logging in or signing up Chiral chemistry aSGuest34608 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 917 Category: Entertainment License: All Rights Reserved Like it (2) Dislike it (0) Added: December 22, 2009 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript Chiral Separations : July 24-27, 2006, San Diego, CA Chiral Separations Outline : July 24-27, 2006, San Diego, CA Outline Stereochemistry Refresher Relationships of Stereoisomers Terminology Chiral Separations Why do we need chiral separations? Different approaches to enantiopure products Chromatographic Chiral Separations What is Chiral Recognition? 3-point rule SFC vs. HPLC Types of CSP’s Screening option Problem solving Absolute Stereochemistry (Oliver McConnell) Relationships of Stereoisomers : July 24-27, 2006, San Diego, CA Relationships of Stereoisomers Courtesy of Brown/Foote, Organic Chemistry, 3/e, Figure 1 Harcourt, Inc. items and derived items copyright 2002 by Harcourt, Inc. and http://www.chem.uic.edu/web1/OCOL-II/WIN/STEREO/ISOMER.HTM Chiral vs Achiral Compounds : July 24-27, 2006, San Diego, CA Chiral vs Achiral Compounds Achiral Molecule: Has no stereogenic center; the carbon atom has less than 4 non-equivalent substituents attached has a plane of symmetry one that is superimposable on its mirror image (the two are identical) i.e. nail, ball, a baseball bat Not optically active Chiral Molecule: Has one stereogenic center (typically C, but can be N, P, etc.), which is attached to 4 different substituents asymmetric one that is not superimposable on its mirror image (the two are not identical) i.e. hands, keys, shoes the two mirror image forms are called enantiomers Optically active http://wps.prenhall.com/wps/media/objects/724/741576/Instructor_Resources/Chapter_05/Text_Images/FG05_01-10UN.JPG Determination of Optical Activity : July 24-27, 2006, San Diego, CA Determination of Optical Activity Each enantiomer has an equal but opposite optical rotation; can be measured using optical rotation polarimeter One enantiomer rotates polarized light in a clockwise direction and is then designed as (+), or dextrorotatory The other enantiomer rotates polarized light in counter-clockwise direction and is the (-) enantiomer, or levorotatory Racemates (1:1 mixture of enantiomers) have no observable optical rotation; they cancel each other out where = observed rotation, l = cell length in dm, c = concentration in g/mL, and D is the 589nm light from a sodium lamp ©1999 William Reusch, All rights reserved (most recent revision 7/14/2006) whreusch@msu.edu Stereochemistry Terms : July 24-27, 2006, San Diego, CA Stereochemistry Terms Isomers: Compounds with the different chemical structures and the same molecular formula Stereoisomers: compounds made up of the same atoms but have different arrangement of atoms in space Enantiomers are the 2 mirror image forms of a chiral molecule can contain any number of chiral centers, as long as each center is the exact mirror image of the corresponding center in the other molecule Identical physical and chemical properties, but may have different biological profiles. Need chiral recognition to be separated. Different optical rotations (One enantiomer is (+) or dextrorotatory (clockwise), while the other is (-) or levorotatory (counter clockwise)) Racemate: a 1:1 mixture of enantiomers. Separation of enantiomers occurs when mixture is reacted with a chiral stationary phase to form 2 diastereomeric complexes that can be separated by chromatographic techniques Diastereomers: stereoisomers that are not enantiomers Have different chemical and physical characteristics, and can be separated by non-chiral methods. Has at least 2 chiral centers; the number of potential diastereomers for each chiral center is determined by the equation 2n, where n=the number of chiral centers Outline : July 24-27, 2006, San Diego, CA Outline Stereochemistry Refresher Relationships of Stereoisomers Terminology Chiral Separations Why do we need chiral separations? Different approaches to enantiopure products Chromatographic Chiral Separations What is Chiral Recognition? 3-point rule HPLC vs. SFC Types of CSP’s Screening option Problem solving Racemate vs. Single Enantiomer : July 24-27, 2006, San Diego, CA Racemate vs. Single Enantiomer http://www.fda.gov/cder/guidance/stereo.htm; C&EN, May 5, 2003, pg. 56 Chiral Blockbuster Drugs : July 24-27, 2006, San Diego, CA Chiral Blockbuster Drugs Note: Sales figures from IMS Health Courtesy of C&EN, September 5, 2005, Volume 83, Number 36, pp. 49-53 Nine of the top 10 drugs have chiral active ingredients Examples : July 24-27, 2006, San Diego, CA Examples Albuterol (anti-asthmatic inhalant) D-albuterol may actually cause airway constriction Levalbuterol (L-albuterol) avoids side effects Allegra (allergy medication) Single enantiomer of Seldane that avoids life-threatening heart disorders of Seldane Fluoxetine (generic name for Prozac, depression medication) R-Fluoxetine – improved efficacy; minimizes side effects, i.e. anxiety and sexual dysfunction. Other indications (eating disorders) S-Fluoxetine – use for treatment of migraines Approaches to Pure Enantiomers : July 24-27, 2006, San Diego, CA Approaches to Pure Enantiomers Courtesy of Christina Kraml, Wyeth Outline : July 24-27, 2006, San Diego, CA Outline Stereochemistry Refresher Relationships of Stereoisomers Terminology Chiral Separations Why do we need chiral separations? Different approaches to enantiopure products Chromatographic Chiral Separations What is Chiral Recognition? 3-point rule HPLC vs. SFC Types of CSP’s Screening option Problem solving Chiral Chromatography : July 24-27, 2006, San Diego, CA Chiral Recognition: Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 interactions through: H-bonding π-π interactions Dipole stacking Inclusion complexing Steric bulk Five general types of CSPs used in chromatography: Polymer-based carbohydrates Pirkle or brush-type phases Cyclodextrins Chirobiotic phases Protein-based Chiral Chromatography http://www.chemhelper.com/enantiomersep.html Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Polymer-based Carbohydrates Chiral polysaccharide derivatives, i.e. amylose and cellulose, coated on a silica support Enantiomers form H-bonds with carbamate links between side chains and polysaccharide backbone Steric restrictions at polysaccharide backbone may prevent access of one of enantiomers to H-bonding site Can be used with normal phase HPLC, SFC, RP-HPLC Limitations: Not compatible with a wide range of solvents other than alcohols Available columns: i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-RH, etc. from Chiral Technologies, Inc. Chiralpak IA and IB…same chiral selectors as AD and OD, respectively, but these are immobilized on the silica; more robust and has much greater solvent compatibilities Courtesy of Chiral Technologies, Inc. Naproxen examples using polymer-based CSPs : July 24-27, 2006, San Diego, CA Naproxen examples using polymer-based CSPs Conditions: Chiralpak AD-H Hexane/IPA/TFA, 80:20:0.1 Flow: 1.0 mL/min Conditions: Chiralpak AS-RH aq. H3PO4 (pH2)/ACN, 60:40 Flow: 0.7mL/min Conditions: Chiralpak AD-H, 100x4.6mm CO2/MeOH, 80:20 Flow: 5.0 mL/min Conditions: Chiralpak AD-H, 100x4.6mm CO2/MeOH, 90/10 Flow: 2.0 mL/min Courtesy of Chiral Technologies, Inc. Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Pirkle or Brush-type Phases: (Donor-Acceptor) Small chiral molecules bonded to silica More specific applications; strong 3-point interactions through 3 classes: π-donor phases π-acceptor phases Mixed donor-acceptor phases Binding sites are π-basic or π-acidic aromatic rings (π-π interactions), acidic and basic sites (H-bonding), and steric interaction Separation occurs through preferential binding of one enantiomer to CSP Mostly used with normal phase HPLC, SFC. May get less resolution with RP-HPLC; compatible with a broad range of solvents Limitations: only works with aromatic compounds Available columns: Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), -Burke 2, β-Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from Regis Technologies, Inc. Phenomenex Chirex phases Courtesy of Regis Technologies, Inc. Naproxen examples using Pirkle-type CSP : July 24-27, 2006, San Diego, CA Naproxen examples using Pirkle-type CSP (Normal phase) (Reversed phase) Courtesy of Regis Technologies, Inc. Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Cyclodextrin CSPs Alpha, beta and gamma-cyclodextrins bond to silica and form chiral cavities 3-point interactions by: Opening of cyclodextrin cavity contains hydroxyls for H-bonding with polar groups of analyte Hydrophobic portion of analyte fits into non-polar cavity (inclusion complexes) One enantiomer will be able to better fit in the cavity than the other Used in RP-HPLC and polar organic mode Limitations: analyte must have hydrophobic or aromatic group to “fit” into cavity Available columns: Cyclobond (-, -, and -cyclodextrins) from Astec, Inc. ORpak CDA (), ORpak CDB (), ORpak CDC () from JM Sciences http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Brush Slide 19: July 24-27, 2006, San Diego, CA Conditions Results Column: CYCLOBOND I 2000 Dimensions (mm): 250x4.6mm Catalog Number: 20024 Mobile Phase: 10/90: CH3CN/1% TEAA, pH 4.1 Flow Rate (mL/min): 1.0 mL/min. Temp (oC): 23°C Chart Speed (cm/min): 0.4cm/min. Detection (nm): 254nm Injection Volume (µL): 2.0µL Sample Concentration (mg/mL): 5.0mg/mL Chlorpheniramine example using Cyclodextrin-type CSP http://www.astecusa.com/applications/result_Mod.asp chlorpheniramine Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Chirobiotic Phases Macrocyclic glycopeptides linked to silica Contain a large number of chiral centers together with cavities for analytes to enter and interact Potential interactions: π-π complexes, H-bonding, ionic interactions Inclusion complexation, steric interactions Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modes Available columns: Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2 (Teicoplanin), Chirobiotic R (Ristocetin A) from Astec http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Macrocyclic Slide 21: July 24-27, 2006, San Diego, CA Conditions Results Column: CHIROBIOTIC V Dimensions (mm): 250x4.6 Catalog Number: 11024 Mobile Phase: 10/90:THF/0.1% TEAA, pH7 Flow Rate (mL/min): 1.0 mL/min. Temp (oC): 25°C Chart Speed (cm/min): 0.5 Detection (nm): 254 Injection Volume (µL): 2 Sample Concentration (mg/mL): 5 Naproxen example using Chirobiotic-type CSP http://www.astecusa.com/applications/result_Mod.asp Naproxen Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Protein-based CSPs Natural proteins bonded to a silica matrix Proteins contain large numbers of chiral centers and interact strongly with small chiral analytes through: Hydrophobic and electrostatic interactions, H-bonding Limitations: Requires aqueous based conditions in RP-HPLC Analyte must have ionizable groups such as amine or acid. Not suited for preparative applications due to low sample capacity Available columns: Chiral AGP (-glycoprotein) from ChromTech HSA (human serum albumin) from ChromTech BSA (bovine serum albumin) from Regis Technologies Slide 23: July 24-27, 2006, San Diego, CA Naproxen examples using Protein-based type CSP http://www.chromtech.se/nap-2x.htm Human Serum Albumin CSP Acid glycoprotein CSP Selecting a CSP : July 24-27, 2006, San Diego, CA Selecting a CSP General use column with no solubility issues Polymer-based phases Specific applications; solubility issues Pirkle-type Chirobiotic phases SFC only Polymer-based, Pirkle-type, Chirobiotic Biological Samples Protein-based phases Slide 25: July 24-27, 2006, San Diego, CA Suggested Applications of CSPs Compiled from Snyder, et. al, “Practical HPLC Method Development”, 2nd ed., John Wiley and Sons, Inc. 1997, p. 549 Chiral SFC vs. HPLC : July 24-27, 2006, San Diego, CA Chiral SFC vs. HPLC Advantages Reduced solvent Amounts (CO2 reduces liquid waste) Reduced toxicity Solvent types (alkanes, chlorinated, etc) CO2 has a net zero environmental impact Safety Reduce flammability Separation speed/efficiency Disadvantages Equipment costs Maintenance/robustness Solubility Slide 27: July 24-27, 2006, San Diego, CA = 1.76 Run time = 20.5 minutes Flow rate = 1.5 mL/min = 1.35 Run time = 10 minutes Flow rate = 0.4 mL/min HPLC (normal phase) SFC (normal phase) Flurbiprofen examples using HPLC and SFC http://www.registech.com/chiral/sfcappguide2006.pdf Chiral Screen : July 24-27, 2006, San Diego, CA Chiral Screen Mobile phases: CO2 + methanol or isopropanol Columns: Chiralpak AD-H, AS-H Chiralcel OD-H, OJ-H Chiralpak IA (immobilized AD) Changing Stationary Phase : July 24-27, 2006, San Diego, CA Daicel Chiralcel OD-H 25% MeOH, 140 bar Daicel Chiralpak AD-H 30% MeOH, 140 bar > LOADABILITY Changing Stationary Phase Problem Solving Approaches : July 24-27, 2006, San Diego, CA Problem Solving Approaches Derivatization of final products and intermediates Use of protecting groups such as t-BOC and CBZ (carbobenzyloxy) CBZ derivatization of chiral primary and secondary amines (common intermediates or final products of enantioselective synthesis) Adding CBZ can improve compound solubility, enables high efficiency purifications through repetitive, stacked injections Enhances chiral recognition and improves 3-point interactions; improves baseline separation ability by either HPLC or SFC CBZ protecting group easily attached and removed during synthetic processes Kraml, Christina et. al ,“Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromato Graphy”, J. of Chrom A, 1100 (2005) 108-115.. CBZ-Derivatization : July 24-27, 2006, San Diego, CA CBZ-Derivatization underivatized Purify: ~2g/hr Low S/N ratio Poor separation Aurigemma, C., BSAT 2005, Boston, MA Slide 32: July 24-27, 2006, San Diego, CA Addition of strong acid additives to mobile phase Especially useful for separation of chiral amines 0.1% ethanesulfonic acid (ESA) added to ethanol, or 0.1% methanesulfonic acid (MSA) added to methanol will cause formation of ion pairs with the amine to increase chances of successful enantioseparation Courtesy of Roger Stringham, Chiral Technologies, Inc Slide 33: July 24-27, 2006, San Diego, CA Use of Basic Additives to Mobile Phase and Sample solvent *Trans()-2-Phenylcyclopropanamine•HCl, CAS No. 1986-47-6 Aurigemma, C., BSAT 2005, Boston, MA Analytical SFC * (S,S) Whelk-O 1, 250x4.6mm, 10u i.d. (Regis Technologies, Inc.) 40% IPA w/ 0.1% IPAm 2.5 mL/min @ 140 bar Isopropylamine in Mobile phase only Preparative Slide 34: July 24-27, 2006, San Diego, CA NO residual base in collected sample No IPAm in sample solvent IPAm added to sample solvent IPAm Use of Basic Additives to Sample Solvent only Aurigemma, C., BSAT 2005, Boston, MA Summary : July 24-27, 2006, San Diego, CA Summary Direct separations of enantiomers achieved by changing CSP’s Solubility issues can be resolved by adding CBZ or another protecting group Poor peak shapes can be overcome by addition of additives to MP, MP + sample solvent, or sample solvent only Questions?? : July 24-27, 2006, San Diego, CA Questions?? You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
Chiral chemistry aSGuest34608 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 917 Category: Entertainment License: All Rights Reserved Like it (2) Dislike it (0) Added: December 22, 2009 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript Chiral Separations : July 24-27, 2006, San Diego, CA Chiral Separations Outline : July 24-27, 2006, San Diego, CA Outline Stereochemistry Refresher Relationships of Stereoisomers Terminology Chiral Separations Why do we need chiral separations? Different approaches to enantiopure products Chromatographic Chiral Separations What is Chiral Recognition? 3-point rule SFC vs. HPLC Types of CSP’s Screening option Problem solving Absolute Stereochemistry (Oliver McConnell) Relationships of Stereoisomers : July 24-27, 2006, San Diego, CA Relationships of Stereoisomers Courtesy of Brown/Foote, Organic Chemistry, 3/e, Figure 1 Harcourt, Inc. items and derived items copyright 2002 by Harcourt, Inc. and http://www.chem.uic.edu/web1/OCOL-II/WIN/STEREO/ISOMER.HTM Chiral vs Achiral Compounds : July 24-27, 2006, San Diego, CA Chiral vs Achiral Compounds Achiral Molecule: Has no stereogenic center; the carbon atom has less than 4 non-equivalent substituents attached has a plane of symmetry one that is superimposable on its mirror image (the two are identical) i.e. nail, ball, a baseball bat Not optically active Chiral Molecule: Has one stereogenic center (typically C, but can be N, P, etc.), which is attached to 4 different substituents asymmetric one that is not superimposable on its mirror image (the two are not identical) i.e. hands, keys, shoes the two mirror image forms are called enantiomers Optically active http://wps.prenhall.com/wps/media/objects/724/741576/Instructor_Resources/Chapter_05/Text_Images/FG05_01-10UN.JPG Determination of Optical Activity : July 24-27, 2006, San Diego, CA Determination of Optical Activity Each enantiomer has an equal but opposite optical rotation; can be measured using optical rotation polarimeter One enantiomer rotates polarized light in a clockwise direction and is then designed as (+), or dextrorotatory The other enantiomer rotates polarized light in counter-clockwise direction and is the (-) enantiomer, or levorotatory Racemates (1:1 mixture of enantiomers) have no observable optical rotation; they cancel each other out where = observed rotation, l = cell length in dm, c = concentration in g/mL, and D is the 589nm light from a sodium lamp ©1999 William Reusch, All rights reserved (most recent revision 7/14/2006) whreusch@msu.edu Stereochemistry Terms : July 24-27, 2006, San Diego, CA Stereochemistry Terms Isomers: Compounds with the different chemical structures and the same molecular formula Stereoisomers: compounds made up of the same atoms but have different arrangement of atoms in space Enantiomers are the 2 mirror image forms of a chiral molecule can contain any number of chiral centers, as long as each center is the exact mirror image of the corresponding center in the other molecule Identical physical and chemical properties, but may have different biological profiles. Need chiral recognition to be separated. Different optical rotations (One enantiomer is (+) or dextrorotatory (clockwise), while the other is (-) or levorotatory (counter clockwise)) Racemate: a 1:1 mixture of enantiomers. Separation of enantiomers occurs when mixture is reacted with a chiral stationary phase to form 2 diastereomeric complexes that can be separated by chromatographic techniques Diastereomers: stereoisomers that are not enantiomers Have different chemical and physical characteristics, and can be separated by non-chiral methods. Has at least 2 chiral centers; the number of potential diastereomers for each chiral center is determined by the equation 2n, where n=the number of chiral centers Outline : July 24-27, 2006, San Diego, CA Outline Stereochemistry Refresher Relationships of Stereoisomers Terminology Chiral Separations Why do we need chiral separations? Different approaches to enantiopure products Chromatographic Chiral Separations What is Chiral Recognition? 3-point rule HPLC vs. SFC Types of CSP’s Screening option Problem solving Racemate vs. Single Enantiomer : July 24-27, 2006, San Diego, CA Racemate vs. Single Enantiomer http://www.fda.gov/cder/guidance/stereo.htm; C&EN, May 5, 2003, pg. 56 Chiral Blockbuster Drugs : July 24-27, 2006, San Diego, CA Chiral Blockbuster Drugs Note: Sales figures from IMS Health Courtesy of C&EN, September 5, 2005, Volume 83, Number 36, pp. 49-53 Nine of the top 10 drugs have chiral active ingredients Examples : July 24-27, 2006, San Diego, CA Examples Albuterol (anti-asthmatic inhalant) D-albuterol may actually cause airway constriction Levalbuterol (L-albuterol) avoids side effects Allegra (allergy medication) Single enantiomer of Seldane that avoids life-threatening heart disorders of Seldane Fluoxetine (generic name for Prozac, depression medication) R-Fluoxetine – improved efficacy; minimizes side effects, i.e. anxiety and sexual dysfunction. Other indications (eating disorders) S-Fluoxetine – use for treatment of migraines Approaches to Pure Enantiomers : July 24-27, 2006, San Diego, CA Approaches to Pure Enantiomers Courtesy of Christina Kraml, Wyeth Outline : July 24-27, 2006, San Diego, CA Outline Stereochemistry Refresher Relationships of Stereoisomers Terminology Chiral Separations Why do we need chiral separations? Different approaches to enantiopure products Chromatographic Chiral Separations What is Chiral Recognition? 3-point rule HPLC vs. SFC Types of CSP’s Screening option Problem solving Chiral Chromatography : July 24-27, 2006, San Diego, CA Chiral Recognition: Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 interactions through: H-bonding π-π interactions Dipole stacking Inclusion complexing Steric bulk Five general types of CSPs used in chromatography: Polymer-based carbohydrates Pirkle or brush-type phases Cyclodextrins Chirobiotic phases Protein-based Chiral Chromatography http://www.chemhelper.com/enantiomersep.html Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Polymer-based Carbohydrates Chiral polysaccharide derivatives, i.e. amylose and cellulose, coated on a silica support Enantiomers form H-bonds with carbamate links between side chains and polysaccharide backbone Steric restrictions at polysaccharide backbone may prevent access of one of enantiomers to H-bonding site Can be used with normal phase HPLC, SFC, RP-HPLC Limitations: Not compatible with a wide range of solvents other than alcohols Available columns: i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-RH, etc. from Chiral Technologies, Inc. Chiralpak IA and IB…same chiral selectors as AD and OD, respectively, but these are immobilized on the silica; more robust and has much greater solvent compatibilities Courtesy of Chiral Technologies, Inc. Naproxen examples using polymer-based CSPs : July 24-27, 2006, San Diego, CA Naproxen examples using polymer-based CSPs Conditions: Chiralpak AD-H Hexane/IPA/TFA, 80:20:0.1 Flow: 1.0 mL/min Conditions: Chiralpak AS-RH aq. H3PO4 (pH2)/ACN, 60:40 Flow: 0.7mL/min Conditions: Chiralpak AD-H, 100x4.6mm CO2/MeOH, 80:20 Flow: 5.0 mL/min Conditions: Chiralpak AD-H, 100x4.6mm CO2/MeOH, 90/10 Flow: 2.0 mL/min Courtesy of Chiral Technologies, Inc. Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Pirkle or Brush-type Phases: (Donor-Acceptor) Small chiral molecules bonded to silica More specific applications; strong 3-point interactions through 3 classes: π-donor phases π-acceptor phases Mixed donor-acceptor phases Binding sites are π-basic or π-acidic aromatic rings (π-π interactions), acidic and basic sites (H-bonding), and steric interaction Separation occurs through preferential binding of one enantiomer to CSP Mostly used with normal phase HPLC, SFC. May get less resolution with RP-HPLC; compatible with a broad range of solvents Limitations: only works with aromatic compounds Available columns: Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), -Burke 2, β-Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from Regis Technologies, Inc. Phenomenex Chirex phases Courtesy of Regis Technologies, Inc. Naproxen examples using Pirkle-type CSP : July 24-27, 2006, San Diego, CA Naproxen examples using Pirkle-type CSP (Normal phase) (Reversed phase) Courtesy of Regis Technologies, Inc. Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Cyclodextrin CSPs Alpha, beta and gamma-cyclodextrins bond to silica and form chiral cavities 3-point interactions by: Opening of cyclodextrin cavity contains hydroxyls for H-bonding with polar groups of analyte Hydrophobic portion of analyte fits into non-polar cavity (inclusion complexes) One enantiomer will be able to better fit in the cavity than the other Used in RP-HPLC and polar organic mode Limitations: analyte must have hydrophobic or aromatic group to “fit” into cavity Available columns: Cyclobond (-, -, and -cyclodextrins) from Astec, Inc. ORpak CDA (), ORpak CDB (), ORpak CDC () from JM Sciences http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Brush Slide 19: July 24-27, 2006, San Diego, CA Conditions Results Column: CYCLOBOND I 2000 Dimensions (mm): 250x4.6mm Catalog Number: 20024 Mobile Phase: 10/90: CH3CN/1% TEAA, pH 4.1 Flow Rate (mL/min): 1.0 mL/min. Temp (oC): 23°C Chart Speed (cm/min): 0.4cm/min. Detection (nm): 254nm Injection Volume (µL): 2.0µL Sample Concentration (mg/mL): 5.0mg/mL Chlorpheniramine example using Cyclodextrin-type CSP http://www.astecusa.com/applications/result_Mod.asp chlorpheniramine Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Chirobiotic Phases Macrocyclic glycopeptides linked to silica Contain a large number of chiral centers together with cavities for analytes to enter and interact Potential interactions: π-π complexes, H-bonding, ionic interactions Inclusion complexation, steric interactions Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modes Available columns: Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2 (Teicoplanin), Chirobiotic R (Ristocetin A) from Astec http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Macrocyclic Slide 21: July 24-27, 2006, San Diego, CA Conditions Results Column: CHIROBIOTIC V Dimensions (mm): 250x4.6 Catalog Number: 11024 Mobile Phase: 10/90:THF/0.1% TEAA, pH7 Flow Rate (mL/min): 1.0 mL/min. Temp (oC): 25°C Chart Speed (cm/min): 0.5 Detection (nm): 254 Injection Volume (µL): 2 Sample Concentration (mg/mL): 5 Naproxen example using Chirobiotic-type CSP http://www.astecusa.com/applications/result_Mod.asp Naproxen Classification of Chiral Stationary Phases (CSP) : July 24-27, 2006, San Diego, CA Classification of Chiral Stationary Phases (CSP) Protein-based CSPs Natural proteins bonded to a silica matrix Proteins contain large numbers of chiral centers and interact strongly with small chiral analytes through: Hydrophobic and electrostatic interactions, H-bonding Limitations: Requires aqueous based conditions in RP-HPLC Analyte must have ionizable groups such as amine or acid. Not suited for preparative applications due to low sample capacity Available columns: Chiral AGP (-glycoprotein) from ChromTech HSA (human serum albumin) from ChromTech BSA (bovine serum albumin) from Regis Technologies Slide 23: July 24-27, 2006, San Diego, CA Naproxen examples using Protein-based type CSP http://www.chromtech.se/nap-2x.htm Human Serum Albumin CSP Acid glycoprotein CSP Selecting a CSP : July 24-27, 2006, San Diego, CA Selecting a CSP General use column with no solubility issues Polymer-based phases Specific applications; solubility issues Pirkle-type Chirobiotic phases SFC only Polymer-based, Pirkle-type, Chirobiotic Biological Samples Protein-based phases Slide 25: July 24-27, 2006, San Diego, CA Suggested Applications of CSPs Compiled from Snyder, et. al, “Practical HPLC Method Development”, 2nd ed., John Wiley and Sons, Inc. 1997, p. 549 Chiral SFC vs. HPLC : July 24-27, 2006, San Diego, CA Chiral SFC vs. HPLC Advantages Reduced solvent Amounts (CO2 reduces liquid waste) Reduced toxicity Solvent types (alkanes, chlorinated, etc) CO2 has a net zero environmental impact Safety Reduce flammability Separation speed/efficiency Disadvantages Equipment costs Maintenance/robustness Solubility Slide 27: July 24-27, 2006, San Diego, CA = 1.76 Run time = 20.5 minutes Flow rate = 1.5 mL/min = 1.35 Run time = 10 minutes Flow rate = 0.4 mL/min HPLC (normal phase) SFC (normal phase) Flurbiprofen examples using HPLC and SFC http://www.registech.com/chiral/sfcappguide2006.pdf Chiral Screen : July 24-27, 2006, San Diego, CA Chiral Screen Mobile phases: CO2 + methanol or isopropanol Columns: Chiralpak AD-H, AS-H Chiralcel OD-H, OJ-H Chiralpak IA (immobilized AD) Changing Stationary Phase : July 24-27, 2006, San Diego, CA Daicel Chiralcel OD-H 25% MeOH, 140 bar Daicel Chiralpak AD-H 30% MeOH, 140 bar > LOADABILITY Changing Stationary Phase Problem Solving Approaches : July 24-27, 2006, San Diego, CA Problem Solving Approaches Derivatization of final products and intermediates Use of protecting groups such as t-BOC and CBZ (carbobenzyloxy) CBZ derivatization of chiral primary and secondary amines (common intermediates or final products of enantioselective synthesis) Adding CBZ can improve compound solubility, enables high efficiency purifications through repetitive, stacked injections Enhances chiral recognition and improves 3-point interactions; improves baseline separation ability by either HPLC or SFC CBZ protecting group easily attached and removed during synthetic processes Kraml, Christina et. al ,“Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromato Graphy”, J. of Chrom A, 1100 (2005) 108-115.. CBZ-Derivatization : July 24-27, 2006, San Diego, CA CBZ-Derivatization underivatized Purify: ~2g/hr Low S/N ratio Poor separation Aurigemma, C., BSAT 2005, Boston, MA Slide 32: July 24-27, 2006, San Diego, CA Addition of strong acid additives to mobile phase Especially useful for separation of chiral amines 0.1% ethanesulfonic acid (ESA) added to ethanol, or 0.1% methanesulfonic acid (MSA) added to methanol will cause formation of ion pairs with the amine to increase chances of successful enantioseparation Courtesy of Roger Stringham, Chiral Technologies, Inc Slide 33: July 24-27, 2006, San Diego, CA Use of Basic Additives to Mobile Phase and Sample solvent *Trans()-2-Phenylcyclopropanamine•HCl, CAS No. 1986-47-6 Aurigemma, C., BSAT 2005, Boston, MA Analytical SFC * (S,S) Whelk-O 1, 250x4.6mm, 10u i.d. (Regis Technologies, Inc.) 40% IPA w/ 0.1% IPAm 2.5 mL/min @ 140 bar Isopropylamine in Mobile phase only Preparative Slide 34: July 24-27, 2006, San Diego, CA NO residual base in collected sample No IPAm in sample solvent IPAm added to sample solvent IPAm Use of Basic Additives to Sample Solvent only Aurigemma, C., BSAT 2005, Boston, MA Summary : July 24-27, 2006, San Diego, CA Summary Direct separations of enantiomers achieved by changing CSP’s Solubility issues can be resolved by adding CBZ or another protecting group Poor peak shapes can be overcome by addition of additives to MP, MP + sample solvent, or sample solvent only Questions?? : July 24-27, 2006, San Diego, CA Questions??