Extraction and identification

Plants produce complex, unique molecules that would be challenging and costly to synthesise. Many of these molecules have medicinal properties, or their structures are easily modified to make a pharmaceutical.

Use this page to revise the following concepts within add page title here:

Medicinal compounds from plants

A photograph of eucalyptus leaves. On one of the leaves there is a graphic of a circle and lines indicating zooming out. These lines connect with a graphic on the left-hand side of the photograph depicting a hexagon. From the top of the hexagon is a line pointing straight up. From the bottom of the hexagon is a line pointing down that splits in two directions to the right and left. From the point of divergence a line points back toward the centre of the hexagon. This line points at a circle within the hexagon. Above this circle is another line that points to the base of the straight line that rises from the top of the hexagon.

Many plants contain compounds that have current or potential medicinal uses. Investigate the examples below.

Consider the tea-tree leaf shown. Among the large number of compounds found in each leaf, it is likely to contain over fifty potential active ingredients.

A coloured drawing of a tea tree (melaleuca alternifolia) branch with leaves attached.

The table shown uses tea-tree oil to illustrate the complex nature of plant extracts.

Component Composition (%)
Terpinen-4-ol 41.1
γ-Terpinene 21.0
α-Terpinene 11.4
1,8-Cineole 4.7
Terpinolene 2.4
ρ-Cymene 2.6
α-Pinene 1.9
α-Terpineol 3.1
Aromadendrene 1.7
δ-Cadinene 1.0
Limonene 0.9

Source: Roy, A. & Tavakolifar, Bahareh & Fallah Huseini, Hasan & Tousi, P. & Shafigh, Navid & Rahimzadeh, Mitra. (2014). Efficacy of Melaleuca alternifolia Essential Oil in the Treatment of Facial Seborrheic Dermatitis: A Double-blind, Randomized, Placebo-Controlled Clinical Trial. Journal of Medicinal Plants. 13, 26-32.

Plants are complex organisms containing thousands of different compounds. To study each of the active components in the tea-tree leaf above:

  • the components in the leaf need to extracted from the leaf
  • each component needs to be separated so that you can study its interaction in the body
  • the structure of the components need to be determined.

The picture below shows the extraction, purification and identification process from a plant sample. After the solvent extraction, the concentrated extract is separated into three active compounds using chromatography. The structure of each compound can be identified using instrumentation, including mass spectrometry, NMR spectroscopy and IR spectroscopy.

A graphic comprising 3 images. On the far left of the graphic is an image labeled step one, grinding/shredding. This image is comprised of two pictures. The first is of a green substance in a mortar with a pestle covered in some of the green substance sitting next to the mortar. Below this picture is another picture of the green substance in a clear glass bottle. To the right of this image is an arrow pointing to the right. To the right of this arrow is a second image labeled step 2 extraction and purification. This image is comprised of an illustration of a plastic vial with liquid filling one third of the vial. To the right of this image is an arrow pointing to the right. To the right of this arrow is a final image labeled step 3 identification structure of active components using instrumental analysis. This image is comprised of a illustration of a chemical process.

Adapted from  Yadav et al, 2021; Source (Step One): ResearchGate

Extraction

Many different extraction methods can be used to release or isolate the active components. For example, First Nations peoples in Australia use the heat of a fire to release active ingredients in the Eucalyptus leaves into the surrounding air.

In modern laboratory and industrial process, solvent extraction, steam distillation and chromatography are often used to obtain active components.

Solvent extraction is the removal of a substance from a mixture by dissolving it in a solvent. Leaves are very tough and many of the components are insoluble in water. Scientists use a range of techniques to improve the efficiency of extraction. These include:

  • shredding or blending the leaves to break down their cell structure and to increase the surface area
  • using heat to improve the solubility of the desired components in the solvent, if the active compound is stable under heat
  • choosing a solvent that has a polarity similar to that of the components of interest

When choosing a solvent, the solubility rule ‘like dissolves like’ is used. A polar solvent, often water, is used to extract polar components while a non-polar solvent is used to extract non-polar components like oils. The diagram below provides a visual of one form of  solvent extraction. Most plant extracts are green in colour due to the chlorophyll content, however, they contain many other components.

A graphic depicting 4 glass vials in sequence. On the left of the image the first glass vial contains a liquid with pink and green small balls in it. This liquid is labeled organic compound in aqueous layer. An arrow points to the right from this glass vial to the next in the sequence. The arrow is labeled add extraction solvent. The second vial contains the same organic compound liquid layer, but above this is another layer of liquid labeled extraction solvent layer (less dense). An arrow pointing to the right connects the second and third vials and is labeled shake and separate layer. In the third vial the two layers of liquid remain, however, the small pink balls remain in the lower layer of liquid, and the small green balls have moved to the upper layer. The bottom layer of liquid is labeled aqueous layer and the upper layer of liquid is labeled organic compound transferred to solvent layer. An arrow pointing to the right connects the third and final, fourth, glass vial and is labeled open stopcock to drain off the aqueous layer. In this vial the two layers of liquid remain divided as in the third vial and the lower layer is still labeled aqueous layer. Below this final glass vial is a glass beaker containing the same aqueous layer liquid and 2 small pink balls.

Steam distillation is a method used to extract compounds from plants. It is suited to thermally stable and volatile non-polar components, such as plant oils. In this process, a flow of hot steam is passed through plant leaves. The steam breaks down the cell structure and carries an extract through to a condenser. The condenser cools the steam and extract, and the non-polar components form a separate layer to the condensed water, which can be collected.

The diagrams below show how steam distillation works and a laboratory example of it in practice to make products such as eucalyptus and lavender oil.

A graphic depicting the steps in the distillation process. To the left of the graphic is an illustration of a metal container with a fire burning underneath. The fire is labeled heat source. Two arrows point to the left from the metal container. One points to an illustration of waves labeled water. The other arrow points to an illustration of short blue lines and is labeled hot steam. A pipe protrudes from the metal container on the right side. Half way along this pipe there is a vertical rectangular shape with an illustration of green leaves in the centre labeled plant raw material. Above this cluster of leaves are short green lines labeled vaporized essential oil and below the cluster of leaves are short blue lines labeled steam. At the end of the pipe, at the far right of the graphic is another rectangular shape containing an illustration of a metal coil labeled condenser. From the bottom of this shape there is an illustration of two drops of water dropping into a bottle filled with a liquid labeled condensed water. Within the water are small yellow balls labeled essential oil.

Source: Adapted from Machado, C. A., Oliveira, F. O., de Andrade, M. A., Hodel, K. V. S., Lepikson, H., & Machado, B. A. S. (2022). Steam distillation for essential oil extraction: An evaluation of technological advances based on an analysis of patent documents. Sustainability, 14(7119). https://doi.org/10.3390/su14127119

High-Performance Liquid Chromatography (HPLC) is a powerful technique used to purify active components from plant extracts. The plant extract is injected into the HPLC system, where it passes through a column containing a stationary phase. As the components interact differently with the stationary phase, they are separated and collected based on their retention times, isolating the desired active compounds with high precision.

Aspirin is manufactured from a component of the bark of the willow tree. A willow tree bark extract mixture is passed through a HPLC column. An example of a chromatogram obtained is shown below.

A graph with the x-axis labeled from 0 minutes on the far left side to 16 minutes on the right hand side. The even numbers between 0 and 16 are displayed at regular intervals along the x-axis between the 0 and 16. Above but parallel with the x-axis is a blue line that spikes at the 4 minute, 6 minute, 9 minute, and 15 minute intervals. The 4 minute spike is labeled acetaminophen, the 6 minute spike is labeled caffeine, the 9 minute spike is labeled benzoic acid, and the 15 minute spike is labeled aspirin. At the top right hand corner of the graphic is a box with the following information. Column, primesep 100, size 3.2 x 150mm, guard primesep 100, size 4.6 x 10mm, mobile phase MeCN-10%, TFA 0.1%, flow 0.5ml/min, detection UV 210nm.

The chromatogram shows that the mixture contained at least four components. The different structures of each component leads to each substance taking a different amount of time to pass through the column. Samples that elute at different retention times can be collected separately and chromatography can be used as a purification method.

After comparing the retention time of peak 4 with that of a pure aspirin sample under the same chromatography conditions, peak 4 matches the retention time of the standard sample and is identified as aspirin. The solvent in the collected peak 4 sample is removed, yielding a pure substance. Its structure can be analysed later using other instrumental analysis methods.

The aspirin concentration can be determined by comparing the area under peak 4 with the area under the curve of other standard solutions of aspirin.

Identification

After extraction and purification, chemical instruments are used to identify compounds extracted from plants by determining their structure. Most compounds have complex structures containing multiple functional groups so the analysis is often complex. The instruments commonly used are mass spectrometer, infrared spectroscopy (IR), and nuclear magnetic resonance spectroscopy (NMR). The tabs below to detail how the structure of aspirin can be determined and confirmed using analytical instruments.

When injecting a purified aspirin sample collected from HPLC, the highest peak on the generated mass spectrum has a m/z ratio of 180, matching a relative molecular mass of 180 and a molecular formula of C9H8O4.

The other significant peaks are caused by fragments of the aspirin molecule.

A graph with the x-axis labeled m/z and the y-axis labeled relative intensity. The x-axis values range from 0 to 240, marked at 20, 40, 60, etc. The y-axis values range from 0 to 100 marked at 20, 40, 60, etc. The are lines rising from various points on the x-axis to varying heights on the y-axis.

Source: AIST: SDBS, 2015

Infrared spectroscopy provides information about chemical bonds through the detection of how infrared light is absorbed by the sample. It gives information about  functional groups. The IR spectrum of aspirin below suggests the existence of a carboxyl group from O-H (acid) at 2500-3500 cm-1 and a carbonyl bond C=O (acid) at 1680-1740 cm-1.

A graph depicting the IR spectrum of aspirin.

Source: AIST: SDBS, retrieved Dec 2024

Carbon NMR

Carbon NMR uses radio waves in a strong magnetic field to detect the chemical environment of each carbon atom in an organic molecule. Carbon-13 atoms first absorb, and then emit, energy from the radio waves. This emitted energy is measured as a shift value. The chemical environment around each carbon atom leads each carbon to have a different chemical shift value, as illustrated by the peaks in the 13C NMR figure. The shift values can be checked against measurements of known molecules to identify the possible connections around the carbon atoms. On the spectrum below, each carbon atom has been numbered so you can match it to the peak it caused. The spectrum shows there are 9 different carbon environments.

A graph with the x-axis labelled ppm (there is no y-axis). The intervals along the x-axis measure from 200 on the left-hand side, to 0 on the right in intervals of 20. There are 9 lines rising from the x-axis at various intervals corresponding to 9 carbon atoms and the spikes they cause along the x-axis. To the right of the graph is a circular diagram with two lines radiating from it. The numbers 0-9 are arranged around this circle and the two lines. Below this diagram is a list of the 9 carbon atoms and their numeric position along the x-axis. Carbon atom 1 corresponds to 170.20 ppm, carbon atom 2 to 169.76 ppm, carbon atom 3 to 151.28 ppm, carbon atom 4 to 134.90 ppm, carbon atom 5 to 132.51 ppm, carbon atom 6 to 126.17 ppm, carbon atom 7 to 124.01 ppm, carbon atom 8 to 122.26 ppm and carbon atom 9 to 20.99 ppm.

Source: AIST: SDBS, 1999

Proton NMR (1H NMR)

Similar to carbon NMR, proton NMR uses radio waves in a strong magnetic field to detect hydrogen-1 atoms in a molecular structure. Each hydrogen atom on the aspirin structure has a letter of the alphabet beside it so you can see which peak corresponds to. Again, these peaks are interpreted by comparison to the functional groups of known molecules.

A graph depicting the hydrogen atoms of the aspirin structure. The x-axis of the graph is labeled ppm and ranges from 14 on the left-side to 0 on the right side, with values labeled at 14, 12, 10, 8, 6, 4, 2 and 0. Along the x-axis lines rise at various intervals. These lines correspond with hydrogen atoms labeled A through F. Atom A corresponds to 11ppm, atom b to 8.125 ppm, atom c to 7.624 ppm, atom d to 7.356 ppm, atom e to 7.142 ppm and atom F to 2.352 ppm. In the top left hand side of the graph is a diagram depicting the 6 atoms surrounding a circle and labeled against spokes coming from the circle.

Source: AIST: SDBS, 1999