What is Chirality?

Chirality describes a three-dimensional object that cannot be superimposed on its mirror image.

The etymology of the word refers to the fact that the right hand and the left hand, although symmetrical on a plane, are not superimposable.

HPLC Chiral columns fig 1

A molecule is chiral when it has no plane of symmetry. This is a necessary and sufficient condition for chirality, whereas an asymmetric carbon in a compound is sufficient but not necessary.

This asymmetry is at the origin of the optical activity of chiral molecules. Two chiral molecules—images of each other—are called enantiomers and have the same physical properties apart from their specific rotational powers, which are opposites. They also have the same chemical properties aside from the chemical reactions involving the chiral centre.

Natural molecules are mostly chiral. Some forms are preferred, like the L (Levorotation, the other chiral version being D for Dextrorotation) which form amino acids.

Why we do chiral analysis

Chiral molecules have the property of deflecting polarized light. One of the isomers deviates it to the right, the other to the left. This property allowed Pasteur to differentiate L-tartaric acid from D-tartaric acid.

Two enantiomers have identical physicochemical properties:

  • same molecular weights
  • same solubilities
  • same melting and boiling points

This makes it difficult to separate and identify them. However, based on their particular spatial configurations, the interactions with other chiral molecules are different.

HPLC Chiral columns fig 2

From a biological standpoint, chiral enantiomers have opposite spatial geometries and may interact differently with their environment as a result. This is of particular importance in perfumery and, to a greater extent, pharmacology.

Two enantiomers won’t necessarily have the same biological activity because they will not react with the same receptors.

HPLC Chiral columns limonene

This is the case with limonene, abundant in citrus essential oils. It has one enantiomer that smells of lemon, while the other smells of orange. Similarly, medical molecules commonly contain one enantiomer that has beneficial properties while the other may be toxic.

We can explain this through interactions between molecules and receptors of the human body, functioning as key-lock systems. One form fits into a given lock and the other into another (or none) but they never fit the same because of their non-superimposable forms.

Chiral separation - how to do it

Developments in the liquid phase (HPLC) and, more recently, in the supercritical phase (SFC) have allowed the development of increasingly efficient separation techniques for the analysis of chiral compounds.

The physicochemical properties of two enantiomers are identical except when they are placed in an asymmetric environment. Achieve this either before the chromatographic column (method 1), in the chromatographic column via the mobile phase (method 2), or the stationary phase (method 3).

  • Method 1: The enantiomers are chemically modified into diastereoisomers and then separated with achiral stationary and mobile phases.
  • Method 2: A chiral agent is added to the mobile phase in which will form labile diastereoisomeric complexes.
  • Method 3: The separation is based on the formation of labile diastereoisomeric complexes between each enantiomer and the chiral stationary phase. The selectivity of the chiral separation is then directly related to the difference in stability of the complexes thus formed.

Early chiral stationary phases (CSP) were developed to separate various classes of chiral compounds. For example:

  • Crown ether columns for basic compounds (using reverse phase mechanisms)
  • Anionic exchanger for acidic compounds (using polar organic or SFC)
  • Zwitterionic exchanger (to cover a larger range of analytes, including amphoteric compounds)

Protein-based columns were introduced to provide higher coverage of analytes; however, they come with two big limitations. 1) They can only be used in reverse phase mechanisms and 2) they only provide a very low surface coverage depending on the size of the protein, meaning low loadability on the column. 

As chiral separation is particularly useful when carrying out purification, loadability is a huge factor in the analytical separation. This means that, while protein-based columns are a great analytical choice, they are poor for purification work.


In the early 2000s, polysaccharides columns were introduced, which are widely applicable. They can be used in all mechanisms (reverse phase, polar organic, normal phase, and SFC) and are an excellent choice for scalability due to their great surface area compared to protein-based columns.

HPLC Chiral columns polysaccharides

Polysaccharides are natural polymers of glucose linked by osidic bridges. These are assemblages of chiral monomers that serve enantiochromatographically as a chiral macromolecule. Today, polysaccharide-based CSPs are the most commonly used columns in Chiral HPLC (> 90%). This type of CSP can also be produced by immobilizing a polysaccharide on a silica gel or monolithic column. 

Cellulose, amylose, chitin, and chitosan are the most common native polysaccharides. They’re abundant and affordable, making them particularly popular in chiral chromatography. Because of their helical structures, amylose derivatives are generally preferred over cellulose. Polar substituents of polysaccharides, such as esters and carbamates, allow selectors to be more efficient than native polysaccharides in terms of chiral recognition. Thus, the polysaccharide derivatives belong to one of the best classes of chiral selectors used in chiral chromatography. However, because of their complex sizes and structures, the mechanism of chiral recognition with this type of selectors remains difficult to understand.

These columns are now available in 2μm particle size, affording fast analysis and scalability for purification work

As previously mentioned, polysaccharide columns are very popular because they work under SFC and in purification analysis. SFC has become a very popular technique for preparative chromatography with 2 main advantages:

  • Speed of analysis: a higher production rate can be obtained since the mobile phase viscosity is very low and fast. Efficient separations can be achieved.
  • Small volume of organic solvent used: between 10% - 20% of the organic solvent needed for an HPLC separation is required when using polysaccharide columns. This not only decreases the total solvent consumption used for the separation but also makes it easier and faster to recover the products from the small modifier volumes remaining after condensation from CO2.

HPLC Chiral columns Supercritical carbon dioxide

Element provide the widest range of polysacchirade columns in the UK.

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