2D-LC in the Analysis of Tea

Tea is one of the most widely consumed beverages worldwide, particularly in China, where tea drinking can be traced back at least 1,500 years. Today, tea is the second most widely consumed drink in the world, after water.

The beverage is produced from the leaves of the shrub Camellia sinensis, which grows indigenously in China, Japan, India, and Thailand. There are two major varieties of the tea plant, Camellia sinensis var. sinensis, known as Chinese tea, and Camellia sinensis var. assamica, which is Assam, or Indian tea.

Over 3 million tons of tea are grown annually, 30 percent of which is produced in India, the world’s largest tea-producing country. Tea consumption is associated with a stimulant effect, due to the presence of caffeine, and a range of health benefits, partially attributed to the antioxidant activity of polyphenols contained in tea. Tea is also the focus of several traditional social rituals, such as the Japanese tea ceremony and British teatime, which contribute to its popularity.

Harvesting and Processing Tea Leaves

Tea is harvested by plucking the bud and the first two leaves of the Camellia sinensis plant. Depending on the methods used to process tea leaves, three different types of tea can then be produced: green, oolong, and black tea.

Generally, after harvesting, the leaves are rolled, disrupting the cellular compartmentation and bringing phenolic compounds into contact with the enzyme polyphenol oxidase. In the production of green tea, the rolled leaves are steamed or dried immediately to inactivate the enzyme and minimize oxidation. In contrast, to produce black tea, the rolled leaves undergo oxidation (fermentation) before drying. Oolong tea is produced similarly to black tea, deploying a shorter fermentation period.

Tea is known to have a highly complex chemical composition comprising diverse polyphenols, purine alkaloids, polysaccharides, amino acids, vitamins, lipids, and volatiles. The predominant polyphenols contained in green tea are catechins (flavan-3-ols) such as gallocatechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. Epigallocatechin gallate is the most abundant catechin present in green tea. In the production of black tea, the monomeric catechins undergo oxidative polymerization to form the condensation products theaflavins and their polymers thearubigins.

Due to the complex composition of tea and the structural similarity of green tea phenolics, complete separation of the phenolic compounds in tea cannot be achieved using conventional one-dimensional liquid chromatography (1D-LC). However, separation power can be greatly increased using comprehensive two-dimensional liquid chromatography (2D-LC). 2D-LC is a technique in which two independent LC separations are applied to the sample. In comprehensive 2D-LC, the complete effluent following a first LC separation is injected onto a second column for further separation. This second dimension greatly increases the peak capacity and, as a result, the resolving power without increasing the analysis time.

This technical article demonstrates the comprehensive 2D-LC analysis of green and black tea. The quantification of the purine alkaloids, caffeine, and theobromine as well as the tea catechins enables a comparison of the composition of green and black tea.

Experimental Methods

Figure 1. Comprehensive 2D-LC separation of purine alkaloids and catechins (10 µg/mL each).

Image Credit: Agilent Technologies

Ten different samples of green tea and black tea were purchased from a German retail market. For this analysis, approximately 2 grams of the finely ground tea were extracted three times in acetone and water. The resulting extracts were each centrifuged to remove any particulate, and the resulting supernatants combined. A 100 microliter sample of this extract was then evaporated using a SpeedVac to dryness, and the resulting residue redissolved in 1 milliliter (mL) acetonitrile/water/acetic acid. The sample was then filtered before LC analysis.