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Chiral e-Times 2011 Volume 2
Have you ever wondered why there are so many different types of chiral columns? An article in the Supelco Reporter Vol. 29 Issue 2 explains why, and provides a succinct overview of modern chiral stationary phases (CSPs) and what they're used for. Through our Astec brand and the columns of our innovative partners in chiral chromatography, Supelco offers analysts the widest selection of HPLC, SFC, and GC chiral stationary phases. Whereas most chiral column suppliers offer slight variations of very similar CSPs, Supelco's range includes CSPs widely varying retention mechanisms and mobile phase systems to maximize your screening success. The Column Screening article in this newsletter is a good example of why it is important to have a variety of types of CSPs in your screening protocol.
The CHIROBIOTIC, CYCLOBOND, CHIRALDEX, and Supelco DEX columns are available in convenient and economical column screening kits.
Even the experts have to screen columns. There is no reliable way to choose a chiral column based on analyte structure alone. Our Chiral Services Lab continually experiments with ways to improve the efficiency of their chiral column screening protocols. The introduction of two new CSPs, the Astec Cellulose DMP and the cyclofructans, presented an opportunity to optimize their system. The result was a 94% success rate on a structurally-diverse, 32-compound screening set (Table 1). The description of our screening protocol and success or "hit" rates are shown in Table 2. It seems complicated, but it is really very simple, and fast! We use two HPLC instruments, one dedicated to normal phase (NP) operation and the other to reversed-phase (RP), polar ionic mode (PIM), and polar organic mode (POM). Polysaccharide and synthetic polymer CSPs are screened in NP and POM systems. Astec CYCLOBOND CSPs are screened in POM and RP. Astec CHIROBIOTIC CSPs are also screened in RP mode, but are most useful in PIM. PIM is unique to CHIROBIOTIC and very valuable for enantioresolution of polar and ionic compounds. When we encounter analytes with a primary amine moiety (especially one at or near the chiral center), we use a third NP and a third POM mobile system and screen three cyclofructan CSPs. All of the columns in our screen are very rugged, and do not have to be dedicated to operation in a single mobile phase system. As expected, the normal phase system gave the highest success rate, as did the CSPs based on polysaccharides and macrocyclic glycopeptides. These are probably the #1 and #2 types of CSPs, respectively, on the market today. This brief description of our screening protocol is meant to demonstrate two points. First, stationary phase selectivity is your biggest lever to affect enantioresolution. Second, having a wide range of mobile phase options, from highly polar ionic and aqueous to nonpolar systems, gives you flexibility in detectors and a means to increase analyte solubility. The latter is a very important consideration for prep chiral separations. Table 1. Compounds Used in the Screening Experiment
Table 2. Screening Protocol Leveraging CSPs Selectivity and Mobile Phase Flexibility
(i) We have an updated Presentations page with posters/talks from PittCon 2011, (accessible under the Chiral Learning Center): "HPLC Enantiomeric Separations of Pharmaceuticals Using Polar Organic Mobile Phases" by Jauh-Tzuoh Lee, William Campbell "Impact of Reversed-Phase Chiral Chromatography on the LC-MS Analysis of Drugs in Biological Fluids" by Dave Bell, Carmen Santasania, Jennifer Claus, Wayne Way, Craig Aurand (ii) We have updated the Product Information & Literature page with links to new brochures, product guides, and technical articles (accessible under the Chiral Learning Center). Printed copies of select brochures are available upon request.
(i) Chirality 2011 Symposium Chirality 2011, the 23rd International Symposium on Chiral Discrimination will be held in Liverpool, UK from July 10 – 14, 2011. Sigma-Aldrich/Supelco events: Oral presentation, Poster Presentations, Vendor Seminar Oral Presentation: Enantioseparation of Pharmaceuticals Using Polar Organic Mobile Phases: An Alternative Approach Abstract: Pharmaceutical and biotech industries place great importance on chirality during drug discovery and development. Since each enantiomer possesses different biological properties in terms of absorption, distribution, metabolism and excretion (ADME), an early understanding of the pharmacological aspects of chiral drugs is imperative. Although chiral separations traditionally use hexane or heptane-based normal phase solvent systems, solubility problems are encountered regularly. The use of polar solvents often eliminates sample solubility issues, and thus, frequently demonstrates improvements in peak shape and resolution. In HPLC, the macrocyclic glycopeptide stationary phases are renowned for their chiral efficacy with the use of polar mobile phases. The polysaccharide chiral stationary phases (CSPs) are typically most effective in normal phase mode; however, they also provide good selectivity towards a number of racemic drugs in polar mobile phases. This study investigates the use of one vancomycin-based and one cellulosic-based CSP to examine the enantioselectivity of a group of chemically diverse, basic pharmaceutical drugs. Methods are made to be LC-MS compatible through the use of methanol-based mobile phases with volatile acidic, basic, or salt additives. Optimization steps providing improvements in resolution and selectivity will be discussed in detail. The complementary performance between vancomycin-based and cellulosic-based CSPs will also be demonstrated. Presented by Jennifer Claus, R &D Chemist, Supelco, Inc. If you can't make the meeting but would like to receive a copy of the presentation, send an email to jennifer.claus@sial.com or visit our Presentations page after the show. Poster Abstracts, Vendor Seminar: To view abstracts for our posters and to register for our vendor seminar which will be held on Monday, July 11, at 1:15pm, please visit our Chirality 2011 page. (ii) Innovations in Chromatography and Sample Prep Seminars We started these in 2009, ran them in 26 cities in 2010, and have expanded to 41 cities in 2011 across the US and Canada. Audience members asked for GC topics in addition to HPLC, and more application focus. So, in 2011, the ½-day complimentary (free) seminars will include: Techniques to Improve Speed, Resolution, and Sensitivity without Investing in Capital Equipment:
Abstracts, registration, and complete details can be found at http://www.sigma-aldrich.com/analytical-seminars.
We talked a little about POM in the services section of this newsletter, how it is an important part of our screening protocol on polysaccharide- and cyclodextrin-based CSPs. This section will go a little deeper into POM.
What is POM? Polar organic mode (POM) mobile phases comprise methanol or acetonitrile or their mixtures. Acetonitrile is the most common POM solvent. Methanol is added to reduce retention of molecules with multiple hydrogen bonding sites. Acids, bases, or salts are often added to control peak shape and retention of certain sensitive analytes. Why is it useful for chiral HPLC & LC/MS? The benefit of POM is realized when dealing with compounds that are poorly soluble in non-polar normal phase mobile phases. For preparative chiral applications, solubility is especially important; analyte concentration per injection influences the throughput. POM mobile phases are also mass spec-friendly because they use methanol-based eluents with some acid and base or volatile ammonium salts as additives. As an additional sensitivity benefit, POM baselines are generally less noisy than normal phase baselines. Column and system equilibration in POM mobile phases is also very rapid, and there is no memory effect. POM is used with what types of CSPs? The POM effect was first described by Armstrong in the early 1990's (1-3) on native and derivatized cyclodextrin-based CSPs (e.g. Astec CYCLOBOND). However, any CSP that can interact with solutes via hydrogen bonding mechanisms can utilize POM. These CSPs include the cyclodextrins, as well as macrocyclic glycopeptides (e.g. Astec CHIROBIOTIC), and polysaccharides (e.g. Astec Cellulose DMP). Can POM be used with the popular cellulose-based CSPs? Yes! Although the polysaccharide-based phases (cellulose and amylose) have proven their worth in normal phase and SFC systems, we have found they have good selectivity towards racemic compounds in POM mobile phases. POM is a viable alternative to normal phase. Separations on POM can be more efficient, and provide more resolution than NP mode. Although this is very compound-dependent, POM should be explored if improvements in solubility, sensitivity, or instrument compatibility are desired. Figure 1 and Figure 2 show the antidepressant mianserin and Tröger's Base on Astec Cellulose DMP (a 3,5-diphenylcarbamate-deriviatized cellulose) in normal phase and POM systems. Note that in both cases, the POM separation gave comparable retention, but better resolution and sharper peaks. Straightforward method development in POM on cellulosic CSPs. Although these CSPs do not have ionic functional groups, ionic additives play an important role in selectivity, efficiency, and peak shape. Figure 3 provides an example of a typical method development routine with POM on Astec Cellulose DMP. Method development on POM comprised testing mobile phases of methanol containing low percentages of various ionic additives. A suggested starting mobile phase is 0.1% w/v ammonium formate in methanol. In the example in Figure 3, the best separation was achieved using methanol/acetic acid/TEA (100/0.2/0.1). Wide applicability of POM. Figure 4 shows a wide variety of chemically diverse, basic pharmaceutical drugs on the Astec Cellulose DMP in POM mode. The mobile phases were also designed to aim for mass spectrometry detection using methanol-based eluents with some acid and base or volatile ammonium salts as additives. Conclusions Polar organic mobile phases (POM and PIM) provide additional opportunities for chiral selectivity should other types of mobile phases fail, or you just want the benefits of this mobile phases, which include:
As noted, the macrocyclic glycopeptides (CHIROBIOTIC), cyclodextrin (CYCLOBOND) and polysaccharide CSPs can be complementary to one another using polar organic mobile phases. For more information on POM, please see J.T. Lee's PittCon 2011 presentation "HPLC Enantiomeric Separations of Pharmaceuticals Using Polar Organic Mobile Phases" available under our Learning Center. References
Figure 1. Comparison of NP and POM on Astec Cellulose DMP (Mianserin) Column: Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 µm particles (51098AST) Mobile phase: See figure  Figure 2. Comparison of NP and POM on Astec Cellulose DMP (Tröger's Base) Column: Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 µm particles (51098AST) Mobile phase: See figure  Figure 3. Example of Method Development of POM on Cellulosic CSP Column: Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 µm particles (51098AST) Mobile phase: See figure  Figure 4. Various POM Separations on Astec Cellulose DMP Column: Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 µm particles (51098AST) Mobile phase: 0.1% w/v ammonium formate in methanol except ketoconazole, 100% acetonitrile 
Astec CLC columns come in two forms to provide elution order enantioreversal of chiral organic acids and amino acids. The copper ligand concept (CLC) was described by Davankov and comprised a chiral bidentate ligand and a copper sulphate-containing mobile phase. The copper ions coordinate with the chiral selector on the stationary phase and functional groups on the analytes to form transient diastereomeric complexes in solution. The technique also has the advantage of giving small acids with no UV chromophore a strong 254 nm signal. Astec CLC columns are ideal for analysis of α-hydroxy acids, like lactic, malic, tartaric, and mandelic acids, amino acids, other amines, and bifunctional racemates, like amino alcohols. Two versions of the column provide elution order reversal (see Figure). On the Astec CLC-D column, the L enantiomer generally elutes before D, with the exception of tartaric acid. The reverse is true on the Astec CLC-L column where D elutes before L. Proline and aspartic acid are particularly suited for low-level detection on the CLC column since the copper complex is detected at 254 nm UV. Both can be resolved on the Astec CLC-D or CLC-L in 5 mM CuSO4 with the usual reversal of elution order from the CLC-D to CLC-L. In theory, any analyte that can complete the coordination with the copper ion can be resolved.  
View Astec CLC flyer (T411062; PDF) Note the promotion on Astec CLC columns described in the Special Offer section below.
Through August 2011, save 50% off list price when you order either form of the Astec CLC column: Get two columns for the price of one! Why would analysts want to have two forms of the CLC column? Perhaps to have a confirmatory column or to effect elution order reversal for better quantification. (Components at trace levels should elute before the peak in large excess.) On the Astec CLC-D column, the L enantiomer generally elutes before D, with the exception of tartaric acid. The reverse is true on the Astec CLC-L column where D elutes before L. Having both forms gives you the flexibility of elution order reversal. And this special limited-time offer makes it a great way to build up your chiral column library! Specify Promo Code Q15 when you order. Offer good through August 31, 2011.
Trademarks: Kromasil, AmyCoat, and CelluCoat are trademarks of EKA Chemicals AB |
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