Chiral Chromatography: Principle, Components, Steps, Types, Uses

Chiral chromatography is a method used to separate enantiomers, which are pairs of chiral compounds that are non-superimposable mirror images of each other. It is also called enantioselective chromatography.

Different methods like crystallization, capillary electrophoresis (CE), and chromatography can be used to separate enantiomers. Among these, chromatography has become the most preferred method for chiral separation.

• Chiral chromatography is especially important in pharmaceutical industries as many pharmaceutically active compounds are chiral and chirality influences the safety and efficacy of drugs. 
• Each enantiomer can show different pharmacological properties within the biological system. One enantiomer is more active called the eutomer and the other may be less active or even harmful called the distomer. 
• A well-known example that shows the importance of chiral separation is the thalidomide tragedy. The thalidomide drug is a racemic drug that was originally commercialized in late 1950s as a sedative. It was also given to pregnant women to treat morning sickness.  
• One enantiomer of thalidomide has sedative effect but the other is a potent teratogen that causes severe birth defects. 
• The use of thalidomide without proper research and safety testing led to over 10,000 children born with birth defects. Even if the safe isomer was administered, it resulted in harmful effects as racemization of the drug occurred inside the body. 
• So, it is important to separate and isolate pure enantiomers to obtain safe and effective chiral compounds.

Principle of Chiral Chromatography

Chiral chromatography separates enantiomers based on their different stereoselective interactions with the chiral selector. Chiral selectors can be used either in stationary phase or in mobile phase. Each enantiomer interacts differently with a chiral selector which causes them to move through the column at a different speed and elute at different times. This separates the enantiomers. 

Enantiomers can be separated using two methods: direct and indirect. Direct separation uses a chiral selector in stationary or mobile phase. Indirect separation involves modifying the enantiomers to form diastereomeric pairs which can then be separated based on their different physical or chemical properties. Commonly used methods for chiral chromatography are gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and supercritical fluid chromatography (SFC). 

Components of Chiral Chromatography

1. Chiral Stationary Phase (CSP)

CSPs are fixed to the chromatography column and interact selectively with the chiral compounds to separate them. There are different types of CSPs based on their separation principle or the type of chiral selector used.

Some common types of CSPs are:

• Pirkle-type CSPs, also called brush-type CSPs, were the first commercially developed CSPs. They separate enantiomers through specific non-covalent interactions like hydrogen bonding, π-π stacking, and dipole interactions. They are effective for separating π-acidic racemates like derivatives of amines and amino alcohols. Further developments have combined π-acidic and π-basic sites to improve separation. Pirkle phases like Welk-O-1 are still commercially available through Regis Technologies.
• Polysaccharide-based CSPs include derivatives of natural polymers like cellulose and amylose. They are the most commonly used CSPs due to high selectivity and ability to separate different compounds. They are used for compounds with functional groups like amides, aromatic rings, hydroxyl, and amino groups.
• Protein-based CSPs use biological macromolecules like enzymes or plasma proteins as chiral selectors. Examples of protein-based CSPs are bovine serum albumin (BSA), ovomucoid, and human serum albumin (HSA). They are particularly used for water-soluble and biologically active compounds. Their chiral recognition mainly involves hydrogen bonding, π–π interactions, dipole–dipole forces, and ionic interactions.
• Cavity-based CSPs separate enantiomers based on inclusion where the guest molecule is selectively hosted inside a chiral cavity. Cyclodextrins (CDs) are the most commonly used CSPs of this type. CDs are suitable for analytes like hydrocarbons, sterols, phenol esters, and aromatic amines. Crown ethers are another example of cavity-based CSPs which are effective for separating primary amines.
• Macrocyclic glycopeptides-based CSPs use antibiotics like vancomycin and teicoplanin as chiral selectors. They are covalently bonded to silica by linkers like carboxylic acid, amine, or isocyanate groups to ensure stability. These CSPs separate enantiomers based on interactions like hydrogen bonding, π–π stacking, polar, and ionic interactions with different functional groups like hydroxyl, amine, and carboxyl groups.

2. Mobile Phase

This is the solvent that carries the sample through the chromatography column. Solvents like methanol, ethanol, or isopropanol are commonly used. The mobile phase can also be modified with chiral mobile phase additives (CMPAs) which are chiral selectors dissolved in the mobile phase. CMPAs form transient diastereomeric complexes with analyte enantiomers and separate them. This allows direct separation on achiral columns.

The most common types of CMPA are:

• Ligand-exchangers involve chiral metal complexes formed from transition metals and chiral ligands like amino acids or their derivatives. These complexes interact with analytes containing functional groups like carboxyl groups, amino groups, or hydroxyl groups.
• Macrocyclic antibiotics are also used as CMPAs. The most commonly used one is vancomycin. These glycopeptides have a complex and unique three-dimensional basket-like structure that allows multiple interactions for chiral recognition and separation.
• Cyclodextrins (CDs) are one of the most commonly used chiral selectors. They form host-guest inclusion complexes where the hydrophobic groups in analytes are included into hydrophobic cavity of CDs. 

3. Derivatizing Agents

Indirect chiral separation methods use chiral derivatizing agents (CDAs) like Marfey’s reagent or Mosher’s acid that convert enantiomers into diastereomers which are easier to separate on achiral columns.

4. Chromatographic System

Most commonly used chromatographic systems for chiral separation are HPLC and SFC. They include components like pumps to move the mobile phase, columns packed with stationary phase, and injection systems to add the sample. These instruments also use detectors such as UV-Vis, mass spectrometry (MS), circular dichroism (CD), and fluorescence. 

Types of Chiral Chromatography

Two main types of chiral chromatography are: 

1. Direct Method

This method uses chiral selectors in the stationary phase (CSP) or mobile phase (CMPA). CSPs are preferred because they work with different types of compounds and are easily available. Direct method does not involve any chemical modification of the analyte. It uses stereoselective interaction between the enantiomers and the chiral selectors to separate them. 

2. Indirect Method

This involves derivatizing the chiral analyte with chiral derivatizing agents (CDAs) to form diastereomers which are then separated using achiral columns. The diastereomers are separated based on their different physical and chemical properties. This method is useful when a suitable chiral selector is not available but it has lower resolution and is more complex due to the derivatization process.

Procedure or Steps of Chiral Chromatography

1. Chromatographic Method Selection: The appropriate chromatography method is selected based on the properties of the sample. Common chiral separation methods used are HPLC, SFC, CE, and GC. Among these methods, HPLC and SFC are the most widely used methods.
2. Chiral Selector Selection: Chiral selectors must be carefully selected as they determine how the enantiomers are separated. Based on the method and the analyte properties, the stationary phase and mobile phase are carefully selected. In direct method, CSPs or CMPAs are used and in indirect method, CDAs are used.
3. Sample Preparation: Then, the sample or analyte is prepared for separation and analysis. The sample is dissolved in a compatible solvent. For indirect methods, derivatization is done to form diastereomers. After preparation, the sample is injected into the chromatographic column.
4. Separation: As the sample passes through the column, the enantiomers interact differently with the chiral selector which results in different retention times and separates the molecules. In direct method, the enantiomer will separate based on their different interactions with the chiral selector. In indirect method, the derivatized diastereomers will separate due to their different physical or chemical properties.
5. Detection and Data Analysis: The separated enantiomers are detected using suitable detectors which generates a chromatogram showing peaks that correspond to each enantiomer. These peaks are detected and the retention time of the enantiomers is also identified which is compared to known values to identify the enantiomers. 

Factors Affecting Chiral Chromatography

1. The properties of analytes like functional groups and molecular size can affect the interactions with the chiral selector. 
2. The type of CSP or the chiral recognition mechanisms affects the separation.
3. The type of mobile phase and CMPAs are also important factors. Mobile phase composition including solvent polarity and pH can influence the chromatographic separation.
4. Temperature also affects the separation. Lower temperature often provides better resolution.
5. Flow rate of the solvent is another factor affecting chromatography. Higher flow rates reduce resolution.

Common Products and Manufacturers of Chiral Chromatography

Applications of Chiral Chromatography

• Chiral chromatography is used in the pharmaceutical industry for chiral drug development and enantiomeric purity testing. 
• It can be used in agriculture to study chiral pesticides and herbicides. It ensures the safety of agricultural chemicals and minimizes risks associated with agrochemical usage.
• It is also used in environmental study to detect and remove chiral pollutants.
• It can be used in quality control of food additives which ensures product safety and quality. 
• Chiral chromatography can be used to produce enantiomerically pure chemicals.

Advantages of Chiral Chromatography

• Chiral chromatography has high selectivity and sensitivity. It can accurately separate and quantify enantiomers.
• The use of different types of CSPs and CMPAs makes chiral chromatography useful for different chiral compounds. 
• Many inorganic molecules and drugs currently in use or under development are chiral and needs chiral chromatography.
• Direct chiral chromatography does not change the structure of the analyte and preserves both enantiomers.
• It helps to identify the therapeutically active enantiomer and remove the harmful ones. This produces safe and effective pharmaceuticals.

Limitations of Chiral Chromatography

• Chiral columns and reagents can be expensive especially for large-scale applications.
• The separation and method development can be time-consuming.
• A single CSP cannot work for all chiral compounds so different phases need to be tested during method development.
• It has difficulty in separating complex mixtures like multiple enantiomers or closely related stereoisomers.

Troubleshooting and Safety Considerations

• Common problems in chiral chromatography include peak overlap, baseline instability, and signal noise which can reduce the accuracy of the results. 
• Peak overlap occurs when enantiomers have similar retention times. This can be fixed by optimizing the mobile phase composition, temperature, or selecting a more suitable CSP.
• Baseline instability and noise may be due to detector fluctuations or issues with instrument. This problem can be resolved by adjusting detector settings and regular maintenance of instruments.
• Some chemicals used may be toxic or hazardous and should be handled in a fume hood. Personal protective equipment (PPE) should be worn to avoid exposure to hazardous chemicals.
• Pressurized instruments like HPLC and GC systems must be handled carefully and regularly checked for pressure safety. 

Recent Advances and Innovations

• Novel CSPs are being developed like zwitterionic materials. Immobilized and hybrid CSPs are also developed with improved stability and selectivity.
• Multidimensional chromatographic methods like two-dimensional liquid chromatography (2D-LC) have improved the separation of complex chiral compounds. 
• Automation and high-throughput screening make method development faster which increases efficiency. 
• Combining chromatography with advanced analytical tools like mass spectrometry (MS) and nuclear magnetic resonance (NMR) has also improved chiral separation.
• Green chromatography methods like SFC are also becoming more common as they reduce solvent use and environmental impact.

Conclusion

Chiral chromatography is used for separation and analysis of enantiomers which is useful in different areas including pharmaceutical, environmental monitoring, and food safety. This ensures the safety of drug development, supports environmental monitoring, and improves the production of pure compounds. Advancements in chiral selectors and methods like HPLC have improved chiral separation and its applications.

References

1. Al-Sulaimi, S., Kushwah, R., Alsibani, M. A., Jery, A. E., Aldrdery, M., & Ashraf, G. A. (2023). Emerging developments in separation techniques and analysis of chiral pharmaceuticals. Molecules, 28(17), 6175. https://doi.org/10.3390/molecules28176175
2. An introduction to Chiral Chromatography. Chromatography Today. Retrieved from https://www.chromatographytoday.com/news/preparative/33/breaking-news/an-introduction-to-chiral-chromatography/31907
3. Chiral chromatography Frequently asked questions. (n.d.). Retrieved from https://www.sigmaaldrich.com/NP/en/technical-documents/technical-article/analytical-chemistry/purification/faq?msockid=04410a7a527f6a7b244f1990537e6bd6
4. Grybinik, S., & Bosakova, Z. (2021). An overview of chiral separations of pharmaceutically active substances by HPLC (2018–2020). Monatshefte Für Chemie – Chemical Monthly, 152(9), 1033–1043. https://doi.org/10.1007/s00706-021-02832-5
5. Libretexts. (2020, August 22). 14.3: Chiral Chromatography. Retrieved from https://chem.libretexts.org/Courses/Providence_College/CHM_331_Advanced_Analytical_Chemistry_1/14:_Liquid_Chromatography/14.03:_Chiral_Chromatography
6. Liu, H., Wu, Z., Chen, J., Wang, J., & Qiu, H. (2023). Recent advances in chiral liquid chromatography stationary phases for pharmaceutical analysis. Journal of Chromatography A, 1708, 464367. https://doi.org/10.1016/j.chroma.2023.464367
7. Tarafder, A., & Miller, L. (2021). Chiral chromatography method screening strategies: Past, present and future. Journal of Chromatography A, 1638, 461878. https://doi.org/10.1016/j.chroma.2021.461878
8. Thalidomide Tragedy. (2024, August 21). World of History. https://worldofhistorycheatsheet.com/thalidomide-tragedy/
9. Understanding Chiral Chromatography: A Comprehensive guide. (n.d.). Retrieved from https://chromtech.com/blog/chiral-chromatography-comprehensive-guide/
10. Zhao, Y., Zhu, X., Jiang, W., Liu, H., & Sun, B. (2021). Chiral Recognition for Chromatography and Membrane-Based Separations: Recent Developments and Future Prospects. Molecules, 26(4), 1145. https://doi.org/10.3390/molecules26041145