By Ted Waszkuc, Ph.D., NOW Foods Method Development Scientist
Botanicals are complex mixtures of natural organic compounds - often referred to as “secondary metabolites” - synthesized by plant species to fulfill their structural and functional needs. This complexity and natural variability is a great challenge to analytical chemists developing tests to determine the identity of herbal ingredients. Natural components have to be separated from each other in order to be identified and determined as individual entities forming a composite phytochemical “fingerprint” characteristic of a single plant species.
Liquid chromatography (LC) is the most powerful separation technique used to identify natural components in plant extracts. Chromatography was originated by the Russian botanist Mikhail Tsvet during research on the plant pigments chlorophylls and carotenoids in Warsaw, Poland at the beginning of the 20th century 1 A solution containing a mixture of colored pigment compounds was allowed to run down through a vertical glass tube filled with powdered calcium carbonate as an adsorbent (a substance that can accumulate compounds on its surface where they can be observed). The various pigments traveled at different rates through the adsorbing material in the tubular column and appeared as colored bands separated by clear spaces of the adsorbent.
The visual output of the separation, called chromatogram, resulted as the yellow carotenoids and green chlorophylls formed distinct visual zones. Tsvet has coined the word chromatography (from Greek: “chromatos”, color and “grafein”, to write) to describe the separation process. 2 Interestingly enough, his surname Tsvet means “color” in Russian.
The principle of chromatographic adsorption has been applied in many different ways since, giving rise to varieties of separation techniques (HPLC, TLC, GC, etc). In Thin Layer Chromatography (TLC) the tubular configuration of the adsorbent was changed to a flat layer covering a glass plate.3 With the advent of high quality adsorbing materials consisting of very small and uniform particle size, TLC has become a sensitive, reproducible, high performance technique and thus the term High Performance Thin Layer Chromatography (HPTLC) was introduced. Increased separation, more sensitive detection and altogether higher reliability of HPTLC plates can be fully realized if all analytical steps are precisely executed with the help of specialized TLC instrumentation. The abbreviation HPTLC is often used as a synonym for advanced instrumental TLC. 4
Identifying species with TLC methods
HPTLC usefulness in plant species identification can be illustrated by the investigation of Acetate species in order to establish authenticity of black cohosh products. Black cohosh, Actea racemosa L. (previously called Cimicifuga racemosa), is an herbaceous plant native to eastern North America, where it grows in shady forests from southern New England to Wisconsin. 5, 6 It has been recognized for benefits in treating symptoms associated with menopausal discomfort. 7
The genus Actea includes several species, some like the black cohosh native to North America and others (A. cimicifuga, A. simplex, A. dahurica) coming from northeast Asia.8 Since all clinical studies that monitored menopause symptoms have been exclusively conducted on A. racemosa, only the plant material coming from North American black cohosh is considered the genuine ingredient.
Actaea species of Chinese origin that are in the same genus as black cohosh, but are not black cohosh, are sometimes used for so-called “economic adulteration”. A high quality and expensive herbal extract may have a similar, but less costly plant species mixed in with it or even substituted for the authentic material. Asian Actea cimicifuga (previously C. foetida) is most frequently used for misbranding.
Differentiating of Acteae species by HPTLC profile has allowed the establishment of cimifugin, a chromone compound related to flavonoids, as a positive marker for A. cimicifuga and other Asian species. Cimifugin is present in poor quality “black cohosh” products and can be detected as a characteristic blue fluorescent zone on the thin-layer chromatogram.9 It is not present in authentic A. racemosa, therefore detection of cimifugin proves that a product is not black cohosh. Using the HPTLC fingerprint methodology, identifying and differentiating of Acteae species can be accomplished.
NOW Foods’ quality control laboratory is dedicated to ensuring both the quality of its products and consumer safety. We use modern, state-of-the-art analytical equipment to conduct multiple tests on raw ingredients and finished products. Botanical identification of plant raw materials is the critical step in the quality control of any herbal preparation. Current Good Manufacturing Practices (cGMP) regulations for botanicals require species identity testing for each raw material that enters the production process. Accidental mix-up or purposeful exchange of the plant species in raw materials affects quality, but adulteration/falsification becomes critical if the wrong species used is toxic.
Liquid chromatography has always been a primary analytical tool employed in NOW Foods’ Quality Control. Our QC laboratory has recently acquired HPTLC automatic sample application instruments and a visualizer equipped with the high-resolution camera for the digital evaluation, documentation and storing of TLC chromatograms. Using the HPTLC, we can expertly screen multiple samples of plant extracts in a fraction of the time and cost that would be required by using other analytical tools. That allows us to ensure the safety and efficacy of NOW products by excluding the possibility that an adulterated raw material may be used in the production process.
Definition of Species
There are three major categories in plant classification: family, genus and species. Each family of plants is divided into different genera (plural of genus). Members of a genus are called species and they share many anatomical and phytochemical features that set them apart from other genera in the family. This is the level that defines an individual plant; e.g. black cohosh is Actea (genus) and racemosa (species), in the family of Ranunculaceae. Genus-plus-species binominal scientific names are in Latin and there can be only a single one that is correct versus sometimes many common names. For example, Actea racemosa is variously known by the common names squaw root, black snakeroot, or black cohosh. Names of genera, as well as families, are capitalized but names of species are not capitalized. Italics are used for the genus and species. They can be abbreviated; for example, A. racemosa.
More about Liquid Chromatography
Modern liquid chromatography can be carried out either on an adsorbent in a column as High Performance Liquid Chromatography (HPLC) or on a two-dimensional plane of adsorbent as Thin Layer Chromatography (TLC). 10 The principle of what we now know as TLC was to get rid of the column and “flatten” out the material. 3 In contrast to the column chromatography techniques where both stationary and mobile phases are enclosed inside a cylindrical column, the TLC stationary phase is configured as a thin, two-dimensional sheet of solid material (usually silica) coated on a glass plate.
In TLC, a tiny drop of a liquid sample is applied along the horizontal spotting line near the bottom of a plate, where it forms a little spot. The plate is then dipped into the thin layer of the liquid mobile phase on the bottom of the special TLC chamber. The mobile phase is drawn up the plate through stationary phase material by the action of capillary forces. As the solvent front moves up the plate passing the spotting line and the spot where sample was applied, the mixture of compounds in the sample will begin to partition between stationary and mobile phase, giving rise to separation. Finally, as the result of that process, various components in the mixture will travel different distances up from the application line.
After the plate is removed from the chamber and separation stops the presence of individual components is shown as narrow “bands” or “zones” on the plate. They might be colored if the individual, separated compounds are colored and visible to the eye, e.g. anthocyanins from fruit berries, carotenoids or chlorophylls. However, many natural compounds are colorless and the bands must be visualized differently (e.g. employing UV light for UV absorbing compounds). There are also chemical methods allowing us to detect separated mixture components as zones with different colors. Chromatographic results can be presented as an image of a sequence of dark, colored or fluorescing zones (“fingerprint” profile) on the plate and documented as a photograph. In that sense TLC clearly demonstrates the term of “chromatography”. 11, 12
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Method for detection of 5% adulteration of black cohosh with Cimifuga foetida, C. heraclifolia, C. dahurica, or C. Americana. J.AOAC International, 2008 91 1257-1264
10. Engelhardt, H., One century of liquid chromatography. From Tsvet’s columns to modern high
speed and high performance separations. J. Chromatogr. B 800, 3-6 (2004)
11. Stahl, E., Bolliger, H. Thin Layer Chromatography: a laboratory handbook. Berlin: Springer; 1962