MXPA06014765A - New chiral columns with broad chiral selectivity. - Google Patents

Abstract

A general chiral column with a multiple proline-based chiral stationary phase.

Description

NEW CHIRAL COLUMNS WITH WIDE QUIRAL SELECTIVITY Government Support This invention was made in relation to the Concession Numbers NIH 1 ROI GM63812-01 and NIH 1 ROI GM60637-01A1, from the National Institute of Health. The government of the United States has the rights of this invention.

Field of the Invention The present invention relates to the field of chiral chemistry. More particularly, the present invention relates to the separation of enantiomers, that is, those isomers in which the distribution of atoms or groups is such that the two molecules are not superimposable. The present invention has been developed in a novel class of chiral columns that can resolve a large number of racemic compounds. These columns are stable and can be used with a certain number of mobile phase solvents.

BACKGROUND OF THE INVENTION Stereoisomers are those molecules which differ from one another only in the way that their atoms are oriented in space. Stereoisomers are generally classified as diastereomers or enantiomers; the last covering those that are mirror images of each other, those mentioned first are those that are not. The particular distribution of atoms that characterize a particular stereoisomer is known as its optical configuration, specified by sequence rules known as, for example, either + or - (also D or L) and / or R or S. Although they differ only in orientation, the practical effects of stereoisomerism are important. For example, the biological and pharmaceutical activities of many compounds are strongly influenced by the particular configuration involved. Currently, many compounds are only of wide utility when employed in a given stereoisomeric form. Living organisms normally produce only one enantiomer of one pair. In this way only (-) - 2-methyl-1-butanol is formed in yeast fermentation of starches; only (+) - lactic acid is formed in muscle contraction; Fruit juices contain only (-) - malic acid, and only (-) - quinine is obtained from the cinchona tree. In biological systems, stereochemical specificity is the rule rather than the exception, since the catalytic enzymes, which are so important in such systems, are optically active. For example, sugar (+) -glucose plays an important role in animal metabolism and is the basic raw material in the fermentation industry; however, its optical counterpart, or antipode, (-) -glucose, is neither metabolized by animals nor fermented by yeast. Other examples in this regard include the Penicillium glaucoma model, which will consume only the (+) - enantiomer of the enantiomeric mixture of tartaric acid, leaving the (-) - enantiomer intact. Also, only one stereoisomer of chloromycetin is an antibiotic; and (+) - ephedrine not only do not have any drug activity, but it interferes with the drug activity of its antipode. Finally, in the world of essences, the enantiomer (-) - carvone provides spearmint oil with its distinctive smell, while its optical counterpart (+) - carvone provides the essence of caraway. Thus, since enzymes and other biological receptor molecules possess chiral structures, the enantiomers of a racemic compound can be absorbed, activated, and degraded by them in different forms. This phenomenon causes that in many cases, two enantiomers of a racemic drug may have different or even opposite pharmacological activities. To recognize these different effects, the biological activity of each enantiomer is often needed to be studied separately. These and other factors within the pharmaceutical industry have contributed significantly to the need for enantiomerically pure compounds and thus the need for chiral chromatography. ThereforeIt is desirable and often essential to separate stereoisomers to obtain the useful version of a compound that is optically active. Separation in this sense is not usually a problem when diastereomers are involved: diastereomers have different physical properties, such as melting points, boiling points, solubilities in a given solvent, densities, refractive indexes, etc. Therefore, the diastereomers are normally separated from one another by conventional methods, such as fractional distillation, fractional crystallization or chromatography. Enantiomers, on the other hand, present a special problem because their physical properties are identical. Thus they can not as a rule-and especially so when in the form of a racemic mixture-be separated by ordinary methods: not by fractional distillation, because their boiling points are identical; not by conventional crystallization because (unless the solvent is optically active) its solubilities are identical; not by conventional chromatography because (unless the adsorbent is optically active) they are also maintained in the adsorbent. The problem of separating enantiomers is further exacerbated by the fact that conventional synthetic techniques almost always produce one. mixture of enantiomers. When a mixture comprises equal amounts of enantiomers having opposite optical configurations, it is called racemate; the separation of a racemate into its respective enantiomers is generally known as a resolution, and is a process of considerable importance. The chiral columns that can resolve a large number of racemic compounds (general chiral columns) have high demand. They are routinely needed in many laboratories, especially in the pharmaceutical industry. Prior to the present invention, Daicel columns, macrocyclic antibiotic columns, and Whel-0 columns were probably known as the industry leaders in this type of general chiral columns. The present invention has been developed in a novel class of general chiral columns based on the use of proline and its analogues. In addition, and notably, the columns of the present invention have the ability to resolve at least a similar or higher percentage of the compounds tested. In addition, the columns of the present invention provide better separation over some of the tested compounds and can resolve certain compounds that could not be resolved with the commonly used commercial columns listed above. The columns of the present invention are stable and can be used with a large number of mobile phase solvents. Accordingly, the columns of the present invention could find important applications as general chiral columns. A large number of chiral columns have been prepared in the past; however, only a few showed broad chiral selectivity. As stated in the above, successful examples include the popular Daicel columns, the Quirobiotic columns, and the Whelk-0 1/2 columns. Daicel columns are prepared by coating sugar derivatives in silica gel. The chirobiotic columns are prepared by immobilizing macrocyclic glycopeptides on silica gel. The Whelk-0 1/2 columns contain both rich electron and deficient electron aromatics. These columns have broad chiral selectivity and have been successfully applied to solve a good number of racemic compounds. They have different profiles of selectivity and stability. Their selectivities complement each other in some cases, while duplicating one another in other cases. Some of the columns are more suitable for reverse phase conditions and others for normal phase conditions. Each column has its own advantages and disadvantages. Despite these developments, there are still many compounds that can not be resolved or solved well using these commercially available columns. Accordingly, there is still a significant need to develop novel columns that have relatively broad chiral selectivity.

SUMMARY OF THE INVENTION The present invention is directed to a chiral selector that represents an improvement in the enantiomeric separation technique. Thus, one embodiment of the present invention is a general chiral column with a multiple chiral selector based on proline. Another embodiment of the present invention is a stationary chiral phase made of peptides with 2 or more prolines, including chiral selectors with 2, 3, 4, 5, 6, or 10 prolines. Also included within the scope of the present invention are the analogs and isomers of prolines, and analogs and isomers of the chiral selector compounds of the present invention. Another embodiment of the present invention is a stationary chiral phase (or column) of the following formula: group plugged at the end-Pro n where n is any integer of 2 or greater, and the analogs and isomers thereof. Another embodiment of the present invention is where n is any integer of 2-10. The separations achieved by analytes are comparable or superior to those achieved in columns Daicel AD, Daicel OD, and Whelk 02. The multiple chiral proline-based columns of the present invention appear promising as a superior general chiral column.

Brief Description of the Drawings Figure 1 shows the structure for L-Proline amino acid and its associated stationary phases Fmoc-Pro- (Me) Ahx-APS (CSP1), Fmoc-Pro2- (Me) Ahx-APS (CSP2), Fmoc -Pro4- (Me) Ahx-APS (CSP3); and Fmoc-Pro6 ~ (Me) Ahx-APS (CSP4). CSP2-4 are embodiments of compounds of the present invention. Figure 2 shows the synthesis of an embodiment of the present invention, stationary chiral phase Fmoc-Pro- (Me) Ahx-APS (CSP3): Stationary chiral phase synthesis Fmoc-Pro4- (Me) Ahx-APS (CSP3): (a) Fmoc- (Me) Ahx-OH, DIC; (b) (1) Piperidine; (2) Fmoc-Pro-OH, HATU; (c) AcOH; (d) aminopropyl silica gel, HATU.

Description of the Invention The present invention has developed a novel chiral column having relatively broad chiral selectivity, when compared to Daicel columns and Whelk 02 columns, as industrial standards or industrial models. Additionally, the chiral columns of the present invention are stable in a number of mobile phase conditions. The success rate of the chiral column of the present invention is compared well with the best of the commercially available general chiral columns developed during the last decades. From 22 racemic compounds chosen based on their availability (see example 4), the Pro4 column (CSP 3) resolved 17 compounds; the Pro2 column (CSP2) resolved 16 compounds; the Pro6 column (CSP4) resolved 15 compounds. In comparison, the Daicel OD column solved 18, Daicel AD resolved 16, and Whelk-02 solved 15 compounds. The monoproline column (CSPl) is much less effective, it can only solve 6 of the 22 compounds tested. The resolutions achieved with the monoproline column are also very modest. Proline is a unique amino acid in many forms (Figure 1). Instead of having a primary amino group as in other α-amino acids, it contains a secondary amine. Due to the cyclic structure, the rotation around the nitrogen-to-carbon bond is restricted. Also due to the cyclic structure, proline is not ideally suited for a-helix or ß-sheet conformation; however, polyproline forms its own unique helical conformation (Polyproline I and polyproline II). The amide linkage in polyproline is spherically prevented compared to other oligopeptides. The distinctly different conformational and structural characteristics of polyprolins suggest that they may behave very differently from other short oligopeptides that have been studied in chiral chromatography. The present invention discovers that chiral selectors based on proline, including the stationary chiral phase 3 mode based on tetraproline (Figure 1), stationary chiral phase 2 based on diproline, stationary chiral phase 4 based on hexaproline has relatively broad chiral selectivity, while mono-proline stationary phase 1 is largely ineffective. The immobilization of the chiral selectors of the present invention by silica gel is completed through a linker group. An example of a linker group of the present invention is an N-alkylamino group. A second example is an N-methylamino group. Another example is 6-N-methylaminohexanoic acid. The amine bond between these linkers and proline residues is more spherically hindered due to the N-methyl or N-alkyl group. The particular linker group can be selected by one of ordinary skill in the art depending on the analyte to be tested. For example, when the Fmoc-Pro-Pro selector is immobilized using 6-N-methylaminohexanoic acid, it can resolve approximately 16 of approximately 22 tested analytes. For the same chiral selector, when immobilized using 6-aminohexanoic acid, it solves only 4 of the same group of analytes. Additionally, the stationary phase compounds of the present invention may comprise several end capped groups as are known in the art. By use of the term proline with respect to the present invention, it is understood that proline analogues and isomers are included. For example, all stereoisomers are included. Additionally, analogs are included. Examples of the analogs included in this are those with the following characteristic basic structure such as in D-proline, hydroxyproline, and pipecolinic acid: wherein n is an integer (such as 1, 2, 3, 4, 5, etc.) and X is a heteroatom such as O, S, N; and other unspecified atoms can be carbon or heteroatoms. These covalently linked columns of the present invention are stable in common organic solvents, including CH2C12 and CHC13. Therefore, a wide selection of mobile phase conditions could be applied in the development of the method. For several analytes, the present invention seeks resolution with CH2Cl2 / hexane as the mobile phase and effective separation was also achieved (example 6). Choosing the broadest solvent has advantages in that some racemic analytes are soluble in only certain solvents and some compounds can be better solved in certain solvents. In terms of modes of potential interaction with the analytes, examples of the chiral selectors of the present invention are those which form attractive hydrogen bonds with the analyte and may also have attractive polar interactions with the analyte. In addition, the spherical interaction between the analyte and the chiral selector could also be important. The following examples and experimental section were designed to be purely exemplary in nature. Thus, this section should not be considered as limiting the present invention.

Examples Throughout this section, various abbreviations will be used, including the following: DIC, diisopropylcarbodiimide; HATU, 0- (7-Azabenzotriazol-1-yl) -N, N? N ', N'-tetramethyluronium hexafluorophosphate; DIPEA, N? N-Diisopropylethylamine; DMF, N, N-Dimethylformamide; DCM, Dichloromethane; DMAP, 4- (dimethylaminopyridine); NMM: N-methylmorpholine; Fmoc, 9- Fluorenylmethoxycarbonyl; (Me) Ahx: 6-methylaminohexanoic acid; Fmoc- (Me) Ahx-OH, 6- [(9H-Fluoren-9-ylmethoxy) carbonyl] methylamino hexanoic acid; Fmoc-Ahx-OH, 6- [(9H-fluoren-9-ylmethoxy) carbonyl] aminohexanoic acid; Fmoc-Pro-OH, N-a-Fmoc-I-proline.

Supplies and General Equipment: Amino Acid Derivatives from? OvaBiochem were purchased (San Diego, CA). All other chemicals and solvents were purchased from Aldrich (Milwaukee, Wl), Fluka (Ronkonkoma,? Y), or Fisher Scientific (Pittsburgh, PA). HPLC grade Kromasil® silica gel (particle size 5 μm, pore size 100 A, and surface area 298 m / g) were purchased from Akzo Nobel (EKA Chemicals, Bohus, Sweden). Selected silica gel (32-63 μm) from Fisher Scientific was used by purification of flash column chromatography of the target compounds. The thin layer chromatography was completed using silica gel EM plates 60 F-254 TLC (0.25 mm, E. Merck, Merck KGaA, 64271 Darmstadt, Germany). Elemental analyzes were conducted by Atlantic Microlab, Inc. (Norcross, GA). The HPLC analyzes were completed with a Beckman analytical gradient system (System Gold). The UV spectrum was obtained with a Shimadzu UV 201 spectrometer (cell volume 3 mL, cell passage length 10 mm).

Example 1: Preparation of stationary chiral phase Fmoc-Pro- (Me) Ahx-APS (CSPl) To 0.80 g of silica (Me) Ahx-APS (the concentration surface (Me) Ahx is 0.64 mmol / g) mixtures were added of Fmoc-Pro-OH (3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20 g) in 8 mL of DMF. After stirring for 6 hours, the resulting silica was filtered and washed with DMF, methanol, and DCM to provide the desired stationary chiral phase. The concentration surface Pro was determined to be 0.57 mmol / g based on the Fmoc dissociation method. The resulting stationary chiral phase was compacted on a 50 x 4.6 mm HPLC column using a standard suspension compaction method Example 2: Preparation of stationary chiral phase Fmoc-Pro2 (Me) Ahx-APS (CSP2) To 0.80 g of silica (Me) Ahx-APS (the concentration surface (Me) Ahx was 0.64 mmol / g) mixtures were added. Fmoc-Pro-OH (3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20 g) in 8 mL of DMF. After stirring for 6 hoursThe resulting silica was filtered and washed with DMF, methanol, and DCM. The concentration surface Pro was determined to be 0.55 mmol / g based on the Fmoc dissociation method. The Fmoc protecting group was then removed by treatment of the silica with 10 L of 20% (V / V) piperidine in DMF for 1 hour. The deprotected silica, Pro- (Me) Ahx-APS, was collected by filtration and washed with DMF, methanol, and DCM. Then another module, Fmoc-Pro-OH, was coupled to the resulting silica following an identical reaction sequence and the desired chiral selector was produced on the silica gel. The Fmoc concentration surface was determined to be 0.52 mmol / g based on the Fmoc dissociation method. The resulting stationary chiral phase was compacted on a 50 x 4.6 mm HPLC column using the standard suspension compaction method.

Example 3: Preparation of stationary chiral phase Fmoc-Pro4- (Me) Ahx-APS (CSP3) To the Rink acid resin (100-200 mesh, 3.0 g, 0.43 mmol / g) pre-filled with DCM (20 mL, 30 min) the mixtures of Fmoc- (Me) Ahx-OH (1.42 g, 3.87 mmol), DMAP (0.16 g, 1.29 mmol), NMM (0.39 g, 3.87 mmol), and DIC (0.49 g, 3.87 mmol) were added. in DCM-DMF (1: 1 V / V, 10 mL). After stirring for 6 hours, the resin was collected by filtration and washed with DMF, DCM, and methanol (20 mL x 3). The Fmoc group was then removed by treatment with 20 mL of 20% (V / V) of piperidine in DMF for 30 min. The deprotected (Me) Ahx-O-Rink resin was collected and washed with DMF, DCM, and methanol (20 mL x 3). To the resin (Me) Ahx-O-Rink were added the mixtures of Fmoc-Pro-OH (1.31 g, 3.87 mmol), HATU (1.47 g, 3.87 mmol), and DIPEA (0.50 g, 3.87 mmol) in 20 mL of anhydrous DMF. After stirring for 3 hours, the resin was filtered and washed with DMF, DCM, and methanol (20 mL x 3). The Fmoc group was then removed and the second, third and fourth modules, Fmoc-Pro-OH, were coupled following exactly the same procedures as described above to provide the resin Fmoc- (Pro) 4- (Me) Ahx- O-Rink desired. The resin was then treated with 1% TFA in DCM (20 mL, 10 min) to release Fmoc- (Pro) 4- (Me) Ahx-OH from the resin. This dissociation reaction was repeated once more to ensure complete reaction. The obtained crude product was purified by flash column chromatography on silica gel (mobile phase: 5% methanol in DCM) to provide the desired Fmoc- (Pro) 4- (Me) Ahx-OH as a white solid (0.90 g , 92%). XH NMR (CD2C12): d 1.2-1.7 (m, 6H), 1.9-2.4 (m, 18H), 2.80 (s, 3H), 3.2-3.6 (m, 10H), 4.2-4.7 (m, 7H), 7.1-7.6 (m, 8H), 9.6 (broad, 1H). ESI-MS: m / z 756.0 (M + H +). A mixture of Fmoc- (Pro) 4- (Me) Ahx-OH (0.90 g, 1.19 mmole), HATU (0.45 g, 1.19 mmole), and DIPEA (0.15 g, 1.19 mmole) in 8 mL of anhydrous DMF was added to 0.7 g of 3-aminopropyl silica gel (APS). The APS was prepared from Kromasil® silica gel (5 μm spherical silica, 100 Á, 298 m2 / g) and 3-aminopropyltriethoxysilane. The amino concentration surface is 0.66 mmol / g, based on elemental nitrogen analysis data (C, 3.11; H, 0.83; N, 0.93). After stirring the mixture for 4 hours, the stationary phase was collected by filtration and washed with DMF, DCM, and methanol (10 mL x 3). The Fmoc concentration surface was determined to be 0.27 mmol / g based on the Fmoc dissociation method. The resulting stationary chiral phase was compacted on a 50 x 4.6 mm HPLC column using the standard suspension compaction method. The following examples establish various chromatographic measurements. In this, the retention factor (k) is equal to (tr-t0) / t0 in which tr is the retention time and t0 is the dead time. The separation factor (a) is equal to k2 / k ?, the ratio of the retention factors of the two enantiomers. The Separation factor of 1 indicates no separation. The largest separation factor is the best separation. The dead time t0 was measured with 1,3,5-tri-t-butylbenzene as the zero volume marker. The flow rate at 1 mL / min., UV detection at 254 nm.

Example 4 This example compares the chromatographic resolution of the racemic compounds with the chiral columns, including embodiments of the present invention (Pro2 (CSP2), Pro4 CSP3), Pr6 (CSP4)). In the following table, ki is the retention factor of the lowest enantiomer retained and the separation factor (a) is previously defined. This example also shows that a chiral mono-proline column does not sufficiently de-water. In addition, this example shows embodiments of the present invention compared to known commercial columns. Table 1. Chromatographic resolution of racemic compounds with chiral columns. Ki is the retention factor of the lowest enantiomer retained. The mobile phases are solutions of specified percentage of IPA and acetic acid in hexanes.

Example 5: Specific modalities, for exemplary purposes, of the stationary phase compounds of the present invention and silica supports. This example establishes the poly-proline compounds of the present invention, including embodiments with different groups capped at the end. The groups plugged at the end are attached to the nitrogen atom which is also far from the support. As noted in the example, some end capped groups such as pivaloyl (PIV) (CSP-β) are more effective for some analytes than others, such as TOP. In general, several different end capped groups useful with the present invention such as Piv, Fmoc, Boc, Cbz, Acá, Dmb, Tpa all work well. CSP-5, which has no group stuck in the end, does not perform equally with respect to some analytes. P? > -Pro-N (Me) -Ahx-APS: CSP-5 PvP Prc-Pro-N (e) -Aí? X-APS: CSP-Ó Boc-Pro-Prc ~ N (Me) -Ahx-APS: CSP-7 Cbz-Prc-Prc ~ N (e) -Ah? -APS: CSP-8 Aca-Pro-Prc-N ¡Vfc) -Ax-APS: CSP-9 apa-Pro-Prc-N (Me) -Ahx-APS: CSP-10 Dmb-Pro-Pro-N. { Me > -Ah? -APS: CSP-U 7 a-Pro-Pro-N < Me) -Ahx-APS: CSP- 12 Table 2. Impact of blocked groups at the end Example 6 This example compares the chromatographic resolution of the racemic compounds with Fmoc-Pro-Pro-Pro-N (Me) Ahx-APS (CSP-3) which is an embodiment of the present invention, in two phase systems mobile. Consequently, this example helps demonstrate the flexibility of the stationary chiral phase of the present invention in different mobile phase systems. Table 3. Chromatographic resolution of racemic compounds with Fmoc-Pro-Pro-Pro-Pro-N (Me) Ahx-APS (CSP-3) in two mobile phase systems The invention being described, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention described herein. It is intended that the Annexes be considered as exemplary only, and is not intended to limit the scope and spirit of the invention. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, experimental results, etc. used in the Specification and Annexes shall be construed as being modified by the term "approximately." Accordingly, unless specifically indicated to the contrary, they are approximations that may vary depending on the desired properties sought to be obtained by the present invention.

References The following references are incorporated for reference in their entirety. (1) Stinson, S.C. Chemical & Engineering News 1995, 73, 44-74. (2) Okamoto, Y .; Kawashima, M.; Hatada, K. Journal of the American Chemical Society 1984, 106, 5357-5359. (3) Yashima, E .; Yamamoto, C; Okamoto, Y. Journal of the American Chemical Society 1996, 118, 4036-4048. (4) Berthod, A .; Chen, X .; Kullman, J.P .; Armstrong, D. W.; Gasparrini, F .; D'Acquarica, I .; Villani, C; Carotti, A. Analytical Chemistry 2000, 72, 1767-1780. (5) Ekborg-Ott, K. H .; Wang, X .; Armstrong, D. W. Microchemical Journal 1999, 62, 26-49. (6) Welch, C. J. Journal of Chromatography A 1994, 666, 3- 26. (7) Dobashi, A .; Dobashi, Y .; Kinoshita, K.; Hara, S. Analytical Chemistry 1988, 60, 1985-1987. (8) Billiot, E .; Warner, I.M. Analytical Chemistry 2000, 72, 1740-1 748. (9) Wang, Y .; Li, T. Analytical Chemistry 1999, 71, 4178-4182. (10) Poole, C. F .; Poole, S. K. Chromatography today; Elsevier: New York, 1991. (11) Creighton, T. E. Proteins. Structures and Molecular Properties. 2nd ed; W. H. Freeman and Company: New York, 1993. (12) Carpino, L .; El-Faham, A .; Minor, C. A .; Albericio, F. Journal of the Chemical Society, Chemical Communications 1994, 201-203.

Claims (16)

1. CLAIMS 1. A stationary chiral phase compound of the following formula: group capped at the end-Pro n where n is any integer of 2 or greater, and analogs and isomers thereof.
2. 2. The stationary chiral phase compound of claim 1, wherein n is any integer from 2 to 10.
3. 3. The stationary chiral phase compound of claim 1, wherein the support is a silica support.
4. 4. The stationary chiral phase compound of claim 1, wherein the linker is an N-alkylamino group.
5. 5. The stationary chiral phase compound of claim 4, wherein the linker is an N-methylamino group.
6. 6. The stationary chiral phase compound of claim 4, wherein the linker is a 6-methylaminohexanoic acid group.
7. 7. The stationary chiral phase of a compound of claim 1, wherein the group capped at the end is a group Piv, Fmoc, Boc, Cbz, Acá, Tapa, Dmb, or a Tpa.
8. 8. The stationary chiral phase of claim 1, wherein the linker is of the following formula: Junction point of chiral selector .... solid wherein n is an integer, and R is an alkyl group including methyl group.
9. 9. The stationary chiral phase compound of claim 1, wherein the linker is of the following formula: Solid junction point
10. 10. The stationary chiral phase compound of claim 1, wherein the group capped at the end is a Piv, Fmoc, Boc group.
11. 11. The stationary chiral phase compound of claim 1, of the following formula: and analogs and isomers thereof.
12. 12. A chiral selector of the formula: group plugged at the end-Pro n where n is any integer of 2 or greater, and analogs and isomers thereof.
13. 13. A process for separating enantiomeric mixtures by liquid chromatography, comprising: providing a racemic mixture; providing a chiral column comprising an optically active multi-proline compound or an analog or isomer thereof; and introduce the mixture to the chiral column. The process of claim 13, wherein the optically active multi-proline compound is of the following formula: in do, and analogs and isomers thereof. The process of claim 13, wherein the optically active multi-proline compound is of the following formula: and analogs and isomers thereof. 16. A stationary chiral phase compound of claim 1, of the following formula: Fmoc-Pro-Pro- (Me) N- (CH2) 5CO-NH (CH2) 3Silice (CSP2); Fmoc-Pro-Pro-Pro-Pro- (Me) N- (CH2) 5CO-NH (CH2) 3Silice (CSP3); Fmoc-Pro-Pro-Pro-Pro-Pro-Pro- (Me) N- (CH2) 5CO-NH (CH2) 3Silice (CSP4); and analogs and isomers thereof.