CD. Yue et al. [69] obtained surface molecularly imprinted silica nanoparticles (MI-SiNPs) in aqueous media, using l-tryptophan (l-Trp) as template molecule. The MI-SiNPs were used as pseudostationary phases in electrokinetic chromatography (EKC) for the enantioseparation of Trp, that can be fully resolved with symmetric peak shapes, mainly due to the fast mass transfer and good accessibility of the sites locating at the surface of the MI-SiNPs with well-defined shape. A silica monolith modified with bovine serum albumin–gold nanoparticles (BSA-GNPs) conjugates as chiral stationary phases for capillary electrochromatography (CEC) was obtained by chemically modifying a bare monolithic silica column with 3mercaptopropyltrimethoxysilane to provide thiol groups, followed by immobilization of gold nanoparticles [70]. By a fine tuning of the experimental conditions using phenylthiocarbamyl d/lTryptophan (PTC-d/l-Trp) as the standard model analytes, ten pairs of PTC-d/l-AAs) were successfully resolved within 18 min by CEC. Magnetic nanoparticles (MNPs), especially iron oxides (Fe3 O4 or ␥-Fe2 O3 ), have attracted great interest because of their strong magnetic properties, regular shapes, uniform sizes, and excellent biocompatibility. In addition an external magnetic field can be used to easily separate magnetic nanoparticles from complex matrices and recycle them. Liang et al. [71] developed a chip-based enantioselective open-tubular capillary electrochromatography (OT-CEC)

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Fig. 5. CE–MS2 extracted ion electropherograms for the 17 FMOC-amino acids enantiomerically resolved. CE conditions: BGE, 50 mM ammonium formate buffer (pH 7.0); partial filling, 150 s × 50 mbar of 10 mM VC in BGE; HDB-coated capillary, 50 ␮m id × 100 cm; injection by pressure at 50 mbar × 15 s, applied voltage, −20 kV; temperature, 25 ◦ C. *Unknown peaks [66].

with ␤-CD conjugated graphene oxide-magnetic nanocomposites (GO/Fe3 O4 NCs) as stationary phase. GO/Fe3 O4 NCs not only have the magnetism of Fe3 O4 NPs that make them easily manipulated by an external magnetic field, but also have a large

surface that can incorporate much more chiral selector molecules. In addition, the successful ␤-CD decorations endowed the nanocomposites with excellent wettability and led to enhanced stability against high ionic strength. Under optimized conditions

Fig. 6. The scheme of the enantioseparation on microchip system and amplified microchannel packed with GO/Fe3 O4 /␤-CD NCs. The sample reservoir (S) was filled with the mixture of d- and l-tryptophan solution. Separation reservoir (B) and injection waste reservoir (SW) were filled with running buffer [73].

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baseline separation of tryptophan enantiomers was achieved in less than 50 s with a resolution factor of 1.65 (Fig. 6). An OT-CEC employing bovine serum albumin (BSA)-conjugated graphene oxide–magnetic nanocomposites (GO/Fe3 O4 ) as stationary phase was developed by the same authors. Successful separation of tryptophan and threonine enantiomers were achieved in less than 80 s with resolution factors of 1.22 and 1.9, respectively, utilizing a separation length of 37 mm coupled with in column amperometric detection [72]. Magnetic mesoporous material was also achieved by grafting teicoplanin on N-(2-aminoethyl)-3aminopropyltrimethoxysilane-modified mesoporous silica Fe3 O4 magnetic nanoparticles (AEAPTMS–MSMNPs) [73]. These versatile magnetic nanoparticles were effective in the direct chiral separation of five racemic compounds in phosphate buffer, as confirmed by CE for the enantioseparation of d,l-Trp. Recently organic nanocrystals from a native cinchona alkaloid, cinchonidine (CCND), were prepared [74]. Their aqueous dispersions were easily and stably immobilized onto the inner surface of a poly(diallyldimethylammonium chloride) (PDDAC) coated fused silica capillary due to electrostatic interactions between cationic PDDAC and the negatively charged organic nanocrystals without any organic synthetic processes. The CCND nanocrystals coated capillary was used in capillary electro chromatography (CEC) obtaining successful enantioseparation of dinitrobenzoyl-dl-Leu and dl-Phe. 6. Concluding remarks The great importance of amino acids in different technological fields has made them the most investigated analytes in the chiral separations area. The different sections that we use in this review to classify the pertinent experimental work identifies four different hypotheses of scientific development. From this point of view, the study of amino acids, besides the inherent interest of obtaining separation procedures more and more simple and efficient, shows an additional interest: in fact it is a significant example of the contribution that these different approaches presently give in this context, as well as of their future possible developments, which indeed appear promising for all four of them. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

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