ESTIMATION OF CAFFEINE CONTENT IN TEA & COFFEE BY UV/VIS SPECTROPHOTOMETER, HPLC WITH UV/VIS DETECTOR & FTIR WITH HATR

Caffeine is a methylxanthine, and methylxanthines are known to have anti-inflammatory properties, is a bitter, white alkaloid and a stimulant. It is the world’s most widely consumed psychoactive drug which is naturally available in coffee plant, tea bush and Kola plant. In biological systems, caffeine acts in the central nervous system and acts as a stimulant. Beverages containing caffeine, such as coffee, tea, soft drinks, and energy drinks, enjoy great popularity.

The structure of caffeine (C8H10N4O2) molecule is given below.

caffeine1

Caffeine belongs to the Xanthine chemical group. Adenosine is a naturally occurring Xanthine in the brain that is used as a neurotransmitter at some synapses. One effect of caffeine is to interfere with adenosine at multiple sites in the brain including the reticular formation. Caffeine also acts at other sites in the body to increase heart rate, constrict blood vessels, relax air passages to improve breathing and allow some muscles to contract more easily.

It is active in cardiovascular transmission, nervous stimulation and other stimulatory activities, but several researches have shown that prolonged consumption of caffeine from different sources may also cause sleep deprivation, nausea, vomiting and depression. The American Academy of Pediatrics recommends that adolescents get no more than 100 mg of caffeine a day

 The content of caffeine varies from different sources available in the market that includes tea and coffee. The percent of the caffeine in tea (Camellia sinensis) and coffee (Coffea spp) varies with the species available which is very much essential for human nutritional purposes and diet.

 The test method applies the evaluator study of the caffeine content in tea and coffee. There are several methods which includes the estimation by HPLC (high performance liquid chromatography) and spectrophotometric assay. But in this study the evaluation of caffeine content is done by FT-IR in coffee and tea samples and simultaneous validation by HPLC and spectrophotometric method. The procedure involves the rapid detection method, by less consumption of the organic solvents with high recovery percentage and hence cost effective. A comparative study of the tea and coffee, caffeine content has been established in the following procedure.

Materials & Methods:

 All the glassware was properly cleaned before use. The chemicals and reagents used in this study were of high quality at least GR grade. Two brands of instant coffee samples and four brands of tea samples were obtained from market. The coffee and tea samples were kept at room temperature throughout the analysis.

Determination of caffeine in coffee and tea samples by High Performance Liquid Chromatography

 Standard solutions

 Caffeine stock solution of 1000 ppm was prepared by accurately weighing 100 mg of pure caffeine and quantitatively transferring it into 100 ml volumetric flask. Working standards of 50, 100, 150, 200, 250 ppm were prepared by serial dilution of the stock solution with Chloroform.

 Sample preparation and analyte determination

 2.5g of tea and coffee samples were taken, caffeine was extracted with chloroform. The standards and the samples were run in the HPLC system (PerkinElmer). The following were the HPLC conditions:

Column, normal phase – Brownie 250 x 4.6 mm; flow rate, 1 ml/min; detector, UV/Vis set at 273 nm; mobile phase – methanol: water (60: 40); sample volume 20 ml.

A calibration curve of peak areas versus concentration of the standards was plotted. The caffeine level of the various samples was calculated using the calibration curve.

Fig- 1

Determination of caffeine in coffee and tea samples by UV/Vis Spectrophotometry

 Caffeine stock solution (1000 ppm) was prepared by dissolving 100 mg of pure caffeine in 100 ml of distilled water. 2, 3, 5, 8 and 10 ppm caffeine working standard solutions were prepared by serial dilution of the stock.

 Sample preparation and analyte determination

 0.2 g of sample is taken in 100 ml volumetric flask and dissolved by distilled water. 10 ml of the dissolved sample is passed through C18 bond elute cartridge under vacuum. The caffeine content is eluted by 10 ml of mobile phase (0.005M Sodium acetate: Tetrahydrofuran – 95: 5 at pH 5.0). Finally the solvent was collected in 10 ml of volumetric flask followed by washing. The resultant solvent is measured at 275 nm in Spectrophotometer (PerkinElmer make). The caffeine levels of the samples were calculated using the standard curve drawn with the standards.

Fig-2

Determination of caffeine in coffee and tea samples by FTIR with HATR

 Standard solutions

 Caffeine stock solution of 10000 ppm was prepared by accurately weighing 1000 mg of pure caffeine and quantitatively transferring it into 100 ml volumetric flask. Working standards of 250, 500, 1000, 2000, 3000 ppm were prepared by serial dilution of the stock solution with Chloroform.

 Sample preparation and analyte determination

 2.5g of tea and coffee samples were taken, caffeine was extracted with chloroform. The standards and the samples were scanned using FTIR attached with HATR (PerkinElmer).

 Spectra were collected of the standards and the sample solutions. A calibration curve of peak height at 1659 cm-1 (the characteristic response of caffeine) versus concentration of the standards was plotted. The caffeine level of the various samples was calculated using the calibration curve.

Fig-3

RESULTS AND DISCUSSION

 The calibration curves (Fig. 1, 2 & 3) illustrate a positive linear relationship between the instrumental signal and the concentration of caffeine standards for HPLC, UV/Vis Spectrophotometry and FTIR with HATR methods.

Table 1

Conclusion

 The caffeine content of the tea and coffee samples was not found to be alarming since it correlated well with values declared in literature. Though the recovery by spectrophotometric method is better than other two methods, FTIR/HATR method is less time consuming, easy and most cost effective as it does not require any expensive solvents like other two methods. From the recovery study it is also clear that this method is equal accurate w.r.t. conventional methods and hence may be used in daily analysis.

 Contributed by: Ms. Atashi Banerjee; Mr. Sourendranath Banerjee and Mr. Bamdev Sinha under guidance of Prof. Barun Gupta
ANALYSIS OF TRACES OF RARE EARTH ELEMENTS IN DIFFERENT MATRIX USING HIGH RESOLUTION ICP-OES
TOXICITY STUDY OF ENVIRONMENTAL SAMPLES COLLECTED FROM DIFFERENT INDUSTRIES

Leave a Reply

Your email address will not be published. Required fields are marked *

RECENT POSTS