ANALYSIS OF CAFFEINE IN GREEN TEA

BY SOLID-PHASE EXTRACTION (SPE)

AND HPLC- UV DETECTOR

NOOR RABIHAH AID

(2018824174)
 ANALYSIS OF CAFFEINE IN GREEN TEA BY SOLID-PHASE EXTRACTION
(SPE) AND HPLC- UV DETECTOR

INTRODUCTION

Caffeine is a natural substance found in more than 60 plant species. These

include tea leaves, coffee beans, guarana and cocoa seeds. Caffeine can also be
produced synthetically and added to certain foods, beverages and medications.
Caffeine is one of a group of drugs called methylxanthines, which have several
effects on the body. For an example, it can increase the frequency of urination and
stimulate the heart muscle.

There are a few functional group in caffeine structure which are ketone, amine
and amide. The IUPAC name for caffeine is 1,3,7-trimethyl-1H-purine-2,6(3H,7H)-
dione 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione. Caffeine has a molecular
formula C8H10N4O2 and its molecular weight is 194.1906 g/mol. Figure below is the
structure of caffeine.
It is a well-established fact that the spectrophotometric determination in UV-vis region
is less expensive, follows a simple procedure, and provides a high accuracy and
reproducibility from a small number of samples. Spectrophotometry is widely used in
all the schools, colleges, universities, and research institutes. Almost all the
researchers are capable of handling this instrument. A wide variety of sophisticated
instruments are available such as HPLC and and are frequently used for the analysis
of caffeine. But every researcher is not able to access these sophisticated instruments.
The contents of this review will boost the knowledge of the researchers working on
caffeine in small scale industries, colleges, and universities.

OBJECTIVE

The objective of this experiment is to extract caffeine from a tea bag by using a
extraction technique.

The objective is to analyse Green Tea by extraction using Solid-Phase Extraction

(SPE) and HPLC Detector

REAGENTS AND SOLUTIONS

Analytical grade Methanol, Acetonitrile and green tea.

APPARATUS

C18 solid phase extraction cartridge (500 mg)

SAMPLE

6.0g green tea prepared in 500 ml distilled water

INSTRUMENT

Liquid chromatography (Agilent G1314A HPLC) equipped with diode array detector
(DAD)
ANALYTICAL PROCEDURE

a) Solid-phase extraction procedure

C18 SPE cartridge was conditioned by passing 10 mL of methanol. The
cartridge was rinse by passing 6 mL of deionized water without applying
vacuum. Then by using vacuum, the cartridge was dried for 15 minutes. The
interference was removed by eluting the column with 10 mL of deionized water
and dried again by vacuum for 10 minutes. The green tea then eluted using 5
mL of acetonitrile. The cartridge then was blower down using gentle Nitrogen
to get 1 mL concentrated green tea and ready for HPLC analysis.

b) Instrument set up
Set up by lab technician. LC parameters as below:
Injected volume: 20µL Flow: 1.5 ml/min
Column temperature: 25 °C Wavelength: 254 nm, Ref: 360 nm
Eluent: A= distilled water, B= Acetone Nitrile (ACN)
Isocratic = 30 dH2O: 70 ACN
Column: Ascentis C18 (150 x 4.6 mm; 5 µm
RESULTS

Figure 1 Standard caffeine

 CALCULATION

Data from Instrumentation value

Standard caffeine = 100 µg/mL Standard area = 1.9251x104

Equation 1 Responds Factor (RF) = Sample Amount / Peak Area

Table 1 Response Factor (RF) and Peak Area (pA*s) for Standard Caffeine

Sample amount
Response factor (RF)
PEAK in Standard Peak area (pA*s)
(Sample amount / Peak area)
(mg/mL)

Standard = 0.1 / 1.9251x104

0.1 1.9251x104
Caffeine = 5.19453 x 10-6 mg/mL

Equation 2 Amount of unknown = Peak area unknown X Response factor (RF)

Table 2 Elution of Green Tea

Peak Area
Standard
Elution by Mean
Deviation
1 2 3

Methanol 0.3432 x10⁴ 0.79419 x10⁴ 1.28705 x10⁴ 0.808147 x10⁴ 0.47208

Acetonitrile 1.7793 x10⁴ 1.90103 x10⁴ 1.70694 x10⁴ 1.79576 x10⁴ 0.09809

Table 3 Amount of unknown eluted by Methanol and Acetonitrile

Peak area Response

Elution by Amount of unknown
(Mean) factor (RF)

= 0.808147 x10⁴ x 5.19453 x 10-6

Methanol 5.19453 x 10-6
0.808147 x10⁴
= 0.041979 mg/mL

= 1.79576 x10⁴ x 5.19453 x 10-6

Acetonitrile 5.19453 x 10-6
1.79576 x10⁴
= 0.093281 mg/mL
 Equation 3 Amount caffeine in Green Tea =

[Amount of unknown x (5 ml/500 ml) x 500ml] / 6 g

Table 4 Amount of unknown eluted by Methanol and Acetonitrile

Elution by Amount of unknown Amount caffeine in Green Tea

= 0.808147 x10⁴ x 5.19453 x 10-6 [0.041979 mg/mL x (5 ml/500 ml) x 500] /6

Methanol
= 0.041979 mg/mL = 0.0349825 mg per gram Green Tea

= 1.79576 x10⁴ x 5.19453 x 10-6 [0.093281 mg/mL x (5 ml/500 ml) x 500] /6

Acetonitrile
= 0.093281 mg/mL = 0.0777342 mg per gram Green Tea

DISCUSSION

Due to time constraints, no further attempts were made to obtain a useable

chromatograph from HPLC. From the result acetonitrile elute more caffeine in green
tea which is 0.0777342 mg per gram green tea compare to methanol 0.0349825 mg
per gram green tea

Rational of chosen HPLC method for the determination of caffeine, because HPLC is
the most widely used qualitative and quantitative determination and separation
method. The method is popular because it is non-destructive and may be applied to
thermally labile compounds (unlike GC); it is also a very sensitive technique since it
incorporates a wide choice of detection methods. With the use of postcolumn
derivatization methods to improve selectivity and detection limits, HPLC can easily be
extended to trace determination of compounds that do not usually provide adequate
detector response. The wide applicability of HPLC as a separation method makes it a
valuable separation tool in many scientific fields.
CONCLUSION

A method has been developed for the extraction, purification of caffeine from Green
Tea. Caffeine from the Green Tea was extracted by Solid phase extraction and
interferences were removed by employing solid-phase extraction (SPE). The purified
caffeine was then analyzed by HPLC. Solid phase extraction prior to HPLC was found
to be an essential step for determining the amount of caffeine by HPLC.
Characterization of caffeine was achieved by determining melting temperature, Rf
value, RI spectrum and UV spectrophotometry.

REFERENCES

1) Nor’ashikin S., Ruziyati T., Mardiana S. (2014). Analytical Separation Methods

Laboratory Guide. UiTM Press.

2. Barone, J.J., Roberts, H.R. ( 1996) “Caffeine Consumption”, Food Chemistry and
Toxicology, McGraw-Hill, Newyork, 34, 119

3. Arnaud, M. J. (1987) The Pharmacology of Caffeine, Prog Drug, 31, 273.

4. Clementz, G. L., Dailey, J. W. Psychotropic effects of caffeine, Amer. Fan Physician.

37 167 (1988)

5. Kirmer, D.A. (1988) Caffeine use and abuse in psychiatric clients. J. Psychosoc
Nurs Ment Health Serv., 26, 20