INTRODUCTION:

Caffeine is a member of the methyl xanthene derivatives family. It is the world's most widely used psychoactive substance¹. At prescribed doses, caffeine has varying effects on learning and memory, although it generally improves reaction time, alertness, focus, and motor coordination, depending on body size and tolerance².

Caffeine acts as antagonist for all adenosine receptor subtypes (A1, A2a, A2b, A3) in the central nervous system. Caffeine increases alertness and decreases drowsiness³.

Coffee is the most popular beverage in the world after water, with approximately 1.6 billion cups consumed every day. Caffeine's behavioral effects are generated by adenosine receptor antagonism, which leads to changes in numerous neurotransmitter systems. Caffeine may be especially effective in low-arousal situations since it improves attention⁴. Tea contains caffeine, which is naturally found in coffee, cola, chocolate, and cocoa, as well as added to numerous energy drinks, pharmaceuticals, and cosmetics⁵. According to the technique of production, commercial tea is split into three categories: fully fermented black tea, semi-fermented oolong tea, and non-fermented green tea. Black tea and green tea are the most well-known and consumed types⁶.

MATERIALS AND METHODS:

Chemicals:

Green tea and black tea leaves were purchased from Local market. Standard caffeine was obtained from SD Fine chemicals, India.

Extraction of Caffeine from Green and Black tea samples⁷:

Caffeine is extracted from Green and black tea samples using two different solvents. To 10g of Tea leaves 100 ml distilled water was added and kept on water bath at 70°C for 40min. Then the solution was filtered to obtain the infusion. To each 15ml volume of infusion 15ml of Dichloromethane and ethyl acetate were added separately and shaken vigorously in two separating funnels. On standing aqueous and oraganic layers were separated and Organic layers of Dicloromethane and Ethylacetate were evaporated to dryness at room temperature. Dry Caffeine crystals from each extract were obtained after 24hrs of drying. All the four samples were stored at cool and dry place till further use.

Qualitative analysis:

Murexide test was performed for each sample. To the sample potassium chlorate and a drop of HCl was added. This sample was evaporated to dryness and the residue is exposed to ammonia vapor then colour was observed.

Melting Point determination:

Purity of compounds was checked by melting point. Melting point of extracted compounds was determined using micro controller-based melting point apparatus.

Thin layer chromatography:

Caffeine in the samples was identified using a thin layer of silica gel G (0.5mm thick) coated on a 12 x 20cm glass plate. For determining the Rf value, dried silica gel G-coated plates were used. The sample was first dissolved in water. As the mobile phase, a mixture of chloroform and ethanol in a 9.5:0.5 ratio was prepared. Ten milliliters of this solvent mixture were poured into a 100ml beaker, which was then covered with aluminum foil to minimize solvent evaporation and left undisturbed on a stable surface. The stationary phase used was a precoated aluminum plate with silica gel. A horizontal line was drawn with a pencil about 1cm from the bottom edge of the plate and labeled A, B, and C. The dissolved sample was spotted onto the designated positions on the plate. The plate was then placed in the beaker containing the solvent, allowing the mobile phase to rise via capillary action. Once the solvent front had moved a sufficient distance, the plate was removed from the chamber, dried in an oven, and observed under UV light. The spots corresponding to caffeine were then analyzed, and their Rf values were calculated.

Distance travelled by Solute

R _f= _{––––––––––––––––––––––––––––––––––––––––––}

Distance travelled by Solvent

FTIR:

FTIR identifies chemical bonds in materials via their infrared absorption spectrum. Transmission and attenuated total reflectance modes permit analysis of a wide range of powders. Potassium bromide was taken and transferred to mortar and pestle triturated until the powder completely mixes. 100mg of sample and potassium bromide was taken and transferred to mortar and pestle, triturated until both mixes properly, the pellet was prepared and used for further analysis. The spectrum was obtained with bonds and bond lengths.

Quantification of Caffeine using UV-Visible Spectroscopy:

Standard solution of Caffeine was scanned in the UV range of (200-400nm) and the spectrum obtained was recorded. From the Spectrum the maximum absorption was observed for the compound and the caffeine is quantified by the method established previously⁷.

RESULTS:

Extraction of Caffeine from Green and Black tea samples:

Caffeine is extracted using two solvents EC and DCM from Green tea and Black tea leaves obtaining Four samples of Caffeine. Caffeine yield was highest from black tea dichloromethane extract and lowest in green tea ethyl acetate extracts.

Table 1: Yield of Caffeine from Green and Black tea

Sample	Solvent	Yield obtained
Green Tea(GTEA)	Ethyl acetate	0.72%
Black Tea(BTEA)	Ethyl acetate	0.82%
Green Tea(GTDC)	Dichloromethane	0.85%
Black Tea(BTDC)	Dichloromethane	1%

Qualitative photochemical testing:

The extracted caffeine was identified by simple chemical tests named Murexide test. Caffeine being a purine alkaloid shown positive result in all the samples and Pure caffeine.

Melting point determination:

The melting point is the temperature at which a pure substance's solid and liquid phases coexist in balance. For crystalline solids, this temperature is a distinct and consistent value, often used to help identify pure elements and compounds. Melting point of extracted compound was determined using micro controller-based melting point apparatus. Caffeine extracted from Green and Black tea leaves using dichloromethane showed melting point very close to the standard caffeine.

Table 2: Melting Point of standard and Sample

Standard	Melting Points	Caffeine Sample	Melting point of caffeine DCM extract
Caffeine (Pure)	237±0.178°C	Green Tea (GTEA)	236±0.571°C
		Black Tea (BTEA)	236±0.378°C
		Green Tea (GTDC)	237±0.241°C
		Black Tea (BTDC)	237±0.123°C

Characterization of standard and sample caffeine extracted from EC and DCM by TLC:

The R_fwas calculated by taking midpoint of the obtained spot. The Rf values caffeine extracted from green and black tea were found to be same as that of standard caffeine (Figure 1). The R_f of Caffeine extracted from Green and Black tea with Ethyl acetate was found to be 0.67cm and 0.66cm and with Dichloromethane was found to be 0.7cm and 0.68cm, respectively.

A. Caffeine from EC extraction B. Caffeine from DCM extraction

a. Pure caffeine b. caffeine from Green tea c. Caffeine from black tea

Figure 1: TLC Chromatogram of Caffeine from Green and Black tea

Characterization of standard and sample Caffeine by Standard by Fourier Transform Infrared Spectroscopy:

In FTIR analyses, Infrared light from the light source passes through a Michelson interferometer along the The infrared (IR) spectra of the various tea extracts and pure caffeine reveal similar functional groups across the samples, with some minor variations. Caffeine extracted from green and black tea with ethyl acetate (GTEA, BTEA) displayed the characteristic absorption bands that include primary amine N-H stretching at 3113cm^-1, sp³-hybridized carbon (sp³-C) stretching at 2848cm⁻¹ and C=O stretching at 1656 cm⁻¹(Figure 2). Caffeine extracted from green and black tea with Dichloromethane (GTDC, BTDC) displayed the characteristic absorption bands that include primary amine N-H stretching at 3383cm⁻¹, sp³-hybridized carbon (sp³-C) stretching at 2901 cm⁻¹and C=O stretching at 1659cm^-1. These findings are in characteristic range of 2850–3000 cm⁻¹ for primary amine N-H stretch, 2750-3000cm⁻¹ for sp³-hybridized carbon (sp³-C) stretch and 1650–1750 cm⁻¹for C=O stretch (Figure 3).

These patterns suggest that the functional groups in the tea extracts remain relatively unchanged despite the use of different solvents (ethyl acetate or dichloromethane). The pure caffeine sample also exhibits these same functional groups, with N-H stretching at 3339 cm⁻¹, sp³-hybridized carbon (sp³-C) stretching 2900 cm^-1, and C=O stretching at 1730 cm⁻¹ (Figure 4). indicating that caffeine retains similar bonding characteristics but with slight shifts in absorption peaks. Overall, the results indicate that both the tea extracts and pure caffeine share common functional groups, with subtle differences in bond environments between the samples.

Figure 2: FTIR Spectrum of Caffeine from Ethyl acetate extraction from Green and Black Tea

Figure 3: FTIR Spectrum of Caffeine from Green tea and Black tea by Dichloromethane

Figure 4: FTIR Spectrum of Standard(Pure)Caffeine

Quantitative analysis

Standard solution of Caffeine was scanned in the UV range of (200-400nm) and the spectrum obtained was recorded (Figure 5). From the Spectrum the maximum absorption was observed 272nm and percentage purity of caffeine samples was estimated from regression equation obtained from standard plot the assay values are represented in Table 3.

Figure 5: Absorption maxima of Caffeine

Table 3: Caffeine % Purity in different green and Black tea samples

Sample	Solvent	% Purity
Green tea	Dichloromethane	98.36%
Black tea	Dichloromethane	99.11%
Green tea	Ethyl acetate	98.12%
Black tea	Ethyl acetate	98.63%

DISCUSSION:

Caffeine is the most versatile compound in that it is consumed in a variety of beverages and medicines by practically everyone. Different varieties of black and green teas are available and many analytical methods have been deployed for the estimation of caffeine singly and simultaneously.⁸ Caffeine present in these beverages is different and it was explored that the higher temperature and longer brewing time result the higher caffeine content.⁹

The percentage yield of caffeine varied depending on both the type of tea and the solvent used for extraction. Dichloromethane proved to be a more efficient solvent compared to ethyl acetate, with caffeine yields of 0.85% and 1.00% from green and black tea respectively. In contrast, ethyl acetate extraction yielded slightly lower amounts—0.72% from green tea and 0.82% from black tea. This difference can be attributed to the higher polarity of dichloromethane, which facilitates more efficient solubilization and extraction of the moderately polar caffeine molecule. The greater yield from black tea in both solvents aligns with previously reported data indicating that black tea generally contains higher caffeine content due to its oxidation level during processing.^10,11 The melting point of the extracted caffeine closely matched the literature value for pure caffeine (~238°C), providing initial confirmation of purity and identity.¹² Phytochemical tests specific to alkaloids further supported the presence of caffeine in the extracts.

Thin layer chromatography provided qualitative confirmation, with Rf values consistent across different solvents and tea types. The Rf values for caffeine extracted with dichloromethane were 0.70cm for green tea and 0.68cm for black tea, while ethyl acetate extracts exhibited Rf values of 0.67cm and 0.66cm, respectively. These values are in close agreement and suggest high consistency and reliability of the extraction and TLC methodology. The slight variation in Rf may result from subtle differences in solvent interaction or matrix effects from the tea samples. Fourier Transform Infrared Spectroscopy (FTIR) was used to identify characteristic functional groups in the caffeine samples. The presence of functional groups such as amine (C–NH₂), aliphatic sp³ carbon (sp³–C), and carbonyl (C=O) groups were observed, all of which are consistent with the molecular structure of caffeine. These peaks further validate the successful extraction and isolation of the correct compound.

Quantitative analysis via UV-visible spectroscopy demonstrated high purity of the isolated caffeine, with percent purity values ranging from 98.12% to 99.11%. This high degree of purity indicates the efficiency of the extraction process and minimal contamination or degradation of the target compound during isolation.

CONCLUSION:

This study demonstrates that dichloromethane is a more effective solvent than ethyl acetate for extracting caffeine from both green and black tea leaves, yielding higher quantities with comparable purity. The successful identification and characterization of caffeine through melting point analysis, TLC, FTIR, and UV-visible spectroscopy confirm the reliability of the extraction process. Variations in caffeine yield between green and black tea highlight the significant impact of tea type and processing on alkaloid content. These findings reinforce the feasibility of using organic solvents for efficient caffeine isolation. The high purity levels achieved suggest that these methods can be adapted for both analytical applications and industrial-scale extractions. Overall, this research provides a strong foundation for future studies aimed at refining natural alkaloid extraction techniques from diverse botanical sources.

CONFLICT OF INTEREST:

The authors have no conflicts of interest.

ACKNOWLEDGMENTS:

Authors are thankful for the management of RBVRR Women's college Barkatpura, Hyderabad for providing necessary facilities to carry out this research.

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