Nano-liquid chromatography applied to enantiomers separation

G Model

ARTICLE IN PRESS

CHROMA-357976; No. of Pages 15

Journal of Chromatography A, xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Review article

Nano-liquid chromatography applied to enantiomers separation Salvatore Fanali ∗ Institute of Chemical Methodologies, Italian National Research Council (C.N.R.), 00015, Monterotondo, Italy

a r t i c l e

i n f o

Article history: Received 5 September 2016 Received in revised form 1 October 2016 Accepted 11 October 2016 Available online xxx Keywords: Chiral Enantiomers Selectors Nano-liquid chromatography Nano-LC Cyclodextrins Glycopeptide antibiotics Polysaccharides Vancomycin Teicoplanin Amylose Cellulose

a b s t r a c t This paper presents the state of the art concerning the separation of chiral compounds by means of nano-liquid chromatography (nano-LC). The enantiomers’ separation and determination are a subject of fundamental importance in various application fields such as pharmaceutical industry, biomedicine, food, agrochemical etc. Nano-LC is a miniaturized chromatographic technique offering some advantages over conventional ones such as low consumption of mobile phase, sample volume and amount of chiral stationary phase, reduced costs etc. This is reported in the first part of the paper illustrating the features of the nano-LC. In addition, chiral resolution methods are briefly illustrated. Some chiral selectors, used in high-performance liquid chromatography have also been applied in nano-LC including cyclodextrins, glycopeptide antibiotics, modified polysaccharides etc. This is discussed in the second part of the review. Finally some examples of the applications available in literature are reported. © 2016 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Key features, instrumentation and usefulness of nano-LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Principles of enantiomers separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Chiral selectors and stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Chiral selectors added to the mobile phase or bonded to the capillary wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Chiral selectors bonded or coated onto particles or monolithic stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2.1. Use of monolithic capillary columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2.2. Use of packed capillaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Nano-liquid chromatography (nano-LC) is a recent developed micro fluidic technique, mainly used for analytical purposes, offering some advantages over conventional high-performance liquid

∗ Correspondence to: Institute of Chemical Methodologies, Italian National Research Council (C.N.R.), Area della Ricerca di Roma I, Via Salaria km 29.300-00015, Monterotondo, Italy. E-mail address: [email protected]

chromatography (HPLC). Because its features, this miniaturized technique has gained more and more interest in various application fields resulting either alternative and/or complementary to HPLC. Analytes separation takes place into capillary columns containing selected stationary phases (SPs) under the effect of a mobile phase (MP) delivered at low flow rates (10–700 nL/min). The SP can be either coated or bonded to i) the capillary wall (open tubular-LC, OTLC), ii) particles (packed) and iii) silica or polymeric (monoliths). The column (usually of fused silica material) has an I.D. in the range 10–100 ␮m. Because of the reduced flow rate, nano-LC results in a higher mass sensitivity when compared

http://dx.doi.org/10.1016/j.chroma.2016.10.028 0021-9673/© 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15 2

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

to HPLC. This feature is related to a lower chromatographic dilution [1]. In addition very small volumes of MP are consumed for experiments resulting in cheaper operation than HPLC. Finally the flow rate allows a perfect coupling with mass spectrometry (MS) that, on the other hand, is the best choice of detectors available. After the pioneering work done by Karlsson and Novotny [2] who demonstrated the high efficiency and shorter analysis time employing columns with small inner diameters, several other authors reported studies dealing with nano-LC considering both theoretical, instrumental and methodological approaches [1,3–11]. Because of its potential advantages over other analytical techniques, nano-LC has been successfully applied to the analysis of a large number of compounds currently studied in different application fields such as proteomics, pharmaceutical, agrochemical, food chemistry, environmental, biomedicine, chiral etc. The separation of enantiomers is a very important issue of great interest in the research field as these compounds are present in nature and are implicated in several biological processes, including those linked to human health. In the pharmaceutical field, many drugs possess chiral centres and therefore can exist as one or more couples of enantiomers with the possibility to interact differently in various biological processes. Often one of the two enantiomers shows greater pharmacological activity, however, in some cases can be even harmful. Therefore, very often, several drugs are marketed as a single enantiomer. Since these compounds possess quite similar physical chemical properties, their structure differs for the spatial orientation of substituent groups at asymmetric centre. Thus their separation is a difficult task. However in presence of a chiral environment, two enantiomers can react in a different way on forming diastereoisomeric complexes exhibiting different properties and thus promoting their separation even in a non-chiral environment. Considering the importance of chiral compounds, especially for human health, there is clearly a need to proceed to their separation, quantitation and characterization. Therefore analytical methods capable to determine accurately and reliably these compounds are required. In addition to conventional techniques such as HPLC, supercritical fluid chromatography (SFC), GC, thin-layer chromatography (TLC), miniaturized techniques such as capillary electrophoresis (CE) and nano-LC are currently applied in this field. Aim of this paper is to report about the use of nano-LC in the specific field of enantiomers separation. The main features of the technique will be briefly discussed. In addition, the currently used chiral stationary phases (CSPs) and the choice of experimental conditions will also be illustrated. Finally some selected applications will also be presented The paper is dedicated to the memory of Prof. Hanfa Zou (Dalian, PRC) who enormously contributed to develop nano-LC in various application fields [10,11].

2. Key features, instrumentation and usefulness of nano-LC Nano-LC is a miniaturized liquid chromatographic technique where analytes separation take place into capillaries, usually of fused silica material, with very thin diameter (10–100 ␮m I.D.) being applied in different fields also including enantiomers separation. In this case, the capillary contains a chiral selector (CS) either bonded or adsorbed on the capillary wall (OTLC) or to packed particles or included/bonded to polymeric material (monolithic). Finally the CS can also be added to the MP. The MP is delivered at low flow-rates (10–700 nL/min) and thus the technique offers a very high mass sensitivity. This feature is

ascribed to the lower peaks chromatographic dilution. As can be observed in the following equation [1].

D=

co cmax

=

εr 2 (1 + k) Vinj

√ 2LH

(1)

With co and c max are the sample concentrations at the injection and at the peak maximum, respectively, r the column radius, L the column length H the plate height,  the column porosity and Vinj is the sample volume injected. It is clear that a decrease of the capillary radius causes a lower value of D. The low flow rates offer additional advantages over LC techniques, e.g., lower consumption of MP with consequent limited costs of organic solvents and waste; perfect coupling with mass spectrometry. The high sensitivity above mentioned needs some more clear information, especially for those that are not familiar with nano-LC. In fact since the injected sample volumes are very low in comparison with HPLC (only 10–60 nL), the sensitivity, especially when analyzing compounds in complex matrices, is lower and therefore it is necessary to consider some pre-concentration steps in the method development. This can be done off-line utilizing, e.g., liquid-liquid extraction (LL), solid-phase extraction (SPE), molecular imprinted polymers (MIP) etc. In addition, one simple method is realized injecting relatively high sample volumes into the capillary columns after selecting carefully the solvent utilized for analytes solution. The sample solvent must have lower elution strength than the MP obtaining a focusing effect. In this way analytes are pre-concentrated at the entrance of the column, as a sharp zone, increasing the sensitivity. In one of our previous work [12] volumes of some ␤-blocker enantiomers in the range 50–2100 nL were injected and analyzed with a capillary packed with vancomycin bonded to silica particles for the enantioresolution. 1500 nL was the optimum volume injected without affecting separation and efficiency and achieving good sensitivity. Other authors reported about focusing approach for other type of applications [8,13–17]. Although nano-LC possesses the above mentioned features it is note worth mentioning that dedicated instrumentation has to be used, e.g., pumping systems capable to deliver flows at levels of nL/min, detectors with reduced cell volumes and at high frequency, nano-injectors, tube connection of thin diameters. Last but not least, appropriate interfaces (nano-spray) for MS connection. These approaches are necessary to minimize extra column band broadening that could cause a decrease of chromatographic efficiency with consequent loose of resolution. Some commercial instrumentation dedicated to nano-LC are available, however it can also be laboratory assembled. Conventional HPLC pumps, equipped with a mechanical split in order to have the nano-flow, could be used to deliver the MP. Detection makes use of capillary electrophoresis (CE) UV-detector. Here the path length of the cell is the radius of the capillary. It has also been demonstrated that nano-LC can also be carried out utilizing CE commercial apparatus. This was done by our group for the separation of racemic loxiglumide employing a short column (7 cm packed) containing silica modified with a teicoplanin [18]. Usually with laboratory assembled instrumentation, isocratic elution mode is not a problem, nevertheless to use a gradient mode is more difficult. However the literature offers some examples where a gradient elution can be carried out. For example Cappiello et al. [19] proposed the use of a ten port valve with several loops (␮L volume) containing MPs of different composition. The valve was moved and controlled by a computer The use of MS is a need to determine the analytes mass and to proceed with the characterization. Although different MS type have been coupled with the nano-LC, e.g., single quadrupole, ion-trap, time of flight (ToF), good results have been achieved with electrospray sources (ESI). Recently electron ionization (EI) has been

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model

ARTICLE IN PRESS

CHROMA-357976; No. of Pages 15

S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

3

Fig. 1. Scheme of a laboratory assembled nano-LC instrumentation.

proposed to analyze compounds that cannot be revealed in ESI [20,21]. The coupling of nano-LC with MS is quite simple; the capillary column is connected through a union to a capillary tip of low ID (<50 ␮m) and positioned in front of the MS orifice. The voltage is applied directly to the union and there is not a need to use nitrogen for nebulization. Fig. 1 shows a scheme of a nano-LC laboratory assembled instrumentation.

3. Principles of enantiomers separation As previously reported, enantiomers are compounds with very similar physical-chemical properties only differing for the space orientation of substituent groups on the asymmetric centres. Therefore their separation can take place in the presence of a chiral environment. Indirect and direct resolution methods can be used for their separation. In the first case the two enantiomers interact on forming stable diastereoisomers. Strong bonding is formed before the chromatographic separation that can take place in non-chiral SP. The method possesses some advantages, e.g. introduction of chromophores useful for UV detection; additional interaction sites etc. However it also exhibits some drawbacks like high purity of the derivatization reagent, time consuming, use of purification steps etc. [1]. Therefore the most employed enantioresolution method is the direct one. Here the CS is present into the column interacting continuously with the enantiomers to be separated. Diastereomeric complexes with the involvement of weak bonding are formed during the nano-LC and then achieving their separation. Some resolution model related to chiral recognition have been proposed, however the most simple one is the “Three point” interaction [22]. A large number of CSs have been studied and applied in GC, HPLC, CE, CEC and nano-LC [23–25], however till now there does not exist a universal CS available. The list of the most applied CS, includes: peptides, proteins, chiral amino acids, cyclodextrins or their derivatives, polysaccharides (modified amylose and cellulose), glycopeptide antibiotics, chiral surfactants, chiral crown-ethers etc [26].

4. Chiral selectors and stationary phases 4.1. Chiral selectors added to the mobile phase or bonded to the capillary wall The above mentioned CSs, utilized mainly with conventional chromatographic techniques have also been used in nano-LC employing minute amount of material. The addition of the CS to the mobile phase is one of the simplest approaches for chiral separation. This approach is particularly simple and cheap in nano-LC but expensive when using HPLC. In fact the miniaturized technique makes use of only a few mL/day of MP. Takeuchi added ␥-cyclodextrin to a mobile phase containing high amount of water (water-acetonitrile, 80:20, v/v) resolving 1,1’binaphtyl-22’dihylhydrogen phosphate enantiomers in a capillary of 350 ␮m I.D. packed with a C18 silica stationary phase [27]. Later on other studies considered this method utilizing packed capillaries of lower I.D., e.g., [28,29]. The enantioseparation of some non-steroidal anti-inflammatory drugs was achieved utilizing a C18 silica–5 ␮m particles and a methylated-␤-cyclodextrin added to the MP. In this study association constants between separated enantiomers and CD were also calculated [28]. A similar approach was done with different SP; Pynnacle II phenyl 3 ␮m silica and sulphated␤ − cyclodextrin were used for the chiral resolution of ofloxacin [29]. The resolution mechanism is based on multiple interactions, e.g., analytes-CD in the MP and adsorbed CD on the SP, analytes-SP. Although the method offers some advantages, e.g., low consumption of CS, over conventional HPLC, the number of reports related to this topic is limited. Schurig’s group reported another very interesting application of nano-LC to chiral separations utilizing derivatized CDs by OTLC. Thin capillaries (50 ␮m I.D.) modified on the wall with mono6-O-octamethylene-permethyl-␤-CD were applied to the chiral resolution of some barbiturates. The same capillary column was successfully used also in CE, SFC and GC [30]. The same group reported about the use of ChirasilDex chemically bonded onto silica capillaries of 50 or 25 ␮m I.D. for the enantiomeric resolution of some drugs and acidic compounds utilizing both OTLC and OT electrochromatography. Authors also demonstrated the possibility to couple OTLC with MS [31]. Bovine serum albumin (BSA) was

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15 4

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Fig. 2. Polysaccharide bonded on the silica capillary wall. Reproduced from [34] with permission of Wiley.

employed in OTLC for successful enantiomeric separation of some derivatized amino acids and 3-hydroxy-1,4-benzodiazepines. The CS was chemically bonded to the capillary wall [32]. Francotte and Yung reported about the separation of some pharmaceutical enantiomers employing a fused silica capillary of 50 ␮m I.D. wall coated with 3,5-dimethylphenylcarbamoyl cellulose and para-methylbenzoyl cellulose in normal and reversed phase modes. The results were compared with those obtained with HPLC and CE [33]. Finally Wakita et al. [34] studied the enantiomers separation utilizing silica capillaries (50–60 cm × 75 ␮m I.D.) where cellulose tris (3,5-dichlorophenylcarbamate) CVDCPC was bonded to the wall. Trans-stilbene oxide, laudanosine, etozolin, piprozolin enantiomers were resolved with hexane/2-propanol (95:5, v/v) or methanol. Fig. 2 shows the scheme of the capillary surface with the CS bonded. Efficiency in the range 25,000–50,000/m was observed and authors concluded that the relatively low values could be ascribed to the large I.D. of the capillary. The column resulted very stable in both reversed-phase and normal phase mode. Although the excellent results achieved, OTLC applications are rare. This is probably due to the need of thin capillaries and to the limited load of sample. 4.2. Chiral selectors bonded or coated onto particles or monolithic stationary phases Although the addition of CSs in the MP offered excellent results in the field of chiral separations, Many studies are related to the use of bonded or adsorbed CSs on particles (mainly silica) or monoliths (silica or polymeric material). Some review papers have been reported on this subject [25,35–43]. Data on this topic, available in literature in the years 2000–2016, are summarized in Table 1. However, some papers published before the mentioned times have also been reported because considered fundamental. In this table, results concerning enantiomers separation achieved by using CEC have not been considered. As can be observed in Table 1 CSs such as cyclodextrins or their derivatives, polysaccharides and glycopeptide antibiotics are the compounds frequently studied. This is due to their large enantioselectivity towards several analytes and most probably to the experience of the different authors. 4.2.1. Use of monolithic capillary columns Monolithic capillary columns have also been used for chiral separations. This material is quite interesting because it can be pre-

pared in situ. Due to the presence of mesopores and macropores of different size, relatively high flow-rates can be applied. In addition the column does not need frits that could be a problem especially when working in CEC. The backpressure measured with monolithic columns is lower than the one with packed particles. Applications done in the field of chiral separations considered either silica sol-gel method or polymerization processes [64,66–68,75]. In this section some examples dealing with the use of monolithic capillary in the field of chiral separations will be reported. A ␤-cyclodextrin phenylcarbamate was immobilized on a silica monolith and used for the chiral separation of various classes of conpounds (␣- and ␤-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminic, anticancer drugs and antiarrhythmic drugs). The results obtained with the silica monolith were compared with the ones obtained with a capillary containing a polymeric monolith methacrylate also modified with ␤-CD. Authors proposed some resolution mechanisms considering experimental conditions and chemical structure of the studied drugs. In addition they concluded that although the silica monolithic column displayed a broader enantioselectivity, the polymeric material offered more reproducible results and was easier to prepare [44]. The same group proposed the combination of nanotubes and methacrylate monoliths contained in a capillary column for the enantiomeric resolution of different classes of drugs by nano-LC. Chiral separations were obtained employing different modes, e.g., reversed-phase, polar organic and normal-phase. Among all studied compounds, baseline resolution was obtained for celiprolol, chlorpheniramine, etozoline, nomifensine and sulconazole utilizing 6% of nanotubes into the monolith material. The encapsulation of the nanotubes into the polymeric monolith was effective for not all analyzed compounds. The enantioresolution was influenced by the concentration of the CS as well as by the elution conditions used. High concentrations of the CS could not be used because the compound formed aggregates that caused poor repeatability [45]. A very interesting study related to the use of silica monolith modified with a CD polymer by both nano-LC and CEC was presented by Schurig’s group. A fused silica capillary was packed with silica material modified with Chira-Dex (permethyl␤-cyclodextrin) and a sol-gel process applied. The column was used with a conventional instrumentation for the separation of several enantiomers including hexobarbital, carprofen and MTH-proline in nano-LC; the applied pressure was 10 bar. Data achieved by CEC indicated that this technique offered higher efficiency and higher resolution factors than nano-LC [52]. In a recent study Zou’s group synthesized a hybrid monolithic capillary column employing a sol-gel methodology also introducing amino groups with the aim to use it for CEC experiments. The monolith was coated with 30 or 60 mg/mL of cellulose tris (3,5-dimethylphenyl-carbamate) (CDMPC). The column was also applied to nano-LC for the chiral separation of some model racemic compounds. Good results were obtained eluting benzoin, transstilbene oxide, flavanone, praziquantel, alprenolol enantiomers with normal and reversed-phase. Good repeatability was obtained and enantioresolution and efficiency were influenced by the concentration of the adsorbed CS. Analytes were eluted in reversedphase mode with a mixture containing acetonitrile/acetate buffer (5 mM, pH 9.7) (30:70, v/v) with flow rate of 100 ␮L/min. Excellent chiral resolution was achieved for benzoin, pindolol and tetrahydropalmantine. Some of the same compounds were also separated by CEC but utilizing a lower concentration of the CS (30 mg/mL). It was reported that in CEC efficiency was lower than in nano-LC and this was ascribed to the presence of a blank section in the column. It can be remarked that also the lower concentration of the CS did not

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

Experimental conditions

Mode/ detection

Refs.

alprenolol, bufuralol, carbuterol, cizolertine, desmethylcizolertine, eticlopride, ifosfamide, 1-indanol, propranolol, tebuconazole, tertatolol and o-methoxymandelic acid celiprolol, chlorpheniramine, etozoline, nomifensine and sulconazole

Standard

immobilized ␤-cyclodextrin phenyl carbamate

Nano-LC/UV detector at 219–270 nm

[44]

Standard

Single-walled carbon nanotubes (SWCNTs)

Nano-LC/UV detection at 240 nm or 219 nm

[45]

Propranolol, ifosfamide, diconazole, ketoprofen, tertatolol, 1-indanol, metoprolol, tebuconazole, 6-hydroxyflavanone, o-methoxymandelic acid, cizolertine, alprenolol, celiprolol nomifensine and naproxen partial resolution for praziquantel, metomidate and 5-methyl-5-phenyl-hydantoin

Standards

2,3,6-Tris(phenylcarbamoyl)-␤ −cyclodextrin-6-methacrylate

CLC/UV 254 nm

[46].

Standards

hydroxypropyl-␤-cyclodextrin (HP-␤-CD)

Nano-LC/UV

[47]

2-bromo-mandelic acid, 4-chloro-mandelic acid and 4-bromo-mandelic acid, ibuprofen, fenoprofen, indoprofen

Standards

␤-cyclodextrin

Nano-LC/UV at 214 nm

[48]

Hesperetin

Urine

phenyl-carbamate-propyl-␤cyclodextrin

Nano-LC/UV at 205 nm

[49]

Flavanone, 7-hydroxy-flavanone, 6-hydroxy-flavanone, 4 -hydroxy-flavanone, 2 -hydroxyflavanone, 7-methoxy-flavanone, 6-methoxy-flavanone, 4 -methocyflavanone, hesperetin, naringenin Ofloxacin

Standards

phenyl-carbamatepropyl–cyclodextrin

Capillary column, silica monolith Capillary column, 150 ␮m I.D, 25 cm length MP: 0.1% TFA in water (v/v) and methanol (v/v) or 0.1% TFA in water (v/v) and acetonitrile Capillary column, methacrylate monolith and 6% nanotubes Capillary, 150 ␮m I.D. and 20 cm length; Folw rate, 0.3 ␮L/min MP: methanol/water (0.1% TFA) 45:55 v/v or (0.1% TFA) 40:60 v/v or (0.1% TFA) 25:75 v/v and Methano/2-propanol at different ratio. Methacrylate polymeric monolithic column with different composition. Capillary, 25 cm x 150 ␮m I.D. MP, methanol/water (0.1% TFA) 10:90, v/v and other composition Flow-rate, 0.3 ␮L/min Capillary continuous bed (monolith) with bonded CD 19 cm x 100 ␮m I.D. MP, 20/80 methanol/water or 20/80 acetonitrile/water, both buffered with 0.1% triethylamine-acetate Polymeric methacrylate monolithic column with bonded cyclodextrin. Capillary, 21 cm x 100 ␮m I.D. MP, acetonitrile/water (95:5, v/v) or methanol/water (70:30, v/v) Flow-rate, 800 nL/min phenyl-carbamate-propyl—cyclodextrin silica particles (5 ␮m) capillary, 22 cm x 100 ␮m I.D. MP, triethylammonium acetate buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v) flow-rate, 400 nL/min phenyl-carbamate-propyl—cyclodextrin bonded to silica particles (5 ␮m) capillary, 22 cm x 100 ␮m I.D. MP, water/acetonitrile or water/methanol at different ratio flow-rate, 240 nL/min

Nano-LC/UV at 205 nm

[50]

Standard

heptakis-(2,3-diacetyl-6sulfo)-␤-cyclodextrin (HDAS-␤-CD)

Nano-LC/UV at

[29]

Standards

heptakis (2,3,6-tri-O-methyl)␤-cyclodextrin (TM-␤-CD)

Nano-LV/UV at

[28]

Naproxen, indoprofen, ketoprofen, flurbiprofen, carprofen, cicloprofen, flunoxaprofen, suprofen

5

Different C18 silica stationary phase − the Cd derivative was added to the MP Capillary, 15 cm x 100 ␮m I.D. MP, 50 mM sodium acetate,pH 3.0/acetonitrile (30: 70, v/v) with 5 mM HDAS-␤-CD; flow-rate 620 nL/min RP18 silica particles (3 ␮m) Capillary, 10 or 25 cm x 100 ␮m I.D. MP, 50 mM sodium acetate, pH 3 in 30% ACN with 30 mM TM-␤-CD; flow-rate, 200 nL/min

ARTICLE IN PRESS

Chiral selector

G Model

Sample

CHROMA-357976; No. of Pages 15

Analytes

S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

Table 1 Enantiomers separation achieved by nano-LC using monolithic or packed capillary columns.

Experimental conditions

Mode/ detection

Refs.

Peopeanolol, Met, synephrine, isoproterenol, Ac.Trp, atenolol

Standards

pernaphthylcarbamoylated ␤-cyclodextrin, peracetylated ␤-cyclodextrin, permethylated ␤-cyclodextrin

Nano-LC/UV

[51]

Hexobarbital, carprofen, MTH-proline,

Standards

Chira-Dex (permethyl-␤-cyclodextrin)

Nano-LC/UV at 240 or 230 nm

[52]

naproxen

Tablets

Methylated-␤-cyclodextrin

Nano-LC/UV at 232 nm and amperometric detection

[53]

hexobarbital, mephobarbital, benzoin, mecoprop methyl, fenoxaprop ethyl, carprofen, and ibuprofen

Standard

Permethylated ␤-CD ChirasilDex

Nano-LC/UV at 230 nm; MP at 50 bar

[54]

Aminoacids (Dansyl-Asp, −Glu, −Leu,-Phe, −Met, −Norleu, −Norval, −Thr, −Val; 9-FluorenylmethyloxycarbonylFMOC-Asp(OtBu)OH −Glu(OtBu)OH, −MetOH, −PheOH, −ValOH)

Standards

Vancomycin

Nano-LC/UV at 205 nm

[55]

Herbicides (dichlorprop, diclofop, fenoprop, fluazifop, haloxyfop, mecoprop); non-steroidal antiinflammatory drugs (carprofen, cicloprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, naproxen, suprofen)

Standards

Vancomycin

Nano-LC/UV at 205 nm

[56]

2-(6-chloro-benzothiazol-2-ylsulfanyl)-, 2(6-methoxy-benzothiazol-2-ylsulfanyl)-, 2-(quinolin-2-yloxy)-, 2-(6-chloro-quinolin-2-yloxy)-, 2-(7-chloro-quinolin-4yloxy)-propionic acid atenolol

Standards

vancomycin

Silica with bonded CS Capillary, 18 cm x 100 ␮m I.D. MP, Triethylammonium acetate/MeOH or phosphate buffer/ MeOH Flow rate, 0.35 mm/s silica monolith Capillary 20 cm (overall length 42 cm) 100 ␮m I.D. MP, 20 mM MES, pH 6/methanol (70:30, v/v) or sodium acetate, pH 4.5/MeOH Pressure 10 bar − Capillary 22 cm x 75 ␮m I.D. packed with C18 , Hypersil–5 ␮m, MP, methylated-␤-CD and sodium acetate, pH 3/acetonitrile (80:20, v/v) Flow-rate, 800 nL/min capillary, silica monolith with chemically bonded CD 17–25 cm length x 10 ␮m I.D. MP, 20 mM morpholino ethane sulphonic acid (MES), pH 6/MeOH (70:30, v/v) vancomycin silica hydride stationary phase column Capillary, 11 cm x 75 ␮m I.D.-1.8 ␮m particles. MP, 500 mM ammonium formate, pH 2.5/H2O/MeOH/ACN (6:19:12.5:62.5, v/v/v/v). Flow-rate 130 nL/min vancomycin silica hydride stationary phase column. Capillary, 11 cm x 75 ␮m I.D.-1.8 ␮m particles. MP, 500 mM ammonium acetate buffer, pH 4.5/H2O/ACN (1:9:90, v/v/v), elution in isocratic mode, flow rate 360 nL/min or 500 mM ammonium acetate buffer pH 4.5/H2O/MeOH (5:10:85, v/v/v), elution in isocratic mode, flow rate 230 nL/min Silica particles with bonded vancomycin Capillary, 22.8 cm x 75 ␮m ID MP, 5% of 500 mM ammonium acetate pH 5.5/water/methanol (5:35:60, v/v/v); flow rate, 130 nL/min.

Nano-LC/UV at 230 nm

[58]

Urine

teicoplanin

Nano-LV/UV at 205 nm and MS

[59]

Pharmaceutical formulations

Hepta-Tyr glycopeptide antibiotic (Teicoplanin derivative)

Nano-LC/UV at 206 nm; CE commercial instrumentation

[18]

Loxiglumide

Silica particles (5 ␮m) with bonded teicoplanin Capillary, 23 cm x 75 ␮m I.D. MP, 500 mM ammonium Acetate, pH 4.5/methanol/acetonitrile (1:60:39, v/v/v); flow-rate, 900 nL/min Silica-bonded Hepta-Tyr; Capillary, 7 cm x 75 ␮m I.D. MP, sodium phosphate buffer, pH 6/acetonitrile (1:1, v/v); flow under 12 bar pressure

ARTICLE IN PRESS

Chiral selector

G Model

Sample

S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Analytes

CHROMA-357976; No. of Pages 15

6

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

Table 1 (Continued)

Chiral selector

Experimental conditions

Mode/ detection

Refs.

Herbicides (Dichlorprop, mecoprop, fenoprop)

Standards

vancomycin

Nano-LC/UV at 195 nm

[60]

Fenoprofen, ibuprofen, ketoprofen, naproxen

Standards

vancomycin

Nano-LV/UV

[61]

alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol, ketoprofen, indoprofen, suprofen, 2-[(4 −benzoyloxy-2 −hydroxy)phenyl] propionic acid, 2[(5 −benzoyl-2 −hydroxy)phenyl]propionic acid Etozoline, praziquantel, troger’s base, flavanone, 4 -methodyflavanone, 7-methoxyflavanone

Pharmaceutical formulation containing metoprolol

vancomycin

Silica particles (5 ␮m) with bonded vancomycin Capillary, 21 cm x 75 ␮m I.D. MP, mixtures of 500 mM ammonium acetate, pH 4.5 or pH 6.0 buffer/methanol/water; flow-rate, 60 nL/min Silica with bonded CS Capillary, 40 cm x 50 ␮m I.D. MP, 6 mM acetate buffer at pH 2.9-6.2, and 10% (v/v) acetonitrile Flow rate, 2 ␮L/min Silica particles (5 ␮m) with bonded vancomycin Capillary, 23 cm x 75 ␮m I.D. MP, 5% 100 mM ammonium acetate, pH 4.5 containing 80% acetonitrile, 5% water and 10% of different organic modifiers (methanol or acetonitrile)

Nano-LC/UV

[62].

Standards

amylose tris(3,5dimethylphenylcarbamate)

Nano-LC/UV at 205 nm Data compared with CEC

[7]

Amlodipine and two impurities

Pharmaceutical formulation

cellulose tris(4-chloro-3methylphenylcarbamate)

Nano-LC/UV at 206 nm

[57]

Etozoline, praziquantel, temazepam, thalidomide, teans-stilbene oxide, troger’s base, warfarin

Standards

Cellulose tris(4-chloro-3methylphenylcarbanate)

Nano-LC/UV at 205 nm, results compared with CEC

[6]

9-Fluorenylmethyl chloroformate-FMOC aminoacids (Cys, Pro, Ile, Pipe, Hys, Thr, Asn, Gln, Leu, Allo, Met, Phe, Val, Pyro, Lys, Cit, Orn, Arg)

Food supplements

cellulose tris(3-chloro4-methylphenylcarbamate)

Nano-LC/UV at 210 and 260 nm performed with CE commercial instrumentation

[5]

Flavanone, ethozoline, temazepam, lopirazepam

Standard

Amylose tris(5-chloro-2methylphenylcarbamate

Silica core-shell particles with coated amylose (1–5%). Capillary 23 cm x 100 ␮m I.D. particles, 2.6 and 2.8 ␮m 100 and 300 A. MP, 500 mM ammonium acetate Buffer, pH 4.5/H2O/methanol/acetonitrile (1:24:20:55, v/v/v/v) Sepapak 4 silica particles with coated cellulose. Capillary, 30 cm x 100 ␮m MP, acetonitrile/water, (90:10, v/v) containing 15 mM ammonium borate, pH 10; flow-rate 100 nL/min Silica porous and core-shell particles coated with cellulose derivative Capillary, 25 cm x 75 ␮m; Fow-rate 50–450 nL/min. MP, acetonitrile/H2O (70:30, v/v) containing 5 mM ammonium acetate, pH 4.5 final concentration Sepapak-2 or Lux Cellulose-2 capillary packed with amino- silica based particles (5 ␮m) coated with cellulose (25%). Capillary, 24 cm x 100 ␮m. MP, 0.5 M ammonium formate/H2O/ACN (1:19:80, v/v/v) at flow-rate applying 12 bar Aminopropylsililica particles (5 ␮m) coated with amylose (5–25%) Capillary 25 cm x 100 ␮m I.D. MP, 500 mM ammonium acetate, pH 5.5/H2O/methanol/acetonitrile, 1:4:25:70 (v/v/v). Flow-rate, 170–210 nL/min

Nano-LC/UV at 214 nm

[65]

ARTICLE IN PRESS

Sample

G Model

CHROMA-357976; No. of Pages 15

Analytes

S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

Table 1 (Continued)

7

Experimental conditions

Mode/ detection

Refs.

Trger’s base, trans-stilbene oxide, benzoin, 1,2,2,2-tetraphenylethanol, 2-phenylcyclohexanone (5); 2,2,2-trifluoro-1-(9anthryl)ethanol, cobalt(III) tris(acetylacetonate), flavanone, trans-cyclopropandicarboxylic acid dianilide, 2,29-dihydroxy-6,69-dimethylbiphenyl.

Standards

Silica monolith with bonded CS. Capillary, 12 cm x 100 ␮m I.D. MP, n-hexane/2-propanol (90:10 v/v) Flow-rate under 2 MPa

Nano-LC/UV at 254 nm

[66]

Troger’s base, trans-stilbene oxide, Benzoin, 1,2,2,2-tetraphenylethanol, 2-phenylcyclohexanone, 2,2,2trifluoro-1-(9-anthryl)ethanol, cobalt(III) tris(acetylacetonate), flavanone, trans-cyclopropandicarboxylic acid dianilide, and 2,2 -dihydroxy-6,6 dimethylbiphenyl. 2,2,2-trifluoro-1-(9-anthryl)ethanol, benzoin, 2,2 9-dihydroxy-6,6 9-dimethylbiphenyl, trans-cyclopropanedicarboxylic acid dianilide, flavanone, trans-stilbene oxide, piprozolin, propranolol, oxprenolol, alprenolol norgestrel

Standards

cellulose 2,3- bis(3,5dimethylphenylcarbamate)-6(3,5dimethylphenylcarbamate)/(2methacryloyloxyethylcarbamate) (CMDMPC), cellulose 2,3bis(3,5dichlorophenylcarbamate)-6(3,5dichlorophenylcarbamate)/(2methacryloyloxyethylcarbamate) (CMDCPC), and amylose 2,3bis(3,5dimethylphenylcarbamate)-6(3,5dimethylphenylcarbamate)/(2methacryloyloxyethylcarbamate) (AMDMPC) amylose tris(3,5dimethylphenylcarbamate)

Silica monolith with adsorbed amylose at different concentration Capillary, 20 cm x 100 ␮m I.D. MP, n-Hexane/2propanol 90:10 (v/v); flow rate at 0.1–12 MPa

Nano-LC/UV at 254 nm

[67]

Standards

cellulose tris(3,5dimethylphenylcarbamate) (CDMPC)

Silica monolith coated with CDMPC. Capillary, 20 or 12 cm x 100 ␮m I.D. MP, n-hexane/2-propanol, 90:10 or 80:20 (v/v); flow-rate under pressure of 0.1–12 MPa

Nano-LC/UV

[68]

Commercial formulation drug

cellulose tris(3,5dichlorophenylcarbamate) (CDCPC).

Nano-LC/UV at 254 nm using a CE commercial instrumentation

[69]

Ambucetamide, aminogluthethimide, etozoline, nifutimox, norgestrel, omeprazole, thalidomide analog, troger’s base

Standards

amylose tris(3,5dimethylphenylcarbamate) (ADMPC)

Nano-LC/UV at 214 nm using a CE commercial instrumentation

[70]

trans-stilbene oxide, warfarin, praziquantel, bendroflumethiazide, benzoin.

Standard

cellulose trisphenylcarbamate

Silica particles coated with cellulose 5–25% (w/w) Capillary 24 cm x 100 ␮m I.D. MP, 5 mM ammonium acetate in methanol; flow-rate at 12 bar Spherical silica gel Daisogel, pore size of 200 nm, surface area 15 m2/g, and particle diameter 5 ␮m. Capillary, 24 cm x 100 ␮m I.D. MP, 5 mM ammonium acetate in acetonitrile/water (60:40, v/v); flow rate at 12 bar 3-aminopropyl silica with bonded cellulose Capillary, 32 cm x 100 ␮m I.D. MP, hexane/2-propanol/THF/acetic acid (70:30:4:1 v/v) or hexane/THF/2-propanol (95:5:1 v/v/v) or hexane/methanol/2-propanol/THF (55:30:10:5 v/v) containing 5 mM acetic acidtriethylamine, pH* 6.5.

Nano-LC/UV at 214 nm

[71]

ARTICLE IN PRESS

Chiral selector

G Model

Sample

S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Analytes

CHROMA-357976; No. of Pages 15

8

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

Table 1 (Continued)

Chiral selector

Experimental conditions

Mode/ detection

Refs.

Piprozolin

Standards

Cellulose tris(3,5dichlorophenylcarbamate)

Nano-LC/UV at 214 nm using a CE commercial instrumentation

[72]

Thalidomide, 5-hydroxythalidomide, 5 -hydroxythalidomide

Standards

Nano-LC/UV at 230 nm

[73]

alimemazine, 3 -amino-6,6 -dimethyl-2-nitrobiphenyl, benzoin, N-benzoyl-phenylglycin-ethylester, cyclopentolate, 2,2 -diamino-6,6 -dimethylbiphenyl, glutethimide, hexobarbital, hydroxyzine, indapamide, ketazolam, lopirazepam, lorazepam, mesuximide, 3-methyldiazepam, metofoline, oxazepam, 1-phenylethanol, pindolole, piprozoline, propranolol, trans-stilbene oxide, trimipramine, verapamil, warfarin, penflutizide, bendroflumethiazide, paraflutizide, chlorthalidone, PTH-leucine, PTH-methionine. Mecoprop, dichlorprop, fenoprop, fenoxaprop, 3,6-dichloro-2-methoxybenzoic acid, 2,4-dichlorophenoxyacetic acid, 2-methyl-4chlorophenoxyacetic acid trans-stilbene oxide, acenaphthenol, warfarn, benzoin, fenvalerate, praziquantel

Standards

cellulose-tris(4methylbenzoate Chiralcel OJ or amylose-tris(3,5dimethylphenylenantiomers of 5-OH-TD. carbamate) (Chiralpak AD) polyacrylamide (poly-N-acryloyl-lphenylalanineethylester) and polysaccharide derivatives (cellulose tris(3,5dimethylphenylcarbamate)

Silica particles pore size (12, 20, 30, 100, and 200 nm) 5 ␮m- aminopropylsilanized Capillary, 24 cm x 100 ␮m I.D. MP, 2.5 mM ammonium acetate in methanol; flow rate at 12 bar aminopropylsilica (5 ␮m) coated with 16% cellulose and 4% cellulose OD amylose Capillary, 30 cm x 100 ␮m I.D. MP, methanol/ethanol (75:25, v/v) (a), methanol/acetonitrile (99:1, v/v). Fow rate, 200 nL/min Silica particles with bonded polyacrylamide or coated with cellulose Capillary 10 or 22 cm x 100 ␮m I.D. MP, n-hexane/2-propanol (90:10, v/v) or other mixtures; flow-rate at 12 bar or 30 bar

Nano-LC/UV at 214 nm using CE commercial instrumentation

[74]

Clenbuterol, sotalol, pronethalol, mefloquine, mefloquine-t-butylcarbamate, rimeterol, talinolol, salbutamol Indapamide, 2-phenylpropionaldehyde, trans-1,2-cyclohexanediol, 1-(1-naphthyl) ethanol, propranolol, warfarin, trans-2-phenylcyclohexanol

commercial herbicide product

Quinidine

Quinidine bonded methacrylate polymeric monolithic column. MP, 0.1 M ammonium acetate, pH 6.0/acetonitrile (40:60,v/v); flow rate 20 ␮L/min

Nano LC/UV at 210 nm

[75]

Standards

Whelk-O1

Nano-LC/UV at 214 nm or MS

[76]

Standards

trans-(1S,2S)-2-(N-4-allyloxy3,5-dichlorobenzoyl)amino cyclohexanesulfonic acid

2.5 ␮m silica particles with chemically bonded CS packed In capillary, 18 or 25 cm x 75 ␮m I.D. with only one monolithic frit MP, MeOH/H2O, 60:40 + 10 mM ammonium acetate or acetonitrile/H2O, 60:40 + 0.1% TFA or hexane/dichloromethane 60:40 + 1% methanol Flow rate, 300 nL/min Silica monolith with bonded CS Capillary 33.5 cm x 100 ␮m I.D. flow rate, 305 nL/min

Nano-LC/UV at 216 nm

[77]

Standards

diaza-18-crown-6-capped ␤-cyclodextrin

Nano-UPLC/UV at 215 nm

[78]

Silica particles (1.5 ␮m) with bonded CS Capillary, 23 cm x 75 ␮m I.D. MP, 5 mM phosphate buffer, pH 7.5/acetonitrile (80:20, v/v) Flow rate at 8000 psi

ARTICLE IN PRESS

Sample

G Model

CHROMA-357976; No. of Pages 15

Analytes

S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

Table 1 (Continued)

9

G Model CHROMA-357976; No. of Pages 15 10

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Fig. 3. Fast enantiomeric separation of 2,2,2-trifluoro-1-(9-anthryl)-ethanol achieved with a monolithic silica column (12 cm × 100 ␮m I.D.) coated with 25 mg/mL solution of CDPC in acetone. Mobile phase, n-hexane/2-propanol, 90:10 (v/v). Linear flow-rate, about 70 cm/min applying 12 MPa. Reproduced from [68] with permission of Wiley.

allow increasing the enantioresolution. In conclusion better results were obtained utilizing nano-LC [64]. The usefulness of monolithic silica columns coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) has been demonstrate previously by Chankvetadze et al. for the chiral separation of 2,2,2-trifluoro-1-(9-anthryl)ethanol, benzoin, 2,2 9-dihydroxy-6,6 9-dimethylbiphenyl, transcyclopropanedicarboxylic acid dianilide, flavanone, trans-stilbene oxide, piprozolin, propranolol, oxprenolol and alprenolol. The silica monolithic material contained in a capillary (20–10 cm × 100 ␮m I.D.) was coated with 50 mg/mL of the cellulose derivative and enantiomers separated with n-hexane/2-propanol (90:10, v/v) MP in less than 4 min. Applying relatively high flow-rates, e.g., 30 cm/min good efficiency was observed. To obtain reproducible results, the CS coating procedure was repeated three times. The monolithic capillary column exhibited better results than the ones obtained with silica packed material of 5 ␮m size. After optimizing all experimental conditions very fast (less than 30 s) enantioseparation was obtained for the enantiomers of 2,2,2-trifluoro-1-(9-anthryl)-ethanol (see Fig. 3) [68]. Later on, the same group employed silica monolithic column (through-pore size of 2 ␮m and skeleton size of 1.5 ␮m) coated with amylose tris (3,5-dimethylphenylcarbamate) for the enantiomeric separation of ten racemic compounds (see Table 1). Methanol/water (80:20, v/v) or n-hexane/2-propanol (9:1, v/v) were the mobile phases. Enantioresolution increased by increasing the concentration of adsorbed CS, while efficiency decreased. However amylose derivative at concentrations higher than 50 mg/mL could not be used due to the increased viscosity of the solution causing problems in filling the capillary column. Some of the studied compounds were resolved in their enantiomers in less than 1 min [67]. Although coated polysaccharides capillaries exhibited very good results for the enantiomers separation of several analytes, these columns may present some drawbacks when working with strong organic solvents such as tetrahydrofuran (THF). In fact the CS is removed from the column observing very low repeatability and no enantioresolution. A solution to this problem is the preparation of silica or polymerized monoliths with chemical bonded

polysaccharides. In the study reported by Chankvetadze et al., cellulose or amylose derivatives were chemically bonded with silica monolithic material and columns used for the enantioseparation of Troger’s base, trans-stilbene oxide, benzoin, 1,2,2,2tetraphenylethanol, 2-phenylcyclohexanone, 2,2,2-trifluoro-1-(9anthryl)ethanol, cobalt(III) tris(acetylacetonate), flavanone, transcyclopropandicarboxylic acid dianilide and 2,29-dihydroxy-6,6 dimethylbiphenyl. Authors studied some experimental parameters such as type and concentration (multiple covalent immobilizations) of the CS on chromatographic performance. The columns resulted to be very stable and resolution could be increased by increasing the amount of the CS bonded. However using higher concentrations of the CS caused a decrease of efficiency [66]. Recently the utility of a quinidine bonded to a methacrylate monolith in nano-LC has been demonstrated by Marina’s group for the chiral resolution of some herbicides. The optimized method was validated and applied to the analysis of herbicide formulations containing R-mecoprop [75]. studies done utilizing monolithic capillary Other columns with bonded different CSs such as Chira-Dex (permethyl-␤-cyclodextrin) [54], 2,3,6-Tris(phenylcarbamoyl)-␤cyclodextrin-6-methacrylate [46], hydroxypropyl-␤-cyclodextrin (HP-␤-CD) [47], ␤-cyclodextrin [48]„2S)-2-(N-4-allyloxy-3,5dichlorobenzoyl)aminocyclohexanesulfonictrans-(1S,2S)-2-(N-4allyloxy-3,5-dichlorobenzoyl)aminocyclohexanesulfonic acid [77] were also reported (see Table 1). 4.2.2. Use of packed capillaries Although the use of monolithic CSPs by nano-LC has demonstrated the great potential of these columns for enantiomers separation, the majority of reported studies employed capillaries packed with silica particles where the CS was either coated or bonded. In general, these CSPs exhibited higher efficiency at least vs. polymerized material. Particles employed mainly considered silica of different diameter, in the range sub-2 ␮m/5 ␮m either fully porous or core-shell. The use of particles with lower diameter permits a higher chromatographic efficiency. In addition a lower value of C term in van Deemter equation is obtained. Just for this a more flat profile of the second part of the curve is observed. Therefore higher flow-rates without sacrificing the efficiency can be achieved. However a higher backpressure is observed with problems in the pumping system. Core-shell particles could be a solution at this problem. In fact the SP occupies only the outside of the particles (a few ␮m). Faster mass transfer is obtained and lower backpressure is generated. Therefore higher flow rates can be used obtaining fast analysis with high efficiency. 4.2.2.1. Cyclodextrins or their derivatives. Gong et al. utilized two new synthesized CSPs, namely 8-amino-quinoline-2-ylmethyl8-amino-quinoline-7-ylmethyl-diaza-18-crown-6-capped and [3-(2-O-␤-cyclodextrin)-2-hydroxypropoxy]propylsilyl silica particles by nano-UHPLC. Some racemic compounds, indapamide, 2-phenylpropionaldehyde, trans-1,2namely cyclohexanediol, 1-(1-naphthyl) ethanol, propranolol, warfarin, trans-2-phenylcyclohexanol were successfully separated in their enantiomers achieving very high efficiency (400,000 N/m). Nonporous 1.5-␮m silica particles were employed and a backpressure of 8000–12,000 p.s.i. was measured. Elution was done using mixtures of acetonitrile/phosphate buffer at different ratio. As expected the two columns offered a low sample capacity. Due to the presence in the CSPs of two different CSs the enantioresolution mechanism was governed by inclusion complexation with the CD and H-H and/or dipolar interaction. This was observed by authors analyzing, e.g., 2-phenylpropionaldehyde and modifying the concentration of the buffer in the MP that also contained Ni

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

ions to positively charge the crown-ether. The increase of the buffer concentration caused an increase of both retention time and enantioresolution [78]. Later on Lin et al. resolved atenolol, propranolol, synephrine, isoproterenol, Ac-Trp and Met enantiomers by nano-LC with porous silica particles modified with pernaphthylcarbamoylated ␤-cyclodextrin (CSP 1), peracetylated ␤-cyclodextrin (CSP 2) and permethylated ␤-cyclodextrin (CSP 3). Authors studied the effect of various MP, e.g., triethylammonium acetate (TEAA)/methanol or phosphate buffer/methanol achieving better results regarding both efficiency and resolution with phosphate. These results were obtained with CSP1, while CSP2 gave the opposite. Authors concluded that best enantioresolutions were, in general, obtained with CSP1 and CSP2 where a carbonyl group was present [51]. Finally the applicability of nano-LC using silica particles containing a chemically bonded cyclodextrin has been demonstrated by Si-Ahmed et al. [49]. Authors employed a CSP porous silica particles studied for the chiral resolution of nutraceutical compounds such as flavanone, 2 -hydroxyflavanone, 4 -hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4 -methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin [50]. The synthesized CSP contained phenyl-carbamate-propyl-␤cyclodextrin and was applied to the determination of hesperetin enantiomers in urine sample. Urines were collected after in ingestion of orange juice. The method was optimized, validated and applied to urine samples. Fig. 4A–C shows the nano-LC separation of urine samples without (panel A), spiked urine with hesperetin (panel B) and hesperetin seven hours after ingestion. As can be observed from panel C, R- and S-HT are present at different ratio indicating that the S-enantiomer was excreted at higher concentration than the R-one. The quantitative data were in accord with previous results by other authors. The proposed method was simple and reliable and considering the beneficial effect of HT, it can be effective in the determination of this compound as a biomarker for juice intake [49]. 4.2.2.2. Glycopeptide antibiotics. Glycopeptide antibiotics are a class of chiral selectors widely used in HPLC [79–81] also tested in nano-LC and CEC. They possess a large number of asymmetric centres (e.g., 18 in vancomycin) with several different groups that can interact with enantiomers, e.g., amino, carboxylic, aromatic rings, amide, sugar moieties etc. They also have a basket moiety offering possibilities for host-guest complexation. However an affinity enantioseparation mechanism is taking place. Vancomycin is stable at pH range 2–6 and at low temperature. Therefore the selection of appropriate experimental conditions is very important for achieving good enantioresolution [82]. Some studies have been performed in nano-LC employing bonded CS (e.g., vancomycin, teicoplanin, Hepta-Tyr antibiotic) to silica or monolithic phases [18,55,56,58–60–62]. These CSs type were applied for the enantiomeric separation of several classes of compounds, e.g., amino acids, ␤-blockers, nonsteroidal anti-inflammatory drugs, herbicides etc. Here just to show the potentiality of the glycopeptide antibiotics a few examples are reported. The first study dealing with the use of vancomycin bonded to silica particles by nano-LC has been reported by Svensson et al. [61]. The CS reacted with silica diol particles after oxidation to aldehyde after reductive amination employing vancomycin. Some non-steroidal anti-inflammatory drugs were tested and resolved in their enantiomers studying some experimental parameters such as composition of the MP, buffer pH, organic modifier and ionic strength. The capillary column resulted to be very stable at the experimental conditions used. Based on this preliminary work and on our experience in CE we synthesized with a quite sim-

11

ilar procedure proposed by Svensson CSPs silica based modified with vancomycin, teicoplanin and Hepta-Tyr antibiotic (a modified teicoplanin). The capillary columns were utilized in nano-LC for the enantiomers separation of some herbicides or loxiglumide [18,60]. The study dealing with the drug chiral separation was done utilizing a commercial CE instrumentation achieving base-line resolution is short time moving the mobile phase by application of 12 bar pressure. This was possible employing a very short packed capillary (7 cm) with 5 ␮m porous silica modified with Hepta-Tyr. Resolution and selectivity were strongly influenced by the type of organic modifier (methanol, acetonitrile or their mixtures). Higher concentration of methanol increased resolution factor. This was due to the competition of the solvent-analyte with the interaction sites of the CS. Acceptable repeatability for retention time and peak areas (5 and 7% respectively) were obtained. The method was applied to the determination of the minor enantiomer eventually present in a pharmaceutical formulation containing only D-loxiglumide [18]. Vancomycin has been bonded to 5 ␮m porous silica particles following the same procedure described in the case of Hepta-Tyr. The CSP was packed into capillaries of 75 ␮m I.D. The column was utilized for the chiral separation of some ␤-blockers and nonsteroidal anti-inflammatory drugs and two related compounds. Some experimental parameters such as organic modifier (type and concentration), buffer (pH and concentration) were studied by nano-LC. Although all basic compounds were base-line resolved in their enantiomers utilizing a MP composed by ammonium acetate pH 4.5/acetonitrile (10:90, v/v), optimum conditions were obtained adding 10% of methanol and reducing at 80% the content of acetonitrile. In the case of acidic compounds, a lower pH (3.5) was effective for all enantiomers studied. The change of the buffer pH influenced the charge of both analytes and vancomycin. The optimum mobile phase resulted to be a mixture of 100 mM ammonium formate pH 3.5/water/metanol–acetonitrile (5:5:90, v/v/v). The organic solvent was 10:80 or 80:10 metanol/acetonitrile. The different mixtures were selected studying their effect on enantioresolution factor [62]. The same vancomycin column type was previously used for the enantiomeric separation of some chlorophenoxy acid herbicides. Racemic mixture of mecoprop, fenoprop, and dichlorprop were resolved in their enantiomers with a MP containing 500 mM buffer, pH 4.5/methanol/water (5:85:10, v/v/v) as mobile phase using a capillary of 75 ␮m I.D. [60]. Teicoplanin was also bonded to silica particles and the CSP applied to the analysis of atenolol present in urine samples. Enantiomers were eluted with a mixture of 500 mM ammonium acetate, pH 4.5/methanol/acetonitrile (1:60:39:, v/v/v). The optimized method was fully validated and applied to urine samples of a patient under atenolol treatment. A liquid-liquid extraction procedure was done to recovery the drug from urine. Both R- and S-atenolol were found in samples even after 300 min from ingestion; this was confirmed also coupling the nano-LC instrument with MS [59]. Because the absence of chromophores in most of investigated AAs, their derivatization can be helpful in increasing the sensitivity using both UV and MS detectors. Based on this consideration D’Orazio et al. [83] separated AA enantiomers after derivatization with fluorescein isothiocyanate (FITC) in a capillary column (75 ␮m I.D.) packed with silica particles modified with vancomycin. For increasing the sensitivity, in addition to the derivatization, sample on column focusing was applied and enantiomers were detected with an ion-trap MS. LODs as low as 8 ng/ mL were obtained. Fresh and commercial orange juices were analyzed for the determination of enantiomeric AAs profiling. Recently, with the aim to perform fast enantiomers separation employing vancomycin bonded to silica particles, Rocchi et al. synthesized a new CSP using 1.8 ␮m silica particles with characteristics different from the ones employed in previous works. They were

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15 12

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

Fig. 4. nano-LC chromatogram of (A) hesperetin-free urine, (B) urine spiked with racemic hesperetin (50 ␮g/mL and the internal standard- IS, 100 ␮g/mL), and (C) human urine sample containing hesperetin and IS 7 h after oral ingestion of juice. Experimental conditions: capillary column (22 cm × 100 ␮m I.D.) packed with phenyl-carbamate2-propyl-␤-CD, mobile phase, 1% (v/v) TEAA pH 4.5 in methanol/water 70:30 (v/v) flow rate 400 nL/min. Reproduced from [49] with permission of Elsevier.

silica hydrate containing a reduced number of free silanol. The reduction of particles diameters had an important role in achieving very good enantioresolution. Acidic compounds belonging to the class of herbicide and non-steroidal anti-inflammatory drugs were successfully resolved (Rs, 1.72–2.95). The capillary column used was 11 cm × 75 ␮m I.D. Employing a column of the same dimension and packed with 5 ␮m particles (ordinary silica previously studied); the same acidic compounds were either not at all or poorly resolved. The better results achieved with the sub-2 ␮m particles were due to both higher efficiency and nature of the silica used [56]. In another study the same novel CSP was used for the chiral separation of some dansyl-amino acids. Also in this case using MP such as mixture of 500 mM ammonium formate buffer, pH 2.5/H2O/acetonitrile (1:9:90, v/v/v) was effective in the chiral resolution of dansyl Asp, Glu, Leu, Phe with Rs in the range 1.46–3.39 [55].

4.2.2.3. Plolysaccharides derivatives.. Cellulose or amylose-based modified polymers were either coated or bonded to silica particles and widely used in HPLC as well as in nano-LC. Carbamate was modified introducing phenyl groups also with different substitution in the aromatic ring in order to have additional and different interaction points. The few examples reported below document the enantioresolution capability of this CSP type. Aminopropylsilica (5 ␮m) coated with 20% cellulose or amylose CSP was used by Meyering et al. for the nano-LC separation of thalidomide and its hydroxylated metabolite enantiomers. Comparing the two CSP, chiralpack AD (amylose-based) exhibited a higher resolving capability than a similar cellulose derivative (chiracel OD or OJ). To optimze the separation of the six compounds a column with mixture (16% AD and 4% OD) polysaccharides was coated obtaining almost baseline resolution of all studied compounds. However the length of the packed capillary was increased at 30 cm [84]. The separation of norgestrel (NG) enantiomers was studied utilizing the same packed column in CEC and nano-LC. Silica particles were coated with cellulose tris (3,5-dichlorophenylcarbamate) (CDCPC) and used for enantiomers separation. Different concentrations of the CS were used showing that enantioresolution and efficiency were strongly influencing the chromatographic performance of the column. Quite interesting that the experiments were carried out with the same CE instrument. Although CEC offered higher efficiency than CLC, the chromatographic technique resulted more effective for the determination of the minor enantiomeric

impurity (1%, w/w). The optimized method was also capable to determine other drugs such as ethinylestradiol present in the pharmaceutical formulation [69]. The high enantiorecognition capability of cellulose tris (3chloro-4-methylphenylcarbamate) coated onto silica particles has been demonstrated by Doninguez-Vega et al. The CSP was packed into capillary columns and used for the enantiomeric separation of several amino acids utilizing both nano-LC. AAs were derivatized with 9-fluorenylmethyl-chloroformate (FMOC) to increase UV sensitivity. Analytes were eluted with a MP composed by 0.5 M ammonium formate, pH 2.5/H2O/ACN (1:19:80, v/v/v). The results were compared with the ones obtained in CEC employing the same column and MP. Better results concerning efficiency and enantioresolution were obtained with CEC. The method was applied to the analysis of citrullin in food supplements [5]. Previously the same CS was coated onto aminopropyl silica particles and the CSP packed. The capillary was used for both CEC and nano-LC for the chiral separation of drug models such as lorazepam, oxazepam, temazepam, thalidomide. The study related to the effect of coated concentration of the CS revealed a very high enantioresolution factors, e.g., 12.9 for temazepam. However efficiency decreased by increasing the amount of coated CS. Finally comparing the results utilizing aminosilca and native silica, authors did not find noticeable differences for neutral compounds [85]. Later on, in order to verify the enantioresolution capability of a different polysaccharide based CSP, amylose tris (5-chloro-2-methylphenylcarbamate) was coated on both native and aminopropyl silica particles. Flavanone, ethozoline, temazepam, lopirazepam enantiomers were studied to verify the effect of both silica nature, particle size and pore diameter. Particles with increased pore size offered higher efficiency and as expected the decrease of particle size (from 5 to 3 ␮m) caused an increase of efficiency. Concerning the coated amylose, it is note worth mentioning that lower concentrations, in general, produced higher efficiency but lower enantioresolution [65]. Additional types of polysaccharide CSPs containing coated cellulose tris (3-chloro-4-methylphenylcarbamate)-Sepapak-2 and cellulose tris (4-chloro-3-methylphenylcarbamate) Sepapak-4 were utilized for the chiral separation of some pesticides including herbicide, insecticides and fungicides. 5 ␮m amino- silica based particles were coated with 25% of the CSs and packed into silica capillaries. The type of CS was fundamental in the chiral resolutions. Seven pesticides were resolved using Sepappak-2, while nine of them were enantioseparated with Sepapak-4. By the comparison with CEC, this technique exhibited better results regarding enantioresolution

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

and efficiency. Therefore CEC was applied to the determination of metalaxyl enantiomers in tap water and soil samples [63]. Cellulose tris (3,5 dimethylphenylcarbamate)-Sepapak-1, (cellulose tris (3-chloro-4-methylphenylcarbamate)-Sepapak-2 and tris (5-chloro-2-methylphenylcarbamate)-Sepapak-3 after coating aminosilica particles were also used for the stereoisomers separation of twelve flavonoids. Several parameters such as content and ratio of organic solvents (methanol-2-propanol) were studied achieving good resolutions. Amylose CSP offered the possibility to resolve eight flavanones eluting with polar organic mode with no effect in the separation of stereoisomers (naringenin and naringin). These last compounds were instead separated with Sepapak-1. Authors concluded that the amylose CSP had the highest enantioresolution capacity towards the studied compounds of nutraceutical interest [86]. Considering the recent trends in the preparation of new stationary CSPs and the potentiality of superficial porous (core-shell) particles, silica particles either porous or core-shell were coated with cellulose tris (4-chloro-3-methylphenylcarbanate) and used in nano-LC. Core-shell particles resulted to be, in general, more efficient than the porous material. The same trend was observed regarding enantioresolution [6]. The study related to the use of core-shell particles has been performed later on utilizing amylose tris (3,5dimethylphenylcarbamate)-based chiral stationary phases. Different concentrations of the CS (1–5%) were used with different supports with pore size of 100 and 200 A. Etozoline, praziquantel, troger’s base, flavanone, 4 -methodyflavanone, 7-methoxyflavanone were resolved in their enantiomers. The increase of CS concentration caused an increase of enantioresolution with a general decrease of efficiency. The different pore size of the particles did not influence the results with nano-LC, while they were noticeable when using CEC [7]. Recently Thurmann et al. demonstrated the usefulness of cellulose derivatives for the separation of some enantiomers by a microfluidic device. The chip was packed with silica particles with adsorbed 25% of cellulose tris (3,5-dimethylphenylcarbamate) obtaining very good enantiomeric resolution of trifluoroanthrylethanol, 6-hydroxyflavanone and 6- methoxyflavanone. Optimizing the experimental conditions very short analysis time were observed (5–30 s). The packing procedure considered the entrapment of particles into the chip preparing monolithic frits. The proposed method was studied using reversed-, normal- and polar organic-elution modes and the results open new horizons for high-throughput chiral analysis [87]. Although the interesting results obtained with CSP containing polysaccharides the use of coated SP with such CSs could cause serious stability problems to columns when MPs with strong organic solvents have to be employed. Therefore bonded CS is a valid solution as proposed by Chankvetadze et al. [67] with silica monolithic material. This has been demonstrated by Zou’s group [71] who synthesized a new CSP containing cellulose tris (phenylcarbamate) bonded to 5 ␮m silica aminopropylsilica. The column was studied separating some model racemic mixtures also including warfarin by nano-LC and CEC. Here analytes were eluted with a mixture of hexane/methanol/2-propanol/THF (50:35:10:5, v/v/v/v) and 5 mM acetic acid/triethanoamine pH* 6. As expected the column was very stable offering good reproducibility. Other bonded CSs have also been used in nano-LC, e.g., SSWhelk-O1 [76], tert-butylbenzoylated tartardiamide [88].

5. Conclusions The separation of chiral compounds is a very important topic in various field including quality control, food, agrochemical, environ-

13

ment, pharmaceutical industry, drugs, bio- medicine etc. Because their quite similar physical-chemical properties they are difficult to be separated. However the use of a chiral environment promoting stereoselective interactions can be effective. Conventional analytical separation techniques such as HPLC, GC, TLC, and SFC are currently used for such purpose. Nano-LC has been recently applied for the enantiomers separation utilizing fused silica capillaries containing CSs either bonded to the internal wall or to particles or to monolithic material contained into the column. Last but not least the CS can also be added to the MP. Several CSs have been used e.g., glycopeptide antibiotics, cyclodextrins, polysaccharides, quinine etc. for successful enantioresolution of amino acids, drugs, nutraceuticals, pesticides etc. Although some applications have been reported, further work is necessary, e.g., new CSP, production of commercial available capillary columns and less expensive instrumentation. Finally, the use of chip technology seems to be very promising. Here dead volumes are minimized, thus providing, in addition to short analysis time, high efficiency and resolution.

References [1] J.P.C. Vissers, H.A. Claessens, C.A. Cramers, Microcolumn liquid chromatography: instrumentation detection and applications, J.Chromatogr. A 779 (1997) 1–28. [2] K.E. Karlsson, M. Novotny, Separation efficiency of slurry-packed liquid chromatography microcolumns with very small inner diameters, Anal. Chem. 60 (1988) 1662–1665. [3] M. Szumski, B. Buszewski, State of the art in miniaturized separation techniques, Crit. Rev. Anal. Chem. 32 (2002) 1–46. [4] J. Hernández-Borges, G. D’Orazio, Z. Aturki, S. Fanali, Nano-liquid chromatography analysis of dansylated biogenic amines in wines, J. Chromatogr. A 1147 (2007) 192–199. [5] E.D. Vega, A.L. Crego, K. Lomsadze, B. Chankvetadze, M.L. Marina, Enantiomeric separation of FMOC-amino acids by nano-LC and CEC using a new chiral stationary phase cellulose tris(3-chloro-4-methylphenylcarbamate), Electrophoresis 32 (2011) 2700–2707. [6] S. Fanali, G. D’Orazio, T. Farkas, B. Chankvetadze, Comparative performance of capillary columns made with totally porous and core-shell particles coated with a polysaccharide-based chiral selector in nano-liquid chromatography and capillary electrochromatography, J. Chromatogr. A 1269 (2012) 136–142. [7] S. Rocchi, S. Fanali, T. Farkas, B. Chankvetadze, Effect of content of chiral selector and pore size of core–shell type silica support on the performance of amylose tris (3,5-dimethylphenylcarbamate)-based chiral stationary phases in nano-liquid chromatography and capillary electrochromatography, J. Chromatogr. A 1363 (2014) 363–371. [8] G. D’Orazio, J. Hernández-Borges, M. Asensio-Ramos, M.A. Rodríguez-Delgado, S. Fanali, Capillary electrochromatography and nano-liquid chromatography coupled to nano-electrospray ionization interface for the separation and identification of estrogenic compounds, Electrophoresis 37 (2016) 356–362. [9] M.F. Mirabelli, J.C. Wolf, R. Zenobi, Pesticide analysis at ppt concentration levels: coupling nano-liquid chromatography with dielectric barrier discharge ionization-mass spectrometry, Anal. Bioanal. Chem. 408 (2016) 3425–3434. [10] J. Bai, H. Wang, J. Oub, Z. Liu, Y. Shena, H. Zou, Rapid one-pot preparation of polymeric monolith via photo-initiated thiol-acrylate polymerization for capillary liquid chromatography, Anal. Chim. Acta 925 (2016) 88–96. [11] H. Zhang, J. Ou, Y. Wei, H. Wang, Z. Liu, H. Zou, A hybrid fluorous monolithic capillary column with integrated nanoelectrospray ionization emitter for determination of perfluoroalkyl acids by nano-liquid chromatography–nanoelectrospray ionization-mass spectrometry/mass spectrometry, J. Chromatogr. A 1440 (2016) 66–73. [12] G. D’Orazio, S. Fanali, Enantiomeric separation by using nano-liquid chromatography with on-column focusing, J.Sep.Sci. 31 (2008) 2567–2571. [13] M.A. Stravs, J. Mechelke, P.L. Ferguson, H. Singer, J. Hollender, Microvolume trace environmental analysis using peak-focusing online solid-phase extraction–nano-liquid chromatography–high-resolution mass spectrometry, Anal. Bioanal. Chem. 408 (2016) 1879–1890. [14] C. Fanali, A. Rocco, G. D’Orazio, L. Dugo, L. Mondello, Z. Aturki, Determination of key flavonoid aglycones by means of nano-LC for the analysis of dietary supplements and food matrices, Electrophoresis 36 (2015) 1073–1081. [15] J. Leonhardt, T. Hetzel, T. Teutenberg, T.C. Schmidt, Large volume injection of aqueous samples in nano liquid chromatography using serially coupled columns, Chromatographia 78 (2015) 31–38. [16] K. Buonasera, G. D’Orazio, S. Fanali, P. Dugo, L. Mondello, Separation of organophosphorous pesticides by using nano-liquid chromatography, J. Chromatogr. A 1216 (2009) 3970–3976.

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15 14

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

[17] S. Héron, A. Tchapla, J.-P. Chervet, Influence of injection parameters on column performance in nanoscale high-performance liquid chromatography, Chromatographia 51 (2000) 495–499. [18] S. Fanali, G. D’Orazio, M.G. Quaglia, A. Rocco, Use of a Hepta-Tyr antibiotic modified silica stationary phase for the enantiomeric resolution of d,l-loxiglumide by electrochromatography and nano-liquid chromatography, J. Chromatogr. A 1051 (2004) 247–252. [19] A. Cappiello, G. Famiglini, C. Fiorucci, F. Mangani, P. Palma, A. Siviero, Variable-gradient generator for micro- and nano-HPLC, Anal. Chem. 75 (2003) 1173–1179. [20] A. Cappiello, G. Famiglini, F. Mangani, P. Palma, A. Siviero, Nano-high-performance liquid chromatography-electron ionization mass spectrometry approach for environmental analysis, Anal. Chim. Acta 493 (2003) 125–136. [21] F. Rigano, A. Albergamo, D. Sciarrone, M. Beccaria, G. Purcaro, L. Mondello, Nano liquid chromatography directly coupled to electron ionization mass spectrometry for free fatty acid elucidation in mussel, Anal. Chem. 88 (2016) 4021–4028. [22] V.A. Davankov, The nature of chiral recognition: is it a three-point interaction? Chirality 9 (1997) 99–102. [23] B. Chankvetadze, Capillary Electrophoresis in Chiral Analysis, John Wiley & Sons, Chichester-New York-Weinheim-Brisbane-Singapore-Toronto, 1997, pp. 1–555. [24] G. Gubitz, M.G. Schmid, Chiral separation principles, in: G. Gubitz, M.G. Schmids, G. Gubitz, M.G. Schmids (Eds.), Chiral Separations Methods and Protocols, Humana Press, Totowa, New Jersey, 2004, pp. 1–28. [25] C. Fanali, L. Dugo, P. Dugo, L. Mondello, Capillary-liquid chromatography (CLC) and nano-LC in food analysis, Trends Anal. Chem. 52 (2013) 226–238. [26] B. Chankvetadze, Liquid chromatographic separation of enantiomers, in: S. Fanali, C.F. Poole, P. Schoenmakers, D. Lloyds (Eds.), Liquid Chromatography Applications, Elsevier Inc., Waltham, 2013, pp. 75–91. [27] T. Takeuchi, Separation of 1,1 -binaphthyl-2,2 -diyl hydrogen phosphate enantiomers by microcolumn liquid chromatography with ␥-cyclodextrin as mobile phase additive, J. High Resolut. Chromatogr. 14 (1991) 560–561. [28] A. Rocco, S. Fanali, Enantiomeric separation of acidic compounds by nano-liquid chromatography with methylated-beta-cyclodextrin as a mobile phase additive, J. Sep. Sci. 32 (2009) 1696–1703. [29] N. Rosales-Conrado, M.E. Leon-Gonzalez, A. Rocco, S. Fanali, Enantiomeric separation of ofloxacin by nano-liquid chromatography using a sulfated-␤-cyclodextrin as a chiral selector in the mobile phase, Curr. Anal. Chem. 6 (2010) 209–216. [30] V. Schurig, M. Jung, S. Mayer, M. Fluck, S. Segura, H. Jakubetz, Unified enantioselective capillary chromatography on a Chirasil-DEX stationary phase advantages of column miniaturization, J. Chromatogr. A 694 (1995) 119–128. [31] H. Jakubetz, H. Czesla, V. Schurig, On the feasibility of miniaturized enantiomeric separation by liquid chromatography (OTLC) and open tubular electrochromatography (OTEC), J. Microcol. Sep. 9 (1997) 421–431. [32] H. Hofstetter, O. Hofstetter, V. Schurig, Enantiomer separation using bsa as chiral stationary phase in affinity otec and otlc, J. Microcol. Sep. 10 (1998) 287–291. [33] E. Francotte, M. Jung, Enantiomer separation by open tubular liquid chromatography and electrocromatography, Chromatographia 42 (1996) 521–527. [34] T. Wakita, B. Chankvetadze, C. Yamamoto, Y. Okamoto, Chromatographic enantioseparation on a wall-coated open tubular capillary column containing covalently bound cellulose (3,5-dichlorophenyl carbamate) as chiral selector, J. Sep. Sci. 25 (2002) 167–169. [35] F.G. Adly, N. Yaantwi, A. Ghanem, Cyclodextrin-functionalized monolithic capillary columns: preparation and chiral applications, Chirality 28 (2016) 97–109. [36] S. Fanali, Z. Aturki, A. Rocco, Forensic drugs analysis: a review of miniaturized separation techniques, LC-GC Eur. 28 (2015). [37] Z. Aturki, A. Rocco, S. Rocchi, S. Fanali, Current applications of miniaturized chromatographic and electrophoretic techniques in drug analysis, J. Pharm. Biomed. Anal. 101 (2014) 194–220. [38] A. Rocco, A. Maruˇska, S. Fanali, Enantiomeric separations by means of nano-LC, J. Sep. Sci. 30 (2013) 421–444. [39] B. Chankvetadze, Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers, J. Chromatogr. A 1269 (2012) 26–51. [40] B.S. Sekhon, Enantioseparation of chiral drugs – an overview, Intern. J. Pharm. Tech. Res. [2] (2010) 1584–1594. [41] X. Dong, R. Wu, M. Wu, Y. Zhu, H. Zou, Recent progress of polar stationary phases in CEC and capillary liquid chromatography, Electrophoresis 30 (2009) 141–154. [42] J. Hernández-Borges, Z. Aturki, A. Rocco, S. Fanali, Recent applications in nano-liquid chromatography, J. Sep. Sci. 30 (2007) 1589–1610. [43] B. Chankvetadze, Monolithic chiral stationary phases for liquid-phase enantioseparation techniques, J. Sep. Sci. 33 (2010) 305–331. [44] A. Ghanem, M. Ahmed, H. Ishii, T. Ikegami, Immobilized ␤-cyclodextrin-based silica vs polymer monoliths for chiral nano liquid chromatographic separation of racemates, Talanta 132 (2015) 301–314. [45] M. Ahmed, M.M. Yajadda, Z.J. Han, D. Su, G. Wang, K.K. Ostrikov, A. Ghanem, Single-walled carbon nanotube-based polymer monoliths for the enantioselective nano-liquid chromatographic separation of racemic pharmaceuticals, J. Chromatogr. A 1360 (2014) 100–109.

[46] M. Ahmed, A. Ghanem, Chiral beta-cyclodextrin functionalized polymer monolith for the direct enantioselective reversed phase nano liquid chromatographic separation of racemic pharmaceuticals, J. Chromatogr. A 1345 (2014) 115–127. [47] A. Rocco, A. Maruˇska, S. Fanali, Enantioseparation of drugs by means of continuous bed (monolithic) columns in nano-liquid chromatography, Chemija 23 (2012) 294–300. [48] Q. Zhang, J. Guo, F. Wang, J. Crommen, Z. Jiang, Preparation of a beta-cyclodextrin functionalized monolith via a novel and simple one-pot approach and application to enantioseparations, J. Chromatogr. A 1325 (2014) 147–154. [49] K. Si-Ahmed, F. Tazerouti, A.Y. Badjah-Hadj-Ahmed, Z. Aturki, G. D’Orazio, A. Rocco, S. Fanali, Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography, J. Pharm.Biomed. Anal. 51 (2010) 225–229. [50] K. Si-Ahmed, F. Tazerouti, A.Y. Badjah-Hadj-Ahmed, Z. Aturki, G. D’Orazio, A. Rocco, S. Fanali, Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-␤-cyclodextrin chiral stationary phase, J. Chromatogr. A 1217 (2010) 1175–1182. [51] B. Lin, S.-C. Ng, Y.-Q. Feng, Chromatographic evaluation and comparison of three beta-cyclodextrin-based stationary phases by capillary liquid chromatography and pressure-assisted capillary electrochromatography, Electrophoresis 404 (2008) 5–4054. [52] D. Wistuba, L. Banspach, V. Schurig, Enantiomeric separation by capillary electrochromatography using monolithic capillaries with sol-gel-glued cyclodextrin-modified silica particles, Electrophoresis 26 (2005) 2019–2026. [53] L.O. Healy, J.P. Murrihy, A. Tan, D. Cocker, M. McEnery, J.D. Glennon, Enantiomeric separation of R,S-naproxen by conventional and nano-liquid chromatography with methyl-beta-cyclodextrin as a mobile phase additive, J. Chromatogr. A 924 (2001) 459–464. [54] D. Wistuba, V. Schurig, Enantiomer separation by capillary electrochromatoraphy on a cyclodextrin-modified monolith, Electrophoresis 21 (2000) 3152–3159. [55] S. Rocchi, C. Fanali, S. Fanali, Use of a novel sub-2 ␮m silica hydride vancomycin stationary phase in nano-Liquid chromatography. II. Separation of derivatized amino acid enantiomers, Chirality 27 (2015) 767–772. [56] S. Rocchi, A. Rocco, J.J. Pesek, M.T. Matyska, D. Capitani, S. Fanali, Enantiomers separation by nano-liquid chromatography: use of a novel sub-2 ␮m vancomycin silica hydride stationary phase, J. Chromatogr. A 1381 (2015) 149–159. [57] R. Auditore, N.A. Santagati, Z. Aturki, S. Fanali, Enantiomeric separation of amlodipine and its two chiral impurities by nanoliquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4-chloro-3-methylphenylcarbamate), Electrophoresis 34 (2013) 2593–2600. [58] M. Fantacuzzi, G. Bettoni, G. D’Orazio, S. Fanali, Enantiometric separation of some demethylated analogues of clofibric acid by capillary zone electrophoresis and nano-liquid chromatography, Electrophoresis 27 (2006) 1227–1236. [59] G. D’Orazio, S. Fanali, Use of teicoplanin stationary phase for the enantiomeric resolution of atenolol in human urine by nano-liquid chromatography–mass spectrometry, J. Pharm. Biomed. Anal. 40 (2006) 539–544. [60] N. Rosales-Conrado, M.E. León-González, G. D’Orazio, S. Fanali, Enantiomeric separation of chlorophenoxy acid herbicides by nano liquid chromatography-UV detection on a vancomycin-based chiral stationary phase, J. Sep. Sci. 27 (2004) 1303–1308. [61] L.A. Svensson, K.-E. Karlsson, A. Karlsson, J. Vessman, Immobilized vancomycin as chiral stationary phase in packed capillary liquid chromatography, Chirality 40 (1998) 273–280. [62] G. D’Orazio, Z. Aturki, M. Cristalli, M.G. Quaglia, S. Fanali, Use of vancomycin chiral stationary phase for the enantiomeric resolution of basic and acidic compounds by nano-liquid chromatography, J. Chromatogr. A 1081 (2005) 105–113. [63] V. Pérez-Fernández, E. Dominguez-Vega, B. Chankvetadze, A.L. Crego, M.Á. García, M.L. Marina, Evaluation of new cellulose-based chiral stationary phases Sepapak-2 and Sepapak-4 for the enantiomeric separation of pesticides by nano liquid chromatography and capillary electrochromatography, J. Chromatogr. A. 1234 (2012) 22–31. [64] J. Ou, H. Lin, S. Tang, Z. Zhang, J. Dong, H. Zou, Hybrid monolithic columns coated with cellulose tris(3,5-dimethylphenyl-carbamate) for enantioseparations in capillary electrochromatography and capillary liquid chromatography, J. Chromatogr. A 1269 (2012) 372–378. [65] S. Fanali, G. D’Orazio, K. Lomsadze, S. Samakashvili, B. Chankvetadze, Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano liquid chromatography and capillary electrochromatography, J. Chromatogr. A 1217 (2010) 1166–1174. [66] B. Chankvetadze, T. Kubota, T. Ikai, C. Yamamoto, M. Kamigaito, N. Tanaka, K. Nakanishi, Y. Okamoto, High-performance liquid chromatographic enantioseparations on capillary columns containing crosslinked polysaccharide phenylcarbamate derivatives attached to monolithic silica, J. Sep. Sci. 29 (2006) 1988–1995. [67] B. Chankvetadze, C. Yamamoto, M. Kamigaito, N. Tanaka, K. Nakanishi, Y. Okamoto, High-performance liquid chromatographic enantioseparations on capillary columns containing monolithic silica modified with amylose tris(3,5- dimethylphenylcarbamate), J. Chromatogr. A 1110 (2006) 46–52.

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028

G Model CHROMA-357976; No. of Pages 15

ARTICLE IN PRESS S. Fanali / J. Chromatogr. A xxx (2016) xxx–xxx

[68] B. Chankvetadze, C. Yamamoto, N. Tanaka, K. Nakanishi, Y. Okamoto, High-performance liquid chromatographic enantioseparations on capillary columns containing monolithic silica modified with cellulose tris(3,5-dimethylphenylcarbamate), J. Sep. Sci. 27 (2004) 905–911. [69] B. Chankvetadze, I. Kartozia, C. Yamamoto, Y. Okamoto, G. Balschke, Comparative study on the application of capillary liquid chromatography and capillary electrochromatography for investigation of enantiomeric purity of the contraceptive drug levonorgestrel, J. Pharm. Biomed. Anal. 30 (2003) 1897–1906. [70] L. Chankvetadze, I. Kartozia, C. Yamamoto, B. Chankvetadze, G. Blaschke, Y. Okamoto, Enantioseparation in capillary liquid chromatography and capillary electrochromatography using amylose tris(3,5-dimethylphenylcarbamate) in combination with aqueous organic mobile phase, J.Sep.Sci. 25 (2002) 653–660. [71] X. Chen, H. Zou, M. Ye, Z. Zhang, Separation of enantiomers by nano liquid chromatography and capillary electrochromatography using a bonded cellulose trisphenylcarbamate stationary phase, Electrophoresis 23 (2002) 1246–1254. [72] B. Chankvetadze, I. Kartozia, Y. Okamoto, G. Blaschke, The effect of pore size of silica gel and concentration of buffer on capillary chromatographic and capillary electrochromatographic enantioseparations using cellulose tris(3,5-dichlorophenylcarbamate), J. Sep. Sci. 24 (2001) 635–642. [73] M. Girod, B. Chankvetadze, G. Blaschke, Enantioseparations in non-aqueous capillary electrochromatography using polysaccharide type chiral stationary phases, J. Chromatogr. 887 (2000) 439–455. [74] K. Krause, M. Girod, B. Chankvetadze, G. Blaschke, Enantioseparations in normal- and reversed-phase nano-high- performance liquid chromatography and capillary electrochromatography using polyacrylamide and polysaccharide derivatives as chiral stationary phases, J. Chromatogr. A 837 (1999) 51–63. [75] Q. Zhang, V. Gil, E. Sánchez-López, M. Á. García, Z. Jiang, M.L. Marina, Evaluation of the potential of a quinidine-based monolithic column on the enantiomeric separation of erbicides by nano-liquid chromatography, Microchem. J. 123 (2015) 15–21. [76] A. Ciogli, G. Pierri, D. Kotoni, A. Cavazzini, L. Botta, C. Villani, J. Kocergin, F. Gasparrini, Toward enantioselective nano ultrahigh-performance liquid chromatography with Whelk-O1 chiral stationary phase, Electrophoresis 35 (2014) 2819–2823. [77] B. Preinerstorfer, C. Hoffmann, D. Lubda, M. Lämmerhofer, W. Lindner, Enantioselective silica-based monoliths modified with a novel aminosulfonic acid-derived strong cation exchanger for electrically driven and pressure-driven capillary chromatography, Electrophoresis 29 (2008) 1626–1637.

15

[78] Y. Gong, Y. Xiang, B. Yue, G. Xue, J.S. Bradshaw, H.K. Lee, M.L. Lee, Application of diaza-18-crown-6-capped beta-cyclodextrin bonded silica particles as chiral stationary phases for ultrahigh pressure capillary liquid chromatography, J. Chromatogr. A 1002 (2003) 63–70. [79] C.L. Barhate, Z.S. Breitbach, E. Costa Pinto, E.L. Regalado, C.J. Welch, D.W. Armstrong, Ultrafast separation of fluorinated and desfluorinated pharmaceuticals using highly efficient and selective chiral selectors bonded to superficially porous particles, J. Chromatogr. A 1426 (2015) 241–247. [80] C.L. Barhate, M.F. Wahab, Z.S. Breitbach, D.S. Bell, D.W. Armstrong, High efficiency, narrow particle size distribution, sub-2 ␮m based macrocyclic glycopeptide chiral stationary phases in HPLC and SFC, Anal. Chim. Acta 898 (2015) 128–137. [81] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 ␮m totally porous silica particles of narrow size distribution, J. Chromatogr. A 1427 (2016) 55–68. [82] S. Fanali, Z. Aturki, G. D’Orazio, A. Rocco, in: A.V. Eeckhaut, Y. Micottes, A.V. Eeckhaut, Y. Micottes (Eds.), Macrocyclic Antibiotics as Chiral Selectors in Chiral Separations by Capillary Electrophoresis, CRC Press, Boca Raton, FL, 2010, pp. 109–137. [83] G. D’Orazio, A. Cifuentes, S. Fanali, Chiral nano-liquid chromatography-mass spectrometry applied to amino acids analysis for orange juice profiling, Food Chem. 108 (2008) 1114–1121. [84] M. Meyring, B. Chankvetadze, G. Blaschke, Simultaneous separation and enantioseparation of thalidomide and its hydroxylated metabolites using high-performance liquid chromatography in common-size columns capillary liquid chromatography and nonaqueous capillary electrochromatography, J. Chromatogr. 876 (2000) 157–167. [85] S. Fanali, G. D’Orazio, K. Lomsadze, B. Chankvetadze, Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography, J. Chromatogr. B 875 (2008) 296–303. [86] K. Si-Ahmed, Z. Aturki, B. Chankvetadze, S. Fanali, Evaluation of novel amylose and cellulose-based chiral stationary phases for the stereoisomer separation of flavanones by means of nano-liquid chromatography, Anal. Chim. Acta 738 (2012) 85–94. [87] S. Thurmann, C. Lotter, J.J. Heiland, B. Chankvetadze, D. Belder, Chip-Based high-performance liquid chromatography for high speed enantioseparations, Anal. Chem. 87 (2015) 5568–5576. [88] S. Fanali, G. D’Orazio, A. Rocco, Use of tert-butylbenzoylated tartardiamide chiral stationary phase for the enantiomeric resolution of acidic compounds by nano-liquid chromatography, J. Sep. Sci. 29 (2006) 1423–1431.

Please cite this article in press as: S. Fanali, Nano-liquid chromatography applied to enantiomers separation, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.10.028