Iraqi J Pharm Sci, Vol.19(2) 2010                                                     Gravimetric estimation of caffeine 

 

 

 

48 

Gravimetric Estimation of Caffeine in Different Commercial Kinds 

of Tea Found in the Iraqi Market 
Maha N. Hamad*

,1
        and    Dhuha A. Abdul-Hussain* 

 
*Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad,Iraq . 

 
Abstract 
          Caffeine (1,3,7-trimethylxanthine), which is the most widely consumed stimulant in the world, 

had been isolated and estimated gravimetrically in fifteen  different commercial kinds of tea found in 

the Iraqi market.The kinds of tea were chosen according to their differences in the degree of 

fermentation and the method of processing i.e. black , gray and  green . The isolated caffeine was 

identified by melting point, sublimation, TLC, chemical tests, UV , IR , HPLC and CHNO analysis.                                                                                                               

Key words: Caffeine, Purine, tea.    

 الخالصة
م, ذم فصهه وذعٍٍه كمٍره تطشٌقح ثالثً مثٍم صاوثٍه( انزي ٌعرثش مه أكثش انمىاد انمىثهح إسرعماالً فً انعان – 7,  3, 1كافاٌٍه )         

وصوٍح فً خمسح عشش وىعاً مخرهفاً مه انشاي انمىجىد فً األسىاق انمحهٍح. ذم أخرٍاس ومارج مه انشاي عهى دسجاخ مخرهفح مه انرخمٍش 

 انزوتان ,. ذم ذشخٍص انكافاٌٍه انمعضول تطشق مخرهفح مىها قٍاط دسجح اي االسىد وانشصاصً واالخضش و طشق مخرهفح نهرحضٍش

 وكشوماذىغشافٍا انطثقح انشقٍقح , وفحىصاخ كٍمٍاوٌح , وطٍف األشعح فىق انثىفسجٍح واألشعح ذحد انحمشاء انرسامً ,

 .وانكشوماذىغشافٍا انسائهح راخ انكفاءج انعانٍح وحساب وسة عىاصش انكشتىن ,انهٍذسوجٍه,انىٍرشوجٍه واالوكسجٍه

 
Introduction 
         Thea or tea consists of the prepared 

leaves and leaf buds of camellia sinensis 

(formerly known as Thea sinensis) of the 

Theaceae family. There are three main 

commercial types of tea: green , oolong (gray) 

and black, depending on the method of 

processing. The leaves may be fermented or 

left    unfermented. Fermented teas are referred 

to black tea, unfermented teas as green tea and 

partially fermented teas as oolong tea.Black tea 

is prepared by heaping the fresh leaves until 

fermentation has begun, then they are rapidly 

dried artificially with heat, while green tea is 

prepared by rapidly drying the freshly picked 

leaves in copper pans over a mild artificial 

heat, or the leaves are often rolled in the palm 

of the hand as they dry. Gray tea is partially 

fermented by heaping then they are dried on 

artificial heat.  Tea contains caffeine (theine) 

and small amounts of adenine, theobromine, 

theophylline, gallotannic acid and volatile oil. 
1,2

Caffeine (1,3,7-trimethylxanthine), the 

molecular formula of which is C8H10N4O2 , is 

the most widely consumed stimulant in the 

world can be considered to be constructed 

from the purine ring system, which is 

important biologically, being found in nucleic 

acids and nucleotide and in few organisms as 

alkaloids.
3 

Caffeine was first discovered in tea 

in 1827, and was named theine , and later it 

was found in mate , coffee and various other 

plants and the term theine was then 

dropped.
4
Purines are considered pseudo 

alkaloids since they are not derived from an 

amino acid precursor.
5
 

 

N

N
N

N
H

NH

H
N

N

N
H

N
N

N

O O

O

H3C-N

CH3

CH3

O

Purine Xanthine Caffeine  
 

 

1Corresponding author E- mail :  manoha_1957@yahoo.com           

Received : 28/4/2010 

Accepted : 8/8/2010 



Iraqi J Pharm Sci, Vol.19(2) 2010                                                     Gravimetric estimation of caffeine 

 

 

 

49 

Caffeine acts as a CNS stimulant , mild 

diuretic, it increases the heart rate and blood 

pressure and stimulate gastric secretions. It 

also acts as a natural pesticide that paralyze 

and kills certain insects feeding on the plants. 
6,7

 Caffeine with UV can kill some kinds of 

algae and there are evidences that it enhances 

mutations in bacteria and viruses and also 

induce chromosome damage. 
8,9,10,11

Caffeine is 

an ingredient of several dozen proprietary 

products, for the most part, these combination 

with acetyl salicylic acid, ascorbic acid, 

codeine, paracetamol, and other analgesics  

and antipyretics.Caffeine is found in a number 

of beverages ingested by people in addition to 

tea as coffee, and to some extent cocoa. Other , 

less commonly used sources of caffeine 

include the plants  guarana, and yerba mate' 

which are sometimes used in the preparation of 

teas, and recently ,energy drinks. Tea leaves 

contain 1-4% caffeine, while coffee 1-2% yet a 

cup of brewed coffee contains about 100-150 

mg caffeine while a cup of tea contains 60-75 

mg. caffeine is also a common ingredient of 

soft drinks such as cola. Soft drinks typically 

contain about 10-50 mg of caffeine per serving 

. The range of caffeine contents in various 

beverages is shown table 1 

 

Table 1 : range of caffeine in various 

beverages 

Approximate caffeine 

content of various 

beverages 

Range of mgs 

of caffeine 

Coffee (5oz. cup)                                                  40-170 

Soft drinks (12 oz. can) 10-50 

Black tea (one tea bag)  25-110 

Oolong tea (one tea bag) 12-55 

Green tea (one tea bag) 8-30 

Decaffeinized tea(one  tea 

bag) 

1-4 

Energy  drinks ( 12 0z. can) 75-90 

  

In this paper we have estimated caffeine 

gravimetrically in fifteen different kind of tea 

found in the market black, gray and green tea . 

 

Materials and  method 
 Samples of tea were chosen randomly to 

represent black, gray and green tea in the 

form of tea bags or unpacked form. 

 All reagents are anhydrous solvents were of 
analar type and generally used as received 

from the commercial suppliers. 

 Silica gel used in the form or ready made 
aluminum plates of silica gel GF254 , Merck 

Co. 

 UV was run in methanol , IR was run in KBr 
disk. 

 HPLC was done using Knauer/ Germany 
HPLC. 

 Standard caffeine is from Evans Medical Ltd 
, Liverpool. 

 

Isolation of caffeine 
         25 gm of tea were boiled with 200ml of 

water for fifteen minutes in a covered beaker . 

The extract was filtered through muslin and the 

mark was re boiled with 120ml of water for 

five minutes, filtered and the mark over the 

muslin was washed with 70ml of boiling water, 

the muslin was then squeezed till exhaustion. 

The combined extracts were cooled and mixed 

with 4gm of sodium carbonate with stirring, 

then transferred to a separatery funnel and 

partitioned with three successive portions of 

methylene chloride each of 50ml (i.e. 

3X50ml), each time the funnel was inverted 

back and forth ten times. The methylene 

chloride layers were combined together and 

dried over anhydrous sodium sulfate , filtered 

and evaporated to dryness under vacuum. The 

obtained caffeine was re crystallized from 

boiling ethanol, filtered and weighed. The 

percentages of caffeine was calculated as w/w. 

The extraction procedure is shown in 

diagram(1). 

 

 

 

 

 

 

 

 

 

 

 

 

 

Diagram 1 : Isolation of caffeine from tea 



Iraqi J Pharm Sci, Vol.19(2) 2010                                                     Gravimetric estimation of caffeine 

 

 

 

50 

Two samples of each kind were used and  the 

average weights of the isolated caffeine were 

taken for calculation of the percentages and 

comparison. 

Identification of Caffeine 
         The isolated caffeine was identified by 

several methods : 

 It was identified by measuring its melting 
point and compare it with standard caffeine 

and also using a mixed sample from isolated 

caffeine and the standard .
12

The results are 

shown in table (2).
 

 

Table 2 : Melting points of isolated and 

standard caffeine 

Melting point Sample 

236.7°C Isolated caffeine 

237.3°C Standard caffeine 

237°C 
Mixed  isolated and 

Standard caffeine 

 

Then caffeine was identified by two chemical 

tests :            

1- Murexide test : Isolated caffeine gave a 
purple color. 

2- Isolated caffeine was treated with hydrogen 
peroxide and 2% HCL. After evaporation 

to dryness , a bright red color was obtained. 

The color turn purple upon addition of 

drops of 5% ammonia. 
13

 

Also caffeine was identified by sublimation 

and this process was achieved by introducing a 

small quantity of caffeine in a porsalen dishand 

covered with a watch glass , the porsalen dish 

was subjected to heat while the watch glass 

was covered with a plastic sack containing ice 

.Upon heating caffeine started to sublime and 

condense on the lower surface of the  watch 

glass. Caffeine was also identified by TLC 

using silica gel GF254 plates developed in three 

different mobile phases and comparing the Rf 

values of isolated caffeine with standard using 

single and mixed spots , and detection was 

done under UV254nm. 
14,15,16,17

 

Mobile phases used are: 

Mobile phase I : Ethyl acetate : acetic acid  

95:5 .  

Mobile phase II : Chloroform : ethyl acetate : 

formic acid  5: 4 : 1 

Mobile phase III : petroleum ether : methylene 

chloride : ethyl acetate 1: 1 : 2.  

The result of TLC are shown in table (3) 

 

Table 3 : Rf values of isolated and standard 

caffeine 

Standard 

Caffeine  Rf  

values 

Isolated 

caffeine       

Rf  value 

Mobile 

phase 

0.257 0.226 I 

0.516 0.490 II 

0.110 0.110 III 

 

 The UV absorption spectrum exhibits a pair 
of absorption bands peaking at (209) and 

(272)nm with a shoulder between them 
18

 . 

The UV spectrum of the isolated caffeine is 

shown in fig.(1) 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1   UV spectrum of the isolated 

caffeine 

Also caffeine was identified by IR
 19

 and the 

spectrum is shown in fig. (2) 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 Figure 2 : IR spectrum of the isolated caffeine 



Iraqi J Pharm Sci, Vol.19(2) 2010                                                     Gravimetric estimation of caffeine 

 

 

 

51 

Also caffeine was identified by CHNO 

analysis ,whereby the percentage of each 

element measured and compared with the 

calculated one.The results are shown in table 4. 

Table 4 : CHNO analysis of caffeine 

Found 

percentages  

Calculated 

percentages 

    Element                    

    

49.877 49.484 Carbon 

5.199 5.155 Hydrogen 

29.109 28.866 Nitrogen 

16.709 16.495 Oxygen 

 

Caffeine was finally identified by HPLC
 18 

 

using C18 5x150mm column and a mobile 

phase composed of methanol/water 90:10  with 

flow rate of 0.8ml/minute and detection with 

UV detector at 275nm. The retention time of 

the isolated caffeine was compared with that of 

the standard. The results are shown in figures 3 

and 4. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure  3 : HPLC  of isolated caffeine 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure  4 : HPLC  of standard 

 
Results and  Discussion 
The average weights and percentages of the 

caffeine isolated from each kind of tea are 

shown in table (5) and  fig. (5). The idea of this 

method of isolation of caffeine is to extract the 

water soluble materials in the tea leaves in a 

hot water . (the solubility of caffeine in water 

is 22 mg/ml at 25
o
c , 180 mg/ml at 80

o
c and 

760 mg/ml at 100
o
c ) . The caffeine is 

extracted from the water after cooling with 

dichloromethane (140 mg/ml) than in water 

(22 mg/ml), it readily dissolves in the 

dichloromethane.However, the tannins are 

slightly soluble in dichloromethane but upon 

addition of sodium carbonate to the extract the 

tannins will be converted to phenolic anions 

(since phenols are acidic enough to be 

converted to phenolic salts i.e. deprotenation of 

OH group ) upon addition of sodium 

carbonate). The phenolic salts thus formed are 

not soluble in dichloromethane , soluble in 

water, as shown below: 

ArOH       +        Na
+

2CO3
-2

   →       ArO- Na+       +   Na+ HCO3- 
           tannins soluble                                         tannins salts 

      in water, dichloromethane                            soluble in water 

                                                                            insoluble in dichloromethane 



Iraqi J Pharm Sci, Vol.19(2) 2010                                                     Gravimetric estimation of caffeine 

 

 

 

52 

Table 5 :The percentage of caffeine in different  kinds of tea 

 

% of caffeine 

Weight of 

caffeine 

In 25 gm of  tea 

Tea brand no 

1.924 

1.40 

1.288 

1.240 

1.176 

0.952 

0.800 

0.720 

0.360 

0.440 

1. 364 

1.308 

0.884 

0.520 

0.480 

0.481 

0.351 

0.322 

0.310 

0.294 

0.238 

0.2 

0.180 

0.090 

0.110 

0.341 

0.327 

0.221 

0.130 

0.120 

Al-Otoor  (black) 

Al-Rabeea (black) 

Mahmood (black) 

Ahmad (black ) 

Al-Wazza (black ) 

Al-Tuffaha (black ) 

Ahmad (black , Tea bags ) 

Ration tea ( black ) 

Lipton (black , tea bags ) 

Al-Okozay ( gray , tea bags ) 

Al-attar ( green , tea bags ) 

Lipton (green , tea bags ) 

Ahmad (green , tea bags ) 

Green tea (un packed) 

Alokozay ( green , tea bags ) 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5 : percentages of caffeine in different kinds of tea 

 

 

 

Caffeine being a xanthine derivative was first 

identified by the murexide test. The core of the 

reaction is apparently that caffeine through a 

number of steps , is oxidized into the 

intermediate that ultimately condenses to 

murexoin. The ammonium salt of murexoin is 

the principal contributor to the purple color of 

the final solution. 
20

The quantitative 

differences obtained in different kind of tea is 

probably due to the method of processing of 

each kind since caffeine sublimes with out 

decomposition upon exposure to heat, 

therefore it should be expected that caffeine 

could be lost during fermentation and 

processing. Also the differences of caffeine 

quantity and consequently the percentage may 

be due to different time of harvesting of leaves 

of the plant.  

Acknowledgement 
         We are deeply greatfull to Mr. Dhulfiqar 

A. Abid ( MSc.) and Mrs. Sahar M. Shakir ( 

MSc. ) and Mr. Zaid M. Abdul-Khalik  for 

running the UV , IR , and HPLC  spectrum. 

 

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Iraqi J Pharm Sci, Vol.19(2) 2010                                                     Gravimetric estimation of caffeine 

 

 

 

53 

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