Materials and Methods
Five different types of brand tea namely Tetley-Premium, Lipton-Taaza, Finlay-Premium, HRC-Clevedon, Ispahani-Blender’s choice were collected from the local market at a time.
Determination of caffeine content in tea samples
Preparation of stock solution
5g tea sample was taken in a 500ml conical flask. 10g crystal of MgO and 200ml of distilled water were added to the sample simultaneously. The mixture was then warmed in the water bath maintained at 40? for 2hrs. Then, it was fettered through whatman- 42 and the filtrate was collected in 200 ml volumetric flask. The volume of the filtrate was made up to the mark by adding distilled water and used as stock solution.
Extraction
200ml of filtrate was taken in 500ml conical flask and 20ml diluted H2SO4 (2ml conc. H2SO4 + 18ml dist. H2O) was added. This mixture of 220ml volume was then heated; at temperature 90? ±2? maintained in a water bath and reduced the volume of the mixture to about 50ml. The concentrated mixture was again filtered through whatman-42 and collected in the separating funnel. Then 20ml chloroform was added with the filtrate in the separating funnel and shaken well for 20 times. The separating funnel was then kept undisturbed on a stand for 10 minutes. The washed chloroform from the bottom of the separating funnel was collected in 50ml conical flask. The same filtrate was then washed thoroughly with different volumes (viz, 20, 15, 10 and 5ml) of chloroform and the total volume of the collected chloroform was washed with 5ml 1% KOH in a clean separating funnel and was collected in a 50ml oven dried conical flask which was previously weighed.
Procedure
Each conical flask was cleaned; dried (oven-dry); marked and weighed in electric balance before receiving the washed chloroform. After receiving the chloroform the respective conical flask was kept in electric oven at 70? for complete dryness. The weight of the dried concial flask was recorded in electric balance.
Calculation Caffeine = (S-B) mg/g Where, S= weight of conical flask with caffeine after dryness, B= weight of conical flask before filtrate collection.
Determination of Polyphenol in tea samples
Preparation of 1000 ppm tannic acid stock solution
1g tannic acid was taken in a 1000ml volumetric flask. Then, small amount of distilled water was added and shaken well to dissolve tannic acid. Then, the volume was made up to the mark with the addition of distilled water. This solution was used as 1000 ppm stock solution. From 1000ppm stock solution 1ppm, 2ppm, 3ppm, 4ppm & 5ppm solution were prepared.
Procedure
100 mg of tea sample was boiled in a water bath with 100 ml of water for 30 minutes and filtered. 0.2 ml of the sample solution and 5ml of the reagent (100mg of FeSO4. 7H2O and 500mg of Rochelle salt in 100ml of water) were taken in a 25ml volumetric flask and filled to the mark with Sorensen’s phosphate buffer of pH 7.5. Then the absorbance was measured at 540 nm against a blank solution substituting water for the reagent and the amount of polyphenol was determined using the correction factor (cf) obtained from the calibration curve made by using different concentration of tannic acid. Black tea samples were analyzed for TF, TPC, TR, HPS and Total Liquor Colour (TLC) by following the method reported by Thanaraj and Seshadri. The briskness and colour indices were worked out as suggested by Ramaswamy [25], [briskness index (BI) = (TFs x 100)/(TF + Caffeine) and colour index (CI) = (TFs x 100)/(TR + HPS)]. Every data was the mean value of three samples (bought one time and split into three samples) and statistical analysis was done using the computer progamme (SPSS). Means were compared using the least significant difference test (LSD) at 0.05.