Background theory

Extraction of Thymol, Carvacrol, and Cymene from Thyme Leaves

Thymol, carvacrol, and p-cymene are compounds found in the leaves of the thyme plant. Thymol and carvacrol are structural isomers. These two phenolic isomers possess medicinal properties, such as the ability to fight bacterial and fungal infections, suppress muscle spasms, and reduce inflammation (Belaqziz, 2010). Because of these therapeutic properties, researchers are interested in the isolation of these oils from plant material, and the analysis of the composition and purity of the extracted compounds.

In this lab experiment, you will carry out the extraction of these compounds from dry thyme leaves using different extraction solvents. You will use thin layer chromatography (TLC) to verify that the extract from the plant material contains one or more of these compounds, and to qualitatively compare the effectiveness of the different extraction protocols. Further analysis of these extracts, using high performance liquid chromatography (HPLC) or gas chromatography (GC) for example, will be used to obtain quantitative data.

Thin Layer Chromatography as an Analytical Technique

Thin layer chromatography is an analytical technique, which allows for the rapid evaluation of the components of a mixture. This technique utilizes a TLC plate, which consists of a thin layer of a stationary phase (such as silica or alumina) bonded to the surface of an inert support material, such as plastic, aluminum, or glass. The mobile phase for this chromatographic separation is a liquid, which is often referred to as the developing solvent or eluent.

Please watch this video, which demonstrates the procedure for TLC analysis, and compare this video to the general procedure for TLC analysis used in this lab experiment. Use this interactive simulation to see the effect of solvent polarity on the outcome of TLC analysis and read more here.

For this experiment, you will use TLC plates with silica on glass as the stationary phase and mixtures of ethyl acetate and heptane as the developing solvents. You will use UV light and iodine vapor to visualize the spots on the TLC plate.

High Performance Liquid Chromatography as an Analytical Technique

High performance liquid chromatography is a technique that can be used to separate and identify compounds in a mixture. Like TLC, HPLC uses a mobile phase and a stationary phase but in HPLC the experimental design is much more sophisticated and controlled, which allows for the quantitative analysis of compounds. Compounds in a mixture are forced through the stationary phase by the mobile phase. The relative affinity of the compounds for the stationary phase and mobile phase is what accomplishes the separation. The stationary phase in HPLC is contained in a rigid stainless steel column that is capable of withstanding high pressures of liquids pushing through. The stationary phase used can also be tailored for optimal separation of compounds. In this experiment you will be using the reverse-phase stationary phase called C18, which is silica modified with a long hydrocarbon chain 18 carbons long. In an HPLC instrument there are several reservoirs for mobile phase solvents. The instrument can be programmed to mix solvents, even during an experiment, to accomplish optimal separation in a short amount of time and this is called a gradient. Pumps push the solvents through the column; the compounds proceed through the column at different rates and are detected when they exit the column. Although there are several types of detectors that can be used for HPLC, the detector used for this experiment is a diode array detector (DAD), which is a type of UV detector. The DAD allows for measurements of multiple wavelengths at once. However, in this experiment you will be using a fixed wavelength of 277 nm because all the analytes of interest absorb light at this wavelength. The Royal Society of Chemistry has an informative video describing HPLC, which you might find helpful to watch.

Gradient mobile phase:

There are two categories of mobile phases that are commonly used in HPLC. When an isocratic mobile phase is used the composition of the mobile phase is kept constant throughout the chromatographic analysis. This type of mobile phase is often used for analytes that are easily separated. In contrast, when a gradient mobile phase is used the composition of the mobile phase varies over the length of the run. A gradient might be used both to improve separation and to speed up analysis if some compounds would otherwise take a long time to elute. In this experiment, you will utilize a gradient mobile phase to separate the compounds in your thyme leaf extract.

The composition of the mobile phase is chosen depending on the strength of interactions between various sample components and stationary phase (which in reverse-phase HPLC is hydrophobic). In this lab, the gradient mobile phase components have already been determined for you (Figure 2). A series of trial runs were performed with the thyme leaf extract in order to find the HPLC parameters that result in separation in a small amount of time.