High Performance liquid Chromatography

(HPLC)

The Determination of Caffeine In Tea

BY

Mkhwanazi L.S (21103379)

Department of Chemical engineering
Mangosuthu University of Technology
ABSTRACT

The aim of the experiment was to determine quantitatively the concentration of caffeine
in tea using a reverse phase High Performance Liquid Chromatography. In this
experiment, the external standard method was used. Series of standards of caffeine
with known concentration of the analyte were prepared and analysed under same
conditions as the sample, which was tea in order to obtain the calibration curve.
Volumes of both standards of caffeine and tea sample were injected and the area
produced by the each standard and sample. The determinations were based on this
calibration curve. The concentration of caffeine in tea was found to be 101.279ppm from
the equation.
INTRODUCTION

Reverse phase HPLC is used to determine the concentration of caffeine in coffee, tea
and Coca-Cola. The traditional method of the determination of the caffeine is via
extraction with spectrophotometric quantitation. Use of the LC system permits a fast and
easy separation of caffeine from other substances such as tannic acid, caffeic acid and
sucrose found in beverages. Five standard solutions of caffeine are prepared and
injected into the HPLC. From the resulting chromatograms, measurements of retention
time and peak areas are made. If the flow rate and pump pressure are held constant
throughout the entire experiment, tR may be used as qualitative measure and peak area
as quantitative measure. A calibration curve for peak area (at respective t R) versus
concentration if the caffeine standards can then be employed to determine the
concentration of caffeine in the beverages
Materials and Methods

Materials

HPLC with UV detector C18 column 7 x 100 cm3

volumetric flasks

AR Grade Caffeine

Acetonitrile: Water (80:20 Mixture) Tea sample

Methodology

Preparation of Caffeine Standards:

1. Into five 100 cm3 volumetric flasks, accurately weigh the following quantities of caffeine:
2.5 mg, 5.0 mg, 7.5 mg, 10 mg, and 12.5 mg.

2. Dilute each flask to the mark with the prepared mobile phase mixture, which is 80:20
Acetonitrile:H2O.

3. Shake each flask adequately to ensure complete dissolution of caffeine.

4. Degas each caffeine solution for five minutes before injection into the LC (Liquid
Chromatography) instrument.

5. Switch on all components of the LC instrument and set the pump flow rate at 1.2
mL/min.

6. Set the detector wavelength to 254 nm.

7. Allow the mobile phase to pass through the column for approximately 5 minutes to
equilibrate the system.

8. Inject each caffeine standard in duplicate, starting with the least concentrated standard
solution, and obtain chromatograms with peak areas for each standard.
Sample Preparation for Tea Analysis: 9. Accurately weigh 2.000 g of the supplied tea leaves
into a 250 cm3 round-bottomed flask.

10. Add 100 cm3 of Milli-Q water to the flask.

11. Set up the flask for a reflux procedure.

12. Reflux the tea leaves for 30 minutes. Be cautious to ensure gentle boiling to prevent
excessive bumping.

13. Allow the flask to cool after the required time has elapsed.

14. Set up a vacuum filtration system and carefully filter the solution to remove the tea
leaves. Retain the filtrate.

15. Transfer the filtrate to a 100 cm3 volumetric flask and dilute it to the mark with Milli-Q
water.

16. Prepare the final tea sample by pipetting 10 cm3 of this diluted solution into a 100 cm3
volumetric flask and diluting it to the mark with Milli-Q water.

17. Inject the final tea sample in duplicate into the LC instrument and obtain a chromatogram
with peak area for analysis.
RESULTS AND DISCUSSION

Masses weighed for Caffeine:

2,5mg; 5,0mg; 7,5mg; 10mg and 12,5mg

Standards Concentrations Peak area 1 Peak area 2 Mean peak area

(ppm)
Std 1 25ppm 549.80804 543.45624 546.63214
Std 2 50ppm 1104.82861 1075.28503 1090.05682
Std 3 75ppm 1555.86804 1606.30066 1581.08435
Std 4 100ppm 2091.78296 2096.59351 2094.188235
Std 5 125ppm 2644.52441 2680.45508 2662.489745
Sample X 814.64355 827.57959 821.11157

Concentration Vs Mean Peak Area

3000

2500 f(x) = 20.9433865 x + 24.1362705000001

R² = 0.99943468668406
Mean Peak Area

2000

1500

1000

500

0
20 40 60 80 100 120 140
Concentrations(ppm)

Concentration from the graph

Y = 20.943x + 24.136

821.11157 = 20.943x + 24.136

X = 38.0545ppm

•[Caffeine]= 38.0545ppm
Discussion

The analysis conducted in this study was primarily quantitative in nature, focusing on
determining the concentration of caffeine in tea. The results indicated a concentration of
38.0545 parts per million (ppm), derived from the equation of the linear graph. To create
caffeine standards for calibration, weights of 2.5mg, 5mg, 7.5mg, 10mg, and 12.5mg
were measured. Due to the small masses involved, a stock solution weighing 2.0019g
was utilized to accommodate all the weights, ensuring minimal impact on result
accuracy. The graph exhibited a linear relationship between the mean peak area and
the concentration of the standards. Notably, an increase in the concentration of the
standards in ppm corresponded to a rise in the peak area.

Conclusion

A quantitative analysis of caffeine in tea was conducted using reverse phase HPLC,
revealing a calculated concentration of 38.0545 ppm. The average peak area observed
in the tea samples fell within the range of the standards, facilitating reliable
extrapolation.
References