Finding Nature’s Molecules: Chromatography and Chemistry


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Japanese chemists first isolated resveratrol from the White Hellebore in 1939. Image by W. S.

Finding, isolating, and characterizing medicines derived from nature is a major sub-discipline within chemistry. One often-used technique for isolating potential medicinals is called chromatography – this is a process that separates similar molecules in a mixture.

Early methods separated molecules by color, so scientists called the process, ‘chromatography.’ How is this technique useful?

Chromatography Uses: Drugs From Food

An instance of a potential ‘drug’ isolated from food came from red wine in the 1990s. The substance known as resveratrol gained widespread acclaim as the possible ‘drug.’

Resveratrol was a part of the French paradox — how can people ingest rich, high calorie foods accompanied with a hearty Bordeaux or Pinot Noir wine without increasing their risk of a heart attack or stroke?

The paradox puzzled researchers, and identifying resveratrol as a preventative agent for heart disease was a stroke of scientific genius.

Resveratrol and chemically-similar compounds occur in wine grapes and are classified as phenolic stilbenes, substances which result from plant biochemistry.

The eventual isolation of resveratrol via chromatography resulted in isolating other similar molecules of importance.

How do scientists come up with the names for molecules? Copyright image by Decoded Science, all rights reserved.

What are Phenolic Stilbenes-or Polyphenols?

The term stilbene originates from the german, stilbein~ to phosphoresce. The original discovery of the ‘stilbene class’ of molecules came in the late 1800s and was named as such because the molecule possessed a peculiar glow. Presently, theoreticians regard the molecule as rather mundane, except for the biochemical properties that were discovered in the mid-1990s.

The primary reasons for the bioactivity of phenolic stilbenes lie in their toxicity to cancer cells and anti-oxidant properties. The dual role of anti-cancer agent and anti-oxidant remain a subject of intense investigation.

Blueberries contain the molecules, resveratrol and pterostilbene. Scientists believe that blueberry plants make these molecules (bio-synthesize them) as response to pathogens-such as plant fungus.

Simply Separate Two (or More) Similar Organic Molecules?

The term chromatography is derived from the ancient Greek: chromato-color and graphica-to write (or roughly~to measure).

The science of chromatography separates organic molecules using their molecular properties: molecular weight, size, or differences in molecular scaffolding (structure), but originally, chromatography separated chemical compounds by their colors: A chemical mixture could have been colored purple and isolating each molecule may have yielded four different molecules- yellow, red, violet, and pale yellow.

Chromatography separates molecules by first dissolving the molecular mixture in a small amount of solvent and letting the molecules separate from one another as the solvent-and-molecules flow across a solid phase (silica gel-silicon oxide or alumina-aluminum oxide).

Silica gel is effective in separating most organic mixtures.  The primary differences between molecules can occur in two or three locations on one of the molecules.

Chromatography exploits the differences in molecular scaffolding (or structure). Silica gel can loosely associate with one molecule-while it less strongly associates with the other. In essence, separating the molecules as the gravity pulls the solvent and molecules apart from one another.

When using gravity-column chromatography to isolate the molecules, experimentation is done first on a small scale: Thin Layer chromatography.

A two- millimeter-thick coating of silica is put on a 10 cm by 20 cm glass plate-the mixture of compounds is placed on a single spot on the plate (near the bottom).

The plate is then ‘spotted’ with the target molecules parallel to the mixture. Then, the plate (with all compounds) is put into a glass container that has a small amount of solvent at the bottom of the container.

The solvent is allowed to rise on the plate via capillary action, or wicking.

In this process, the compounds separate from each other as the solvent rises to the end point near the top of the plate.

The researchers improve the technique until they discover the best solvent, which can thoroughly separate the molecules.

Once optimized, scientists transfer the small-scale technique to a larger silica gel column-a glass tube approximately 0.5  meter in length and 3 centimeters in diameter. The mixture of compounds is placed atop of a column of silica gel,  and  solvent passes through the column of silica gel by gravity. The technique separates the chemical compounds in the same manner as the glass plates in the Thin Layer experiment.

The molecules: stilbene, resveratrol, and pterostilbene appear from left to right (above) The subtle differences between the molecules appear in red and gray. Resveratrol has (red) oxygens bonded exclusively to H’s (Hydrogens). Pterostilbene lacks two hydrogens (but are replaced with methyl groups —solid lines). Copyright image by John A. Jaksich. All Rights Reserved.

Chromatography of Resveratrol and Pterostilbene in Blueberries?

One literature reference from 2004, published in the Journal of Agriculture and Food Chemistry, describes the separation of resveratrol from pterostilbene.

Researchers freeze-dried the berries and mixed approximately 200 milliliters of an appropriate solvent with the freeze-dried berries and homogenized them with a laboratory-grade blender.

A published technique for the isolation of resveratrol and pterostilbene is as follows:

The two component mixture, resveratrol and pterostilbene, were sequestered from blueberries. The mixture of resveratrol and pterostilbene was condensed into a paste and separated by C 18 column chromatography.

C 18 methods utilize a high-pressure flow of solvent through tubing and columns that are millimeters to centimeters in diameter. This type of separation is known for speed and accuracy.

Non-pressurized chromatography utilizes gravity to assist in the separation, which is slower – pressurized chromatography can reduce 4 hours of laboratory time to 1 hour.

Natural Products: A Wave of Future Chemistry?

Medicinal chemistry is a mature science-its origins came about as early hominids experimented with plants and their fruits. The eventual maturation of chromatography allows scientists to isolate and understand medicinal substances. Further advancements will come as biochemistry and sub-disciplines like chromatography probe biology for clues to our chemical past.

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