Determination of Caffeine in Tea Samples

Caffeine
(trimethylxanthine coffeine theine mateine guaranine methyltheobromine) is a
stimulant drug and mild diuretic. In pure form, caffeine is a white crystalline solid.
An average 5 ounce cup of tea (Thea/camellia sinensis) can contain 10-ll0 mg,
making it the next highest source of caffeine in beverages. How much caffeine is in
your cup of tea will depend upon the type of tea used, the tea leaf cut, and how
long it was brewed.
TEA BREWING
EFFECT
(5-oz. cup)
1 min. brew
3 min. brew
5 min. brew
Instant tea
Iced tea (12-oz.
cup)

CAFFEINE CONTENT
(mg)
9-33
20-46
20-50
12-28
22-36

[compiled from data supplied by Consumers' Union, the Food and Drug
Administration, National Coffee Association of the U.S.A., and National
Confectioner's Association of the U.S.]

Because of difference in fermentation and brewing techniques, caffeine varies by

types of tea as well:
TYPE OF
CAFFEINE CONTENT
TEA
(mg)
(5-oz. cup)
Black tea 50-60
Oolong
40-50
tea
Green tea 20-30
10
White tea

Traditionally, many people have thought of teas' caffeine levels as being associated
with tea "types," such as black tea, green tea and white tea. More recently,
scientific testing has shown that variations in caffeine levels of different tea types
have more to do with how they are brewed than how they were processed into tea.
For example, if you brew your white tea at a low brewing temperature for a short
infusion time, then it will be much lower in caffeine than if you brew it like a black
tea. In fact, brewing a white tea as you would brew a black tea (with boiling or nearboiling water for four to five minutes) could produce a cup of white tea that is
HIGHER in caffeine than black tea.

Caffeine & Brewing Style

Brewing methods and styles can have a large impact on a tea's caffeine level. Using
a higher water temperature, longer brewing time or higher ratio of tealeaves to
water will increase the caffeine level of your brew.
Caffeine Levels in Different Tea Grades
Tea grades are categories assigned to teas based on how whole or broken the
leaves are. Generally speaking, broken leaves will impart more caffeine into your
brew faster than whole leaves. Teabags often hold very broken grades of tea, so
they tend to have higher levels of caffeine. (For more information, see teabags vs.
whole-leaf tea.) Tea grades also assess how "tippy" a tea is. The ratio of tips in a tea
can also impact its caffeine level.

Tea Tips, Tea Stems & Caffeine Levels

Tea tips / buds (the newly formed leaves of the tea plant that are often used to
make white tea) are generally known to be higher in antioxidants and nutrients than
older tea leaves. However, few people realize that they are also higher in caffeine
than older tea leaves.
In terms of the pure leaf, many white teas from outside of Fujian, China are higher in
caffeine than black teas simply because they are made with more tips / buds.
Similarly, tippy black teas and green teas will be higher in caffeine than their leafy
counterparts.
Conversely, tea stems contain very little caffeine. Teas like Hojicha and Kukicha are
made from "twigs" (stems) and are naturally very low in caffeine.
Caffeine Levels of Tea Varietals
The Assamica tea varietal is higher in caffeine than other tea varietals. The
Assamica varietal is primarily grown in Assam, India and used to make bold, tannic
black teas, such as English Breakfast tea.
The so-called "white tea varietals" (China's tea varietals #1 and #2) are naturally
lower in caffeine (and higher in antioxidants) than other varietals. For this reason,
white teas grown from these varietals (such as Fujian Silver Needles and White
Peony) are also lower in caffeine and higher in antioxidants than many other teas.
However, there are some "white teas" made from other varietals in other parts of
the world, and these white teas are not as low in caffeine. One example of this is
White Darjeeling, which is made from varietals with higher caffeine levels, and is
mostly made of tea tips (which naturally contain more caffeine than opened leaves
or stems).
Caffeine in Shade-Grown Teas
Generally speaking, shade-grown teas (such as Gyokuro Green Tea) will have higher
levels of caffeine than other teas. This phenomenon has to do with a shift in
chlorophyll and other chemicals that occurs when netting is used to shade the
leaves from sun in the days or weeks prior to harvest.
Caffeine Levels of Powdered Teas

Powdered teas (such as Matcha Green Tea) are usually very high in caffeine. This is
because you consume the entire leaf rather than just an infusion of the leaf, so you
consume all of its caffeine instead of just some of it.
Matcha powdered tea is especially high in caffeine because it is shade grown (see
"Caffeine in Shade-Grown Teas" above).
Caffeine Release in Twisted / Rolled Teas
Teas that are highly rolled or twisted may release caffeine more slowly than leaves
that are flat or open. This tends to apply to certain types of oolong teas, which are
typically brewed many times in a gaiwan or yixing teapot. It is not known whether
the overall release of caffeine over multiple infusions is comparable to the caffeine
release of single infusion a similar, but less twisted / rolled, tea.
Tea Blends & Caffeine Levels
Teas that have been blended with other ingredients (such as mint or masala chai
spices) will often have lower caffeine levels than unblended teas. This is because
people often brew them with the same ratio of tea to water (such as one teaspoon
per cup), but the total amount of tea leaf used is lower, as it has been partially
replaced by herbs.
Extraction of caffeine from tea requires a thorough understanding of the chemistry
of caffeine and other
substances, mainly tannins, present in tea. For instance, tea contains 10~30 % of
tannins by mass but only 2~4 % of caffeine by mass. The separation of caffeine
from tannin impurities is the most crucial step in the extracting process. Tannins are
usually divided into two groups, namely hydrolyzable tannins (HTs) and
proanthocyanidins (PAs). The commonly known tannic acid is an example of
hydrolyzable tannins.
HTs can be hydrolyzed by mild acids or mild bases to yield carbohydrate, phenolic
acids or their salts. Under the same conditions, proanthocyanidins do not hydrolyze.
Therefore, by varying the nature of the extracting solvents, caffeine extracted from
tea can be purified from a mixture containing HTs and PAs.

METHOD 1
Procedure
Preparation of Tea Infusion
1. In the first set of samples, three tea bags from each of the seven brands were
steeped in 150 cm3 of boiling deionized water for 1 minute in a beaker containing
5.5 g of sodium carbonate and 12.5 g of sodium chloride.

2. Step 1 was repeated by steeping tea samples for 5 minutes and 30 minutes
respectively.
Sample
Set 1
Set 2
Set 3
Steeping Time (min)
1
5
30
3. The tea remained in the tea bags were squeezed back to the beaker by watch
glasses and the used tea bags were discarded.
Solvent Extraction
4. The tea samples were cooled to room temperature and then introduced into
separating funnels.
5. The beakers formerly contained the tea samples were rinsed with 20cm3 of
dichloromethane. The rinsings were then added to the separating funnels.
6. The mixtures in the funnels were then shaken gently. The vapour produced was
released after a few shakes. Any emulsions were broken up by agitation with a glass
rod. The mixture in each funnel was allowed to separate for 10 minutes.
7. The lower layer together with the emulsion, if any, was transferred to the original
beaker.
8. The tea samples left in the separating funnels were extracted twice with two 20
cm3 portions of
dichloromethane.
9. The three dichloromethane extracts were combined and washed twice with 20
cm3 of cold 6M sodium hydroxide and once with 20 cm3 of cold deionized water.
10. The dichloromethane extract was transferred to a clean beaker and dried over
anhydrous sodium sulphate. The clear solution was then decanted into another
clean beaker.
Evaporation and Sublimation
11. The dried samples were heated gently over a hot plate to evaporate the excess
solvent until about 5 cm3 of solution was left in each beaker.
12. A clean soda glass test tube was weighed and the mass was recorded as ().
13. The sample was added into the pre-weighed test tube using a dropper. The
beaker and the dropper were rinsed with a little dichloromethane and the rinsing
was transferred to the test tube. (This is to minimize the loss of caffeine). The
solvent was evaporated completely by heating the test tube over a hot plate. The
test tube containing crude caffeine was then weighed again and the mass was
recorded as (). The value ( ) represents the mass of crude caffeine.
14. *A cotton wool plug was inserted at the mouth of the test tube and the test tube
was heated in an oil bath for about 2 hours. Pure caffeine was condensed as

sublimate at the upper part of the tube. The test tube was cut into two pieces at
about 3 cm from the bottom with a red glow nichrome wire.
15. The upper part of the tube that contained pure caffeine was weighed and the
mass was recorded as ().
16. A little caffeine was removed and tested for purity by melting point
determination.
The upper part of the tube was cleaned and dried completely. It was then weighed
and the mass was recorded as (). The value ( ) represents the mass of pure
caffeine obtained from the tea sample.
* In the first attempt of sublimation, cotton wool plugs were not inserted in the test
tubes.
This resulted in a contamination of the final product with vapour of oil from the oil
bath.
In the final set-up, this error had been avoided by insertion of cotton wool plugs that
absorbed the oil.

The factors affecting the extent of caffeine extraction were investigated and the
results were shown below.
a. The time factor
Results: The longer the steeping time, the more caffeine and tannins extracted.
Moreover, over 50% of caffeine was extracted in the first-minute steeping. It was
also found that the steeping time of tea should not be long; otherwise, more tannins
would dissolve which formed stubborn emulsion making the solvent extraction more
difficult.
b. The nature of tea
Results: The more the tea was fermented, the higher was its caffeine content. For
example, Black Tea (fully fermented) was found to contain more caffeine than Pu
Erh (partially fermented), which contained more caffeine than Green Tea (not
fermented).
a. The yield of caffeine was significantly improved by allowing the extraction
mixture to stand overnight.
b. The amount of emulsion was substantially reduced when sodium chloride and
sodium carbonate were not used in preparing tea infusion.
c. The amount of caffeine extracted increased sharply in the first 5-minutes
steeping and remained more or less the same in the next 25 minutes.

METHOD 2
Sample preparation.
Five types of tea in loose leaf form: Pai Mu Tan-superior (white tea), Sencha (green
tea), Formosa Fine Oolong (oolong tea),Lingia (black tea), Yin Zhen (yellow tea) and

two types of loose leaf mate tea (mate tea and roasted mate tea) were purchased
on a local market. In order to simulate household brewing conditions, teas were
prepared using an aqueous extraction. Tea samples (2.5 g) were poured with 200 ml
of boiling water and stirred for 10 minutes. Extracts were filtered through a cotton
wool, cooled at a room temperature, diluted to 250 ml with distilled water, and used
for spectrophotometric analyses.
Caffeine isolation with chloroform.
The caffeine isolation procedure was performed according to a modified method
described by Rapi (1994). Briefly, 20 g of tea and 90 ml of distilled water was
refluxed for 30 min, and filtered under vacuum. The residue was again refluxed and
filtered. Obtained filtrates were combined, 12.5 ml of Pb(CH 3COO)2 solution was
added, boiled (5 min), and filtered through a Buchner funnel with silica gel layer.
The filtrate was extracted four times with chloroform (40 ml). Combined chloroform
phases were washed with KOH solution and then with distilled water. Chloroform
was removed from extracts by rotary evaporator. After evaporation, extracted
caffeine was weighed and expressed in mg/l.
Caffeine determination using the lead acetate solution.
This procedure is based on international standards with some modifications (Yao et
al.1992, 1993). Tea extract was treated with HCl solution (5 ml), Pb(CH 3COO)2 and
H2SO4 solution. Absorbance of obtained extracts was measured at 274 nm. The
content of caffeine (mg/l) was calculated using a standard curve derived from
caffeine (0250 mg/l). All measurements were performed in triplicate.
The micromethod for the determination of caffeine.
The teas were also analyzed for their caffeine content according to the method
reported by Groisser (1978). Briefly, tea extracts (pH =89) were extracted with
benzene and H2SO4. Absorbance of extracts was read at 273 nm against a blank
(H2SO4). Results, obtained from triplicate analyses, were calculated using a standard
curve and expressed as mg/l.
HPLC analysis of caffeine.
Filtered tea extracts and caffeine solutions were injected for HPLC analysis
according to the method reported in our previous study (Hori et al. 2009).
Equipment used consisted of a Varian Pro Star Solvent Delivery System 230 and a
Photodiode Array detector Varian Pro Star 330 (Varian, Walnut Creek, USA) with a
reversed-phase column Pinnacle II C-18 (Restek, USA) (250 4.6 mm, 5 m i.d.).
Caffeine was identified by comparing the retention times and spectral data with
those of authentic standards. All analyses were repeated three times.

METHOD 3
Method
Materials.
All chemicals and equipment used are of analytical grades and are used without
further purification.
Caffeine standards of 2.5, 5.0, 7.5, and 10.0 g/m1 are prepared with 5 N H 2S04. A
model DB spectrophotometer is used.
Procedure
Make 180 ml (6 ounces) of tea to desired strength. Adjust pH of the beverage to 8 to
9 using 1 N NaOH at 20 C. Pipet 0.1 ml of tea into a 20 ml Teflon capped tube. Pipet
10 ml benzene into the tube and add about 0.5 g NaCI. Shake tube for I min using a
vortex super mixer. Centrifuge for 10 min at 3500 rpm. Carefully pipet S ml from the
benzene (top) layer into a Teflon capped test tube containing 5 ml of 5 N H 2S04.
Shake for 30 sec. Centrifuge for 5 min. carefully pipet some of the H 2S04 (bottom)
layer into a quartz cuvet. Read the optical density of the sample versus blank H 2S04
cuvet at 273 nm. Calculate the caffeine concentration as mg/h80 ml of beverage.
Using the previously described method, various strengths and brands of tea were
studied for caffeine
content. The comparison was made of teas prepared in two ways: 1) on the basis of
strength (color); and, 2) on the basis of package directions for brewing.

Tea prepared by color comparison

Tea bags or porous metal tea bags containing 2.35 g of loose tea leaves (the
amount of tea found in most bagged brands) were dunked at a uniform rate into
uniform glass jars containing boiling water. Against a uniform background, weak,
medium, and strong cups of tea were made by color matching with these standards.
The standards were in the same type of jar and were at the same temperature as
the samples. Black and Oolong teas were made in this fashion. Green tea, which
yields a pale green color, cannot be compared with the color of Black and Oolong.
These latter teas were analyzed for caffeine content only by following package
directions for preparation.
Tea prepared by package instruction
In addition, all teas tested were made by standard package directions. Bagged tea,
loose tea in porous metal bags, and unencased loose tea were left to steep for 4
min with the jar covered. (Package directions on most brands call for a 3 to S min
steep.) Instant, ice, and automatic tea were made by individual package
instructions.