Recently, a reader sent a question about how to get started with a method that required separation of enantiomers by reversed-phase HPLC. Unfortunately, the problem is a non-starter in this context. Enantiomers cannot be separated by reversed-phase techniques – they require some chirality in the system.
Enantiomers are compounds that are mirror images of each other that cannot be superimposed. The most common image of this is to compare your right and left hands. They are exactly the same, but mirror images. Reversed-phase HPLC separates primarily by sample polarity, and selectivity often is controlled by secondary interactions, such as ionic or hydrostatic attractions. Reversed-phase does not recognize any difference between enantiomeric forms of a compound, because they have the same polarity and other chemical properties important to reversed-phase separation.
To separate chiral compounds, you must have some chirality in the separation. There are three ways this can be achieved. The first is to selectively form a derivative of one enantiomer. This would be accomplished with a reagent that would react only with one enantiomer and not the other. The result would be two significantly different compounds – one of the original enantiomers and a derivative of the second enantiomer. Such compound pairs usually have a significant difference in structure and can be separated using conventional reversed-phase or other chromatography techniques. However, derivatization is tedious, and in some cases, the reaction conditions can cause racemization, where one enantiomer is converted to the other.
A second option is to add a chiral reagent to the mobile phase. This would affect the interactions of one enantiomer vs the other and allow for separation in a reversed-phase system. A couple of problems exist with this approach. First, such additives usually are expensive, so flow rates of 1-2 mL/min can eat up a lot of expensive reagent. Second, they aren’t available for all applications. Third, they often interfere with detection, especially UV- or MS-based detectors. When very small flow rates are used, such as microbore or nanoflow HPLC or capillary electrophoresis, where flow rates are in microliters, not milliliters, this approach is more attractive.
The third option is one that most people prefer – the use of a chiral column. There are several types of chiral columns on the market, but they all feature a surface that favors interactions with one enantiomer vs the other. The most popular chiral columns are based on a fixed size of cavity, a synthetic surface that selectively interacts with one enantiomer at two or more sites, or a biomolecule surface, such as a protein. I won’t go into chiral separations any more in the present discussion, but when you take the leap into chiral separations, be prepared -- it isn’t as simple as reversed-phase. Columns tend to be 2-4 times more expensive and much less robust. An accidental selection of the wrong solvent can ruin some chiral columns. There is as much art as science in development a good chiral separation, so it may be worthwhile to take advantage of a free or low-cost column screening service offered by many chiral column manufacturers.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com