Application Review

No. 001/2020
Purification of Natural Compounds & Selection of Chromatography
Solvents
1. Abstract

Natural compounds are unique in their molecular diversity and biological functionality. Due to their
enormous variety, the purification of these compounds can be very different, including the type of
solvents used for the chromatographic separation. Generally, the selection of the solvents is done
based on the separation power, but other factors are as important to ensure an efficient
purification.

2. Background Information Natural Compounds

Natural compounds are defined as chemical substances produced by living organisms, such as
plants, microbes or animals. Generally, a natural product is produced by primary or secondary
metabolism pathways. Metabolites that are required for the growth of the organism are
called primary metabolites, whereas metabolites that are the end products of primary
metabolism are called secondary metabolites. Secondary metabolites fulfill important functions in
the interaction between the organism and their environment, for example as defense compounds
against herbivores and pathogens, as flower pigments that attract pollinators, or as hormones or
signal molecules. Many of these secondary metabolites are high-value-added chemicals that are
frequently used as ingredients in food, cosmetics and pharmaceuticals. They are also important
sources for drug development and are used asanti-cancer agents and anti-infectives.

Plant Metabolites

Primary Metabolites Secondary Metabolites

Carbohydrates, Lipids, Proteins, Alkaloids, Phenolics, Sterols,

Nucleic acids Steroids, Essential oils, Lignins

Involved directly in growth Produced for defence purposes

3. General Method Overview Purification

The purification of secondary metabolites is usually a rather time intensive process. Prior to
biochemical characterization or structural analysis, the target compounds need to be extracted,
separated and concentrated or dried. The separation process is certainly the most challenging
step in the whole process.
Extraction is the first step needed to separate the desired compounds from the raw materials. The
user can decide between solvent extraction, distillation methods, or pressing and sublimation
according to the extraction principle. Solvent extraction is the most widely used method.
The final sample mixture, which includes the target compound among other compounds, contains
various types of natural products. To obtain the pure target compound, a separation step, such
as crystallization, filtration, liquid-liquid extraction or chromatography, is needed. Chromatography
is the only method which allows the separation of all compounds of a mixture and is therefore the
most preferred method. Most widely used due to its simplicity, high capacity and low cost of
adsorbents, is the adsorption column chromatography.
Between extraction, separation and analysis steps, the sample needs to be concentrated or
completely dried. There are several solutions available, such as rotary evaporation, freeze or
spray drying. The choice depends on several criteria, such as the amount of sample, the type of
solvent, the stability of the compound and others. Since a rotary evaporator is available in nearly
any lab, it is more frequently used than any other technique.

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 PRO CON
Normal-Phase (NP)

Organic solvents are more

Organic solvents can be easily
Mobile Phase expensive and feature safety and
evaporated after separation
environmental concerns

Silica media is only suitable for

Stationary Phase Silica media is cheap
single use

Reversed-Phase (RP)
Water/ alcohol mixtures Water needs a long time for
Mobile Phase
are rather cheap concentration (evaporation)
Bonded Silica (C18) can Bonded Silica (C18) is rather
Stationary Phase
be used multiple times expensive

5. Additional Tips & Tricks Selection of Chromatography Solvents

The most frequent choice of stationary phase is either, silica (NP) or C18 (RP). The choice of
solvents (mobile phase) is more various. Besides polarity, which plays the main role in the
separation results, solvents differ in terms of their toxicity, viscosity, costs, UV limit and boiling
point. All these parameters are important to understand and to briefly discuss further.

Toxicity: in general, normal-phase solvents, especially hexane and dichloromethane (DCM),

are more toxic than reversed-phase solvents. It is recommended to replace hexane by
heptane and DCM by acetone if possible.

Boiling point: solvents with low boiling points make the post-run sample concentration step
faster and easier, especially when working with high sample volumes. Also, solvents with
low boiling points are advantageous for evaporative light scattering detection (ELSD), as
they allow work at lower temperatures, which is beneficial for compound stability.

UV limit: when using a UV detector, the solvents should not absorb UV light at the wavelength
used by the detector to avoid interference and to allow for proper collection. Acetone and
ethyl acetate are the most critical solvents in this respect. It is recommended to use a
combination of UV with another detector, preferably ELSD.

e
column or cartridge back pressure. Viscosity is especially important when using mixtures
with specific ratios of water and organic solvents with high viscosity. Ethanol is the most
viscous solvent, followed by propanol and methanol. Whenever possible, it is advisable to
use acetonitrile due to its low viscosity.

Costs: Solvents are one of the major cost contributors in adsorption chromatography,
especially when working with big size cartridges or columns. Generally, NP solvents are
more expensive than RP solvents and it is worth testing low cost solvents. A good example
is acetone, which has a similar polarity to ethyl acetate but is available at much lower costs.

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 Normal-Phase Polarity Toxicity Boiling Point UV limit Viscosity Costs
(NP) °C nm
Hexane 0 high 69 200 low high
Heptane 0 medium 98 200 low high
Dichloromethane
0.42 high 40 245 low medium
(DCM)
Acetone 0.56 medium 56 330 low low
Ethyl acetate 0.58 medium 78 260 low medium
Methanol 0.95 medium 64 205 medium medium
Reversed-Phase
(RP)
Acetonitrile 0.65 medium 81 200 low high
Propanol 0.71 low 82 210 high low
Ethanol 0.88 low 78 210 high high
Methanol 0.95 low 64 210 medium low
Water 1 low 100 190 medium low

6. Application Examples

The purification protocols for natural products are as diverse as the natural products themselves.
Below are some examples of how to extract and separate several different types of natural
products.

https://www.buchi.com/en/content/separation-quercetin-ginkgo-biloba-mother-tincture-using-
prepchrom-c-700

https://www.buchi.com/en/content/isolation-curcumin-natural-extract-flash-chromatography-and-
prephplc

https://www.buchi.com/en/content/separation-citral-isomers-lemongrass-oil

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