The Half Decent Pharmaceutical Chemistry Blog

Tea, what else? Xanthines are old friends of this blog. This time, however, I'm not going to talk about their properties: it's time to go deeper and see how much of these molecules is actually in your daily cup of tea.

The tea we chose for the extraction looked pretty old and wasn't placed in proper tea bags, either: a gram of tea from an old-fashioned bottle was ground in a mortar and 1.0511 g were accurately weighed.

Then, it was time to prepare a cup of tea. The pulverized leaves were put in a round-bottom flask, with 30 mL of water and nearby 0.5 g of calcium carbonate. The latter reagent is needed because xanthines (bases) tend to bind to tannins (acids): so, the carbonate displaces either caffeine and theophylline.

The mixture was heated to 120°C for 20 minutes. Meanwhile, I set to clean and regenerate the SM-2 column I would have utilized for the extraction. For this solid phase extraction, we selected a resin with the appropriate ratio of styrene to divinylbenzene (XAD-4) for the two lipophilic xanthines.

In a XAD-4 resin, in fact, the aromatic rings of either styrene and divinylbenzene are the perfect environment for lipophilic molecules such as caffeine, which can bind tightly, thanks to π-π interactions.



First of all, we poured 40 mL of ethanol and, then, cleaned the resin with 20 mL of water and 20 mL of bleach. From now on, the column will never get dry.

The procedure went on as follows: 40 mL of water, NaOH (1M) 20mL, water (40 mL), HCl (1M) 20 mL and then water until the pH of the column reached neutrality.

Once the tea was ready, we filtered it twice, in order to completely remove the carbonate.



The column was therefore loaded (twice) and the extraction of the alkaloids was underway, as we began to add the first amount (25 mL) of ethanol. We will carry out four extractions, with 25 mL each time.

Actually, the first 14 mL of (hopefully) only aqueous solution were collected separately in a couple of test tubes (which we were told to keep, just in case something had gone wrong), whereas the rest of the alcoholic solution was placed in a 100 mL volumetric flask.

Although (according to the protocol) the volume used for the four extractions should have been 100 mL, some more ethanol had to be added to reach the final volume.



In order to fill the column, I let the last quantity of ethanol go through the column: this increased the efficiency of the extraction and, in the end, this method proved to be more practical than doing the same operation with a pipette.

This solution was then diluted (1:10), to get a signal, from a spectrophotometric titration (at 276 nm), that could be compared with that of a 11.5 μg/mL solution of caffeine in ethanol,  our standard.

So, our 1:10 solution gave us an absorbance of 0.5632. This means the concentration of xanthines was 13.61 μg/mL; so, goin back to our sample, it  contained 1.30 % of xanthines.

Unfortunately, since the tea was rather old, nobody could find the original data from the manufacturer. Still, our professor said it was a plausible, albeit rather high, percentage.

Finally, to assay which xanthines we were measuring, we carried out a TLC (mobile phase: methylene chloride/methanol 9:1), once 25 mL of the first alcoholic solution had been concentrated to 2 mL at the rotavapor.