Caffeine

Isolation Method 1

• Around 50 grams of tea powder is taken and extracted with alcohol for around 6 hours in a Soxhlet extractor.

• The extract is transferred into a porcelain dish that already contains magnesium oxide (30 grams in 200 ml water).

• Heat it on a steam bath with continuous stirring and evaporate it.

• Boil the obtain residue three times with 100 ml water.

• Filter it in the hot condition through the Buchner funnel.

• Add the 10 percent sulphuric acid (30 ml) in combined filtrate.

• Concentrate the above filtrate to one third of the volume.

• Filter the solution when hot and extract it with 5 times with chloroform (25 ml).

• Add sodium hydroxide (1 percent, few ml) in chloroform extract so that the decolourisation takes place.

• An equal quantity of water is added to this and separate the chloroform layer.

• Evaporate the chloroform extract to obtain crude caffeine which can be recrystallized with hot water.

Isolation Method 2

• Damaged tea leaves are powdered and boiled with water, preferably alkaline, and filtered hot.

• To the filtrate, basic lead acetate is added to precipitate out the tannins and albuminoids.

• Sulphuric acid is used for removing the excess lead (as lead sulphate) from the filtrate.

• The filtrate is decolourised with animal charcoal, and caffeine is extracted with chloroform.

• The solvent is recovered by distillation and caffeine is purified by re-crystallisation with water.

Isolation Method 3

• Finely or coarsely powdered tea leaves are extracted with ethanol in Soxhlet extractor.

• The extract of caffeine obtained is adsorbed on magnesium oxide.

• Caffeine is then desorbed after treatment with 10% H 2SO4.

• It is then extracted with chloroform and re-crystallised.

Isolation Method 4

• For the isolation of caffeine from coffee beans the leaching process with water is followed.

• When the coarse coffee powder is extracted with water at 75°C, it will yield 90% of caffeine.

• The extraction procedure will take about 30 minutes with water/coffee ratio of 9:1.

• Caffeine can be also extracted by supercritical fluid extraction technique by using liquefied carbon dioxide.

• Liquefied carbon dioxide absorbs the caffeine through moist coffee.

• Then it is passed through another pressurized vessel containing absorbing media like resin or activated carbon which retain the caffeine.

• Separate this caffeine by extracting with chloroform.

Thin layer chromatography of Caffeine:

• Dissolve caffeine (1 mg) in chloroform (1 ml) or methanol.

• Then spot the sample on TLC plates and eluted in solvent system ethyl acetate: methanol: acetic acid (8:1:1).

• Then visualize the spots on dried TLC plate by exposure to iodine vapour.

• The Rf value was 0.41.

Identification Caffeine can be identified by the following tests:

1. Murexide Test:

• Caffeine is taken in a petri dish and added with hydrochloric acid and potassium chlorate.

• The mixture is then heated to dryness.

• The residue is exposed to vapours of dilute ammonia.

• A purple colour appears, which disappears on adding fixed alkali.

2. On treatment with tannic acid solution , caffeine produces a white coloured precipitate

By UV/Vis Spectrophotometry Calibration Standards:

1. Caffeine stock solution (1000ppm) is prepared by dissolving 100mg of pure caffeine in 100ml of distilled water.

2. Working standards of 0, 10, 20, 40, 60 and 80ppm are prepared by serial dilution of the stock in 25ml volumetric flasks with the addition of 1.0ml hydrochloric acid before making up the volume up to the mark with distilled water.

Sample Preparation and Analytic Determination:

• 0.25gm of accurately weighed sample is dissolved in water and volume is made up to 20ml with distilled water; this is the sample solution.

• 20ml of this solution is pipetted into 250ml flask, and added with 10ml 0.01M hydrochloric acid and 2ml basic lead acetate solution. The volume is made up to the mark with distilled water, shaken, and filtered to clarify.

• 50ml of the filtered solution is pipetted into 100ml flask, and added with 0.2ml sulphuric acid.

• The volume is made up to the mark with distilled water, shaken, and filtered.

• The Absorbance of the working standards and samples are measured on a UV/Vis spectrophotometer at 274nm wavelength using 10mm quartz cuvette.

• The caffeine levels in the samples are calculated from the regression equation of the best line of fit of the standards.