Chiral Stationary Phases for Liquid Chromatography Based on Chitin-and Chitosan-Derived Marine Polysaccharides

The development of chiral stationary phases (CSPs) for liquid chromatography (LC) revolutionized the enantioseparation and, nowadays, different types of CSPs are commercially available. Polysaccharide-based CSPs are one of the most versatile and widely used for both analytical and preparative applications and they are able to resolve several classes of racemates. Phenylcarbamates of amylose and cellulose derivatives are the most successful; however, polysaccharide-based CSPs comprising marine-derived polysaccharides are also described revealing high chiral recognition abilities and wider range of mobile phases. A literature survey covering the report on chitin and chitosan based CSPs is presented. The chemical structure of the chiral selectors, their development and applications in chiral LC are emphasized.

Polysaccharides are polymers comprising several units of monosaccharides linked to each other by a glycosidic bond [5].There are several types of polysaccharides, and some of them have been studied as possible chiral selectors for LC (Figure 1).

Marine Polysaccharide-Derived CSPs
Marine-derived polysaccharides have also been exploited as chiral selectors, and some of them proved to be good alternatives to amylose and cellulose derivatives.
Braconnot discovered chitin, a marine polysaccharide obtained by isolation from shells of crustaceans and mollusks, in the early 19th century [46].Chitin is one of the most abundant polysaccharides comprising N-acetyl-D-glucosamine units linked by β- (1,4).
Chitosan is a 2-deoxy-2-glucosamine polysaccharide, discovered in 1859 by Rouget after deacetylation of chitin by boiling in concentrated potassium hydroxide solution [47].Both marine-derived polysaccharides have diverse applications whether in medicine as wound healing agents [48], as drug carriers [49,50], in bone tissue regeneration [51] as well as in the food industry as clarification agents [52], among others.Another important application is their use as suitable chiral selectors for LC [26].In fact, they have been used as CSPs, since Okamoto et al., in 1984, introduced the first phenylcarbamate of chitosan.Considering chitin, the first reported study was published by Cass et al., in 1996, describing the chiral discrimination ability of two arylcarbamates of chitin [53].
A literature survey covering the report on chitin and chitosan based CSPs is the main objective of this review.Different CSPs were developed allowing the enantioresolution of several different analytes (α > 1.00) (Tables 1-6).
The structures of the separated analytes (A1-A73) are shown in Figures 3 and 4.
Considering that the chitin derivatives have a very low solubility, the possibility to perform enantioseparations under reversed phase as well as using different solvents in normal phase, such as chloroform and ethyl acetate, was also studied (Table 1).Both chromatographic elution conditions were tested for 3,5-dimethyl-(2) and 3,5-dichlorophenylcarbamates (3) and, in some cases, the racemates were more efficiently resolved under reversed phase mode [55].
Recently, the same group developed a new strategy to enhance the chromatographic performance of chitin-based CSPs [57,58].The aim was combining amylose or cellulose with chitin derivatives and coated on silica gel to improve the chiral recognition as well as their stability and solvent resistance.The first report of this type of biselector as CSPs comprised amylose tris-3,5-dimethylphenylcarbamate and chitin bis-3-chloro-4-methylphenlcarbamate (18) blended at different molar ratios [58].Although the chiral recognition of the blended CSPs did not improve significantly, comparing to the single selector CSPs, there was a great improvement in the solvent tolerance and stability.Interestingly, the biselector CSPs prepared by blending chitin bis-3,5-dimethylphenylcarbamate (3) with cellulose bis-4-methylbenzoate and cellulose bis-3,5-dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.

Chitin-Based CSPs
The bis-phenylcarbamate (1) and bis-3,5-dimethylphenylcarbamate (2) (Table 1), coated on microporous aminopropyl silica, were the first described chitin-based CSPs [53].Two distinct sources of chitin (commercial and noncommercial) were used for the preparation of both polysaccharide derivatives.Interestingly, the results obtained demonstrated that the chiral discrimination of both aryl carbamate derivatives was significantly affected by the source of chitin used.For example, from the series of racemates tested, only (E)-1-chloro-1,2-diphenylethane oxide was resolved on the CSPs prepared using a commercial chitin, with α values of 1.5 and 2.0 in CSPs 1 and 2, respectively.The similar bis-aryl carbamate derivatives of a noncommercial chitin presented higher resolution power compared with commercial chitin.These results are due to the differences related to the resource and  dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.dimethylphenylcarbamate sowed better chiral recognition capabilities compared to the corresponding single selectors [57].They can also work in a wider range of mobile phases.
All the described chitin-based CSPs were prepared by coating method and, to the best of our knowledge, there is no studies reporting immobilized chitin derivatives as well as commercially available chitin-based CSPs.

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on the chromatographic support.

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on the chromatographic support.

Chitosan Tris-Carbamate CSPs
As previously mentioned, the first study of a chitosan derivative as a CSP was published by Okamoto et al., in 1984 [7].In this study, they compared the chiral discrimination ability of various polysaccharide phenylcarbamates as CSPs.Chitosan tris-phenylcarbamate derivative (21) coated on macroporous aminopropyl silica was found to resolve the enantiomers of 1-(9-anthryl)-2,2,2trifluoroethanol with a α value of 2.25 (Table 2) [7].

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on the chromatographic support.

Chitosan Tris-Carbamate CSPs
As previously mentioned, the first study of a chitosan derivative as a CSP was published by Okamoto et al., in 1984 [7].In this study, they compared the chiral discrimination ability of various polysaccharide phenylcarbamates as CSPs.Chitosan tris-phenylcarbamate derivative (21) coated on macroporous aminopropyl silica was found to resolve the enantiomers of 1-(9-anthryl)-2,2,2trifluoroethanol with a α value of 2.25 (Table 2) [7].

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on the chromatographic support.

Chitosan Tris-Carbamate CSPs
As previously mentioned, the first study of a chitosan derivative as a CSP was published by Okamoto et al., in 1984 [7].In this study, they compared the chiral discrimination ability of various polysaccharide phenylcarbamates as CSPs.Chitosan tris-phenylcarbamate derivative (21) coated on macroporous aminopropyl silica was found to resolve the enantiomers of 1-(9-anthryl)-2,2,2trifluoroethanol with a α value of 2.25 (Table 2) [7].

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on the chromatographic support.

Chitosan Tris-Carbamate CSPs
As previously mentioned, the first study of a chitosan derivative as a CSP was published by Okamoto et al., in 1984 [7].In this study, they compared the chiral discrimination ability of various polysaccharide phenylcarbamates as CSPs.Chitosan tris-phenylcarbamate derivative (21) coated on macroporous aminopropyl silica was found to resolve the enantiomers of 1-(9-anthryl)-2,2,2trifluoroethanol with a α value of 2.25 (Table 2) [7].

Chitosan-Based CSPs
Several studies regarding chitosan-based CSPs are found in literature.The first studies were focused mainly on tris-phenylcarbamates of chitosan.In the last decades, an increasingly number of bis-phenylcarbamates of chitosan have been described.Furthermore, besides the traditional coating method, same chitosan-based CSPs were prepared by immobilization of the chitosan-derivatives on the chromatographic support.

Chitosan Tris-Carbamate CSPs
As previously mentioned, the first study of a chitosan derivative as a CSP was published by Okamoto et al., in 1984 [7].In this study, they compared the chiral discrimination ability of various polysaccharide phenylcarbamates as CSPs.Chitosan tris-phenylcarbamate derivative (21) coated on macroporous aminopropyl silica was found to resolve the enantiomers of 1-(9-anthryl)-2,2,2trifluoroethanol with a α value of 2.25 (Table 2) [7].In 1998, the same group compared the chiral recognition performance of 3,5-dichloro-and 3,5dimethylphenylcarbamate derivatives of several polysaccharides, including chitosan (22, 23) (Table 2) [60].These two chitosan derivatives presented a relatively high chiral recognition for the tested racemates, setting their potential use as CSPs [60].In the same year, Franco et al., described another strategy to obtain new chitosan-based CSPs by bonding the chitin-carbamate derivatives on chromatographic support [44].The obtained bonded CSPs allowed the use of a larger panel of solvents in the mobile phases compared to coated ones.Accordingly, the 3,5dimethylphenylcarbamate derivative of chitosan (23i) was mixed with 10-undecenoyl and covalently immobilized on allyl silica gel, which demonstrate to be very useful in the separation of several racemates, such as lormetazepam and temazepam with a α value of 1.80 (Table 2).The mobile phases comprising either different proportions of heptane/2-propanol and heptane/chloroform mixtures allowed the best enantioresolutions [44].
In another study, the synthesis and chromatographic evaluation of the chitosan derivatives 22 and 23 as well as four new chitosan derivatives (25-28) were described (Table 2) [62].All derivatives were coated on macroporous silica gel and evaluated as CSPs.Among them, the 3,5-dichloro-( 22 In 1998, the same group compared the chiral recognition performance of 3,5-dichloro-and 3,5dimethylphenylcarbamate derivatives of several polysaccharides, including chitosan (22, 23) (Table 2) [60].These two chitosan derivatives presented a relatively high chiral recognition for the tested racemates, setting their potential use as CSPs [60].In the same year, Franco et al., described another strategy to obtain new chitosan-based CSPs by bonding the chitin-carbamate derivatives on chromatographic support [44].The obtained bonded CSPs allowed the use of a larger panel of solvents in the mobile phases compared to coated ones.Accordingly, the 3,5dimethylphenylcarbamate derivative of chitosan (23i) was mixed with 10-undecenoyl and covalently immobilized on allyl silica gel, which demonstrate to be very useful in the separation of several racemates, such as lormetazepam and temazepam with a α value of 1.80 (Table 2).The mobile phases comprising either different proportions of heptane/2-propanol and heptane/chloroform mixtures allowed the best enantioresolutions [44].
In another study, the synthesis and chromatographic evaluation of the chitosan derivatives 22 and 23 as well as four new chitosan derivatives (25-28) were described (Table 2) [62].All derivatives were coated on macroporous silica gel and evaluated as CSPs.Among them, the 3,5-dichloro-( 22 In 1998, the same group compared the chiral recognition performance of 3,5-dichloro-and 3,5dimethylphenylcarbamate derivatives of several polysaccharides, including chitosan (22, 23) (Table 2) [60].These two chitosan derivatives presented a relatively high chiral recognition for the tested racemates, setting their potential use as CSPs [60].In the same year, Franco et al., described another strategy to obtain new chitosan-based CSPs by bonding the chitin-carbamate derivatives on chromatographic support [44].The obtained bonded CSPs allowed the use of a larger panel of solvents in the mobile phases compared to coated ones.Accordingly, the 3,5dimethylphenylcarbamate derivative of chitosan (23i) was mixed with 10-undecenoyl and covalently immobilized on allyl silica gel, which demonstrate to be very useful in the separation of several racemates, such as lormetazepam and temazepam with a α value of 1.80 (Table 2).The mobile phases comprising either different proportions of heptane/2-propanol and heptane/chloroform mixtures allowed the best enantioresolutions [44].
In another study, the synthesis and chromatographic evaluation of the chitosan derivatives 22 and 23 as well as four new chitosan derivatives (25-28) were described (Table 2) [62].All derivatives were coated on macroporous silica gel and evaluated as CSPs.Among them, the 3,5-dichloro-( 22 In 1998, the same group compared the chiral recognition performance of 3,5-dichloro-and 3,5-dimethylphenylcarbamate derivatives of several polysaccharides, including chitosan (22, 23) (Table 2) [60].These two chitosan derivatives presented a relatively high chiral recognition for the tested racemates, setting their potential use as CSPs [60].In the same year, Franco et al., described another strategy to obtain new chitosan-based CSPs by bonding the chitin-carbamate derivatives on chromatographic support [44].The obtained bonded CSPs allowed the use of a larger panel of solvents in the mobile phases compared to coated ones.Accordingly, the 3,5-dimethylphenylcarbamate derivative of chitosan (23i) was mixed with 10-undecenoyl and covalently immobilized on allyl silica gel, which demonstrate to be very useful in the separation of several racemates, such as lormetazepam and temazepam with a α value of 1.80 (Table 2).The mobile phases comprising either different proportions of heptane/2-propanol and heptane/chloroform mixtures allowed the best enantioresolutions [44].
To our knowledge, the most recent study with chitosan tris-phenylcarbamates was published by Guntari et al., in 2014 [63].In this study, they developed and evaluated a new way of immobilization of chitosan tris-3,5-dimethylphenylcarbamate (23) using continuous assembly of polymers techniques.These techniques employed a catalyst immobilized on silica particles to produce stable CSPs suitable to be used in a wide range of mobile phases.The obtained CSP proved to be effective in separating the enantiomers of Trögers base and trans-stilbene oxide [63].

Chitosan Bis-Carbamate CSPs
The first study related to bis-carbamate derivatives as chiral selectors for LC was described by Son et al., in 2006, which reported the development of a CSP based on chitosan bis-3,5-dimethylphenylcarbamate in which the amine group of the chitosan was modified with N-nicotinoyl-L-phenylalanine (36) (Table 3) [65].The bis-phenylcarbamate derivative 36 demonstrated a high solubility in several organic solvents and, consequently, was easily coated on aminopropyl silica.The LC performance of the obtained CSP was evaluated using different mobile phases and all the tested racemates were enantioseparated.The best chromatographic result was achieved for flavanone with α and Rs values of 4.70 and 4.33, respectively, using a mixture of hexane/2-propanol 80:20 as mobile phase [65].
In 2008, Yamamoto et al., prepared several bis-carbamate derivatives with the amino group of chitosan replaced by an imide moiety (37-45) (Table 4) [62].This study showed interesting results of enantioresolution for all the CSPs based on imide-chitosan derivatives.Examples include the resolution of trans-cyclopropanedicarboxylic acid dianilide in CSPs 42 and 45 with α values of 1.78 and 1.63 respectively, and the resolution of cobalt(III) tris (acetylacetonate) in CSP 41 with a α value of 1.84.L-phenylalanine (36) (Table 3) [65].The bis-phenylcarbamate derivative 36 demonstrated a high solubility in several organic solvents and, consequently, was easily coated on aminopropyl silica.The LC performance of the obtained CSP was evaluated using different mobile phases and all the tested racemates were enantioseparated.The best chromatographic result was achieved for flavanone with α and Rs values of 4.70 and 4.33, respectively, using a mixture of hexane/2-propanol 80:20 as mobile phase [65].dimethylphenylcarbamate in which the amine group of the chitosan was modified with N-nicotinoyl-L-phenylalanine (36) (Table 3) [65].The bis-phenylcarbamate derivative 36 demonstrated a high solubility in several organic solvents and, consequently, was easily coated on aminopropyl silica.The LC performance of the obtained CSP was evaluated using different mobile phases and all the tested racemates were enantioseparated.The best chromatographic result was achieved for flavanone with α and Rs values of 4.70 and 4.33, respectively, using a mixture of hexane/2-propanol 80:20 as mobile phase [65].In recent studies (2016), Tang et al., prepared several bis-phenylcarbamate derivatives in which the amine moiety of chitosan was modified by an isobutyrylamide moiety (46-57) [66,67].The synthesized chitosan derivatives were coated on aminopropyl silica resulting in a series of new CSPs for LC.Considering their poor solubility, they were able to withstand operations with other mobile phases than the typical hexane/2-propanol (Table 5).They demonstrated high solvent tolerance and could still work after being flushed with chloroform (100%), ethyl acetate (100%) or tetrahydrofuran/n-hexane (70:30 v/v) without significant loss of enantioseparation.Furthermore, the CSPs presented chiral recognition performance for some of the tested racemates, including Troger's base in CSPs 47, 49, 50, 55 and 57, with α values of 1.40, 1.46, 1.54, 1.53 and 1.30 respectively, using n-hexane/2-propanol (90/10 v/v) as mobile phase (Table 5) [66,67].In recent studies (2016), Tang et al., prepared several bis-phenylcarbamate derivatives in which the amine moiety of chitosan was modified by an isobutyrylamide moiety (46-57) [66,67].The synthesized chitosan derivatives were coated on aminopropyl silica resulting in a series of new CSPs for LC.Considering their poor solubility, they were able to withstand operations with other mobile phases than the typical hexane/2-propanol (Table 5).They demonstrated high solvent tolerance and could still work after being flushed with chloroform (100%), ethyl acetate (100%) or In recent studies (2016), Tang et al., prepared several bis-phenylcarbamate derivatives in which the amine moiety of chitosan was modified by an isobutyrylamide moiety (46-57) [66,67].The synthesized chitosan derivatives were coated on aminopropyl silica resulting in a series of new CSPs for LC.Considering their poor solubility, they were able to withstand operations with other mobile phases than the typical hexane/2-propanol (Table 5).They demonstrated high solvent tolerance and  for LC.Considering their poor solubility, they were able to withstand operations with other mobile phases than the typical hexane/2-propanol (  synthesized chitosan derivatives were coated on aminopropyl silica resulting in a series of new CSPs for LC.Considering their poor solubility, they were able to withstand operations with other mobile phases than the typical hexane/2-propanol (Table 5).They demonstrated high solvent tolerance and could still work after being flushed with chloroform (100%), ethyl acetate (100%) or tetrahydrofuran/n-hexane (70:30 v/v) without significant loss of enantioseparation.Furthermore, the CSPs presented chiral recognition performance for some of the tested racemates, including Troger's base in CSPs 47, 49, 50, 55 and 57, with α values of 1.40, 1.46, 1.54, 1.53 and 1.30 respectively, using nhexane/2-propanol (90/10 v/v) as mobile phase (Table 5) [66,67].
Symmetry 2017, 9, 190 17 of 28  In the same year, some bis-phenylcarbamate derivatives with different substituents in both phenylcarbamate and amine moieties (58)(59)(60)(61) were obtained by the same group (Table 5) [69].The synthesized chitosan derivatives were coated on aminopropyl silica, and showed chiral recognition for the majority of the tested racemates.These new CSPs also proved to be stable when used with other mobile phases than the typical hexane/2-propanol [69].
Other CSPs based on the substitution of the amine of chitosan with an alkyl moiety, prior to the derivatization of the hydroxyl groups with different isocyanates were described [68,70] In the same year, some bis-phenylcarbamate derivatives with different substituents in both phenylcarbamate and amine moieties (58)(59)(60)(61) were obtained by the same group (Table 5) [69].The synthesized chitosan derivatives were coated on aminopropyl silica, and showed chiral recognition for the majority of the tested racemates.These new CSPs also proved to be stable when used with other mobile phases than the typical hexane/2-propanol [69].
Other CSPs based on the substitution of the amine of chitosan with an alkyl moiety, prior to the derivatization of the hydroxyl groups with different isocyanates were described [68,70]       Other chitosan bis-3,5-dimethylphenylcarbamates with different moieties linked to the chitosan amine group (83-87) were developed by Wang et al., (Table 6) [73].The obtained CSPs showed good chiral recognition abilities, especially the CSP comprising the chitosan-derivative 87, which was able , postulated that the development of a chitosan CSP would be an excellent tool to be used in chiral ligand-exchange chromatography (CLEC), considering the high binding capacity of chitosan to heavy metals [75].Consequently, they described the immobilization of chitosan into silica gel and the application of the obtained CSP (Figure 5) in CLEC to achieve enantioresolution of a variety of α-hydroxycarboxylic acids and α-aminoacids using CuSO 4 100% or CuSO 4 /MeOH (80:20 v/v) as mobile phases [75].To the best of our knowledge, this is the only report related to the application of chitosan-derived CSPs for this type of study.
published by Liang et al., [74].In this study, several CSPs based on chitosan N-isobutylurea (88a-91b) were prepared (Table 6).Two types of chitosan with different molecular weights were used.In this study, the CSPs developed with higher molecular weight chitosan (88a, 89a, 91a) showed lower chiral recognition ability than their low molecular weight chitosan counterparts (88b, 89b, 91b), with the exception of derivative 90a that showed higher chiral recognition ability than derivative 90b.These CSPs were also able to withstand organic solvents such as ethyl acetate (100%) and chloroform (100%) [74].

Chitosan Amine-Carbamate CSPs
Liu et al., in 2006, postulated that the development of a chitosan CSP would be an excellent tool to be used in chiral ligand-exchange chromatography (CLEC), considering the high binding capacity of chitosan to heavy metals [75].Consequently, they described the immobilization of chitosan into silica gel and the application of the obtained CSP (Figure 5) in CLEC to achieve enantioresolution of a variety of α-hydroxycarboxylic acids and α-aminoacids using CuSO4 100% or CuSO4/MeOH (80:20 v/v) as mobile phases [75].To the best of our knowledge, this is the only report related to the application of chitosan-derived CSPs for this type of study.

Conclusions
Polysaccharide-based CSPs are of great value and are being recognized as highly successful for both analytical and preparative separations.Among them, amylose and cellulose carbamate derivatives are the most widely used CSPs for the efficient resolution of several racemates revealing very high chiral recognition abilities.
Although several efficient polysaccharide-based CSPs are described in the literature and many of them are commercially available, studies on new and improved polysaccharide-based CSPs are still being conducted.The research is mainly focused on the immobilization of the chiral selectors on chromatographic support, allowing the use of a wider range of mobile phases and, consequently, increasing the range of their applications.Polysaccharide-based CSPs comprising other natural polymers and derivatives such as tris-phenylcarbamates of chitin and chitosan as well as bis-phenylcarbamates of chitosan also showed high chiral recognition abilities being able to resolve diverse types of racemates.Most of these CSPs were obtained by the traditional coating method; however, regarding their poor solubility they were able to perform enantioseparations under reversed phase as well as using different solvents as components of the mobile phases in normal phase, such as chloroform and ethyl acetate.Some chitosan-based CSPs were also prepared by immobilization of the chiral selector on the chromatographic support.

Figure 1 .
Figure 1.Structures of different types of polysaccharides studied as selectors for liquid chromatography (LC).

Figure 1 .
Figure 1.Structures of different types of polysaccharides studied as selectors for liquid chromatography (LC).

Figure 1 .
Figure 1.Structures of different types of polysaccharides studied as selectors for liquid chromatography (LC).

Figure 3 .
Figure 3.Chemical structures of the analytes A1-A40 separated in chitin and chitosan based CSPs.Figure 3. Chemical structures of the analytes A1-A40 separated in chitin and chitosan based CSPs.

Figure 3 .
Figure 3.Chemical structures of the analytes A1-A40 separated in chitin and chitosan based CSPs.Figure 3. Chemical structures of the analytes A1-A40 separated in chitin and chitosan based CSPs.

Figure 4 .
Figure 4.Chemical structures of the analytes A41-A73 separated in chitin and chitosan based CSPs.

Figure 4 .
Figure 4.Chemical structures of the analytes A41-A73 separated in chitin and chitosan based CSPs.

Table 3 .
Chitosan bis-carbamate CSP with the amine group of the chitosan modified by N-nicotinoyl-L-phenylalanine.

Table 3 .
Chitosan bis-carbamate CSP with the amine group of the chitosan modified by N-nicotinoyl- L-phenylalanine.

Table 3 .
Chitosan bis-carbamate CSP with the amine group of the chitosan modified by N-nicotinoyl- L-phenylalanine.

Table 4 .
Chitosan bis-carbamate CSPs with the amine group of the chitosan replaced by an imide moiety.

Table 4 .
Chitosan bis-carbamate CSPs with the amine group of the chitosan replaced by an imide moiety.Symmetry 2017, 9, 190 13 of 28

Table 4 .
Chitosan bis-carbamate CSPs with the amine group of the chitosan replaced by an imide moiety.

Table 5 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an alkylamide moiety.

Table 5 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an alkylamide moiety.

Table 5 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an alkylamide moiety.

Table 5 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an alkylamide moiety.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.

Table 6 .
Chitosan bis-carbamate CSPs with the amine moiety of chitosan modified by an N-alkyl urea.