Purifying natural product extracts? Why you should use ELSD with UV detection

I like working with natural products, at least from a chromatography perspective. The sheer breadth of compounds extractable from a plant makes separations a challenge but overcoming that challenge with a good, robust method for their separation and isolation can be very rewarding.

In order to have a good chromatographic method, one needs to not only be able to separate the extracted compounds but also detect them. This means having a chromatography system designed with a PDA UV (or UV-vis) detector and an inline evaporative light-scattering detector (ELSD). These detectors work differently and will “see” compounds based on dissimilar properties.

We all know that a UV or UV-vis detector sees compounds based on their ability to absorb light at a specific wavelength or within a wavelength range. An ELSD, however, detects compounds that are oils or solids when dry (hence evaporative) through light diffraction and reflection (scattering).

Not all natural product compounds absorb UV so the use of an ELSD can help to find UV transparent compounds otherwise not detectable. Likewise, not all extracted compounds can be detected as an oil or solid with ELSD but may absorb UV light. So together, these two detectors work synergistically to find and fractionate the vast majority of extracted molecules.

One of the most popular natural products talked about in recent years is hemp as source of CBD. Hemp extracts contain many compounds most of which can be separated by reversed phase flash chromatography. While the vast majority of the extractable cannabinoids do absorb UV, many of the more lipophilic extractables (terpenes, etc.) are present in low concentration making UV detection a challenge. By adding an ELSD to the flash system, detection of these minor compounds can be greatly enhanced, Figure 1.

Figure 1. Hemp extract purification by reversed phase flash chromatography. The ELS detector found and cased fractionation of compounds undetectable by UV.

The ELSD was able to find many extracted compounds with little to no UV absorption throughout the purification.

Another example where using tandem UV/ELS detection is helpful is the isolation of terpenes and terpenoids from botanicals such as a plant called Ylang Ylang. Extracts from this flower are used as perfumes in a variety of products but are a complex mixture of terpenes. Higher boiling terpenes are found in what is called Ylang Ylang III [1], Figure 2.

Figure 2. Ylang Ylang III flash purification using both UV and ELS detection. The ELSD found two additional compounds (blue arrows) not easily detected by UV. 

So, if you are extracting and purifying natural product extracts using flash chromatography, consider adding an ELSD to your flash system to help improve your success and productivity.

Products used for this post included:

• - Biotage® Selekt
• - Biotage® Selekt ELSD
• - Biotage ® Sfär C18 column, 6-gram
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Interested in learning more about ELSD? Check our website for more information.

[1] What’s the difference in Ylang Extra, I, II, III and Complete? (birchhillhappenings.com)