Experiment Report: Spectrophotometric Analysis of Caffeine and Benzoic Acid in Soft Drink

8/10/2019 Experiment Report: Spectrophotometric Analysis of Caffeine and Benzoic Acid in Soft Drink

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UNIVERSITI TUNKU ABDUL RAHMAN

FACULTY OF SCIENCE

BACHELOR OF SCIENCE (HONS) CHEMISTRY

UDEC1224 CHEMISTRY LABORATORY II

Name: Nitthia A/P Meyappan

ID no.: 1106171

Group members: Christine Choong Jing Wei, Ching Li Sin

Experiment no.:11

Title of the Experiment: Spectrophotometric Analysis of Caffeine and Benzoic Acid in Soft

Drink

Date of the experiment: 30th

July 2012

Lecturer: Dr. Neo Kian Eang



Table 4: Concentration of Standard Benzoic acid and Absorbance under λ’= 229nm

Calculation 1:

From the Least-Squares Regression,

a = y-intercept of the graph

a =()

= -0.00524

b = slope of the graph

b = = 0.0912 mL/mg

y’ = bx + a

Equation of the graph: y’ = 0.0912 x - 0.00524

r =linear correlation coefficient

Solution Concentration, x (mg/mL) Absorbance under λ’=

229nm

Corrected absorbance, y

blank 0.0 0.051 0.00

A 2.00 0.217 0.166

B 4.00 0.408 0.357

C 6.00 0.606 0.555

D 8.00 0.776 0.725

E 10.00 0.953 0.902

Unknown 1 3.70 0.389 0.338

Unknown 2 6.75 0.664 0.613

Ʃx (mg/mL)

Ʃx (mg/mL) Ʃy

Ʃy Ʃxy (mg/mL) n

220 30.0 1.802259 2.705 19.91 6



r =

√

r = 0.9996982675 ≈ 0.9997

r 2 = coefficient of determination

r 2

= (0.9996982675)2

r 2 = 0.9994

If r = 0.9997, then r2 = 0.9994, which means that 99.94% of the total variation in absorbance

under λ’ = 229nm, y can be explained by the stronger negative linear relationship between

concentration, x and absorbance, y (as described by the regression equation). The other 0.06% of

the total variation in absorbance, y remains unexplained.

Prediction:

When the absorbance of unknown 1 is 0.338,

y = 0.0912 x - 0.00524

Then, 0.338 = (0.0912) x -0.00524

The concentration of unknown 1, x = 3.76 mg/mL


0.613 = (0.0912) x – 0.00524

Then, the concentration of unknown 2, x = 6.78 mg/mL

Table 5: Concentration of Standard Caffeine and Absorbance under λ’’= 273nm

Solution Concentration(mg/mL)

Absorbance underλ’’= 273nm

Correctedabsorbance

blank 0.0 0.043 0.00

A 4.00 0.417 0.374

B 8.00 0.789 0.746



Calculation 2:

From the Least-Squares Regression,

a = y-intercept of the graph

a =()

= 0.0118

b = slope of the graph

b =

= 0.0821 mL/mg

y’ = bx + a

Equation of the graph: y’ = 0.0821 x + 0.0118

r =linear correlation coefficient

r =

√

r = 0.9798556039 ≈ 0.9798

r 2 = coefficient of determination

C 12.00 0.989 0.946

D 16.00 1.160 1.117

E 20.00 1.855 1.812

Unknown 1 2.60 0.254 0.211

Unknown 2 5.00 0.457 0.414

Ʃx (mg/mL)

Ʃx (mg/mL) Ʃy

Ʃy Ʃxy (mg/mL) n

880 60.0 6.122341 4.995 72.928 6



r 2

= (0.9798556039)2

r 2 = 0.9601170044 ≈ 0.9601

If r = 0.9798, then r2 = 0.9601, which means that 96.01% of the total variation in absorbance

under λ’’= 2.73nm, y can be explained by the strong positive linear relationship betweenconcentration, x and absorbance, y. The other 3.99% of the total variation in absorbance, y

remains unexplained.

Prediction:


y = 0.0821x + 0.0118

Then, 0.211 = 0.0821 (concentration, mg/mL) + 0.0118

The concentration of unknown 1, x = 2.43 mg/mL


0.414 = 0.0821 (concentration, mg/mL) + 0.0118

Then, the concentration of unknown 2, x = 4.90 mg/mL

Discussion:

This experiment was conducted to obtain the absorbance of the caffeine and benzoic acidin soft drinks through spectrophotometric analysis. Spectrophotometry is one of the mostcommonly used instrumental methods in all of science. While the term is used to refer to any

technique that uses light to measure the concentration of a chemical, most spectrophotometric

measurements rely on the absorption of light. The basic components of an absorptionspectrophotometer are shown in Fig. 1. Light passes into a monochromator where one small

band of wavelengths is selected. This monochromatic light then passes through the sample

compartment and on to a detector where it is converted into an electrical current, i, proportionalto the intensity of the light. Intensity is more correctly referred to as irradiance, P .



Some of the light passing through the sample may be absorbed by the molecules in the

sample. When a molecule absorbs a photon of light, the energy of the molecule increases. If the

light is from the ultraviolet (UV) or visible portions of the electromagnetic spectrum, thisabsorption results in electrons being promoted to the higher energy levels. We say the electron

has gone from the lowest energy state (the ground state) to a higher energy state (an excited

state). This process is illustrated in Fig. 2 below. A photon of light can be absorbed when it hasenergy (hν) exactly equal to the energy difference between the ground state ( E 1) and the excitedstate ( E 2).

Figure 2: The absorption process

To begin an absorbance measurement we must first measure the initial irradiance by

placing solvent in the sample holder. This is called the blank measurement. The solvent is then

replaced with the real sample, and the irradiance is measured. Finally, we see the relationship

between absorbance and concentration, c, by the familiar Beer’s law equation:

Where c is the concentration of the analyte in the sample, ε is the molar absorptivity of the

analyte, and b is the sample cell path length shown in Fig. 1. You should recognize that Beer’s

law predicts a linear relationship between absorbance and concentration. Experimentally, this is

shown by generating a Beer’s law plot of A (y-axis) vs. c (x-axis).

Figure 3: Beer's Law plot

In this experiment, the amount of caffeine and benzoic acid in a soft drink sample were

determined. Caffeine is a stimulant, and benzoic acid is commonly used as a food preservative

since it inhibits the growth of mold, yeast, and bacteria. The UV spectra of caffeine and benzoic

acid overlap, although there are wavelength regions where one component dominates. It’s

important that we don’t use a diet drink since aspartame absorbs in the same region of the

UV. Also, Mountain Dew generally doesn’t work well since a colorant absorbance band extends

too far into the UV. Each compound exhibits absorbance at two different wavelengths: 229 nm

and 273 nm.



From the calculation 1, b = 0.0912 indicates that every addition of concentration of

standard benzoic acid in mg/mL leads to an additional 0.0912 of absorbance under 229nm of

wavelength. If the slope were positive, we would expect absorbance to increase as the

concentration of the sample increases. Thus, for every additional concentration, absorbance will

increase by an average of 0.0912 units. Besides that, a = -0.00524which means that at zero

concentration, the absorbance is estimated to be -0.00524. In most applications, the intercept has

no useful practical interpretation. It just serves to fix the line. The dependent variable (in this

case Absorbance under λ’=229nm) is plotted on the vertical axis and the explanatory (or

independent or regressor) variable (in this case concentration in mg/mL) is plotted on the

horizontal axis.

From the graph 1, it is seen that there is a clear positive relationship between the

variables, since as concentration of the standard benzoic acid increases, absorbance increases.

The relationship can also be seen to be linear since the plotted points scatter around a straight

line. The equation of the regression line for predicting absorbance can be found to be y’ = 0.0912

x - 0.00524 or absorbance = 0.0912(concentration, mg/mL) - 0.00524. Here R 2 = 99.94%. This

indicates that 99.94% of the variation in the dependent variable, absorbance at λ’=229nm, is

explained by the explanatory variable, concentration of the sample in mg/mL. In other words,

99.94% of the variation in the absorbance at λ’=229nm on these 6 samples can be attributed to

the fact that different concentrations of the samples were made. This leaves only 0.06% to be

explained by other factors (including pure chance effects). This suggests that the model gives a

good fit to the data. From the line of best fit, the predicted the concentration of unknown 1is 3.70

mg/mL when the absorbance of the unknown 1 at the wavelength is 0.338. When the absorbance

of the unknown 2 is 0.613, then the concentration of the unknown 2 is 6.75mg/mL .The

predictions are examples of interpolation and so should be acceptable since R

2

is high. However,when substitute the absorbance of the unknown 1 and unknown 2 in the regression equation,

absorbance = 0.0912(concentration, mg/mL) - 0.00524, we got the concentration of the unknown

1 and unknown 2 are 3.76 mg/mL and 6.78 mg/mL respectively.

From the calculation 2, b = 0.0821 indicates that every addition of concentration of

standard benzoic acid in mg/mL leads to an additional 0.0821 of absorbance under 273 nm of

wavelength. If the slope were positive, we would expect absorbance to increase as the

concentration of sample increases. Thus, for every additional concentration of the standard

benzoic acid, absorbance will rise by an average of 0.0821 units. Besides that, a = 0.0118 which

means that at zero concentration, the absorbance is estimated to be 0.0118. Again, this is not particularly useful. The dependent variable, Absorbance under λ’=273 nm is plotted on the

vertical axis and the independent variable, concentration in mg/mL is plotted on the horizontal

axis.

It is clear from the diagram that there is a positive relationship between the variables. As

the concentration of the sample increases so does the absorbance. It is also reasonably clear that

there is a linear trend in the data. The equation of the regression line for predicting absorbance



can be found to be y’ = 0.0821 x + 0.0118 or y’ = 0.0821 (concentration, mg/mL) + 0.0118.

From the further calculation, r 2 is determined again. The r

2 shows that 96.01% of the variation in

the absorbance at λ’=273nm is explained by the concentration of the sample in mg/mL. The

other 3.99% of the total variation in absorbance, y remains unexplained. This advocates that the

model gives a good fit to the data. From the line of best fit, the predicted the concentration of

unknown 1is 2.60 mg/mL when the absorbance of the unknown 1 at the 273 nm of wavelength is

0.211. When the absorbance of the unknown 2 is 0.414, then the concentration of the unknown 2

is 5.00 mg/mL. Since R 2 is high, the predictions are acceptable. The concentrations of the

unknown 1 and 2 are 2.43 mg/mL and 4.90 mg/mL by calculating with the regression equation,

absorbance = 0.0821 (concentration, mg/mL) + 0.0118.

There can be many sources of error with a spectroscopic method. Electronic noise in the

detector is the most common. Mistakes with the cuvette can also be important- improper

positioning, touching with fingers or allowing dust on the cell all could introduce errors.

Safety precautions:

1. Some that must be treated with care in order to avoid damage to yourself or your

surroundings.

2. None of these chemicals should be ingested. Safety goggles, lab coat and gloves are

required at all times.

3. There are also no toxic chemicals present, so disposal directly down the drain is

allowable and safe.

4.

Use the hydrochloric acid solutions in the FUME HOOD. If any acid or base solutionsplashes on your skin, wash it off immediately with copious amounts of running water.

Conclusion:

The spectrophotometry is used to obtain the absorbance of the caffeine and benzoic acid in soft

drink, Mountain Dew. From the graph (i), the concentration of the benzoic acid in the unknown 1

and 2 are 3.70mg/mL and 6.75mg/mL correspondingly at the 229nm of wavelength. The

concentrations of the caffeine in the unknown 1 and 2 are 2.60mg/mL and 5.00mg/mL

respectively at the 273nm of wavelength from the graph (ii).

___________________ 23rd

July 2012.

(NITTHIA A/P MEYAPPAN)



Reference:

1. Amit Choudhury (2010). Correlation and Regression. Retrieved 12 Aug. 2012 from

Experiment Resources: http://www.experiment-resources.com/correlation-and-regression.html

2. Harris, D.C. Quantitative Chemical Analysis, 7th ed.; W.H. Freeman & Co.; New York:

2007; pp 228-237 and 303-318.

3. “Laboratory Experiments for Chemistry; A Basic Introduction’, 4th edition, by Wynn

and Joppich, Wadsworth Inc., 1987.

4. McDevitt, V. L.; Rodriguez, A.; Williams, K. R. Analysis of Soft Drinks: UV

Spectrophotometry, Liquid Chromatography, and Capillary Electrophoresis. J. Chem. Ed.

1998, 75, 625-629. pdf

http://www.experiment-resources.com/correlation-and-regression.html




http://jchemed.chem.wisc.edu/Journal/Issues/1998/May/PlusSub/V75N05/p625.pdf








Graph 1: Absorbance under λ’= 229nm

y' = 0.0912x - 0.00524

R² = 0.9994

-0.2

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12

A b s o r b a n c e u n d e r λ ' = 2 2 9 n m

Concentration (mg/mL)

A graph of Absorbance against Concentration



Graph 2: Absorbance under λ’’= 273nm

y = 0.0821x + 0.0118

R² = 0.9601

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25

A b s o r b a n c e u n d e r λ ' ' = 2 7 3 n m

Concentration (mg/mL)

A graph of Absorbance against Concentration

Experiment Report: Spectrophotometric Analysis of Caffeine and Benzoic Acid in Soft Drink

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