Chiral Product Care and Use Guide
TABLE OF CONTENTS
- π-Acceptor HPLC Column: Nature, Applicability, and Column Selection
- π-Donor HPLC Column: Nature, Applicability, and Column Selection
- π-Acceptor/π-Donor HPLC Column: Nature, Applicability, and Column Selection
- Mobile Phase Selection
- Care of the Pirkle-Concept Columns
- Column Installation
- Technical Support
- Chiral Column Selection Chart
I. Pirkle*-Concept π-Acceptor HPLC Column: Nature, Applicability, and Column Selection
(*Developed by Dr. William H. Pirkle, of the University of Illinois)
A. Nature
The first Pirkle-Concept chiral stationary phases (CSP's) developed were the p-electron acceptors. These CSP's resolve enantiomers that contain π-donor groups, the π-electrons being supplied by the aromatic groups within the enantiomer. These packings consist of spherical silica particles, to which the CSP's are covalently bound.
Most of the interaction sites for chiral recognition on Pirkle-Concept CSP's are classified as π-basic or π-acidic aromatic rings, acidic sites, basic sites, or steric interaction sites. π-π interactions occur between aromatic ring systems within the enantiomers and those in the CSP. Acidic sites supply protons for potential hydrogen bonding and are usually generated from an amide, carbamate, amine, urea, or alcohol. Basic sites supply π-electrons, usually from a functional group containing oxygen, such as alcohols, ethers, or amides. Steric interactions occur between large bulky groups.
B. Applicability
The π-electron acceptor Pirkle Chiral Stationary Phases can be used to separate a wide range of enantiomers without derivatization, as demonstrated for the following classes of solutes: secondary benzyl alcohols, mandelic acid analogs, a-hydroxy-a-aryl phosphates, a-tetralol analogs, propranolol analogs, β-hydroxy-aryl sulfoxides, alkyl-aryl sulfoxides, diaryl sulfoxides, aryl-substituted cyclic phthalides, aryl-substituted lactams, aryl-substituted succinimides, aryl-substituted hydantoins, bi-b-naphthol and its analogs, and a-aryl acetamides.
C. Column Selection
Phenylglycine
Phenylglycine is based on the 3,5-dinitrobenzoyl derivative of phenylglycine, bound covalently to aminopropyl silica. Phenylglycine columns are available in the L-, and D- configurations.
This CSP resolves a wide variety of compounds which contain π-basic groups. These include the following: aryl-substituted cyclic sulfoxides, bi-b-naphthol and its analogs, a-indanol and a-tetralol analogs, and aryl-substituted hydantoins.
Leucine
The leucine CSP is based on the 3,5-dinitrobenzoyl derivative of leucine, bound covalently to aminopropyl silica. Columns derived from either L- or D-leucine are available.
This π-acceptor phase demonstrates enhanced enantioselectivities for several classes of compounds, including benzodiazapines.
β-Gem 1
β-Gem 1 is derived from N-3,5-dinitrobenzoyl-3-amino-3-phenyl-2-(1,1-dimethylethyl)-propanoate, covalently bound to silica through an ester linkage.
For a great many analytes, this chiral phase considerably out performs its widely used analog, phenylglycine. It can separate anilide derivatives of a wide variety of chiral carboxylic acids, including nonsteroidal anti-inflammatory agents.
α-Burke 2
The α-Burke 2 CSP is derived from dimethyl N-3,5-dinitrobenzoyl- -amino-2,2-dimethyl-4-pentenyl phosphonate covalently bound to mercaptopropyl silica.
The α-Burke 22 CSP has been specifically designed to directly separate the enantiomers of β-blockers without chemical derivatization. It also resolves the enantiomers of many compounds separated on π-acceptor Pirkle type chiral stationary phases.
Pirkle 1-J
The Pirkle 1-J CSP is based on 3-(3,5-dinitrobenzamido)-4-phenyl-β-lactam. This unusual β-lactam structure significantly alters its molecular recognition properties.
This CSP is useful for the direct separation of underivatized -blocker enantiomers, and can also be used for the separation of the enantiomers of arylpropionic acid NSAID's as well as other drugs.
II. Pirkle-Concept π-Donor HPLC Column:
Nature, Applicability, and Column Selection
A. Nature
The second generation of Pirkle-Concept CSP's are pi-electron donors. They are designed to separate amines, amino acids, alcohols, and thiols. The chiral recognition mechanisms are the reciprocal of the π-acceptor CSP's.
B. Applicability
The chiral analyte used with p-donor Pirkle-Concept CSP's are usually derivatives of chiral alcohols, amines, thiols, amino acids, amino alcohols, and carboxylic acids. Derivatization is typically required for these analytes, which lack adequate interaction sites, such as aromatic substituents. While many analytes contain p-basic aryl groups and can often be resolved on π-acidic CSP's without derivatization, relatively few analytes originally possess π-acidic groups. Hence, incorporation of such a group by derivatization is a usual first step if a p-donor column is to be used.
C. Column Selection
Naphthylleucine
The naphthylleucine CSP is based on the N-(1-naphthyl) derivative of leucine, covalently bound to 11-undecanyl silica through an ester linkage.
This CSP resolves the amides and esters of 3,5-dinitro-benzamide amino acids with enantioselectivities that typically range between 10 and 40. It also resolves the 3,5-dinitrophenylcarbamate; 3,5-dinitrophenylurea; and 3,5-dinitroanilide derivatives of a wide range of amines, alcohols, diols, carboxylic acids, amino acids, thiols, and hydroxy acids.
III. Pirkle-Concept π-Acceptor/π-Donor HPLC Column: Nature, Applicability, and Column Selection
A. Nature
This third generation of the Pirkle-Concept CSP, developed specifically for the separation of 2-arylpropionic acid, is a combination of both π-donor and π-acceptor phase attributes. As with the other Pirkle-Concept CSP's, multiple interaction sites between analyte and phase continue to be crucial to chromatographic separations.
B. Applicability
The nature of the π-donor/π-acceptor phase offers unlimited applications. This CSP uses the aromatic ring and hydrogen bonding acceptor sites to separate epoxides, sulfoxides, amides, alcohols, and esters without derivatization.
C. Column Selection
Whelk-O 1
The Whelk-O 1 CSP is derived from 4-(3,5-dinitro benzamido)tetrahydrophenanthrene covalently bound to silica.
The Whelk-O 1 was originally designed for the separation of underivatized racemates from a number of families including amides, epoxides, esters, ureas, carbamates, ethers, aziridines, phosphonates, aldehydes, ketones, carboxylic acids, alcohols and non-steroidal anti-inflammatory drugs (NSAIDs). This π-electron acceptor/π-electron donor phase exhibits an extraordinary degree of generality. This broad versatility observed on the Whelk-O 1 column, compares favorably with polysaccharide-derived chiral stationary phases. In addition, because of the Whelk-O 1's covalent nature, this chiral phase is compatible with all commonly used mobile phases, including aqueous systems - a distinct advantage over polysaccharide-derived chiral stationary phases. Other advantages include column durability, excellent efficiency, elution order inversion allowing availability of both enantiomeric forms, and excellent preparative capacity.
Whelk-O 2
The Whelk-O 2 is the covalent trifunctional version of the Whelk-O 1. The Whelk-O 2 retains the same chiral selector but modifies the support to silica from a monofunctional linkage to a trifunctional. In most cases, the enantio- selectivity remains the same allowing the separation of the analogous family of racemates as does the Whelk-O 1.
Whelk-O 2 was designed to enhance the stability of the stationary phase due to hydrolysis while using strong organic modifiers such as trifluoroacetic acid.
ULMO
The ULMO chiral sationary phase was developed by Austrian Researchers, Uray, Lindner, and Maier. This CSP has a general ability to separate the enantiomers of many racemate classes, and is particularly good at separating the enantiomers of aryl carbinols.
The ULMO CSP is based on a 3,5-Dintrobenzoyl derivative of diphenylethylenediamine and is covalently bound to spherical silica.
DACH-DNB
The innovative DACH-DNB CSP was designed by Italian chemists Dr. Francesco Gasparrini, Misiti and Villani at Rome University "La Sapienza".
The DACH-DNB CSP; which contains the 3,5-dinitro- benzoyl derivative of 1,2-diaminocyclohexane, has been found to resolve a broad range of racemate classes including amides, alcohols, esters, ketones, acids, sulfoxides, phosphine oxides, selenoxides, phosphonates, thiophosphineoxide, phosphineselenide, phosphine-borane, beta-lactams, organometallics, atropisomers and heterocycles.
IV. Mobile Phase Selection
The composition of the mobile phase should be adjusted to produce a capacity factor of about 3 or 4 for the second eluting enantiomer. The capacity factor k' can be measured using the following equation:
k'=(tr-to)/to
Whereas, tr is the retention time for the second eluting enantiomer and to is the retention time for an unretained substance, such as 1,3,5-tri-tert-butyl benzene.
A. Covalently bound CSP's
The Regis line of CSP's are all covalently bound. They can easily be used for both reversed-phase and normal-phase applications. In reversed-phase modes, mobile phases for these CSP's can consist of any polarity but must remain within a pH range of 2.5 to 7.5.
Components for normal and or reversed-phase mobile phases can consist of IPA, hexane, heptane, ethanol, water, acetonitrile, methanol, 1,2-dicholoroethane and THF. Common mobile phase additives include acetic acid, triethylamine, diethylamine and ammonium acetate.
However, in order to maintain the integrity of the column, it is critical that TFA not be a mobile phase component.
NOTE: TFA may be used with the Whelk-O 2 stationary phase only.
V. Care of the Pirkle-Concept Columns
A. Testing
Each Pirkle-Concept column is shipped with a test chromatogram and performance report detailing test conditions and results.
B. Reversibility
The Regis Pirkle-Concept columns are Rexchrom Reversible columns proprietarily packed with spherical silica. The column may be used equally well in either direction.
Routine reversal of the flow direction leads to the following benefits of cleaner outside frit surfaces, deposit-free columns, and fine-free inside frit surfaces, all of which can extend column life.
C. Storage
Do not allow water to remain in these or any silica-based columns. Remove any buffers by washing with 20 mL of water and then flush the column with at least 20 mL of compatible organic solvent before leaving the column overnight.
If used only under normal phase conditions, the column may be stored in that mobile phase. If an acidic modifier is present, flush the column with a pure organic solvent prior to storage.
The column is to be kept wet during storage. Each column is shipped with two removable end plugs, to prevent the drying of the column bed.Save these plugs and re-install them whenever the column is removed from the HPLC system.
VI. Column Installation
Column connections are an integral part of the chromatographic process. If ferrules are overtightened, not set properly, or are not specific for the fitting, leakage can occur. Nuts and ferrules are provided with each column and should be used for all column connections.
Set the ferrules for column installation to the HPLC system as follows:
1. Place the male nut and ferrule, in order, onto a 1/16" o.d. piece of tubing. Be certain that the wider end of the ferrule is against the nut.
2. Press tubing firmly into the column endfitting. Slide the nut and ferrule forward, engage the threads, and fingertighten the nut.
3. While continuing to press the tube firmly into the endfitting, use a 1/4" wrench to tighten the nut 90 degrees past fingertightness.
4. Repeat this coupling procedure for the other end of the column.
VII. Technical Service
Customer satisfaction is our goal.
If you have any questions concerning the performance or application of the column, contact the Regis Technical Service department at (800) 323-8144, (847) 583-7661 or (847) 967-1214 FAX.
Trademarks:
Pirkle-Concept Regis Technologies, Inc.
Rexchrom Regis Technologies, Inc.
Regis has made every effort to present accurate information in this booklet. However, users are cautioned to use their own judgment and make their own determination of the suitability of any of the products, data, or procedures presented.
Note: The products presented are for research use only and are not intended for food or drug purposes; nor are they to be resold for such use. Due care should be exercised in their handling and use.
VIII. Pirkle-Concept Chiral Selection Chart
π-Acceptors
| D-Phenylglycine | 25 cm x 4.6 mm i.d. | 731021 |
| 25 cm x 10.0 mm i.d. | 731221 |
| L-Phenylglycine | 25 cm x 4.6 mm i.d. | 731024 |
| 25 cm x 10.0 mm i.d. | 731224 |
| D-Leucine | 25 cm x 4.6 mm i.d. | 731054 |
| 25 cm x 10.0 mm i.d. | 731254 |
| L-Leucine | 25 cm x 4.6 mm i.d. | 731041 |
| 25 cm x 10.0 mm i.d. | 731241 |
| (S,S)-Beta-GEM | 25 cm x 4.6 mm i.d. | 731029 |
| 25 cm x 10.0 mm i.d. | 731229 |
| (R,R)-Beta-GEM | 25 cm x 4.6 mm i.d. | 731043 |
| 25 cm x 10.0 mm i.d. | 731243 |
| (R)-Alpha-Burke | 25 cm x 4.6 mm i.d. | 735035 |
| 25 cm x 10.0 mm i.d. | 735235 |
| (S)-Alpha-Burke | 25 cm x 4.6 mm i.d. | 735037 |
| 25 cm x 10.0 mm i.d. | 735237 |
| (3R,4S)-Pirkle 1-J | 25 cm x 4.6 mm i.d. | 731044 |
| 25 cm x 10.0 mm i.d. | 731244 |
| (3S,4R)-Pirkle 1-J | 25 cm x 4.6 mm i.d. | 731045 |
| 25 cm x 10.0 mm i.d. | 731245 |
π-donors
| L-Naphthylleucine | 25 cm x 4.6 mm i.d. | 731034 |
| 25 cm x 10.0 mm i.d. | 731234 |
π-acceptor/π-donor
| (S,S)-Whelk-O 1 | 25 cm x 4.6 mm i.d. | 786101 |
| 25 cm x 10.0 mm i.d. | 786102 |
| (R,R)-Whelk-O 1 | 25 cm x 4.6 mm i.d. | 786201 |
| 25 cm x 10.0 mm i.d. | 786202 |
| (S,S)-Whelk-O 2 | 25 cm x 4.6 mm i.d. | 786415 |
| 25 cm x 10.0 mm i.d. | 786425 |
| (R,R)-Whelk-O 2 | 25 cm x 4.6 mm i.d. | 786315 |
| 25 cm x 10.0 mm i.d. | 786325 |
| (R,R)-ULMO | 25 cm x 4.6 mm i.d. | 787200 |
| 25 cm x 10.0 mm i.d. | 787201 |
| (S,S)-ULMO | 25 cm x 4.6 mm i.d. | 787100 |
| 25 cm x 10.0 mm i.d. | 787101 |
| (R,R)-DACH-DNB | 25 cm x 4.6 mm i.d. | 788101 |
| 25 cm x 10.0 mm i.d. | 788102 |
| (S,S)-DACH-DNB | 25 cm x 4.6 mm i.d. | 788201 |
| 25 cm x 10.0 mm i.d. | 788202 |
NOTE: All columns listed contain chiral stationary phases that are covalently bound on 5 µm or 10 µm 100 Å spherical silica. A large variety of column dimensions and/or particle sizes are available upon request.
For a complete listing of chiral applications, go to our web site at:
Email questions: teds@registech.com