Choosing Chiral Separation Mode: Normal Phase or Reverse Phase?

Hello, folks! Do you still remember the “Thalidomide Tragedy” that shocked the world in the 1950s and 1960s? Thalidomide, whose chemical structure is shown below, was originally marketed as a remedy for morning sickness during pregnancy. However, it was later discovered that the R-isomer of Thalidomide was the effective antiemetic component, alleviating symptoms like morning sickness and vomiting. In contrast, the S-isomer of Thalidomide was found to cause severe birth defects, resulting in a tragic epidemic of malformed babies in regions like West Germany, the Netherlands, and Japan.

With the advancement of biotechnology and life sciences, scientists have come to realize that chiral compounds, such as the enantiomers of chiral drugs, exhibit significant differences in physiological activity and pharmacological effects within the human body, despite their nearly identical physical and chemical properties except for their optical activity. As a result, in the pharmaceutical industry, it is imperative to separate enantiomers of active ingredients rather than selling them as mixtures, recognizing the importance of their distinct biological behaviors and pharmacological actions.

Using liquid chromatography to separate chiral compounds is currently one of the preferred methods, and it involves considering the type of chiral stationary phase, separation mode, and mobile phase solvent. There are various types of chiral stationary phases available in the market. Chiral chromatography can be performed using either normal phase separation or reverse phase separation modes, and the choice of mobile phase solvent depends on the stationary phase, sample properties, and the desired separation mode. Today, I’ll discuss how to choose the appropriate chiral separation mode.

Normal Phase Separation Mode

When the sample is dissolved in a non-polar solvent, the normal phase separation mode is chosen. The commonly used mobile phase consists of hydrocarbons and alcohols. In this mode, alcohols are used for elution, while hydrocarbons are employed to adjust the elution strength of the mobile phase.

For hydrocarbon solvents, options include n-hexane, n-pentane, isohexane, and n-heptane. These solvents have minimal impact on sample separation, and they do not significantly alter selectivity and resolution. Among them, n-hexane is often the preferred choice.

The alcohols in the mobile phase play a crucial role in the separation of the target compound. Different alcohols exhibit varying selectivity, and as the proportion of alcohols in the mobile phase increases, the retention time of the target peak decreases. The most commonly used alcohols are ethanol and isopropanol, with their elution capabilities ranking as follows: ethanol > isopropanol. Methanol has poor solubility in alkanes, and the maximum allowable concentration of methanol in n-hexane, for example, is typically limited to 5%. If methanol is to be added to alkanes, it is advisable to include a certain amount of ethanol to enhance the solubility of the mobile phase.

In chiral chromatography, to improve the peak shape of the target compound, it is necessary to add acid or base to the mobile phase. For acidic compounds, acidic additives such as trifluoroacetic acid, acetic acid, or formic acid are often added to the mobile phase. If the target compound is basic, alkaline additives like diethylamine, triethylamine, butylamine, or ethanolamine are introduced into the mobile phase. The typical concentration of acidic or alkaline additives ranges from 0.1% to 0.3%, with a maximum limit of 0.5%. It is essential to consider the pH tolerance range of the chromatographic column when incorporating acidic or alkaline additives.

Important Considerations:

1. Pre-column Flushing: Before installing a normal phase chromatography column, it is crucial to thoroughly flush the entire instrument’s tubing and the sample injector with an appropriate mobile phase. Some solvents like acetone, chloroform, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, dichloromethane, and tetrahydrofuran (THF) can damage the structure of chiral stationary phases. Please do not use these solvents as components of the mobile phase or as sample diluents. If you are unsure, consult the manufacturer for guidance.
2. Choice of Solvents: Chromatography columns can be used with pure methanol or pure acetonitrile as the mobile phase. If you intend to replace n-hexane with methanol, acetonitrile, or other polar solvents, you should ensure a thorough transition by using pure isopropanol as a solvent.
3. Avoiding Aqueous Mobile Phases: Normal phase chiral columns should be kept away from mobile phases that contain water.