product review
Chiral Stationary Phases for HPLC Choosing the right stationary phase can be daunting. Daniel W. Armstrong Bo Zhang
T
he development of widely useful, high-efficiency enantiomeric separations is a tremendous success story. In a little more than a decade (starting in the early 1980s), this field went from an academic curiosity to an extraordinarily useful collection of related techniques, which are routinely used today in many branches of science and technology. Indeed, the U.S. Food and Drug Administration (FDA) issued guidelines for the development of stereoisomeric drugs in 1992 (1) largely because of the tremendous advances in enantiomeric separations that facilitated enantioselective pharmacokinetic and pharmacodynamic studies, as well as rapid, sensitive chiral assays. This completely altered the nature of chiral drug development. Today, the FDA may be considering additional guidelines in this area (2). In addition, greater attention is being paid to the stereochemical properties of compounds in foods, fragrances, agrochemicals, and the natural and work environments. Enantiomeric impurities are now measured routinely to the 0.01% level. HPLC can be used to measure an amino acid enantiomeric impurity near the parts-per-million level (3, 4).
Types of phases Chiral selectors can be classified in many different ways. One of the more useful classification formats is by structure (Box 1). Knowing the structure and
O C T O B E R 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
557 A
product review
Table 1. Selected CSPs for HPLC. Company
Chiral selector type
Trade name
Mode
Comments
Advanced Separation Technologies 37 Leslie Ct. P.O. Box 297 Whippany, NJ 07981 973-428-9080
[email protected] http://www.astecusa.com
Cyclodextrins (3 types) Derivatized cyclodextrins (7 types)
Cyclobond (I, II, III) Cyclobond (RSP, SP, DM, AC, RN, SN, DMP) apHera (ACD, BCD, GCD)
RP, PO RP, NP, PO
Chirobiotic (V, T, R, TAG) Chiral (AGP, CBH, HSA) CLC Cyclobond (RN, SN, DMP) Chiraldex
RP RP RP NP, RP, PO GC
In Europe, contact denise@ asteceuro.com; V, vancomycin; T, teicoplanin; R, ristocetin A; TAG, teicoplanin aglycone; Chirobiotic T may be the most useful CSP; Cyclobond I-RSP may be the most useful RP cyclodextrin-based CSP.
ChromTech, Ltd. Unit 2, The Courtyard Greenfield Farm Trading Estate Congleton, CW12 4TR Cheshire, U.K. 44-1260-270-153
[email protected] http://www.chromtech.se
Proteins (3 types)
Chiral-(AGP, CBH, HSA)
RP
AGP may be the most useful of the protein CSPs.
Daicel Chemical Industries CPI Division Chiral Separations Kasunigaseki-Chrome Chizoda-ku Tokyo 100, Japan 81-3-3507-3151
[email protected] http://www.daicel.co.jp/chiral
Derivatized cellulose (14 types)
NP, PO RP for (-R) only NP, PO RP RP
In the U.S. and Europe, contact Chiral Technologies (http://www. chiraltech.com/use.htm); AD and OD are the most useful of the amylosic and cellulosic CSPs, respectively.
Crown ethers (2 types) Synthetic polymer (2 types)
Chiracel (OA, OB, CA-I, OBH, OC, OD, OD-H, OD-R, ODRH, OF, OG, OJ, OJ-R, OK) Chiralpak (AD, AS) Chiralpak (WH, WM, WE, MA + coating) Crownpak (CR) Chiralpak (OT, OP)
Eka Chemicals Separation Products SE-445 80 Bohus, Sweden 46-31-587000
[email protected] http://www.ekachemicals.com
Derivatized tartaric acid amide polymer (2 types)
Kromosil CHI-DMB Kromosil CHI-TBB
NP, PO NP, PO
CHI-TBB = O,O'-bis (4-tert-butylbenzoyl)-N,N'-diallyl-L-tartardiamide; CHI-DMB = O,O'-bis (3,5-dimethyl-benzoyl)-N,N'diallyl-L-tartardiamide
IRIS Technologies 3008 Oxford Rd. Lawrence, KS 66049 785-842-8499
[email protected] http://www.iristechnologies.net/ index.html
π–π Association (6 types)
Chiris D1, D2, D3 Chiris AD1, AD2, AD3
NP NP
π–π Association CSPs contain a small chiral selector covalently bound to the silica surface via a spacer.
Macherey-Nagel P.O. Box 101352 D-52313 Duren Germany 49-2421-969-0
[email protected] http://www.macherey-nagel.com/
Protein (2 types, BSA) Ligand exchange (1 type) Cyclodextrins (4 types) π–π Association (2 types) Derivatized cyclodextrins for GC (9 types) Derivatized amino acids for GC (1 type)
Resolvosil BSA-7, BSA-7PX Nucleosil Chiral-1 Nucleodex -OH; ␣-, -, ␥-PM Nucleosil Chiral-2, Chiral-3 Lipodex, Hydrodex Permabond L-Chirasil-Val
RP RP RP NP, PO GC GC
Merck P.O. Box 64271 Frankurter Str. 250 64293 Darmstadt Germany 49-6151-72-0
[email protected] http://www.merck.de
Ligand exchange (2 types) Polymeric (2 types) π–π Association (2 types) Microcrystalline cellulose (1 type) Cyclodextrins (2 types)
LiChrospher 100 RP-8, RP-18 ChiraSpher, ChiraSpher NT ChiraSep DNBPG, Whelk-O 1 Cellulose triacetate ChiraDex, ChiraDex Gamma
RP NP, RP NP NP NP
Polymer (bonded carboxymethyl cyclodextrins, 3 types) Macrocyclic glycopeptide (4 types) Protein (3 types) Ligand exchange (1 type) π–π Association (3 types) Derivatized cyclodextrins for GC (15 types)
Derivatized amylose (2 types) Ligand exchange (4 types)
RP, NP, PO
NP
Organic polymer CSPs have more limited pressure stability and can swell in some chromatographic eluents.
RP, reversed-phase; NP, normal-phase; PO, polar organics phase
properties of a chiral selector is the first step in understanding how they function and what they will separate. Chiral selec-
558 A
tors also can be classified according to their source or origin (Box 1). Today, the semisynthetic chiral selectors domi-
A N A LY T I C A L C H E M I S T R Y / O C T O B E R 1 , 2 0 0 1
nate the field of enantiomeric separations, although many important contributions are still made (particularly in
product review
Table 1. Selected CSPs for HPLC. Company
Chiral selector type
Phenomenex 2320 W. 205th St. Torrance, CA 90501 310-212-0555
[email protected] http://www.phenomenex.com/ phen/home.htm
Trade name
Mode
Comments
π–π Association Chirex (11 types) Ligand exchange (1 type) Chirex
NP
These CSPs are similar to the Sumichiral CSPs produced by Sumika.
Regis Technologies 8210 Austin Ave. Morton Grove, IL 60053 847-967-6000
[email protected] http://www.registech.com/ chiral/index.htm
π–π Association (9 types) Phenylglycine, leucine Whelk-O 1 -Gem 1, ␣-Burke 2, Pirkle 1-J Naphthylleucine Ulmo, Dach-DNB Ligand exchange (1 type) Davankov Protein (3 types) Chiral-(AGP, CBH, HSA)
NP, PO
Showa Denko 5-1, Ougimach, Kawasaki-ku Kawasaki-city Kanagawa 210-0867 Japan 81-44-329-0733
[email protected] http://www.sdk.co.jp/shodex/ english/contents.htm
Cyclodextrins derivatives ORpak cyclodextrin series (4 types) Protein (1 type) BSA AFpak ABA-894
RP
Ligand exchage (1 type)
RP
Sumika Chemical Analysis Service 1-135, Kasugabe-Naka 3chome, Konohana-ku Osaka 554-0022 Japan 06-6466-5243
[email protected] http://www.scas.co.jp/english/ index.html
π–π Association (4 types) Sumichiral OA-2000 series NP π–π Association Sumichiral OA-3000, 4000 series NP (11 types) Ligand exchange Sumichiral OA-5000, 6000 series RP (4 types)
Thermo Hypersil 320 Rolling Ridge Dr. Penn Eagle Industrial Park Bellefonte, PA 16823 800-292-6088
[email protected] http://www.thermohypersil.com
Protein (2 types) Keystone HAS, BSA -Cyclodextrins (2 types) Keystone -OH, PM
RP RP, PO
Tosoh 3-8-2, Shiba, Minato-ku Tokyo 105-8623, Japan 81-3-5427-5103
[email protected] http://www.tosoh.com/ EnglishHomePage/tchome.htm
Protein (1 type) Ovamucoid (1 type)
RP
YMC Karasuma-Gojo Bldg. 284 Daigo-cho Karasuma Nisihiiru Gojo-dori Shimogyo-ku Kyoto 600-8106 Japan 81-75-342-4567
[email protected] http://www.ymc.co.jp/en/ index.html
Cyclodextrins (3 types) YMC Chiral -cyclodextrins BR π–π Association (2 types) YMC Chiral NEA (R) (S)
ORpak CRX-894
TSKgel Enantio L1 TSKgel Enantio-OVM
RP
RP RP
L-amino acid derivatives as ligands; the π-acceptor leucine CSP is based on 3,5-dinitrobenzoyl leucine, covalently bonded to 5-mm aminopropyl silica; Gem 1 is a π-acceptor CSP and is prepared by covalently bonding N-3,5-dinitrobenzoyl-3-amino-3phenyl-2-(1,1-dimethylethyl)-propanoate; ␣-Burke 2 is derived from dimethyl N-3,5-dinitrobenzoyl-3-amino2,2-dimethyl-4-pentenyl phosphonate covalently bound to 5-mm particles; Whelk-O 1 may be the most broadly applicable of the π-association CSPs.
Polymer-based gel to which cyclodextrin derivatives are bonded as the ligand. Packed with polymer-based gel bonded with BSA as the ligand. Polymer-based gel bonded with L-amino acid derivatives as the ligands. Polymer-based CSPs have more limited pressure stability and can swell in some solvents.
RP
Most CSPs are π–π association types based on derivatized amino acids.
The ovamucoid CSP has selectivities similar to the Chiral AGP.
RP NP
RP, reversed-phase; NP, normal-phase; PO, polar organics phase
O C T O B E R 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
559 A
product review
LC) by natural and synthetic compounds. Attempts to classify chiral selectors on the basis of perceived function were somewhat arbitrary because a single type of chiral selector can function differently in different situations and environments. For example, the Cyclobond-I-SN (i.e., -cyclodextrin functionalized with S-naphthylethylcarbamate groups) chiral stationary phase (CSP) can form inclusion complexes in the reversed-phase (RP) mode, act as a -complex CSP in the normalphase (NP) mode, or show only external surface adsorption in the polar organic (PO) mode. In a few cases, the structure and exact function of a chiral selector is not well understood. Arguably, the macrocyclic class of chiral selectors has had the biggest impact on analytical enantiomeric separations. The vast majority of all GC and CE enantiomeric separations are done with macrocyclic chiral selectors, predominantly derivatized cyclodextrins. However, in LC, the field is much more divided. Early on, the macrocyclic and protein-based CSPs were used for the majority of RP and PO mode separations, while the derivatized carbohydrates and -complex CSPs were used for the majority of NP separations. More recently, these lines have blurred.
For example, the macrocyclic glycopeptide and aromatic-derivatized cyclodextrins are highly effective in the NP mode, while some of the linear derivatized carbohydrate CSPs have been conditioned to work in the RP mode (they’ve also been shown to work in the PO mode).
Selecting the proper columns Choosing the best CSP for a separation can be a daunting task for the novice. There are hundreds of columns and many manufacturers to choose from (Table 1). Fortunately, choosing a column can be made less difficult because many of the CSPs simply duplicate, in structure or function, other CSPs, and despite the large number of CSPs, there are relatively few classes (Box 1). Usually only one or two columns from the first three major classes are needed for most LC chiral separations. Two of the earliest commercialized CSPs provide good examples of column duplication. The 3,5-dinitriobenzoylphenylglycine CSP (a -complex-type CSP) was first marketed by Regis Technologies, and cyclodextrin CSPs were first marketed by Advanced Separation Technologies (Astec). Afterward, both types of CSPs were mimicked, copied,
Box 1. Structure-based classification and origin of chiral selectors. Class* Macrocyclic
Examples Cyclodextrins and their derivatives, glycopeptides (macrocyclic antibiotics), and chiral crown ethers
Polymeric
Naturally occurring (derivatized linear or branched carbohydrates and proteins) and synthetic polymers
– Association
-Electron acceptor (-acid), -electron donator (-base), and combination types (containing both p-electron acceptor and donator groups)
Ligand exchange (bidentate)
Hydroxyproline, penicillamine
Miscellaneous and hybrid
Cross-linked tartaric acid derivatives
Origin Naturally occurring
Examples Cyclodextrins, macrocyclic glycopeptides, amino acids, and proteins
Semisynthetic
Derivatized cyclodextrins, derivatized linear carbohydrates, modified macrocyclic glycopeptides, derivatized amino acids and alkaloids (with -acid or -basic groups), and tartaric acid derivatives
Synthetic chiral selectors
Methacrylate polymers, a few – complex compounds, and chiral crown ethers
*Pertinent references for each class and type of chiral selector can be found in Refs. 5 and 6.
560 A
A N A LY T I C A L C H E M I S T R Y / O C T O B E R 1 , 2 0 0 1
and reproduced worldwide. In a similar vein, CSPs based on the protein serum albumin have been produced and marketed by different companies. Many of the large number of CSPs based on – associations have analogous or overlapping selectivities. Columns containing analogous or identical chiral selectors are sold under different trade names that sometimes have no obvious connection to the type of chiral selector involved. To make an informed column choice, the user must know what chiral selector is involved, what it is selective for, and how it is used. However, it is possible that, even though two different companies produce columns with the same chiral selector, they can have somewhat different selectivities and stabilities because of factors such as type of silica gel used, linkage chemistry, and coverage. Since the advent of commercial CSPs, there have always been a few “dominant” columns that accomplished the majority of separations. These so-called preferred columns have evolved or changed with time as new and better CSPs have been developed. In 1985, for example, three of the best columns were based on 3,5dinitrobenzoylphenylglycine (Regis’s phenylglycine), -cyclodextrin (Astec’s Cyclobond I), and ␣1-acid glycoprotein (LKB). Of course, there were far fewer choices at that time. By 1990, the Daicel Chiracel OD (3,5-dimethylphenylcarbamolated cellulose) proved to be the most widely applicable of the cellulosic columns, and the Crownpak chiral crown ether column’s broader selectivity and ease of use were favored for amino acid separations. Also at this time, derivatized cyclodextrin CSPs (Cyclobond I-RSP, Cyclobond I RN, etc.), a more stable and efficient second-generation ␣1-acid glycoprotein column (ChromTech’s Chiral AGP), and new – association CSPs with better selectivity became available. By 1995, the newest chiral selectors, macrocyclic glycopeptides (i.e., the Chirobiotic series from Astec), were having a significant impact because of their wide utility and high efficiency. Also, the most broadly useful -complex CSP (Whelk-O 1 from Regis) and the most widely useful derivatized linear carbohydrate CSP (Chi-
product review
ralpak AD from Daicel) were introduced around this time. It is only when these few “mainstays” don’t work that the sometimes arduous task of surveying the vast array of other columns presents itself. Irving Wainer of the National Institutes of Health says, “We accomplish most of our separations on the Chirobiotic T (teicoplanin) and Chiralpak AD columns. Only in a few cases, where these don’t give the desired separation, are other CSPs considered.” Sometimes enantiomeric separations can be achieved on two or more CSPs. This added flexibility increases the possibility that analysts can achieve a separation under their preferred experimental conditions and with the desired efficiency, selectivity, and run time. Many of the major column manufacturers offer practical background information on their columns as well as technical assistance for those interested in developing analytical and/or preparative enantiomeric separations. For example, Astec, Daicel (a.k.a. Chiral Technologies in the United States and Europe), Regis Technologies, and ChromTech AB have accumulated substantial libraries of LC enantiomeric separations. They have handbooks and/or tables of separation data along with information on the use and care of their columns. Much of this information is available on their respective Web sites. The material is usually current and often contains the latest strategies for obtaining good enantiomeric separations. In addition, the major manufacturers will develop customized analytical and/or preparative separations for interested customers, and they will contract to do the separations in-house, if the customer prefers.
Trends and future directions Manufacturers and vendors see several trends in enantioselective LC that will continue in the near future. Louis Gluntz of Regis Technologies sees an increase in the use of bulk media for simulated moving-bed applications. He also points out, “Information about chiral separations is available and exchanged much more readily today. I think that this is due to the increase in communication through the Web.” Gluntz also sees some increase
The macrocyclic class of chiral selectors has had the biggest impact on enantiomeric separations. in the use of small scout columns. Tom Beesley of Astec agrees that large-scale chiral LC separations have increased and will continue to do so in the foreseeable future. Also, he sees a tremendous increase in activity in specific areas of analytical enantiomeric separations. “Pharmaceutical companies are converting their LC chiral separations to LC/MS-compatible formats for both drug metabolism work and routine enantiomeric separations,” says Beesley. “We are seeing a lot more demand for microbore columns [2-mm i.d. ⫻ 150 mm], many of which are being used in LC/MS, as well as demand for short scout columns [0.44-mm i.d. ⫻ 100 mm] for rapid screening and methods development. There seems to be some smaller demand for CSPs in a capillary electrochromatography [CEC] column format, as well as for help in interfacing these columns to instruments.” As yet, it is difficult to predict the future of chiral CEC from a manufacturer’s standpoint. Tom Lewis of Chiral Technologies (the U.S. arm of Daicel) notes that not only are demands for large-scale, highproductivity separations increasing, but “the trend is to use the optimal CSP for a preparative chiral application rather than more general or broad selectivity media.” Lewis also sees an increase in the use of preparative supercritical fluid chromatography (SFC). Indeed, SFC is making some inroads into chiral separations, largely as an alternative to many NP separations, but not RP separations. The same packed-column CSPs that are used in LC are used in SFC. CSPs that work best in NP LC also tend to work well in SFC. Lewis notes, “In the case of Daicel’s CSPs, the cellulosic derivatives tend to be better for SFC, while the analogous amylosic derivatives are often preferred for LC.” The types of chiral compounds that are of interest are changing as well. “Today we are seeing more polar compounds, and compounds with multiple chiral centers,” says Beesley. “Further-
more, there seems to be greater interest in chiral agrochemicals and compounds of environmental importance.” This may be the result of other regulatory agencies (e.g., EPA, OSHA, and other international equivalents) finally beginning to understand the biological importance of chiral molecules.
Perspective When choosing a column, in addition to its selectivity, the mode of operation, ruggedness, efficiency, loadability, and reproducibility should be considered. If a separation can be achieved on more than one CSP, then these are the factors that dictate the choice of columns. Finally, it should be noted that complementary methods are available for strictly analytical enantiomeric separations. For example, many volatile or semivolatile compounds (including those with limited functionality and/or multiple stereogenic centers) often are easily separated using GC CSPs. Likewise, CE is an option for compounds that can be dissolved in water, hydro-organic, and some polar organic solvents. In the field of chiral separations, one thing is clear: What was once thought to be impossible is considered routine today.
References (1) (2)
(3) (4) (5) (6)
U.S. Food and Drug Administration. Chirality 1992, 4, 338–340. Miller, S. P. FDA Perspective on Quality Control for Chiral Pharmaceuticals. Abstract and Presentation at ISCD 13, Chirality 2001, Orlando, FL, July 2001. Armstrong, D. W.; Lee, J. T.; Chang, L. W. Tetrahedron Asymmetry 1998, 9, 2043–2064. Armstrong, D. W.; He, L.; Yu, T.; Lee, J. T.; Liu, Y.-S. Tetrahedron Asymmetry 1999, 10, 36–60. Armstrong, D. W. Anal. Chem. 1987, 59, 84 A–90 A. Armstrong, D. W. LC-GC 1997, 59 (Supplemental issue), S20–S28.
Daniel W. Armstrong is a professor and Bo Zhang is a graduate student at Iowa State University.
O C T O B E R 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
561 A
