Aim: Extraction, Isolation & detection (by TLC) of Caffeine 

from tea dust




Reference: 

1.      Kokate, C. K. (1994). Practical pharmacognosy. Vallabh Prakashan, New Delhi, 4.

2.      Indian herbal pharmacopoeia. (2002). Revised new edition. Indian Drug Manufacturers Association, Mumbai, 106-113.

3.      Dr. Prabood shukla (2019). Pharmacognosy and Phytochemistry-II, Nirali Prakashan. Pune.

Requirements: 

Vacuum distillation unit, Soxhlet extractor, porcelain dish, ethanol (95%), sulphuric acid, chloroform, sodium hydroxide, nitric acid, amnionium hydroxide, tea powder.


Synonym: Camellia thea, Tea leaves

B. S.: Caffeine is a purine alkaloid, obtained from the leaf unfold buds and leaves of Thea sinensis.

Family: Theaceae


Extraction and Isolation

Method-I

About 50gms of finely powdered   tea leaves are taken in a 500ml beaker. Add 10gms of sodium carbonate and 250ml of water. The mixture is boiled till a decoction of tea is formed. Take care to avoid thickening of liquid while boiling. Filter the hot solution and neutralize it with 10% sulphuric acid solution. The neutralized filtrate is then extracted with dichloromethane in successive quantities. The dichloromethane extracts are combined and concentrate the solvent. Allow the extract to crystalline to form silky needles of caffeine

Method-II

Powder the tea leaves to coarse size and extract with water for half an hour. Filter the extract; treat with lead acetate to precipitate tannins. Filter the solution and add dilute sulphuric acid to solution to remove the excess lead acetate in the form of lead sulphate. Filter; add charcoal to remove the colouring matter. Filter this solution. Take this filtrate in separating funnel and extract with 3 portions of chloroform with mild shaking. Separate and combine the chloroform layers and evaporate to precipitate white crystals of caffeine. Re-crystallise with alcohol.

 

Method -III

Weigh 10 gm of tea leaves and transfer to 250 ml distilled water. Boil the water for 30 minutes with occasional stirring. After complete decoction is effected, allow to cool and filter the solution. Take filtrate in a separating funnel to which add 100 ml chloroform. Shake vigorously so that total caffeine will be transferred to chloroform. Separate chloroform layer. Evaporate chloroform over water bath. White caffeine crystals will collect at the bottom.

Chemical Constituents: Caffeine (1-3%)

It is also contain theophylline and theobromine in minor quantities. The color of the tea leaves is due to the presence of gallotannic acid. The agreeable odor is due to the presence of volatile oil. The leaves also contain an enzymatic mixture is called thease.

Observation

1.     Total amount of tea powder used (a) =      gm

2.     Total amount of caffeine obtained (b) =    gm

% yield = (100 X b)/ a

 

Identification Test of Caffeine

Murexide Test:

A few milligrams of Caffeine and 3-4 drops of nitric acid are placed in a small porcelain dish and evaporated to dryness. Addition of few drops of ammonium hydroxide imparts a purple coloration indicates the presence of caffeine.

UV-Spectral data :  λmax 278nm normal purine absorption at 260nm

Mass Spectral data  :  Molecular wt. of Caffeine 194.

m/e :  194  127 (C-Co) 165 (M-CH3) 109 (tropylium ion)

 

 Identification Test by TLC Method- I

Theory: TLC is  simplest technique  of chromatography, it  is  based on the  principle  of affinity chromatography. The separation relies on the relative affinity of compounds towards both the phases. Separation of compounds occurs based on their affinity towards the stationary phase, the compound having higher  affinity towards stationary phase moves slowly while the compounds having less affinity will travels fasters. No two compounds possess the same affinity towards the stationary phase at the combination of specific stationary  and mobile phase. Thus separation  occurs. The distance travelled  by compounds can be measured and used for analysis as retention factor (Rf) value.  

Procedure:

*      Prepare the mobile phase with the combination of given solvent.

*      Allow the saturate the developing chamber with the vepours of solvents of mobile phase by keeping this chamber aside for 30 min.

*      Apply the spot on the activated silica gel plate using capillaries and mark it with pencil

*      Place this plate into the developing chamber and put lid on it.

*      Allow to run the mobile phase on the silica gel plate (stationary phase) to its 80% of height

*      Mark the level of solvent front immidiatly after removing of plate from developing chamber

*      Dry the plate to room temperature and observe the plate either in iodine chamber of UV chamber at 254 nm.

TLC Protocol

Stationary Phase         : Absorbent – Pre-coated Silica gel

Mobile Phase               : Ethyl acetate: MeOH : H2O (100: 13.5:10)

Sample                                     :  Leaf extract

Rf values                     : 0.45

Detecting Agents         :  Iodine : HCl spray

                                                                              Distance travelled by solute 

                                    Rf value =                -----------------------------------------------------

                                                                               Distance travelled by solvent


Utilization

Caffeine

            Increases the blood pressure

            Stimulates the central nervous system 

            Promotes urine formation

            Stimulates the action of the heart and lungs

 

Uses:

            Treats migraine

            Increases the potency of analgesics

            Relieves asthma attacks


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