8.7 Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

8.7 Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers HT Rasmussen and K Huang, Vertex Pharmaceuticals Inc., Cambridge, MA, USA r 2012 Elsevier Ltd. All rights reserved.

8.7.1 8.7.2 8.7.3 8.7.4 8.7.4.1 8.7.4.1.1 8.7.4.1.1.1 8.7.4.1.1.2 8.7.4.1.1.3 8.7.4.1.2 8.7.4.1.2.1 8.7.4.1.2.2 8.7.4.1.2.3 8.7.4.1.3 8.7.4.1.4 8.7.4.1.5 8.7.4.2 8.7.4.2.1 8.7.4.2.2 8.7.4.2.3 8.7.4.2.4 8.7.4.3 References

Overview of the Historical Development of Chromatography Chiral Separations: Chromatographic Derivatization in Chiral Chromatography Chiral Stationary Phases (CSPs) for High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Supercritical Fluid Chromatography (SFC) Chiral Stationary Phases for High-Performance Liquid Chromatography Polymeric chiral stationary phases Polysaccharide chiral stationary phases Protein-based chiral stationary phases Synthetic polymeric chiral stationary phases Macrocyclic chiral stationary phases Crown-ether-based chiral stationary phases Cyclodextrin-based chiral stationary phases Macrocyclic glycopeptide chiral stationary phases p-Complex chiral stationary phases Ligand exchange chiral stationary phases Other chiral stationary phases Chiral Stationary Phases for Gas Chromatography Derivatized cyclodextrin chiral stationary phases Amino acid-based chiral stationary phases Metal–ligand complex chiral stationary phases Ionic liquid chiral stationary phases (IL-CSPs) Chiral Stationary Phases for Supercritical Fluid Chromatography

Abbreviations CD CSP

8.7.1

cyclodextrin chiral stationary phase

GC HPLC SFC

96 96 98 100 101 101 101 102 102 102 102 104 105 105 106 107 107 108 110 111 111 111 113

gas chromatography high-performance liquid chromatography supercritical fluid chromatography

Overview of the Historical Development of Chromatography

Chromatography is a family of separation techniques for the resolution of individual components in mixtures. The word is derived from Greek and literally translates as ‘color to write.’ It was named in 1903 by Tswett, who used the technique for the separation of colored plant pigments such as chlorophyll. In 1941, British biochemists Martin and Synge established the basic techniques and theoretical foundation of partition chromatography.1 This milestone led to the rapid development of various chromatographic techniques, such as paper chromatography by Gordon and Consden in 19442 and reverse-phase chromatography by Howard and Martin in 1950.3 In 1952, again based on the theory of partition chromatography, the first successful use of gas chromatography was demonstrated by James and Martin.4 Further development of GC was enabled by Golay’s work on open tubular capillary columns in the mid 1950s. All of these developments provided the basis for enantiomeric separations. Over the past 30 years, chiral chromatography has been established as a well-developed and mature tool for the separation of optically active entities in various industries including pharmaceutical, biomedical, agricultural, environmental, food and beverage, etc.

8.7.2

Chiral Separations: Chromatographic

As a basis for separation, the fundamental prerequisite for chiral recognition in chromatography is the formation of diastereomers. Chiral discrimination will then occur due to thermodynamic selectivity resulting from different Gibbs free energies between the two diastereomeric complexes. Diastereomer formation can be attained in two ways. Either formal diastereomers are formed via precolumn derivatization of enantiomers or transient uncovalent chiral diastereomers are produced by the direct use of chiral

96

Comprehensive Chirality, Volume 8

http://dx.doi.org/10.1016/B978-0-08-095167-6.00831-4

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

97

selection media. The first approach is usually followed by separation on conventional achiral columns. This could be advantageous in that separations on achiral stationary phases generally show better behavior than on chiral stationary phases. However, stringent requirements are placed on the enantiopurity of the chiral derivatization reagents, and the chemistry must be demonstrated to show that it does not induce racemization of the enantiomers. Extra work-up is also sometimes needed postderivatization to extract the diastereomeric products from the reaction matrix. For these reasons, the direct use of chiral media is a much more practiced methodology and will be the focus of discussion for this section. When chiral compounds come in contact with chiral selection media, a complex array of nonstereospecific and stereospecific interactions can occur simultaneously. The observed chiral separation is the exhibited net result of all these interactions. The construction of the chiral separation mechanism is difficult. Presently, the most prominent and widely used model is the threepoint intervention theory. The key to this theory is that a minimum of three simultaneous interactions should take place and they should occur with three different substituents emanating from the stereogenic center. Figure 1 illustrates favored and unfavored binding between a bonded chiral stationary phase and a racemic mixture of R and S configurations. Bound state

Unbound state Support material

A Spacer

Selector, CS C

B

(R )

CS

C

b

d

c

(S )-SA

‘Ideal fit’

a b

a

A B

(R)

d c (S)-Selectand, (S)-SA A a

c b

(R)

d (R)-Selectand, (R )-SA

CS

a B

C

c

d b (R)-SA

‘Non-ideal fit’ ....Non-covalent bond Figure 1 Three-point interaction illustration between a silica gel-based bonded chiral stationary phase and a racemic mixture of R and S stereoconfiguration. Reproduced with permission from Laemmerhofer, M. J. Chromatogr. A 2010, 1217(6), 814–856.

The historical development of the three-point interaction model dates back to the early 1930s. In 1933, Easson and Stedman postulated a three-point geometric model to explain stereoselective interactions between chiral molecules and protein receptors and the impact on physiological activity between a dissymmetric drug and its target.5 In 1952, Dalgiesh proposed a three-point interaction model for elucidating the separation of D/L-amino acids using paper chromatography. In the original model, all three interactions are attractions. However, it was later proved by a series of studies that not all interactions need to be attractive. In 1997, Davankov established that when one sufficiently strong attractive force is existent, the other forces could be either attractive or repulsive.6 Still, the three-point attachment model is challenged in that the original foundation on which the model was built projects a rather simplified scenario. For example, the model was founded on the premises that only one stereogenic center is located in the structure and neglects the conformational mobility of chiral analytes and chiral selectors. Subsequent debate, extension on the theory, and proposals of alternative models have been provided in numerous publications.7–11 Despite the criticism and challenges, the three-point interaction model still has practical validity for many enantioselective systems. For example, the design and development of Pirkle-type LC chiral stationary phases adhered to and utilized this theory. They have proved effective for the enantioseparation for several classes of compounds. In a successful case of enantiomeric separation, one primary docking interaction is required to bring the analyte to the proximity of the chiral selector. These are often stronger interactions, such as ionic interactions, inclusion complexation, or p acidic–p basic interactions. A combination of secondary interactions usually then take place to determine chiral recognition. These tend to be weaker forces such as hydrogen bonding, dipolar interactions, steric interactions, and dispersion interactions. Indeed, the forces involved in the process of enantiomeric recognition do not need to be strong. Typically, a difference in the free energy of interaction of 0.03 kJ mol1 between the two enantiomers and the stationary phase will suffice for enantiomeric discrimination. The spatial location of the stereospecific interaction is another important factor. The stereogenic centers on the enantiomers need to be within certain proximity of where the primary docking event occurs. For example, there are two requirements for enantiomers to obtain reverse-phase chiral separation on cyclodextrin-based CSPs. First, at least one aromatic functionality needs to be present to ensure the formation of inclusion complexation. Second, interactions must take place between the analyte substituent group on or near the stereogenic center and the cyclodextrin functionality at the mouth of the cyclodextrin cavity. Note that both

98

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

the primary docking sites and the secondary interaction locales can be altered based on the separation environment. Different separation modes induce the most drastic changes in separation mechanisms when different structural groups are involved. In addition, pH variation can cause ionization in certain functionalities and a consequent change in the selector–selectand interaction as well.12 As was mentioned earlier, from a thermodynamic perspective, chiral separation is intrinsically a manifestation of the different Gibbs free energies of the diastereomers formed between each enantiomer and the chiral selector. The Gibbs–Helmholtz equation for the dynamic formation of noncovalent diastereomers between chiral selectors and optical isomers is DG ¼ DH  TDS ¼ RT ln K ¼ RT lnðk0 bÞ ¼ RT ln k0  RTln b

ð1Þ

The equation shows that the complexation process involves enthalpy (DH) and entropy (DS) contributions. The two factors combined are dependent on the partition coefficient K. R is the universal gas constant (8.3144 J mol1 K1) and T is the absolute temperature in Kelvin. k0 is the capacity factor and b is the phase ratio. A van’t Hoff plot can be used to determine the enthalpy and entropy effects on chiral recognition. For example, Figure 2 shows the relationship between ln k and 1/T for the separation of 2-(N-isopropylamino)-1-phenylethanol on an 18-crown-6 based chiral stationary phase. DH can be derived from the slope and DS can be calculated from the y-intercept.

In k1(kR) In k2(kS) 9 TIso: −60 °C

In k

6

3

2

4

1/T × 1000

6

8

Figure 2 Van’t Hoff plot for ln k vs. 1/T for 2-(N-isopropylamino)-1-phenylethanol on crown-ether-based CSP. Reproduced with permission from Choi, H. J.; Park, Y. J.; Hyun, M. H. J. Chromatogr. A 2007, 1164(1–2), 235–239.

Enantioselectivity (a) exhibited by the chiral selector for two enantiomers is related to the difference in the change of Gibbs free energy on binding with the chiral selector. This relationship can be stated as DDG ¼ DDH  TDDS ¼ RT lnðDKÞ ¼ RT ln a

8.7.3

ð2Þ

Derivatization in Chiral Chromatography

Derivatization in chromatography serves several different purposes. First, in indirect separation of optical isomers, precolumn chiral derivatization is required. Enantiomers of interests are derivatized with a single enantiomer of the chiral derivatizing agent to form pairs of diastereomers before their introduction into the chromatographic system. Conventional achiral media can then be used for the separation of the formed diastereomers. For example, Figure 3 shows the separation of chiral amines on a reversephase ODS column after chiral derivatization with DBD-b-proline.13 However, as mentioned, there can be complications associated with the entire precolumn derivatization methodology. It is important to ensure that the derivatization agent is optically pure and no racemization of the target analyte is caused by the derivatization. Another concern is that a mixture of multiple products may be formed when the analyte has more than one functional group that may be reactive to the derivatization agent (e.g., amino alcohols). Also, to ensure the completion of derivatization, an excess of chiral derivatization agent is usually used and will not be totally consumed by the chemistry. The residual amount of starting material, together with any by-products, can confound the chromatography (see Chapter 8.15).14 The second purpose of derivatization is to improve sample suitability for chromatographic analysis. In this case, achiral reagents are often used and hence the optical properties of the original molecules are maintained. One of the most commonly

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

99

R1 CONH C R2 R3

COOH N

N EDC/pyridine

N

N

R1 H2N C R2 R3

O N

O N SO2N(CH3)2 DBD--Pro-amide

SO2N(CH3)2 DBD--proline (A)

0.7 0.4

1

0.6

0.1

0.1

Volts

0.2

Volts

Volts

2 0.2

0.0

0.0 12.0

12.5

12.0

(a)

13.5 Minutes

140

14.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0.0

0.0

15.0

12.5 (b)

0.036

4

0.5

0.3

0.3

0.6

3

15.0 Minutes

Volts

0.4

0.7

17.5

0.036 5 6

0.026

0.016

0.016

0.006

0.006

−0.004

−0.004

(c)

(B)

35

40

45 Minutes

50

Volts

Volts

0.026

55

Figure 3 Separation of amines on an ODS column after their chiral derivatization with 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole-bproline (DBD-b-proline). For separation conditions, see (A) Chiral derivatization schematics. (B) Chromatograms representative of the separation after chiral derivatization. (a) (R) and (S)-1-phenylethylamine derivative. (b) (DL-1-phenylalanine ester derivative. (c) (R) and (S)-1-(1-naphthyl)ethylamine derivative. Reproduced with permission from Min, J. Z.; et al. Biomed. Chromatogr. 2005, 19(1), 43–50.

used forms of this scenario is the derivatization of amino acids to form various derivatives with less polarity that reduce deleterious interactions between the molecule and the chiral resolving media.15 Derivatization in GC is based largely on the same concerns. Modification of compounds containing active hydrogens (–COOH, –OH, –NH, –SH) is frequently necessary to avoid their undesirable interactions with GC-CSPs and silanol groups on the solid supports. The derivatization often yields a shorter analysis time, improved peak shape and efficiency, enhanced detection sensitivity, and varied enantioselectivities. This is depicted in Figure 4. Another advantage of the derivatization of polar groups is the increased compound volatility and thermal stability. These are the crucial factors that determine compounds’ amenability for GC analysis. The various derivatization schemes adopted for GC analysis generally fall into three categories: silylation, acylation, and akylation. Various derivatization agents are available for individual types of analytes. A list of the methods and the corresponding agents applicable for achiral derivatizations of alcohols and phenols is provided in Table 1. Further information on the derivatization methodology and agents for other structures is available elsewhere.16–19

100

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

A

OH Cl

O O

B

C

0

1

2

3

4 5 Chiraldex® G-TA, 80 °C Time (min)

6

7

D

8

Figure 4 Chromatogram illustrating the enantiomeric separation of underivatized ethyl-4-chloro-3-hydroxybutyrate (C and D) and their derivatized products (A and B). Peaks A and D represent the S-enantiomers and Peaks B and C represent the R-enantiomers. Column: Chiraldexs G-TA; Carrier gas: Helium; Temperature: 120 1C; Detection: FID. Reproduced with permission from Huang, K.; Breitbach, Z. S.; Armstrong, D. W. Tetrahedron: Asymmetry 2006, 17(19), 2821–2832.

Table 1 Methods and corresponding agents for derivatizing alcohols and phenols for gas chromatographic analysis24 Method

Reagent

Silylation

BSA (bis(trimethylsilylacetamide)) BSTFA (bistrimethylsilyltrifluoroacetamide) MTBSTFA (N-methyl-N-t-butyldimethylsilyltrifluoroacetamide)

Acylation

TFAA (trifluoroacetic anhydride) MBTFA (N-methyl-bis(trifluroacetamide)) HFBI (heptafluorobutyrylimidazole) HFBA (heptafluorobutyric anhydride) PFPA (pentafluoropropionic anhydride)

Alkylation

DMF (dialkylacetals) PFB-Br/TBA-H-SO4 (pentafluorobenzyl bromide) TBH (tetrabutylammonium hydroxide)

Additionally, precolumn derivatization and postcolumn derivatization are used in LC or CE to enable or improve the detection of certain structures. A large number of agents and derivatization schemes have been used to generate or enhance an analyte’s UV, fluorescence, or electrochemical sensitivity.20 For example, o-phthalaldehyde (OPA), cysteine, and 2-methyl-3-oxo-4-phenyl-2,3dihydrofuran-2-yl acetate have been reported to selectively tag primary amines with fluorescent labels.21,22 Figure 3 shows an example for fluorescent labeling of amines using DBD-b-proline as the labeling agent. Derivatization used in GC also increases compound detectability, by either increasing the bulkiness of the molecule or by introducing appropriate atoms and functional groups that can be sensitively measured using the detection method. For example, halogen atoms can be added to the analyte for electron capture detection (ECD).23,24

8.7.4

Chiral Stationary Phases (CSPs) for High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Supercritical Fluid Chromatography (SFC)

Two approaches can be adopted for the introduction of chiral media in chromatography systems: (1) as a mobile phase additive and (2) as an immobilized stationary phase. The former option allows the use of a conventional solution-based separation mechanism, such as CE. However, the approach is not as popular in the other chromatographic techniques due to the waste of costly chiral selectors. In HPLC, GC, and SFC, application of immobilized chiral selector is the method of choice and accounts for

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

101

99% of chiral separations.25 Accordingly, the use of chiral mobile phase additives will not be discussed herein. References are available elsewhere for readers’ interests.26–29 Although ultra performance liquid chromatography (UPLC) has the advantages of fast speed, high resolution and sensitivity, and relatively low solvent consumption, its application in chiral separation remains rare.30,31 Therefore, it will not be discussed here either.

8.7.4.1

Chiral Stationary Phases for High-Performance Liquid Chromatography

HPLC has proven to be one of the most effective methods for enantiomeric separations. It is widely used in chiral separations in pharmaceutical, agricultural, chemical, and biomedical applications. It gains its predominant popularity due to its broad applicability, great versatility, wide selectivity, good reproducibility, and ease of scaling up. Chiral stationary phases for HPLC are produced by immobilizing chiral selectors on solid supports by either coating or covalent bonding. The following section summarizes the main classes of chiral stationary phases in use today for enantiomeric HPLC separations: polymeric chiral stationary phases, microcyclic chiral stationary phases, [p]-complex chiral stationary phases and ligand exchange chiral stationary phases.

8.7.4.1.1

Polymeric chiral stationary phases

8.7.4.1.1.1 Polysaccharide chiral stationary phases Polysaccharides are among the most common naturally occurring chiral macromolecules. They are polymeric structures of (D) þ glucose units linked by (1–4) glycosidic bonds. The first report on using polysaccharides as HPLC-CSPs was presented in the early 1980s.32 In the mid-1980s, research by Okamoto et al. demonstrated high enantioselectivities and broad applicability of various derivatized cellulose and amylase-based chiral selectors.33–35 This work generated worldwide attention and caused the polysaccharide-based CSPs to thrive for the following decades. Today, the tri-ester and tri-carbamate derivatives are most commonly used for HPLC due to their remarkable selectivities, sensitivities, and reproducibilities. Cellulose and amylose tris(3,5-dimethylphenylcarbamate) (Daicel Chiralcels OD and Chiralpaks AD, Figure 5) are currently the most used versions. Okamoto reported an enantioresolution of 64% out of 483 racemic compounds using cellulose tris(3,5-di-methylphenyl carbamate), and chiral separation of 80% when the corresponding amylose carbamate was used in combination.36 Figure 6 demonstrates the separation of atenolol enantiomers on a Chiracel OD column.37 The effectiveness of these polysaccharide derivatives is believed to arise from the helical twist in their structures, which leads to inclusion complexation to the range of possible interaction mechanisms.

OR

O O OR

R=

C

H N

n

OR

(a)

OR

O R=

OR (b)

OR O

C

H N

n

Figure 5 Structure of Chiralcels OD and Chiralpaks AD chiral stationary phases. (a) Chiralcels OD, cellulose tris(3,5-dimethylphenylcarbamate). (b) Chiralpaks AD, amylose tris(3,5-dimethylphenylcarbamate).

Chiralcels OD and Chiralpaks AD are generated by first derivatizing the native cellulose and amylose and then coating onto the silica support. However, since carbohydrates are partially or fully soluble in certain solvents, these coated versions are restricted to normal phase separations, with limited solvent options. For example, chlorinated solvents should be avoided due to their tendency to strip the carbohydrates off the solid support. Since 2004, bonded phases have become commercially available. They have the same carbohydrate derivatives as their normal phase analogs, but are chemically bonded to the silica support. This allows the chiral selectors to maintain viability, even when using solvents that swell or destroy the coated polymers. Better durability is achieved on these columns with almost no solvent restrictions. However, the configurational alteration of the chiral selector due to the bonding causes enantioselectivity differences from the coated counterparts. Another approach used to enable the use of an aqueous-organic mobile phase is to coat the chiral selectors on a high-quality hydrophobic silica gel. Currently, a good variety of polysaccharide-based CSPs are carried by a number of commercial suppliers including Daicel, Kromasil, Macherey Nagel, Knauer, and Sepaserve (see Chapter 8.11).

102

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

1

2

min s

Figure 6 Enantiomeric separation of atenolol on Chiralcel OD. Mobile phase: hexane:ethanol:diethylamine 75:25:0.1 v/v/v; Flow rate: 0.7 ml min1; Detection: 276 nm; Temperature: ambient; Peak label: 1. (R)-atenolol 2. (S)-atenolol. Reproduced with permission from Santoro, M. I. R. M.; Cho, H. S.; Kedor-Hackmann, E. R. M. Drug Dev. Ind. Pharm. 2000, 26(10), 1107–1110.

8.7.4.1.1.2 Protein-based chiral stationary phases Protein molecules contain a large number of stereogenic centers that make them suitable chiral selectors for many classes of chiral analytes. The essential mechanisms of enantioseparation for these CSPs are hydrophobic and polar interactions. Some examples of commercially available protein-based CSPs are bovine serum albumin (BSA), human serum albumin (HSA), a1-acid glycoprotein (AGP), ovomucoid (OVCHI), and avidin (AVI). All are reverse-phase columns with natural protein bonded to the silica gel matrices. Figure 7 shows the use of AGP for the separation of ethotoin, an anticonvulsant drug used in the treatment of epilepsy. However, proteins are known to be the most labile and most easily overloaded CSPs. When operating with these columns, precautions with the mobile phase need to be taken to avoid the denaturing of the stationary phase. The separation also needs to be strictly controlled for pH (an example of a pH effect is given in Figure 8), ionic strength, and added organic solvent or buffer. All these factors significantly lower column ruggedness. Also, the chiral selector loading of these CSPs is limited due to the large size of protein molecules (40 000–70 000 Da). Consequently, these CSPs exhibit low sample capacity. Together with the low applicable flow rate of these columns, this class of CSPs is frequently unsuitable for large-scale applications. Due to these limitations, protein-based CSPs play a minor role in enantiomeric HPLC separations (see Chapter 8.9). 8.7.4.1.1.3 Synthetic polymeric chiral stationary phases Due to their fully synthesized nature, synthetic polymeric CSPs encompass a vast number of structures including helical polymethacrylates, polyamides, polyolefin, poly(vinyl ether)s, polychloral, polyurethanes, polyacetylene derivatives, etc. Only a few commercial CSPs are available, all in the form of either bonded or adsorbed phases, such as Astec’s P-CAPs and P-CAP-DP. Synthetic CSPs tend to have a capacity for higher sample loadings. The first reason for this is that they usually have high chiral selector loading. Secondly, often, one single chiral selector molecule could interact with several molecules of analytes simultaneously along its length. An example of a high-capacity separation based on an (R,R)-P-CAP-DP column is provided in Figure 9. Other advantages of this class of chiral selectors include the richness of chemical structures and the ease of structural design. However, to date, the impact of the synthetic polymeric CSPs remains relatively limited. Structures providing wider enantioselectivities are still in development.

8.7.4.1.2

Macrocyclic chiral stationary phases

8.7.4.1.2.1 Crown-ether-based chiral stationary phases Chiral crown-ether CSPs were introduced in 1974 by Cram et al. by immobilizing bis-(1,1-binaphthyl)-22-crown-6 on polystyrene or silica gel.38 Subsequent development has given rise to three subclasses: a binaphthyl type, a tartaric acid type, and a

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

0

103

10 Time (min)

Figure 7 Enantiomeric separation of ethotoin on AGP. Column: 2.0  100 mm i.d.; Mobile phase: 10 mM phosphate buffer (pH 5.0); Detection: 210 nm; Temperature: 25 1C; Sample loading: 100 ng. Reproduced with permission from Haginaka, J.; Matsunaga, H. Enantiomer 2000, 5(1), 37–45.

pH 7.0

0.0

pH 6.0

2.0 min

0.0

2.0 min

pH 5.0

5.0

0.0

2.0 min

Figure 8 pH effects on chiral separation for an AGP column. From http://www.mz-at.de/pdf/ChromTech_AGP.pdf

5.0

104

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

O

H N

H N

(R)

(R)

O

(a)

8 µg injection

OH

mAU

60

Peak 1 = 10.4 min.

O

*

40

O

O

GOO4030 Peak 2 = 13.2 min.

20

0 0

5

Min

10

15 GOO4031

1000 µg injection Peak 1 = 9.4 min

mAU

1000

Peak 2 = 11.2 min

0 0 (b)

5

10 Min

15

20 GOO4032

Figure 9 High-capacity separation of furoin enantiomers on synthetic polymeric (R,R)-P-CAP-DP. (a) Structure of P-CAP-DP75 CSP. (b) Chromatogram of the separation. Reproduced with permission from http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/General_Information/ 1/t410060.Par.0001.File.tmp/t410060.pdf

sugar type. For example, the commercialized Crownpaks-CR, which is available in both enantiomeric forms, is based on a binaphthyl-derivatized 18-crown-6-polyether structure. The size and structural configuration of 18-crown-6 enables the ring-shaped ether backbone to form specific host–guest complexation with cations the size of ammonium and potassium. Figure 10 illustrates the complexation between Crownpaks-CR and guest cations. Although the host–guest binding acts as the primary docking force of interactions, secondary enantioselective interactions such as steric and hydrophobic interactions occur to induce chiral recognitions. Crown-ether-based CSPs are highly specialized in the enantioresolution of chiral primary amines. Acidic buffer such as perchloric acid is necessary to ensure the protonation of the amines. Also, the system needs to be potassium free to avoid competitive binding to the crown-ether cavities (see Chapter 8.13).

8.7.4.1.2.2 Cyclodextrin-based chiral stationary phases Cyclodextrins (CDs) constitute an importance class of HPLC-CSPs. CDs are D-(þ)-glycopyranoside units linked in a ring structure via a-1,4 linkages. They are produced by partial digestion of starch, followed by coupling of cleaved glucose units using cyclodextrin glycosyltransferase (CGTase) and bacillus macerans amylase. The enzymatic coupling step generates mixtures of CDs of various sizes. The most commonly used are a, b, and g-CDs with 6-, 7-, and 8-memberred rings, respectively. CDs show a toroidal shape in space with a hydrophobic cavity. On the exterior, it extends hydrophilic rims of primary and secondary hydroxyl groups and stereogenic centers. Due to the structural characteristics, cyclodextrin-based CSPs are capable of various types of interactions

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

105

O O

O A

R O

O O R = K+, NH4+

Silica gel s

Figure 10 Complexation between Crownpak -CR and guest cations.

such as inclusion complexation, dipolar interactions, hydrogen bonding interactions, steric hindrance, etc. Indeed, CDs have proved to be good chiral selectors for a wide range of chiral molecules. Both native and functionalized CDs have been used as bonded stationary phases in HPLC chiral separations. These CSPs excel in different chromatographic modes depending on the type of derivatization. For example, native CDs will most likely be effective in the reverse-phase mode, in which inclusion complexation constitutes the driving force for enantiomeric recognition. A few types of derivatives with aromatic functionalities are suitable for normal phase operations, where p–p interactions are the essential interactions. All columns can be used in the polar organic mode, in which the analytes interact exclusively with the exterior groups of the CDs due to the occupation of the hydrophobic cavity by the solvent molecules in the mobile phase. Figure 11 provides an overview of some important commercial cyclodextrin columns. Their appropriate operation modes are also included (see Chapters 8.10 and 8.14).

8.7.4.1.2.3 Macrocyclic glycopeptide chiral stationary phases Macrocyclic glycopeptides are an important class of CSPs for chiral HPLC analysis. They are manufactured by chemically bonding fermentation-produced antibiotics onto a silica gel. Their effectiveness can be attributed to their basket-like structural region, abundance of stereogenic centers, and ample groups of various functionalities. Since their introduction as HPLC-CSPs by Armstrong et al. in the mid-1990s,39,40 four CSPs of this class have been commercialized based on the structure of vancomycin (Chirobiotics V and V2), teicoplanin (Chirobiotics T and T2), ristocetin A (Chirobiotics R), and teicoplanin aglycone (Chirobiotics TAG). The structures of the four molecules are shown in Figure 12. Each of these Chirobitotic CSPs exhibits unique enantioselectivities. Meanwhile, they are highly complementary to each other. Usually, partial separation obtained on one type of Chirobiotics CSP indicates great possibility of baseline separation when switching to another type of structurally related antibiotic CSP, with the exact same mobile phase (see Figure 13). The reason for this is the subtle difference in binding sites among these antibiotics. The structures of these antibiotics show that they can provide a range of interaction forces including polar, hydrogen bonding, inclusion complexation, p–p, and ionic interactions. This makes these chiral structures appropriate CSPs for all separation modes. A detailed review on the separation mechanism in each chromatographic mode has been published elsewhere.41 Currently, these ‘multi-modal’ chiral columns have been established as valuable tools for the separation of a wide variety of chiral molecules, including applications in drug analysis, metabolism studies, bioconversion processes monitoring, etc. Figure 14 shows a Chirobiotics V-based HPLC method for the quantitative determination of promethazine enantiomers in pharmaceutical formulations. The macrocyclic glycopeptide class is particularly suitable for enantioresolution of chiral amino acids. Direct separations of amino acid racemates with no buffer addition or precolumn derivatization have been achieved in many cases (see Table 2). This capability not only simplifies the separation but also facilitates preparative scale applications. Table 2 lists the CSPs and separation parameters (retention factor k0 and enantioresolution Rs) for the separation of underivatized natural a-amino acids (see Chapter 8.12).

8.7.4.1.3

p-Complex chiral stationary phases

As is indicated by the name, the major types of interactions the p-complex CSPs, or charge transfer CSPs, provide are p–p acceptor–donor interactions. Also known as Pirkle-type CSPs due to the series of works by Pirkle and coworkers,42,43 this type of CSP finds most effective applications in normal phase separations where the p–p interactions are more pronounced. Over

106

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers OR OR

RO RO

OR

RO Silica gel

Cyclobond suffix

Functionality

R-

I 2000, II 2000, III 2000 I 2000 DM I 2000 AC I 2000 SP or RSP

Native Dimethylated Acetylated Hydroxypropyl Ether

H

I 2000 RN or SN

Naphthylethyl ether

I 2000 DMP

3,5dimethylphenylcarbamate

CH3 COCH3

Operation mode RP, PO RP, PO RP, PO

CH2CH(OH)CH3

RP, PO RP, NP, PO

CONHCH(CH3)

CH3

RP, NP, PO

CONH CH3

I 2000 DNP

2,6-dinitro-4trifluromethylphenyl ether

O2N

RP, NP, PO

CF3

O2N Note: NP, normal phase; RP, reverse phase; PO, polar organic mode. Figure 11 Examples of cyclodextrin-based commercial CSPs.

the past few decades, p-acidic (p-electron-acceptor), p-basic (p-electron donator), and mixed mode (p-electron-acceptor and p-electron-donator) CSPs have been developed.44–46 The functionalized chiral selectors can be coated or bonded onto the silica gel support. They have been proven effective in the separation of many types of compounds including profens, aryl-amides, arylepoxides, aryl-sulfoxides and b-blockers. An example of commercialized CSP is Whelk-Os 1 (see Figure 15 for structure). Figure 16 shows a chiral HPLC application for the separation of ibuprofen enantiomers. The advantages of the p-complex-based CSPs are the ease of synthesis, high chiral selector loading, and cost effectiveness. However, due to the separation mechanism, p-basic types of CSPs can only resolve p-acidic compounds and vice versa. Therefore, aromatic rings and p-donator or p-acceptor functionalities are required in the analyte structure to enable separation. Otherwise, the compound must be derivatized with appropriate ‘interaction groups.’

8.7.4.1.4

Ligand exchange chiral stationary phases

Chiral ligand exchange chromatography (CLEC) was first introduced by Davankov and Rogozhin.47,48 In CLEC, enantiomeric resolution is achieved via the formation of transicient diastereomeric metal–ligand complexes. Generally, Cu(II) complexes with a-amino acids or their derivatives are used as chiral selectors. The chiral selectors are bound to a silica gel either by chemical bonding or by hydrophobic interactions. Alternatively, a copper ion can be included in the mobile phase to avoid loss of copper. Other transition metals have also been investigated, but copper is still the most widely used. These stationary phases have demonstrated very specific applicability in the resolution of chiral amino acids. The direct separation of baclofen on a commercial Chirexs 3126 column is shown in Figure 17 (Chapter 8.8).

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers HO

OH HO

OH

HO

H3C NH

HO

NH

O

NH O

HO

COOH NH

O

Cl

NH

CH3

O O H

O O

O H H H N

H

N H HN COOCH3

O

HO

O

O

O

CH3

CH3

OH

O OH

HO HO NH2

(a)

NH2

O

OH

CH3

OH H O N H

OH H N

HO

O

HO

N H

O

O

HO

O

O

HO

O

Cl

O H2N

O

OH CH3

O

OH

NH

O

O

O

OH

O

O

NH

OH

O

OH

NH2 O

OH HO OH

O

HO

107

OH O

(b)

OH OH HO

OH NHR

CH2OH O Cl O CH2OH O HO

O

Cl

O O

O

H

O N HNCOCH3 OH H H HN H

O

O H H N O

N H H

H H N

HOOC

HO

O

HO

OH

OH

HO

O N O H H H

NH2

O

O HO

OH

H

O H H N

HO

O H

Cl O

C

B

N H H A HN H HOOC

CH2OH OH

O (c)

H O

Cl

O N O H H

H H N

O N O H H H

NH2

D OH

HO

O HO

(d)

Figure 12 Structures of macrocyclic glycopeptide. (a) Vancomycin. (b) Ristocetin A. (c) Teicoplanin. (d) Teicoplanin aglycone. Reproduced with permission from Chirobiotics Handbook, A Guide to Using Macrocyclic Glycopeptide Bonded Phases For Chiral LC Separations, 5th Ed. 2004.

8.7.4.1.5

Other chiral stationary phases

Other chiral stationary phases include cinchona alkaloid,49 molecular imprinted polymers (MIPs),50 cyclofructan derivatives,51 etc. These all operate with very specific mechanisms or for very specific types of molecules. Research is still ongoing for the discovery and development of new chiral stationary phases with fast speed, universal selectivity, and improved robustness.

8.7.4.2

Chiral Stationary Phases for Gas Chromatography

Open tubular gas chromatography features the unique advantages of fast analysis, high efficiency, high sensitivity, and virtually universal detection (FID). It also provides high resolution capability and good peak capacity, which makes it suitable for the separation of complex mixtures, such as those originating from biological or environmental sources. It is regarded as a pivotal complementary technique to HPLC for enantiomeric separations. For example, unlike LC, GC often demonstrates effectiveness in the resolution of a non-aromatic chiral compound lacking functionalities.52 However, not all compounds are suitable for GC analysis. GC-amenable species need to have sufficient volatility and thermal stability. The compound has to maintain both chemical composition and stereo-configurations at the high temperatures typically used for GC separations. Another disadvantage of GC is its limited sample capacity. Unlike HPLC, the only interactions present inside the GC column are between the stationary phase and the analytes. The mobile phase solely functions as a carrier gas instead of having any actual interactions with the analyte or the stationary phase. Therefore, in gas chromatography, the choice of stationary phase is the key to successful chiral recognition. This section discusses the major types of GC-CSPs currently in use (see Chapter 8.17).

108

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers O

O

O

CHCH2COOH3

11.6

9.6

14.9

14.1

OH

Mobile phase: 10/90: CH3CN/1% TEAA, pH 4.1 Chirobiotic® T Chirobiotic® V

3.807

Figure 13 Complementary separation of warfarin achieved on Chirobiotics T vs. Chirobiotics V, using the same mobile phase. Reproduced with permission from Chirobiotic Handbook, A Guide to Using Macrocyclic Glycopeptide Bonded Phases For Chiral LC Separations, 5th Ed. 2004.

Internal standard

10

12.845

mAU

15 Enantiomer 1

13.962

20

Enantiomer 2

14

16

5 0 0

2

4

6

8 min

10

12

Figure 14 Chromatogram for the separation of promethazine enantiomers on Chirobiotics V column. Mobile phase: methanol/acetic acid/ triethylamine 100/0.1/0.1 v/v/v; Flow rate: 1.0 ml min1; Detection: 254 nm. Reproduced with permission from Saleh, O. A.; et al. Drug Dev. Ind. Pharm. 2009, 35(1), 19–25.

8.7.4.2.1

Derivatized cyclodextrin chiral stationary phases

The cyclodextrin type of chiral selector enjoys dominant popularity in GC. However, unlike in HPLC, GC negates the use of native CDs due to their low solubility in common solvent matrices and limited enantioselectivity. Historically, native forms have only been used in a few cases, for the separation of a- and b-pinenes and a few other molecules, and with poor selectivities and efficiencies.53 Konig et al. discovered lowered melting points in some acylated and alkylated CDs and consequently developed derivatized CDs for chiral GC analysis54–56 Currently, modified CDs account for greater than 95% of GC enantiomeric separations. They are generally unsurpassed in the resolution of structural isomers, geometrical isomers, organic sulfonates, and sulfates. There are two types of immobilization schemes to generate GC-CSPs, via coating or via bonding. For the coated CSPs, the chiral selector is either coated neat (if in a liquid state at room temperature) or dissolved in appropriate matrices to yield coating mixtures (if they have high boiling points). Polysiloxane-based matrices have been the most prevalent solvent so far for the second approach. Polyethylene glycol and ionic liquid have been used as alternative solvents.57,58 Some important commercialized coated-type CSPs are listed in Table 3.

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

Table 2

109

Enantioseparation of underivatized natural a-amino acids on Chirobiotics CSPs.

R CH COOH

Chirobiotics T(c1)

Chirobiotics TAG(c2)

Chirobiotics R(c3)

NH2 a-Amino acid

R-Moiety

k0

Rs

k0

Rs

k0

Rs

Alanine Arginine Aspartic Asparagine Cysteine Glutamic Glutamine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Tryptophan Valine

–CH3 –(CH2)3–NH–CNH–NH2 –CH2–COOH –CH2–CO–NH2 –CH2–SH –CH2–CH2–COOH –(CH2)2–CONH2

0.56 1.17 1.49 0.58 0.45 1.15 1.13 3.10 0.40 0.47 0.81 0.55 0.87 0.58 0.69 0.75 0.60 1.01 0.56

2.9 2.1 1.9 2.1 1.6 2.2 1.6 1.5 2.5 3.5 2.2 3.3 2.0 2.5 1.5 1.4 1.9 2.0 1.9

0.16 2.17 0.95 0.29 0.20 0.64 0.82 3.96 0.18 0.60 1.21 0.47 0.98 0.43 0.11 0.46 0.76 2.05 2.48

4.0 3.0 2.0 3.7 1.8 2.5 3.5 1.5 3.0 5.5 2.5 3.5 7.2 6.2 1.9 4.0 2.9 3.5 4.5

0.30 N/A N/A 1.45 1.78 N/A N/A 1.13 1.03 0.27 1.27 1.23 0.64 2.00 1.13 0.19 0.52 1.12 1.22

1.7 N/A N/A 1.56 1.50 N/A N/A 1.45 2.9 2.2 1.97 2.5 2.5 3.24 0.8 1.0 1.0 2.0 2.0

–CH(CH3)–CH2–CH3 –CH2–CH–(CH3)2 –(CH2)4–NH2 –CH2–CH2–S–CH3

–CH2OH –CHOH–CH3

–CH(CH3)2

Source: Reproduced with permission from Chirobiotic Handbook, A Guide to Using Macrocyclic Glycopeptide Bonded Phases For Chiral LC Separations, 5th Ed. 2004.

NH Silica gel

Si

O

O

NO2

O2N s

Figure 15 Structure of Whelk-O

1 CSP, 4-(3,5-ditritrobenzamido) tetrahydrophenanthrene.

CH3 OH

CH3 H3C

O

min Figure 16 Separation of ibuprofen on a Whelk-O 1 column. Mobile phase: hexane: isopropanol: acetic acid 98:2:0.05; Flow rate: 0.9 ml min1; Detection: 254 nm. From http://www.chromtech.com/Catalog7/PDF_Indvpge/108.pdf s

110

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

H2NCH2-CH-CH2-COOH

CH3 CH3

O Si O

(CH2)7

(CH2)17 CH3

S

C

CH3

*CH

(CH2)7

NH

CH3

Cl

COO− . 1/2 Cu2+

0 (a)

10 min

(b)

Figure 17 Separation of Baclofen on a Chirexs 3126 column. (a) Structure of Chirexs 3126. (b) Chromatogram of the separation of alanine. Mobile phase: 2 mM copper(II) sulfate in water/isopropanol (85:15); Flow rate: 1.0 ml min1; Detection: 254 nm. From https:// phenomenex.blob.core.windows.net/documents/7797552e-5664-4668-a608-637537fc4678.pdf. 2001 by Phenomenex. Reproduced with permission. All rights reserved.

Table 3

List of commercial cyclodextrin-based GC-CSP and their maximum allowable operation temperatures (MAOTs)82

Name (Chiraldexs)

A-TA, B-TA, G-TA B-DP, G-DP G-PN G-BP B-DM. G-DM B-PM A-DA, B-DA, G-DA B-PH

Derivative

MAOT (1C)

2,6-di-O-pentyl-3-trifluoroacetyl 2,3-di-O-propionyl-6-t-butyl silyl 2,6-di-O-pentyl-3-propionyl 2,6-di-O-pentyl-3-butyryl 2,3-di-O-methyl-6-t-butyl silyl 2,3,6-tri-O-methyl 2,6-di-O-pentyl-3-methoxy (S)-2-hydroxy propyl ether

Isothermal

Programmed

180 200 200 200 200 200 200 200

180 220 220 220 220 220 220 220

An example of a bonded type of GC-CSP is the commercialized Chirasil-Dex (see Figure 18). A mono-6-octamethylene spacer links the permethylated cyclodextrin to the dimethylpolysiloxane backbone.59 Column operation temperatures of  20–220 1C have been achieved on this stationary phase.60 O

O Si

O Si

n

(CH2)8 O

O

-CD

O

O

Figure 18 Structure of the Chirasil–Dex chiral stationary phase.

8.7.4.2.2

Amino acid-based chiral stationary phases

In 1966, Gil-Av et al. demonstrated the first direct GC enantiomeric separation by using an N-trifluroacetyl (N-TFA)-L-isoleucine lauryl ester stationary phase for the enantiomeric resolution of several N-trifluroacetyl amino acid esters.61 Hydrogen bonding interactions were the dominant interactions for chiral recognition. The first commercialized amino acid-type GC-CSP was Chirasil-Val, with the chemical structure shown in Figure 19. It is based on the work of Frank et al. in 1977 on the binding of N-propionyl-L-valine-tert-butylamide onto a polysiloxane copolymer.62 The thermal stability of this column was extended to 250 1C and the column was found to be effective in the separation of derivatized amino acids, amino alcohols, and peptides. Currently, both the Chirasil-L-Val and the Chirasil-D-Val are commercially available.

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

111

O O

O

O

Si

H N

O

Si

R

N H

R=

Si

O

R

Figure 19 Chemical structure of a commercialized amino acid-based GC-CSP Chiralsil-Val.

It is commonly observed that Chirasil-L-Val selectively shows stronger retention for D-amino acid derivatives and Chirasil-D-Val tends to retain L-amino acid derivatives longer.

8.7.4.2.3

Metal–ligand complex chiral stationary phases

Metal–ligand complex CSPs realize chiral recognition primarily through metal–ligand coordination. The difference in p-complexation energies between the chiral selector and the enantiomers is the key to chiral discrimination. In 1982, chiral olefin 3-methylcyclopentene was resolved from its enantiomers on an optically active dicarbonylrhodium(I) 3-trifluoroacetyl-(1R)camphorate structure.63 This was the first abiotic selector–selectand system used for enantiomeric separation. Following this work, Schurig et al. continued to report on studies using manganese(II), cobalt(II), and nickel(II) bis[3-(heptaflurobutanoyl)(1R)-camphorate] to enrich the library of chiral transition metal-based GC-CSPs. The early developed versions exhibited narrow operation temperature windows of 25–120 1C. Improvement was observed with Chirasil-Metal (Figure 20) through coupling the metal–ligand complex with the polysiloxane backbone.59 Immobilization of the CSP to the capillary wall has also been investigated to extend the column operation temperature. However, metal–ligand complex CSPs still have poor column efficiencies, peak tailing, and limited enantioselectivities. Therefore, although they are potentially suitable for the separation of p-electron and lone pair electron-rich species, their application in enantiomeric separation is currently limited.

O O

O Si

O

Ni/2

Si n

R

R=

O

Figure 20 Structure of bonded phase metal–ligand complex GC-CSP Chirasil-Metal.

8.7.4.2.4

Ionic liquid chiral stationary phases (IL-CSPs)

Ionic liquids can contribute to the generation of a GC-CSP in two ways. They can be either used as the nonchiral solvent matrices for the dissolution of conventional chiral selectors or they can be designed as optically active structures to be used as chiral selectors directly. However, very limited success has been reported so far using either approach. In 2001, dimethylated and permethylated b-CDs were dissolved in 1-butyl-3-methylimidazolium chloride (BMIM-Cl) to investigate their enantiomeric selectivities.58 These stationary phases accomplished enantiomeric recognition for several chiral molecules evaluated, but exhibited insufficient compound retentions, limited enantioselectivities, and low thermal stabilities. In 2010, Armstrong et al. attached an ionic pendent on permethylated CDs before the dissolution in dicationic ionic liquids.64 High column efficiencies, high thermal stabilities, and unique enantioselectivities were achieved using this approach, re-establishing the potential of ionic liquids as alternative matrices to polysiloxane type polymers. The structure of the charged cyclodextrin is shown in Figure 21. The exploration of using neat ionic liquids as chiral selectors for enantiomeric separation has been evaluated as well. The first report was on the use of N,N-dimethylephedrinium-bis(trifluoromethanesufon)imidate (Figure 22),65 for the enantioresolution of several classes of molecules, including chiral alcohols, chiral sulfoxides, and chiral acetylate amines. Minimal additional research has been conducted in this field.66,67

8.7.4.3

Chiral Stationary Phases for Supercritical Fluid Chromatography

Through decades of development, SFC is known today as a ‘green technology,’ which boasts unique features of high-speed separation and method development, high column efficiency, easy solvent removal and rapid sample recovery, and compatibility with both HPLC and GC detectors (see Chapter 8.18). It is well suited for chiral separations, especially for preparative scale

112

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers O O

O

O O

O

O

O

OO O

O

O

O

O O

O

O O O O

O O

O

O

O O

OO O

O

O

O

O O O O O

O

O

O

O

O

O

O O

O

O

OO O

O

O

O

O

O

O O

O O O

O

O

OO O

O N

O

A−

+ N

+ P

A−

A− = I−, NTf2−, TfO− Figure 21 Structures of charged CD used to yield ionic liquid matrix-based GC-CSPs. Reproduced with permission from Huang, K.; Zhang, X.; Armstrong, D. W. J. Chromatogr. A 2010, 1217(32), 5261–5273.

OH O

N

O S

S F3C

N O

O

CF3

Figure 22 Chiral ionic liquid GC-CSP based on N,N-dimethylephedrinium bis(trifluoromethanesulfon)imidate.

purification of enantiomeric products. For all these reasons, SFC has secured a niche in pharmaceutical applications, either in early-phase drug screening or in pharmaceutical process development. A chromatogram demonstrating typical kilogram-scale enantiopurifications of a development intermediate is shown in Figure 23.

Absorbance (AU)

1000 708

Target: Enantiomer 2

Enantiomer 1

596 Elution 394 192 −10

Inject

−1

0

1

2

3

4

5 6 7 Time (min)

8

9

10

11

12

13

Figure 23 Preparative scale purification of an intermediate single enantiomer. Column: 25  11 cm DAC Chiralpaks AD; Mobile phase: 75/25 heptane/ethanol; Flow rate: 600 ml min1; Detection: 285 nm. Injection: 150 ml racemates at 83 mg ml1 in mobile phase. Reproduced with permission from Welch, C. J.; et al. LCGC North Am. 2004, 23(1), 16, 18, 22, 24, 26–29.

In terms of mechanism and selectivity, SFC separations resemble normal phase HPLC. Carbon dioxide, as the major mobile phase component, is inherently nonpolar. Alcohols or acetonitrile are usually added to aid solubility of polar molecules in the

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

113

mobile phase and facilitate elution of polar compounds. Most normal phase suitable chiral HPLC columns are applicable for SFC separations. The classes that have reported usage today are polysaccharide-based CSPs, macrocyclic glycopeptide antibiotic CSPs, Pirkle type p-complex CSPs, cyclodextrin-based CSPs, and synthetic polymeric CSPs, with the polysaccharide-based types being the most dominant of all.68 Successful separation on Chirasil-Metal and Chirasil-Dex has also been reported.59,69

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

Martin, A. J. P.; Synge, R. L. M. Biochem. J. 1941, 35, 1358–1368. Consden, R.; Gordon, A. H.; Martin, A. J. P. Biochem. J. 1944, 38, 224–232. Howard, G. A.; Martin, A. J. P. Biochem. J. 1950, 46, 532–538. James, A. T.; Martin, A. J. P. Biochem. J. 1952, 50, 679–690. Easson, L. H.; Stedman, E. Biochem. J. 1933, 27, 1257–1266. Davankov, V. A. Chirality 1997, 9(2), 99–102. Booth, T. D.; Wahnon, D.; Wainer, I. W. Chirality 1997, 9(2), 96–98. Mesecar, A. D.; Koshland, D. E., Jr. Nature (London) 2000, 403(6770), 614–615. Pirkle, W. H.; Pochapsky, T. C. Chem. Rev. 1989, 89(2), 347–362. Bentley, R. Arch. Biochem. Biophys. 2003, 414(1), 1–12. Jozwiak, K.; Moaddel, R.; Ravichandran, S. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 875(1), 200–207. Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1996, 68(8), 1369–1374. Min, J. Z.; Toyo’oka, T.; Kato, M.; Fukushima, T. Biomed. Chromatogr. 2005, 19(1), 43–50. Stalcup, A. M. Annu. Rev. Anal. Chem. 2010, 3, 341–363. Einarsson, S.; Josefsson, B.; Moeller, P.; Sanchez, D. Anal. Chem. 1987, 59(8), 1191–1195. Husek, P.; Simek, P. Curr. Pharm. Anal. 2006, 2(1), 23–43. Catanese, M. Lab. 2000 2004, 18(5), 46–49. http://www.registech.com/Library/gcderrev.pdf http://www.sigmaaldrich.com/img/assets/4242/fl_analytix3_2002_new_.pdf Toyo’oka, T. Modern Derivatization Methods for Separation Sciences. Chichester, UK: Wiley. 1999. Aswad, D. W. Anal. Biochem. 1984, 137(2), 405–409. Chen, P.; Novotny, M. V. Anal. Chem. 1997, 69(14), 2806–2811. Jia, M.; Wu, W.; Yost, R.; et al. Anal. Chem. 2003, 75(16), 4065–4080. Carpinteiro, M. I.; et al. J. Chromatogr. A 2009, 1216(14), 2825–2831. Ali, I.; Saleem, K.; Hussain, I.; Gaitonde, V. D.; Aboul-Enein, H. Y. Sep. Purif. Rev. 2009, 38(2), 97–147. Gavioli, E.; Maier, N. M.; Minguilon, C.; Lindner, W. Anal. Chem. 2004, 76(19), 5837–5848. Ma, S.; Grinberg, N. Am. Pharm. Rev. 2009, 12(3), 12, 14, 16–18. Rosales-Conrado, N.; Leon-Gonzalez, M. E.; Rocco, A.; Fanali, E. Curr. Anal. Chem. 2010, 6(3), 209–216. Salvador, A.; Herbreteau, B.; Dreux, M.; Gyllenhaal, O. J. Chromatogr. A 2001, 929(1-2), 101–112. Ai, F.; Li, L.; Ng, S.; Tan, T. J. Chromatogr. A 2010, 1217(48), 7502–7506. Cancelliere, G.; Ciogli, A.; D’Acquarica, L.; et al. J. Chromatogr. A 2010, 1217(7), 990–999. Lindner, K. R.; Mannschreck, A. J. Chromatogr. 1980, 193(2), 308–310. Okamoto, Y.; Kawashima, M.; Yamamoto, K.; Hatada, K. Chem. Lett. 1984, (5), 739–742. Okamoto, Y.; Kawashima, M.; Hatada, K. J. Am. Chem. Soc. 1984, 106(18), 5357–5359. Okamoto, Y.; et al. Chem. Lett. 1987, (9), 1857–1860. Okamoto, Y.; Kaida, Y.; Aburatani, R.; Hatada, K. ACS Symp. Ser. 1991, 471, 101–113 (Chiral Sep. Liq. Chromatogr.). Santoro, M. I. R. M.; Cho, H. S.; Kedor-Hackmann, E. R. M. Drug Dev. Ind. Pharm. 2000, 26(10), 1107–1110. Helgeson, R. C.; Timko, J. M.; Moreau, P.; et al. J. Am. Chem. Soc. 1974, 96(21), 6762–6763. Armstrong, D. W.; Tang, Y.; Chen, S.; et al. Anal. Chem. 1994, 66(9), 1473–1484. Armstrong, D. W.; Liu, Y.; Ekborgott, K. H. Chirality 1995, 7(6), 474–497. Berthod, A. Chirality 2009, 21(1), 167–175. Pirkle, W. H.; House, D. W. J. Org. Chem. 1979, 44(12), 1957–1960. Hyun, M. H.; Pirkle, W. H. J. Chromatogr. 1987, 393(3), 357–365. Mikes, F.; Boshart, G.; Gil-Av, E. J. Chromatogr. 1976, 122, 205–221. Mikes, F.; Boshart, G. J. Chromatogr. 1978, 149, 455–464. Pirkle, W. H.; Welch, C. J. J. Liq. Chromatogr. 1992, 15(11), 1947–1955. Davankov, V. A.; Rugozhin, S. V.; Semechkin, A. V.; Sachkova, T. P. J. Chromatogr. 1973, 82(2), 359–365. Davankov, V. A. Adv. Chromatogr. (N.Y.) 1980, 18, 139–195. Laemmerhofer, M.; Lindner, W. J. Chromatogr. A 1996, 741(1), 33–48. Lu, Y.; Li, C.; Zhang, H.; Liu, X. Anal. Chim. Acta 2003, 489(1), 33–43. Sun, P.; Wang, C.; Padivitage, N.; et al. Analyst (Cambridge, U.K.) 2011, 136(4), 787–800. Huang, K.; Armstrong, D. W. Org. Geochem. 2009, 40(2), 283–286. Smolkova-Keulemansova, E. J. Chromatogr. 1982, 251(1), 17–34. Koenig, W. A.; Lutz, S.; Mischnick-Luebbecke, P.; Brassat, B.; Wenz, G. J. Chromatogr. 1988, 447(1), 193–197. Koenig, W. A.; Lutz, S.; Wenz, G. Angew. Chem. 1988, 100(7), 989–990. Armstrong, D. W.; Li, W.; Pitha, J. Anal. Chem. 1990, 62(2), 214–217. Grisales, J. O.; Lebed, P. J.; Keunchkarian, S.; Gonzalez, F. R.; Castells, C. R. J. Chromatogr. A 2009, 1216(40), 6844–6851. Berthod, A.; He, L.; Armstrong, D. W. Chromatographia 2001, 53(1/2), 63–68. Schurig, V.; Schmalzing, D.; Schleimer, M. Angew. Chem. 1991, 103(8), 994–996; Schurig, V.; Schmalzing, D.; Schleimer, M. See also Angew. Chem. Int. Ed. Engl. 1991, 30(8), 987–989. 60. Schurig, V.; Jung, M.; Mayer, S.; et al. J. Chromatogr. A 1995, 694(1), 119–128. 61. Gil-Av, E.; Feibush, B.; Charles-Sigler, R. Tetrahedron Lett. 1966, (10), 1009–1015. 62. Frank, H.; Nicholson, G. J.; Bayer, E. J. Chromatogr. Sci. 1977, 15(5), 174–176.

114

63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82.

Chromatographic Separations and Analysis: Chromatographic Separations and Analysis of Enantiomers

Schurig, V.; Buerkle, W. J. Am. Chem. Soc. 1982, 104(26), 7573–7580. Huang, K.; Zhang, X.; Armstrong, D. W. J. Chromatogr. A 2010, 1217(32), 5261–5273. Ding, J.; Welton, T.; Armstrong, D. W. Anal. Chem. 2004, 76(22), 6819–6822. Kimaru, I.; Clifford, B.; Smith, T. Synthesis and Characterization of Chiral Ionic Liquids for use in Enantioseparations. Abstracts of Papers, 239th ACS National Meeting, San Francisco, CA, United States, March 21–25, 2010, 2010; p. ANYL-190. Zhao, L.; Ai, P.; Duan, A.; Yuan, L. Anal. Bioanal. Chem. 2011, 399(1), 143–147. Felix, G.; Berthod, A.; Piras, P.; Roussel, C. Sep. Purif. Rev. 2008, 37(3), 229–301. Armstrong, D. W.; Tang, Y.; Wand, T.; Nichols, M. Anal. Chem. 1993, 65(8), 1114–1117. Laemmerhofer, M. J. Chromatogr. A 2010, 1217(6), 814–856. Choi, H. J.; Park, Y. J.; Hyun, M. H. J. Chromatogr. A 2007, 1164(1–2), 235–239. Huang, K.; Breitbach, Z. S.; Armstrong, D. W. Tetrahedron: Asymmetry 2006, 17(19), 2821–2832. Haginaka, J.; Matsunaga, H. Enantiomer 2000, 5(1), 37–45. http://www.mz-at.de/pdf/ChromTech_AGP.pdf Han, X.; Berthod, A.; Wang, C.; Huang, K.; Armstrong, D. W. Chromatographia 2007, 65(7/8), 381–400. http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/General_Information/1/t410060.Par.0001.File.tmp/t410060.pdf Chirobiotic Handbook, A Guide to Using Macrocyclic Glycopeptide Bonded Phases For Chiral LC Separations, 5th Ed. 2004. Saleh, O. A.; El-Azzouny, A. A.; Aboul-Enein, H. Y.; Badawy, A. M. Drug Dev. Ind. Pharm. 2009, 35(1), 19–25. http://www.chromtech.com/Catalog7/PDF_Indvpge/108.pdf https://phenomenex.blob.core.windows.net/documents/7797552e-5664-4668-a608-637537fc4678.pdf Welch, C. J.; Leonard, W. R.; DaSilva, J. O.; et al. LCGC North Am. 2004, 23(1), 16, 18, 22, 24, 26–29. Chiraldex GC Columns, A Guide to Using Cyclodextrin Bonded Phases for Chiral Separations By Capillary Gas Chromatography. http://www.sigmaaldrich.com/etc/ medialib/docs/SAJ/Brochure/1/j_astec.Par.0001.File.tmp/j_astec.pdf