Purification of natural compounds can come this naturally to you. Here is how.

Natural compounds are some of the most essential, interesting and exciting compounds to purify and analyze. But the purification of natural compounds is no easy feat. But I am here to help where I can. In this blog post, I try to help navigate you through the purification process, choice of type of chromatography and most suitable solvents for this application. Naturally, you must be very curious by now, so don’t wait, but read on.

I have a confession to make. I really dislike ginger. A little bit of ginger in some nice tasting dishes, I can tolerate. But raw ginger, ginger tea, forget about it. There is a back story to this profound war my tastebuds have declared against the root. You see, I get severe motion sickness. On one unfortunate bus ride, I had decided to arm myself with ginger as a natural compound against feeling ill. As soon I bit into it, I felt my stomach rebel like never before. It took my entire concentration to keep my lunch down. My wife still laughs about my green, sweat-covered face that to this day I blame on the horrible taste of ginger.

Yes, this natural product did not work so well for me, but a colleague of mine swears in its health power. It is true that natural products are often beneficial and very popular, so I am sure that you would agree with me that a post on how to purify natural compounds is way overdue.

Let us first see what we mean with the term natural compounds.

Natural compounds are defined as substances produced by living organisms, such as plants, microbes or animals. Frequently, these natural compounds are metabolites that play an important function, such as pathogen defense or signaling functions. We often use these metabolites as ingredients in food, cosmetics and pharmaceuticals.

Due to their incredible variety, purification of natural compounds can follow very different protocols, including the types of solvents used for chromatographic separation.

But let us see an overview of the general process.

Purification of natural compounds is frequently a long process. You need to extract, separate, concentrate, dry your extracts, then perform biochemical characterization and structural analysis. During extraction, you separate the desired compounds from the raw materials. You can apply methods such as solvent extraction, distillation methods, or pressing and sublimation, where solvent extraction tends to be the most popular choice.

Your sample mixture at this point contains the target compound among other compounds. To obtain the pure target compound, you need to perform a separation step, such as crystallization, filtration, liquid-liquid extraction or chromatography . Adsorption column chromatography with a chromatography system is often the preferred method, as the technique is simple, with high capacity and low cost of adsorbents.

In between the extraction, separation, and analysis steps, you also need to concentrate or completely dry your sample. You can use techniques such as rotary evaporation , freeze drying or spray drying to achieve this.

These are the steps required in the purification of natural compounds. Make no mistake, the chromatographic separation of natural compounds is certainly one of the most challenging steps of the process. It is worth the effort to understand and perfect the method for this application.

Let us first consider if normal or reversed-phase chromatography is better suited to the purification of natural compounds.

This is a difficult question without a single answer. This is because, due to their enormous variety, natural products can be of any polarity, from extremely low to highly polar. Generally, you can choose between normal- and reversed-phase chromatography based on the extraction solvent. Hexane, DCM, EtAc and other non-water miscible solvent extracts are best purified by normal-phase. Alcohol and water extracts are best purified by reversed-phase. Both of these types of chromatography have advantages and disadvantages when it comes to purification of natural compounds, as seen in the table below:

PROCON
Normal-Phase
Mobile phase		Organic solvents can be easily evaporated after separationOrganic solvents are more expensive and feature safety and environmental concerns
Normal-Phase
Stationary phase		Silica media is cheapSilica media is only suitable for single use
Reversed-Phase
Mobile phase		Water/alcohol mixtures are rather cheapWater needs a long time for concentration/evaporation
Reversed-Phase
Stationary phase		Bonded silica (C18) can be used multiple timesBonded silica (C18) is rather expensive

Now, let us discuss the choice of chromatography solvents for the purification of natural compounds. Selection of the stationary phase is relatively straight-forward. You can either go with silica when performing normal-phase chromatography or C18 when performing reversed-phase chromatography.

The choice of mobile phase tends to be more complex. Besides polarity, which is the parameter that exerts the greatest influence on separation results, solvents differ in terms of their toxicity, viscosity, cost, UV limit and boiling point. Let us take a closer look at these parameters and how they affect your purification of natural compounds.

  • Toxicity – normal-phased solvents, such as hexane and DCM are more toxic than reversed-phase solvents. If possible, replace hexane by heptane and DCM by acetone.
  • Boiling point – Using solvents with low boiling points can speed up and simplify your post-run sample concentration step. Such solvents are preferred for evaporative light scattering detection (ELSD). Since you can work with low-boiling point solvents at lower temperatures, they are also beneficial for compound stability.
  • UV limit – The solvents you select should not absorb UV light at the wavelength used by the detector to avoid interference and to allow for proper collection. Pay particular attention to acetone and ethyl acetate as the solvents most likely to cause you problems with this parameter. Consider using a combination of UV with another detector, such as ELSD to eliminate any obstacles regarding UV limits.
  • Viscosity – Select solvents with low viscosity, as they are less “thick”, so they minimize column or cartridge back pressure. I recommend to use acetonitrile whenever possible, as a solvent with low viscosity
  • Cost – Typically, normal phase chromatography solvents are more expensive than reversed-phase solvents. You could always try to test cheaper alternatives. For example, acetone has similar polarity to ethyl acetate and it is available at much lower costs.

Ok, ok, I better stop here before you get queasy with information overload. I hope that just like with my previous post on protein and peptide purification , this post has offered you some nice tips on how to adapt your chromatography process to a particular application. If you’re a fan of these application-focused blog posts, let me know in the comments and I will do my best to deliver you with some more. Either way, keep dropping by to see where the wind takes me next!

Till next time,

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