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OBJECTIVE

:
 To determine the concentration of caffeine in tea bag by using UV-VIS
spectrophotometric method.
 To study the right techniques in handling UV-VIS Instrument.

INTRODUCTION:

Caffeine is a central nervous system stimulant
of the methyl xanthine class. It is the world most
widely consumed psychoactive drug, but unlike
many other psychoactive substances it is legal
and unregulated in nearly all parts of the world.
Caffeine is most fond in the bean of coffee plant
and the leaves of bushes. Up to 400 milligrams
Figure 1 : Structure of caffeine (mg) of caffeine a day appears to be safe for
most healthy adults. That’s roughly the amount
of caffeine in four cups of brewed coffee, 10 cans
of cola or two ‘energy shot’ drinks. Although
caffeine use may be safe for adults but not a
good idea for children. Adolescents should limit
themselves to no more than 100mg of caffeine per day.

Even among adults, heavy caffeine use can cause unpleasant side effects. Caffeine are not
good for those who take certain medications. Heavy daily caffeine use more than 500 to
600mg a day may cause side effects such as insomnia, nervousness, restlessness, irritability,
stomach upset, fast heartbeat and muscle tremors. Some people may be more sensitive to
caffeine than others. If you are susceptible to the side effects of caffeine, just small amounts
of it may prompt unwanted effects, for example sleep problems. How you react with caffeine
may be determined in part of how much caffeine you consumed. People who did not take
caffeine regularly tend to be more sensitive to its negative effects. Others factors include
body mass, age, medication use and health conditions such as anxiety disorder. Research also
suggest that men may be more susceptible to the effects of caffeine than women.

Caffeine works by stimulating the central nervous system (CNS), heart, muscles and the
centre that control blood pressure. Caffeine can also rise the blood pressure, but might not
have this effect in people who are used to it. Caffeine can also act like a ‘water pill’ that’s
increase the urine flow. But again, it may not have this effect in people who consume caffeine
regularly. Also, drinking coffee during moderate exercise is not likely to cause dehydration.

Glass and plastic can be used in the visible region as well. photodiode. Containers of quartz or fused silica are necessary in the UV range and can be used in the 700 -3000nm infrared region. UV-VIS spectra can be used to detect the presence of absorbing functional groups or chromophores. a radiation detector which converts transmitted radiation into a useable signal and a signal processor or readout. photomultiplier tube (PMT). This wavelength will allow valid absorption measurements to be made on analyte samples that contain mixture of materials. The detector are phototube. At around 350nm. UV-VIS spectroscopy is valid. This is to ensure that the absorbance will be high and constant in narrow range around the chosen wavelength. The sample container. UV-VIS Molecular Absorption Spectrometry is the most common analytical technique in the analytical laboratory in which involves the absorption of ultraviolet and visible radiation for quantitative purposes. Absorptions commonly occurs with many organic molecules. Many UV-VIS spectrophotometer use a deuterium lamp for the UV region and switch to tungsten filament lamp at 350nm for the visible region. The analysis should be done at a wavelength of maximum absorbance and located in relatively flat region of the spectra. absorption involves bonding (outer valence) electrons. UV-VIS spectrophotometer are made up of stable source of radiant energy. . wavelength range and stability for detectable and reproducible results. a transparent sample container. simple and cost effective method in determining the concentrations of absorbing species if applied to pure compounds and used with the appropriate standard curve. The optimal wavelength should be selected on the good absorbance of the analyte and low absorbance by other species presence in the solution. The electrical excitation of deuterium at low pressure results in continuous spectrum of emitted radiation from 160nm to the beginning of visible region which is in 375nm. metals and metal-organics complexes. the instrument switches its radiation source that a black body and thus is temperature dependant. a device for isolating specific wavelength. In UV-VIS. that we called cuvette or cell. The wavelength selector are filters and monochromators. photodiode array. must be constructed of a material that is transparent to radiation in the wavelength range of interest. A radiation sources of spectroscopy must generate a beam with sufficient power. A standard curve relating absorbance to concentration can be developed for any compounds and used to determine the concentration of sample containing the same compound.

50 ml V1 (1000ppm) = 30 ppm (25ml) V1 = 0. V1 (1000ppm) = 10 ppm (25ml) V1 = 0.RESULT AND CALCULATIONS: Table 1: Preparation of Standard Caffeine Solution Standard Concentration (ppm) Volume From 1000ppm Caffeine Stock Solution (ml) Blank 0 0 1 10 0.75 4 40 1.75 ml V1 (1000ppm) = 40 ppm (25ml) V1 = 1.25 ml Table 2: Absorbance of Caffeine Standard Solution at λ max .25 2 20 0.25 ml V1 (1000ppm) = 20 ppm (25ml) V1 = 0.00 5 50 1.00 ml V1 (1000ppm) = 50 ppm (25ml) V1 = 1.50 3 30 0.25 Calculation of preparation Standard Caffeine: 100mg = 100 x 10-3g 100 x 10-3g = 1000ppm 100mg Dilution Equation: M1V1=M2V2 thus.

4 0.2 f(x) = 0x + 0.055 30 0.91 1 0.130 2 0.105 Calibration Curve of Caffeine Standard Solution 1.8 Absorbance 0.96 R² = 0.055 3 0.6 0.105 50 0.019 20 0.4 1.2 0 0 20 40 60 80 100 120 140 160 Concentration Figure 1: Calibration curve of caffeine standard solution Table 3: Absorbance of Tea Sample at λ max .130 Tea Sample Absorbance 1 0.085 40 0.Concentration of Standard Solution (ppm) Absorbance 10 0.

105 = 0.370 Calculation of tea sample concentration based on the Beer’s Law: y= mx + c where.130 48. m = gradient.0027x – 0.0013 X = 20.370 ppm Average concentration of tea sample: (48.105 39.0. Table 4: Concentration of tea sample based on the calibration curve equation Tea Sample Absorbance Concentration (ppm) 1 0.130 = 0.0027x – 0. y = absorbance.630 + 20. x = concentration.0013 X = 48.055 = 0.284 ppm .055 20.630 ppm Tea sample 2: Y = mx + c 0.0027x – 0.0027x .630 2 0.851 3 0.0013 X = 39.370) ppm = 36.851 + 39. Tea sample 1: Y = mx + c 0. c = y-axis From graph: y = 0.851 ppm Tea sample 3: Y = mx + c 0.0013 thus.

42 mg L Percentage weight (w/w) of caffeine in triplicate sample: (Mass of solute ÷ mass of solution) x 100 % (181. A = absorbance. 3 Actual concentration of tea sample: Average concentration x dilution factor = 36.67 x 10-3 Tea sample 2: 0.66 x 10-3 Calculation for the tea sample molar absorptivity using the equation: A = ɛbc where.00) (48.64 x 10-3 3 0.00) (39.630) ɛ = 2.5L = 181.851) ɛ = 2.84 ppm Amount of caffeine in triplicate sample: 362. ɛ = molar absorptivity.284 ppm x 10 = 362. c = concentration of analyte Tea sample 1: 0.105 = ɛ (1.370) .630 2.84 mg x 0.07 % Table 5: Molar absorptivity of tea sample Tea sample Absorbance Concentration (ppm) Molar Absorptivity (ɛ) 1 0.370 2.055 20.105 39.851 2.055 = ɛ (1.00) (20.64 x 10-3 Tea sample 3: 0.130 = ɛ (1. b = length of cuvette.130 48.67 x 10-3 2 0.42 mg ÷ 2000 mg) x 100% = 9.

the energy happened in this type of transition is rather a high energy level and their positions are sensitive to the presence of substitution. Therefore. Secondly. this is because the λ max is the wavelength that gives the maximum value applied in the experiment. After calculations had been made.105 respectively. 0. ɛ = 2.105 and 0. In this experiment. 0. the absorbance value that take was taken are 0.055 and 0. Kriz and Vyvyan (2015).π* transition. the selection of the wavelength should be made at the λ max. Beer’s law applies monochromatic radiation with use of continuous source and a monochromator which will provide a very narrow band with an effective bandwidth. According to Pavia. 0. Beer’s Law typically applied in this experiment. Meanwhile.130. we can observed that there are few chromophores presence in the caffeine molecule by the presence of carbonyl group (C=O) in it. the spectrum for standard caffeine and caffeine from the tea sample were obtained. Beer’s law is strictly valid only where the molar absorptivity changes little wavelength. The absorbance values that we obtained from the spectrum of standard caffeine was 0.019.055.085. Lampman.66 x 10-3 DISCUSSION: In this experiment. there were possibilities that the structure undergo π . This energy transition was due to the presence of double bond and will occur in the near UV region. A chromophore is the functional group of an organic molecule that responsible for the molecule to be coloured. for the sample we analysed. we obtained the percentage weight (w/w) . The linearity of the Beer’s Law is limited to chemical and instrumental factors. solute-solute interaction and hydrogen bonding which will affect the analyte concentration and its absorptivity. Beer’s law describe absorption behaviour of relatively low analyte concentration in a solution as a limiting law. Therefore. This particular chromophore will undergo an n – π * transition around 280-290 nm.130 according to their respective concentration. 0. by this fact it is clearly stated why the peak of 285nm wavelength was chosen as the taken into account. The maximum peak of absorption was measured at peak 285nm. At high concentration will involve solute-solvent interaction. The peak maximum correspond to the maximum value of molar absorptivity and hence the sensitivity of the analysis.

3. Lampman. Cengage learning. (2007). F. but we cannot said it is totally succeed even though from the graph we obtain. Fong.R. If the solution contains many solute. In liquids.. Singapore: Cengage learning Asia Pte Ltd.org/10. CONCLUSION: Caffeine in the tea sample was successful analyse by using UV-VIS spectrophotometer. REFERENCES: 1. to get a good result of the Beer’s law plot.0013. Skoog. Holler. D. & Ghollap. Belmont.9926 is nearly to 1..V. S. the R2 = 0. This is because. African Journal of Pure Applied Chemistry. Principle of Instrumental Analysis (6th Edition).. The rate of change in absorbance with wavelength is the smallest at the maximum peak of absorbance curve. (5th Edition). International Journal of Analytical Chemistry.R. T. Dobrinas. CA: Brooks/Cole.J. The experiment been made. the extinction coefficient will usually changes slowly with the wavelength. Therefore. G.M. D.S.1155/2015/170239. The Beer’s law equation for this analysis is y = 0. Scientific study &Research. The average concentration of caffeine in triplicate sample is 362. Coffee And Other Beverages. S..N. S. Atomssa. Optimization of a UV-VIS Spectrometric Method for Caffeine Analysis In Tea Bag. G.doi. 5. the sample container or the cuvette is not matched and it is not clear and transparent to let the light to penetrate through it completely. it could lead to a greater scatter effect of the source of radiation. Bhawani. the concentration of caffeine in the tea sample was successful determined by graph. Biotechnology. A.S.A.0027x + 0. Characterization of Caffeine and Determination of Caffeine in Tea leaves Using UV-VIS Spectrometer. the concentration of the sample analysed must be diluted to become a sample solution that is very low concentration. (2013). & Crouch. (2010). S.42mg.. Pavia.. Chemistry & chemical engineering. 1-8. & Mohamad Ibrahim. A. (2015). M. 2.07%. . Spectrometric Analysis of Caffeine.A.84ppm and the amount of caffeine presence in it is 181. Other than that. 14(2). J. This came from the result in plotting the Beer’s law graph to figure out the quantitative value of the caffeine from the tea samples we had prepared.with the value of 9. 4. food industry. 71-78.. (2015). the measurement will be made at this particular peak to minimize the errors produced by errors in wavelength in the instruments that is errors due to having a different extinction coefficient than assumed. Kriz. Soceanu. Introduction to Spectroscopy. & Vyvyan. Popescu.. Next. V. 5(1). Retrived from http://dx.L.

2. What is the purpose of this method? Background-correction method is to minimize the derivations and enhancing the signal from the analyte. Background-correction methods have been developed for the determination of caffeine in beverage. it is also make peak appears sharper and easier to take information. . This is because there are other signal detected by the detector from things other than sample resulting a loss in spectral detail such as broadening of peak and peaks in place other than where the sample absorb.6nm.QUESTIONS: 1. The longer the tea bag in hot water. coffee and tea by second derivative ultraviolet spectrometry. The concentration of caffeine (along with the flavour molecules and everything else) will slowly trend towards an equal concentration in the leaf and in the water. 3. What is the advantage of applications of derivative spectra? Derivative spectrometry is very useful to separate the signal of analytical compound from background or other compound signal. the closer to equilibrium you will get. The second derivatives of caffeine is measured at 298. Why does caffeine of tea increase as we boil it for more time? Leaving the tea bag longer will make a stronger cup of tea. Besides.