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The main purpose of this experiment is to determine the amount of caffeine in percent
weight in tea samples using background-correction and second derivative ultraviolet-visible
spectrophotometry. Background-correction methods were used to remove the interferences on
the reading and the second derivative for caffeine were measured at 292.92nm. 2.4675g of tea
sample were boiled to extract the compounds in the sample and the standard solutions of
10ppm, 20ppm, 30ppm, 40ppm and 50ppm were prepared from 100ppm by using dilution
technique. 3 samples of caffeine were prepared from the boiled sample. Then, the samples
and standard solutions are analysed with a blank sample using second derivative of UV
spectrometry. Copper acetate and sodium hydroxide were added into the standard and sample
solution. The instrument used is Ultraviolet-visible spectrometer Perkin Elmer Lambda 35. In
order to ensure the amount of caffeine in samples is in the range of standard solutions, the
samples were diluted with dilution factor of 10. The average concentration of caffeine in the
triplicate samples is with relative standard deviation of 3.13%. The amount of caffeine in
the samples is 37.82mg with 1.533w/w%.
OBJECTIVE
To determine the amount of caffeine in percent weight in tea samples using
background-correction method and second-derivative Ultraviolet-visible spectrometry.
SAMPLES
Tea bag (2.4675g)
CHEMICALS
1. Caffeine standard (100ppm)
2. Copper acetate monohydrate (0.025M)
3. Sodium hydroxide (1.0M)
APPARATUS
1. Volumetric flask
2. Filter paper
3. Conical flask
4. Centrifuge tube
5. Pippete
6. Test tube
INSTRUMENT
Ultraviolet-visible Spectrometer Perkin Lamda 35. The Lambda 35 is a double beam UV/Vis
spectrophotometer from Perkin Elmer, packing pre-aligned Tungsten and Deuterium Lamps.
It has a wavelength range of 190-1100nm and a variable bandwidth range of 0.5 to 4nm.
EXPERIMENTAL PROCEDURE
A. Preparation of reagents
1. Preparation of 0.025M Copper(II) Acetate solution
a. About 0.25g Cu(CH3COO)2 solid was weighed and then was diluted in distilled water.
b. The solution was then transferred into a 50ml volumetric flask and distilled water was
added until the mark.
C. Preparation of sample
a. 2.4675g of tea was added into 200mL distilled water and was heated for 15 minutes.
b. The mixture was let to cool at room temperature.
c. The cooled sample was then transferred into 250mL volumetric flask and distilled
water was added until the calibration mark (working sample solution).
e. The working sample solution was prepared for analysis by mixing 1mL of 1.0M
NaOH solution, 4mL of 0.0025M Cu(CH3COO)2 and 5mL of standard solution in 3
different vials.
d. All vials were shaken and centrifuged for 10 minutes at 20rpm.
RESULTS
Table of result
Sample Concentration Weight of caffeine Percent weight (w/w
(ppm) (mg) %)
1 14.61 36.53 1.480
2 15.24 38.10 1.544
3 15.53 38.83 1.574
Average 15.13 37.82 1.533
DISCUSSION
The analysis is done at maximum wavelength where the highest second derivative from
the absorbance can be read which is at 292.92nm. We use the value of second derivative from
the absorbance curve as they are more accurate compared than the original value from the
absorbance curve. The maximum wavelength is in the range of UV region which we desired
for this experiment. Interference from tannins in tea was minimized by treating the sample
with 0.1mol dm-3 sodium hydroxide and by precipitating tannins with copper (II) acetate
Based on the spectra obtained from this experiment, we have calculated the value of m and c
by performing a least square method. Then, by using the formula y=mx+c, we have obtained
the value of y theoretical. A graph of absorbance against standard concentration was plotted
based on both value of y data and y theoretical. From that, we can see a small difference
between the absorbance from the data and theoretical. Extrapolation of the graph was done to
get the concentration of the caffeine in samples.
The actual concentration of the sample in average after taking the dilution into account is
equal to 15.13ppm. With that value, the amount of caffeine in the triplicate sample calculated
to be 37.82 mg with the weight percent of 1.533 w/w%. This amount of caffeine is in the
range of the amount of caffeine in a tea bag by most brands of tea producers. The amount of
caffeine analysed also in the range of safe consume-able amount with less probability of
overdose. In order to ensure the accuracy between the triplicate samples, the relative standard
deviation was calculated. The calculated value of relative standard deviation of the triplicate
sample is 3.13%. The smaller the value of relative standard deviation to zero indicates the
more accurate sample preparation. Thus, the value of confidence level is higher For this
triplicate sample, there still need to be improved in the sample preparation as there might be
some error during performing this experiment that affect the accuracy of the result.
QUESTIONS
1. Write the wavelength ranges for the ultraviolet and visible regions of the spectrum.
Ultraviolet (UV) light has wavelength of 200 - 400nm while visible light covers the
range of wavelengths from 400 - 800nm.
2. List the molecular or structural features that give rise to absorption of the
ultraviolet/visible (UV/VIS) radiation in organic species.
- multiple covalent bonds
- unbonded electrons (N, O, Cl)
- hexane
Give specific example of an organic compound that would not absorb UV/VIS
radiation.
- valence electrons
- NaCl
CONCLUSION
The developed UV-Vis spectroscopy method was suitable for the determination of
caffeine in tea sample at the trace level. The amount of caffeine in percent weight in tea
samples using background-correction method and second-derivative Ultraviolet-visible
spectrophotometry was determined. The actual average concentration of caffeine in tea bag
sample is 15.13ppm that correspond to 37.82 mg with 1.533 w/w%. The amount of caffeine
is in the range of suggested amount.
REFERENCES
1. Skoog, Douglas A.; Holler, F. James; Crouch, Stanley R. (2007). Principles of
Instrumental Analysis (6th ed.). Belmont, CA: Thomson Brooks/Cole. pp. 169–
173. ISBN 9780495012016
2. Misra, Prabhakar, Dubinskii, Mark, eds. (2002). Ultraviolet Spectroscopy and UV
Lasers. New York, Marcel Dekker. ISBN 978-0-8247-0668-5.
3. Mettler-Toledo International Inc. "Spectrophotometry Applications and
Fundamentals". Retrieved 2018-07-10.